Secretions are central to the ability of the salmon louse Lepeophtheirus salmonis to parasitize salmonid hosts by promoting immunomodulation and feeding. Previous characterizations of secretory and excretory products (SEPs) have relied on pooled individuals, preventing assessment of inter-individual variation and responses to host-derived cues. A novel SEP collection method was developed to obtain samples from individual adult female L. salmonis, enabling evaluation of secretory profiles following exposure to conditioned seawater from a susceptible host, Atlantic salmon (Salmo salar), or a resistant host, coho salmon (Oncorhynchus kisutch). SEP concentrations ranged from 328 to 1597 µg mL⁻¹ , with no effects of host species or conditioning treatment on protein yield. Across 16 individuals, a mean of 101.4 proteins were detected per replicate, including 61.2 secretory proteins, indicating substantial inter-individual variation. Conditioning did not alter protein richness, yet conditioned treatments showed clear qualitative differences in composition. S. salar-conditioned lice uniquely secreted 40 proteins, including proteases, protease inhibitors, C-type lectins, gamma crystallins, and labial gland factors, whereas controls yielded a single unique protein. O. kisutch conditioning was further associated with additional proteases, protease inhibitors, epidermal growth factor-like proteins, and other putative virulence factors. Detection of chitin deacetylase-7 and an LY6/uPAR domain protein across multiple conditions highlights previously uncharacterised candidates relevant to louse-host interactions. In total, these findings establish individual-level SEP collection as a robust and sensitive approach for resolving secretomic diversity in L. salmonis and detecting host-associated modulation of parasite secretory activity obscured by pooled analyses.

Vaccination is crucial for Atlantic salmon farming, protecting against bacterial and viral infections, such as infectious salmon anemia (ISA) caused by infectious salmon anemia virus (ISAV). Salmon immune responses are highly temperature-dependent and optimal water temperatures are expected to impact vaccine efficacy, while those out of the optimal range may weaken immunity and compromise protection. Additionally, vaccination regime impacts the length of protection against most common pathogens. To assess the impact of temperature and dosing regime on commercial vaccination, we evaluated two commercial multivalent vaccines in salmon reared at 8°C, 12°C, and 15°C. Fish were intraperitoneally injected with 100 μL of either vaccine 1 (V1) or vaccine 2 (V2). Half of each group received a booster after, between 700 and 750-degree days (dd), including groups that were boosted with the opposite vaccination (V1/V2; V2/V1). After an additional ∼650 dd post boost (∼1400 dd post first vaccination), ten fish from each group were sampled for serum IgM detection. One week later, donor fish previously infected with ISAv (ISAV-HPR4 at TCID50 of 1 × 105/ml) were introduced to cohabitation tanks at a 6.5:1 ratio of cohabitants to donors. Post-infection, survival rate was recorded, and serum samples were collected for specific-IgM detection, as well as head kidney to determine ISAv load. The results show that elevated rearing temperatures (12-15°C) consistently enhanced specific antibody responses against ISAv, A. salmonicida, and V. anguillarum, whereas cold conditions (8°C) limited or delayed antibody-mediated immune responses in pre-exposed fish. Booster vaccinations and higher temperatures effectively increased and maintained IgM levels in pre-exposed Atlantic salmon, compensating for low-temperature suppression. Survival and viral load data further highlight the interaction between temperature and immune protection, with fish hold at warmer temperatures during vaccination exhibiting higher survival and more efficient ISAv clearance. These findings demonstrate that water temperature and vaccination strategy, including regimen and formulation, critically influence adaptive immunity in Atlantic salmon. Aligning vaccination protocols with seasonal and environmental conditions can maximize protection and limit pathogen persistence.

Infectious disease outbreaks remain a major driver of antibiotic use in global aquaculture, with an estimated 10,259 tons applied annually, predominantly through group-level delivery of medicated feeds. However, behavioural restructuring during disease can disrupt alignment between infection status and therapeutic delivery. Infected fish commonly reduce or cease feeding, while healthy conspecifics disproportionately consume medicated diets, reducing treatment efficiency and increasing unnecessary antibiotic exposure. Using an in vivo Tenacibaculum maritimum challenge model, custom thermal-gradient enclosures, and high-resolution computer vision-assisted behavioural analyses, we tested whether natural behavioural thermoregulation could overcome this constraint. Access to warmer zones induced behavioural fever and revealed a reproducible temporal window during which healthy and diseased fish self-segregated spatially. Thermal enrichment, even without complete segregation, restored feed uptake in diseased individuals to levels statistically comparable to those of healthy conspecifics, enabling more precise therapeutic delivery and demonstrating operational feasibility within existing production systems. These findings establish behavioural thermoregulation as a practical and scalable mechanism for improving treatment precision during aquaculture disease outbreaks, with direct implications for antimicrobial stewardship, sustainable production, and global food security.

Eyes are essential sensory organs needed by teleost Atlantic salmon for high visual acuity and survival in both the wild and in aquaculture settings. In this work, we assessed the ocular manifestations of Infectious Salmon Anaemia Virus (ISAV) infection in Atlantic salmon by a cohabitation-mediated infection assay and histological and immunohistochemical approaches. The findings reveal that Atlantic salmon with a systemic ISAV infection displayed ISAV antigen accumulation in specific ocular tissues and significant ocular morphological changes that could compromise visual acuity. Immunohistochemical analyses showed that ISAV-related ocular pathological changes correlate with changes in expression levels and/or spatial localizations of IgM, Sox6, Sox9, collagen type 1, Gata1, and CD10 in specific compartments of the eye. The cornea is likely one of the first ocular tissues to be exposed to the cohabitation-mediated ISAV infection. The ISAV-infected Atlantic salmon showed corneal dysplasia, which correlated with increased corneal stromal ISAV antigen expression, dysregulated IgM, Sox6, and collagen type 1 expression, and an induction of Sox9 expression at the corneal surface. The choroid rete mirabile of ISAV-infected eyes showed a decreased complement of erythrocytes, consistent with anaemia, while Gata1 expression, a key mediator of erythropoiesis, was upregulated compared to non-infected eyes, suggesting a potential erythropoietic compensatory response. These findings provide a basis for further study of ISAV-mediated ocular pathological changes and new insight into the pathogenesis of orthomyxoviruses in the eye.

This study explored transcriptomic alterations in the skin and head kidney of Atlantic salmon (Salmo salar) from multiple families in response to infestation with the salmon louse (Lepeophtheirus salmonis) at the chalimus and adult stages, under elevated (20 degrees C) versus standard (10 degrees C) temperature conditions. Family-specific transcriptomic comparisons identified several candidate genes potentially associated with variation in host responses among families to sea lice, as reflected by differential expression patterns between families with varying lice densities. These biomarkers were primarily involved in immune function (e.g., cd209, cd22, btn2a1, h2-q9, c1qb, muc2, muc5ac, sntx-b, kbas), tissue repair (e.g., krt13, tgm2, mmp9, mmp13), inflammation (e.g., nlrp1, il1b, nlrc3, nlrp12), iron homeostasis (e.g., hbb1, ftm) and stress response (e.g., hspa8, herc3). Under elevated temperature conditions (20 degrees C), 124 and 324 shared differentially expressed genes (DEGs) were respectively identified in the skin and head kidney of lice-infested salmon. RNA-seq analysis revealed consistent modulation of several thermal stress biomarkers (e.g., serpinh1, hsp90aa1, hspa8, cirbp, fkbp10) in both chalimus- and adult-infested salmon under elevated temperature conditions, along with pronounced differences in the diversity of transcriptomic alterations and biological pathways compared to fish parasitized under standard temperature. Our results provide insights into molecular signatures that can help to differentiate more and less susceptible salmon families, supporting the development of selective breeding strategies for improved sea lice management. The temperaturerelated transcriptomic changes highlighted the climate adaptation strategies adopted by salmon, as well as their potential vulnerability to long-term shifts in water temperature.

Nutrient deficiency can cause increased susceptibility to infectious diseases in fish, thus leading to high rates of morbidity and mortality. Thiamine deficiency complex (TDC) in fish can lead to low reproductive success and high mortality rates. Columnaris disease in salmonids, caused by Flavobacterium columnare, has resulted in devastating losses in aquaculture production and wild populations of Pacific salmon particularly associated with climate change and high water temperatures. There is growing awareness that both TDC and columnaris are emerging diseases of salmonids on the west coast of North America; however, it is unknown whether fish that survive from low/intermediate thiamine level eggs will experience latent mortality due to susceptibility to infectious diseases like columnaris. To investigate the interaction of TDC survivors and columnaris, Chinook salmon Oncorhynchus tshawytscha fry reared from either thiamine-deficient (n = 120) or thiamine-replete (n = 120) eggs were challenged with F. columnare using an immersion challenge model of infection, and morbidity/mortality, immune responses, and bacterial load were evaluated. The cumulative mortalities between the treatment groups were significantly different, with the thiamine-deficient, F. columnare-exposed fry ending the challenge with an 80.3% survival rate and the thiamine-replete, F. columnare-exposed fry ending with a 29.03% survival rate (p < 0.0001). Different transcript abundance was detected in gills and spleen of thiamine-deficient and thiamine-replete fry exposed to F. columnare. This study demonstrated that fry reared from eggs low in thiamine have an altered immune response and warrants further studies to better understand interaction with potential pathogens at different life stages.

Non-medicinal approaches for controlling sea lice (Lepeophtheirus salmonis) infestations, such as freshwater (FW) treatments, have been increasingly implemented to support more sustainable parasite control in aquaculture. This study evaluated the efficacy of FW treatment against sea lice under controlled experimental conditions and assessed the potential development of resistance across multiple generations. Sea lice populations were maintained over five consecutive generations, and FW efficacy was evaluated by quantifying lice abundance on Atlantic salmon before and after treatment. Resistance assessment involved comparative bioassays using copepodid and adult life stages from two distinct populations, one exposed to FW and the other a saltwater (SW) control group. Our results demonstrated that a 4 h FW treatment significantly reduced total lice abundance, while an extended 18-day exposure resulted in complete detachment of all lice from salmon. Importantly, bioassay comparisons revealed no evidence of resistance development, as both FW-exposed and SW-control populations maintained similar susceptibility across generations. The absence of measurable changes in salinity tolerance over multiple generations suggests that FW protocols may remain a viable and sustainable management strategy. However, the potential for genetic or epigenetic variation in FW tolerance among lice populations warrants further investigation to better understand long-term implications for treatment efficacy.

ABSTRACT Declines in wild Atlantic salmon populations in the North Atlantic with migratory routes adjacent to commercial salmonid aquaculture operations in coastal marine environments have raised concerns about the potential for direct or indirect effects this industry may have on local wild populations. To better understand potential impacts on wild Atlantic salmon in Newfoundland, Canada, we used six moored sentinel cages stocked with wild Atlantic salmon smolt to compare smolt health in an area with Atlantic salmon and steelhead trout aquaculture (Conne River/Bay d'Espoir) and without (Campbellton River/Notre Dame Bay). Average survival for wild Conne River smolt stocked in sentinel cages in Bay d'Espoir was 64.1% but varied from 35.5% to 84.8%. Average survival for wild Campbellton River smolt stocked in sentinel cages in Notre Dame Bay was higher at 73.5%, less variable and ranged from 69.7% to 78.1%. Differences in environmental variables and body condition loss among sentinel cages showed no significant association with survival. Health assessments revealed no external parasites, including the absence of sea lice on the smolt. Kidney and heart tissue samples from both locations were negative for infectious salmon anaemia virus. Prior to stocking, histological analysis revealed no epicarditis in the Conne River smolt and mild epicarditis in 6.7% of the Campbellton River smolt. Following deployment, histological analysis revealed mild epicarditis in 5.7% of the Conne River smolt and 7.7% of the Campbellton River smolt. Results suggest that local stressors, where present, are likely transient and spatially variable, highlighting the need to consider exposure duration and microhabitat influences when interpreting sentinel cage experiments. This research supports the use of sentinel cages in Newfoundland, and likely elsewhere, as a method for evaluation of wild smolt health along coastal migratory routes, whereas outcomes must be interpreted relative to short‐term site‐specific environmental conditions.

Wound healing is a complex multi-system process that enables organs and tissues to re-establish their form and function post-injury. Exposure to stress and nutritional restrictions results in energetic trade-offs at a physiological level, usually at the expense of immunity and tissue repair. Relatively few studies have focused on how stress and nutrition mediate immune function and healing capacity in fishes. We subjected wild-caught pumpkinseed (Lepomis gibbosus (Linnaeus, 1758)) to a subcutaneous incision (a standardized wound) while experimentally manipulating plasma cortisol levels (via a slow-release cortisol implant) and nutrition (via restricting food intake) to evaluate how these affected wound healing over time (2, 7, and 14 days). Wound healing was poorest for cortisol-treated fed fish at 7 days post-implant. While cortisol implants resulted in lymphocyte apoptosis and neutropenia, which are well-described immunological responses to increased cortisol concentrations, fasting had little effect. Reproductively active males had different leukocyte counts than non-reproductive males, though the direction varied with leukocyte type, suggesting a trade-off in immune response during reproduction. The negative influence of cortisol on tissue repair and inflammation aligns with the wound healing literature across taxa. However, the ability of fasting to reduce these negative effects was unexpected and deserves further study.

Gill regeneration in fish varies inter- and intra-specifically. The latter may be associated with myriad factors including capacity of energy metabolism. This study investigated whether mitochondrial respiration capacity influences the degree of gill regeneration and features of mitochondria in regenerated tissue by feeding fish an experimental diet aimed at modulating mitochondrial efficiency. Atlantic salmon reared on standard and experimental diet were subjected to 50% filament resection on a subset of filaments on the ventral and dorsal regions of the first gill arch. Mitochondrial respiration and citrate synthase activity (CSA) were measured in the resected tips of filaments (week-0) and then in the regenerated tissue at 20 weeks post-resection (week-20). The degree of filament regeneration was measured at week-20. The experimental diet reduced CSA and respiratory control ratio (RCR), and increased proton leak at week-0, which was associated with a 30% reduction in tissue regeneration compared with fish on standard diet. While CSA increased in the regenerated tissue of experimental diet fish, there was a decline in other metrics of mitochondrial respiration including state 3, proton leak and RCR irrespective of diet. Overall, mitochondrial respiration efficiency at week-0 was positively correlated with the degree of subsequent gill tissue regeneration. Additionally, state 3 respiration and proton leak at week-20 were positively correlated with tissue regeneration, whereas CSA exhibited a negative relationship. Our results indicate that the capacity of mitochondrial respiration may at least partially explain the interindividual variation in tissue regeneration, but mitochondrial function in the regenerating tissue may be limited.

Hydrogen Peroxide is an oxidative chemical used as a topical treatment via bath exposures in salmonid aquaculture to mitigate external infections such as sea lice and amoebic gill disease. Protocols can depend on each individual site treatment, which varies in environmental temperature, dose concentration used and exposure time. Treatments such as this, in addition to environmental insults, increasing ocean temperatures and other farm management practices occurring simultaneously and/or chronically can lead to Complex Gill Disease (CGD). The purpose of this study was to assess the effects of hydrogen peroxide on the gills of Atlantic salmon (Salmo salar) following acute and chronic repeated exposure and dose and temperature dependent exposure. Our focus was to examine biological responses influencing gill function such as immune, healing, and redox reactions while understanding the cellular damage occurring in the gill. Histology and targeted gene expression through RT-qPCR analysis was used to examine these effects. Results showed minor to severe morphological changes in the gill, increasing damage in a linear relationship with increasing temperature and dose. The main morphological changes observed in the gill were lamellar epithelial lifting, lamellar edema, and lamellar fusion. Gene expression analysis revealed upregulation of genes associated with healing, redox, oxygen transport, cell death and DNA repair whereas suppression of immune responses genes was evident for both experimental groups. Differential regulation of genes associated with apparent adaptation to repeated hydrogen peroxide treatment could be useful in identifying markers to differentiate acute and chronic impact of treatment(s) that may not always de discerned through standard histological assessment.

Flesh colour is a critical quality criterion for consumer purchasing of salmonid products. As fish cannot synthesize pigments endogenously, they must be provided in their diet, and in Atlantic salmon farming, synthetic astaxanthin is the most widely used dietary supplement. This study evaluated two astaxanthin products derived from the microalga Haematococcus lacustris against a commercial synthetic product. Seven dietary treatments were tested in duplicate tanks using Atlantic salmon reared in a commercial freshwater recirculation system. Diets included: a negative control with no astaxanthin (C); two synthetic astaxanthin positive control treatments at 40 and 80 mg Ax [kg diet]⁻1, Low and High, respectively; and two natural astaxanthin products, oleoresin (O) and powder (P), each at the same two concentrations. Fish were assessed monthly for production parameters, and after six months, ten fish per tank were sampled for further analyses. Diet significantly influenced fillet pigmentation and tissue astaxanthin content, while most production parameters, biological indices, fillet proximate composition, structural parameters, and shelf life were not significantly affected. Initial fish size and stocking density significantly influenced several response variables. Oleoresin resulted in fillet pigmentation comparable to the synthetic product and superior to the powder product, indicating that it has strong potential as a natural alternative to synthetic astaxanthin in Atlantic salmon farming.

Sea lice (Lepeophtheirus salmonis) infestation continues to pose a persistent and escalating challenge to the global salmon aquaculture industry. Given the complexity of host-parasite interactions, family-based transcriptomic studies provide crucial insights into genetic variation in host responses to sea lice, potentially guiding the development of selective breeding programs to manage parasite resistance in Atlantic salmon. This study investigated global gene expression (transcriptomic) responses of the skin and head kidney of Atlantic salmon (Salmo salar) from different families following infestation at two distinct stages of sea lice, chalimus II and adult, under varying temperature conditions (10°C and 20°C). RNA sequencing results revealed consistent expression of lice-responsive genes across different families under varying thermal conditions, which allowed the identification of potential biomarkers associated with adult-stage compared to chalimus-stage infestations. Our findings highlight critical physiological disruptions in salmon infested with advanced (adult) stages of lice, including uncontrolled and persistent inflammation, dampened/dysregulated immune responses, and impaired tissue repair at attachment sites. This study provides a comprehensive analysis of the transcriptomic responses of Atlantic salmon to different developmental stages of sea lice under specific temperature conditions (10°C and 20°C), and identifies several novel molecular markers from RNA-seq analysis that may be instrumental in developing targeted control strategies for this economically important parasite.

Acanthocephalans collected from the stomach of the mackerel tuna (Euthynnus affinis Cantor) in the Persian Gulf off Iran were identified as Neorhadinorhynchus nudus Harada, 1938 based on morphological characters and molecular sequence data from mitochondrial (cytochrome c oxidase subunit I, COI) and nuclear ribosomal DNA (small and large subunit ribosomal DNA, 18S and 28S rDNA). Phylogenetic analyses of the order Echinorhynchida Southwell & Macfie, 1925 were performed based on the concatenated (18S+28S+COI) sequence data using maximum likelihood (ML) and Bayesian inference (BI) analyses. The results showed that N. nudus grouped together with species of Rhadinorhynchus Lühe, 1911, supporting the proposal that Neorhadinorhynchus Yamaguti, 1939 is a synonym of Rhadinorhynchus. Rhadinorhynchus mariserpentis Steinauer et al., 2019 was also treated as a junior synonym of Rhadinorhynchus nudus. Haplotype network analysis revealed no correlation between haplotype distribution and geographical origin of R. nudus.

The study investigates the susceptibility of Atlantic salmon (Salmo salar) and Pacific salmon species (pink salmon, Oncorhynchus gorbuscha; coho salmon, Oncorhynchus kisutch; and chum salmon, Oncorhynchus keta) to the parasitic salmon lice (Lepeophtheirus salmonis). The research had two main objectives: to characterize the morphology of the scaly skin in four salmonid species and to compare the cellular response at the louse attachment site in coho salmon and Atlantic salmon. Three consecutive challenge trials were conducted, with successful louse infestation only achieved across all four species in the third trial using mild anesthesia with tricaine methanesulfonate. Skin and fin samples were collected at 12, 24, 36, 48, 60, and 168 h post-infestation (hpi) for histological, proteomic, and spatial transcriptomic analyses. Results showed that chum salmon had significantly higher mucous cell coverage (30–40

The Atlantic salmon (Salmo salar) aquaculture industry must mitigate the impacts of rising ocean temperatures and the increased prevalence/severity of marine heat waves. Therefore, we investigated the genetic architecture and gene expression (transcriptomics) responsible for determining a salmon’s upper thermal tolerance. A genome-wide association study (GWAS) was conducted using fin clips of salmon from a previous incremental thermal maximum (ITMax) challenge (n = 251) and the North American 50 K SNP chip. ITMax was a highly polygenic trait with low/moderate heritability (mean SNP-based h2 = 0.20 and pedigree-based h2 = 0.25). Using data from the same fish, a separate GWAS assessed thermal-unit growth coefficient (TGC). Five significant SNPs were detected on chromosomes three and five, and high heritability estimates were calculated for TGC measured as fish grew from 12 to 20 °C (mean SNP-based h2 = 0.62 and pedigree-based h2 = 0.64). RNA-seq analyses of liver samples (n = 5–6 family-1 temperature-1) collected from the four most and four least tolerant families at 10 and 20 °C were also used to provide insights into potential mechanisms modulating this species’ thermal tolerance. Between the top and bottom families, 347 and 175 differentially expressed transcripts (FDR-adjusted p < 0.01; fold-change ≥|2.0|) were identified at 10 and 20 °C, respectively. GO term enrichment analysis revealed unique responses to elevated temperature between family rankings (e.g., ‘blood coagulation’, ‘sterol metabolic process’ and ‘synaptic growth at neuromuscular junction’). qPCR analyses further confirmed differences pertaining to cholesterol metabolism (lpl), inflammation (epx, elf3, ccl20), apoptosis (htra1b, htra2, anxa5b), angiogenesis (angl4, pdgfa), nervous system processes (insyn2a, kcnj11l) and heat stress (serpinh1b-1, serpinh1b-2). Three differentially expressed transcripts (i.e., ppp1r9a, gal3st1a, f5) were located in close proximity (± 120 kbp) to near-significant SNPs from the GWAS. Interestingly, ppp1r9a and gal3st1a have putative neurological functions, while f5 regulates blood coagulation. These analyses provide several putative biomarkers of upper thermal tolerance in salmon that could prove valuable in helping the industry develop more temperature-tolerant fish. Further, our study supports previous reports that ITMax has low/moderate heritability in this species, and suggests that TGC at elevated temperatures is highly heritable.

Acipenserid herpesvirus 2 (AciHV-2) is a large double-stranded DNA virus in the family Alloherpesviridae that causes catastrophic outbreaks in young naive white sturgeon (Acipenser transmontanus) populations, with mortalities of up to 80%. Survivors of these infections are suspected to remain latently infected. The gram-positive zoonotic bacterium Streptococcus iniae is another important sturgeon pathogen that causes severe myositis and up to 50% mortality during natural outbreaks. Throughout the last decade, co-infections of AciHV-2 and S. iniae have been reported in cultured white sturgeon in California resulting in severe presentations of piscine streptococcosis. This phenomenon of herpesvirus and streptococcus co-infection appears to span multiple taxa since in humans, it is recognized that a Human herpesvirus 3 infection (VZV) is a negative prognostic indicator for pediatric Invasive Group A Streptococcal infections (IGASI). While a decrease in humoral immunity caused by VZV has been hypothesized as a potentially important factor in IGASI cases, no natural animal model exists to study this process. Moreover, no studies have investigated these reported co-infections in white sturgeon. Therefore, the goal of this study was to investigate the effects of a recent AciHV-2 infection on the outcome of a subsequent S. iniae challenge in white sturgeon fingerlings. When fish were infected with 108 colony forming units (CFU) of S. iniae intramuscularly (IM), a statistically significant decrease in survival of 41% was detected in the co-infection group compared to the S. iniae group (p-value < 0.001). This difference was not observed when fish were infected with 106 CFU of S. iniae IM. At this lower infection dose, however, a statistically significant downregulation of tnfα was observed in the spleen of fish in the co-infection group compared to the S. iniae group (p-value = 0.0098). Analysis of serum from survivors revealed a statistically significant reduction in anti-S. iniae serum IgM and serum serotransferrin in fish from the co-infection group compared to the S. iniae group (p-value = 0.0134 and p-value = 0.0183, respectively). Further studies are indicated to determine what interactions lead to the decreased production of pathogen-specific IgM, serotransferrin, and TNFα in the host.

Sea lice infestations and winter ulcer disease caused by Moritella viscosa are two major challenges for the Atlantic salmon (Salmo salar) aquaculture industry. Despite their common tropisms for the skin/muscle we know very little about the interaction between these two significant pathogens. The objective of this study was to elucidate the transcriptomic response of Atlantic salmon to sea louse (Lepeophtheirus salmonis) and M. viscosa under single infection and different scenarios of co-infection (i.e. first, L. salmonis and then, M. viscosa and vice versa). After exposure, sampling was performed from the infestation site as well as the adjacent area on fish skin, followed by transcriptome analysis. RNA-seq analysis revealed that infection with either L. salmonis or M. viscosa induced a significant immune response from the skin and resulted in extensive transcriptomic changes. More differentially expressed genes (DEGs) were detected at M. viscosa lesion sites compared to L. salmonis attachment sites. In addition, the effect of the infection order was evaluated. Interestingly, we found that primary infection with lice was associated with a significantly higher number of DEGs during the co-infection process on fish skin compared to the fish first infected with M. viscosa (721 and 5336 DEGs from lice attachment and M. viscosa lesion sites, respectively, vs 291 and 3601 DEGs from lice attachment and M. viscosa lesion sites, respectively). We also found that lice infection caused localized effects on the skin of Atlantic salmon, while single infection with M. viscosa caused a moderate systemic impact and inhibited the tissue repair function of the skin, leading to severe ulceration. In addition, the C-type lectin receptor signaling pathway was drastically activated at the lice attachment sites during both lice alone- and co-infections, regardless of the order of infections, and similar results were observed in M. viscosa infection at both lesion and adjacent sites, indicating that this pathway played an essential role for immunity in Atlantic salmon. A more intense inflammatory and immune response was also observed at M. viscosa lesion sites. These results will promote our understanding of the immune interactions between L. salmonis and M. viscosa during the co-infection process and provide insights for the development of preventive and treatment strategies for these pathogens in salmon aquaculture.

BackgroundSalmonid species have followed markedly divergent evolutionary trajectories in their interactions with sea lice. While sea lice parasitism poses significant economic, environmental, and animal welfare challenges for Atlantic salmon (Salmo salar) aquaculture, coho salmon (Oncorhynchus kisutch) exhibit near-complete resistance to sea lice, achieved through a potent epithelial hyperplasia response leading to rapid louse detachment. The molecular mechanisms underlying these divergent responses to sea lice are unknown.ResultsWe characterized the cellular and molecular responses of Atlantic salmon and coho salmon to sea lice using single-nuclei RNA sequencing. Juvenile fish were exposed to copepodid sea lice (Lepeophtheirus salmonis), and lice-attached pelvic fin and skin samples were collected 12 h, 24 h, 36 h, 48 h, and 60 h after exposure, along with control samples. Comparative analysis of control and treatment samples revealed an immune and wound-healing response that was common to both species, but attenuated in Atlantic salmon, potentially reflecting greater sea louse immunomodulation. Our results revealed unique but complementary roles of three layers of keratinocytes in the epithelial hyperplasia response leading to rapid sea lice rejection in coho salmon. Our results suggest that basal keratinocytes direct the expansion and mobility of intermediate and, especially, superficial keratinocytes, which eventually encapsulate the parasite.ConclusionsOur results highlight the key role of keratinocytes in coho salmon's sea lice resistance and the diverged biological response of the two salmonid host species when interacting with this parasite. This study has identified key pathways and candidate genes that could be manipulated using various biotechnological solutions to improve Atlantic salmon sea lice resistance.