A comparative analysis of alternative splicing patterns in Atlantic salmon (Salmo salar) in response to Moritella viscosa and sea lice (Lepeophtheirus salmonis) infection.

Moritella viscosa (M. viscosa) and sea lice (Lepeophtheirus salmonis) are severe pathogens that primarily infect the skin of Atlantic salmon (Salmo salar), which cause significant economic losses in the farming industry. However, the pathogenesis and molecular mechanisms underlying the host's immune defence at the post-transcriptional level remain unclear. Alternative splicing (AS) is an evolutionarily conserved post-transcriptional mechanism that can greatly increase the richness of the transcriptome and proteome. In this study, transcriptomic data derived from skin tissues of Atlantic salmon after M. viscosa and sea lice infections were used to examine the AS profiles and their differential expression patterns. In total, we identified 33,044 AS events (involving 13,718 genes) in the control (CON) group, 35,147 AS events (involving 14,340 genes) in the M. viscosa infection (MV) group, and 30,364 AS events (involving 13,142 genes) in the sea lice infection (LC) group, respectively. Among the five types of AS identified in our study (i.e., SE, A5SS, A3SS, MXE, and RI), SE was the most prevalent type in all three groups (i.e., CON, MV, and LC groups). Decreased percent-spliced-in (PSI) levels were observed in SE events under both MV- and LC-infected conditions, suggesting that MV or LC infection elevated exon-skipping isoforms and promoted the selection of shorter transcripts in numerous DAS genes. In addition, most of the differential AS genes were found to be associated with pathways related to mRNA regulation, epithelial or muscle development, and immune response. These findings provide novel insights into the role of AS in host-pathogen interactions and represent the first comparative analysis of AS in response to bacterial and parasitic infections in fish.

Morphological and molecular characterization of Stomachicola muraenesocis Yamaguti, 1934 (Digenea: Hemiuridae) from the daggertooth pike conger Muraenesox cinereus (Forsskål).

Hemiurid digeneans conspecific with Stomachicola muraenesocis Yamaguti, 1934 (the type species of the genus Stomachicola Yamaguti, 1934) were collected from the stomach of the daggertooth pike conger Muraenesox cinereus (Forsskål) off the Persian Gulf of Iran. This study aimed to provide a detailed characterization of Stom. muraenesocis, including measurements, illustrations and scanning electron microscopy (s.e.m.) representations. Comparisons with the original and previous descriptions revealed morphological and metrical variations in several features (i.e. body size and shape, arrangement of reproductive organs, soma to ecsoma length ratio, position of genital opening, number of vitelline tubules and extension of uterine coils) between Stom. muraenesocis from different hosts and localities. This study presents the first molecular sequence data associated with the small (18S) and large (28S) subunit nuclear ribosomal RNA genes (rDNA) for Stom. muraenesocis. Phylogenetic analyses of the 18S dataset placed Stom. muraenesocis as sister lineage to a clade formed of a group of species of Lecithaster Lühe, 1901 (Lecithasteridae Odhner, 1905). In contrast, phylogenetic analyses based on the 28S consistently recovered a sister relationship between Stom. muraenesocis and representatives of the Hemiuridae Looss, 1899. Further comprehensive phylogenetically based classification in light of morphology and taxonomic history of the Hemiuridae and Lecithasteridae is required to infer phylogenetic affinities and historical biogeography of Stomachicola. A comprehensive list of previously reported species of Stomachicola together with their associated hosts, localities and morphometric data is provided.

Transcriptome analysis revealed immune responses in the kidney of Atlantic salmon (Salmo salar) co-infected with sea lice (Lepeophtheirus salmonis) and infectious salmon anemia virus.

Sea lice (Lepeophtheirus salmonis) and infectious salmon anemia virus (ISAv) are two of the most important pathogens in Atlantic salmon (Salmo salar) farming and typically cause substantial economic losses to the industry. However, the immune interactions between hosts and these pathogens are still unclear, especially in the scenario of co-infection. In this study, we artificially infected Atlantic salmon with sea lice and ISAv, and investigated the gene expression patterns of Atlantic salmon head kidneys in response to both lice only and co-infection with lice and ISAv by transcriptomic analysis. The challenge experiment indicated that co-infection resulted in a cumulative mortality rate of 47.8 %, while no mortality was observed in the lice alone infection. We identified 240 differentially expressed genes (DEGs) under the lice alone infection, of which 185 were down-regulated and 55 were up-regulated, while a total of 994 DEGs were identified in the co-infection, of which 206 were down-regulated and 788 were significantly up-regulated. The pathway enrichment analysis revealed that single-infection significantly suppressed the innate immune system (e.g., the complement system), whereas co-infection induced a strong immune response, leading to the activation of immune-related signaling pathways such as Toll-like receptors and NOD-like receptors pathways, as well as significant upregulation of genes related to the activation of interferon and MH class I protein complex. Our results provide the first global transcriptomic study of gene expression in the Atlantic salmon head kidney in response to co-infection with sea lice and ISAv, and provided the baseline knowledge for understanding the immune responses during co-infection.

Rejection of Lepeophtheirus salmonis driven in part by chitin sensing is not impacted by seawater acclimitization in Coho salmon (Oncorhynchus kisutch).

There is tremendous variation in life-history strategies among anadromous salmonids. Species that enter the ocean environment at small sizes (< 20 g) are likely under more physiological pressure from pathogens; however, little data is available on responses at these early stages. With this in mind, we performed salmon louse challenges with Coho salmon either immediately after seawater entry (SW; ca. 10 g) or after 30 days in SW (ca. 20 g). Irrespective of size or time in SW, parasites were rapidly rejected by the host, with > 90% of all parasites lost by 16 days post-infection (dpi). Rejection was concomitant with host epithelial granulomatous infiltrations that initially targeted the embedded frontal filament (4 dpi) and the entire parasite by 10 dpi. Illumina sequencing, followed by functional enrichment analysis, revealed a concerted defense response in the fin within 1 dpi that included multiple innate and adaptive immunity components. Strikingly, early indications of an allergic-type inflammatory response were associated with chitin sensing pathways orchestrated by early overexpression of the IgE-receptor, fcer1g. Additionally, there was profound overexpression of several classes of c-type lectin receptors, including dectin-2, mincle, and dc-sign at 1 dpi onward. These profiles and upregulation of cellular effector markers were corroborated by histopathological evaluation, revealing the simultaneous presence of mast cell/eosinophilic granular cells, sacciform cells, macrophages/histiocytes, and granulocytes in fin. At 10 dpi and concurrent with parasite expulsion, there was evidence of immunoregulation in addition to tissue remodelling pathways. At 16 dpi, the response was effectively abrogated. Simultaneous profiling of the parasite transcriptome revealed early induction of chitin metabolism and immunomodulation, toxin production and ECM degradation; however, after 7 dpi, these were replaced with overexpression of stress and immune defense genes. These data present the first evidence for Coho salmon demonstrating chitin- and sugar moiety-sensing as key drivers of salmon louse rejection.

Description, molecular identification and pathological lesions of Huffmanela persica sp. nov. (Nematoda: Trichosomoididae: Huffmanelinae) from the daggertooth pike conger Muraenesox cinereus.

BACKGROUND: The genus Huffmanela Moravec, 1987 (Nematoda, Trichosomoididae, Huffmanelinae), represents a group of nematodes that infect both marine and freshwater fish, and the main gross feature of infection with different species of the genus is the presence of noticeable dark spots or tracks within the parasitized tissues. The purpose of this study was to describe morphologically and morphometrically the eggs of a new marine species of Huffmanela (Huffmanela persica sp. nov.), which was found in the form of black spots in the ovary and the tunica serosa of the stomach of the daggertooth pike conger (Muraenesox cinereus). The new species differs from Huffmanela hamo, another species reported from musculature of this host in Japan, in egg metrics, eggshell features and targeted organ. Molecular identification and pathological examination of the lesions caused by the new species are also reported. METHODS: Nematode eggs with varying degrees of development were separated from the infected tissues (ovary and tunica serosa of stomach) and investigated using light and scanning electron microscopy. Different species-specific markers (small subunit ribosomal DNA, 18S; large subunit ribosomal DNA, 28S; internal transcribed spacer, ITS) were used for molecular identification and phylogenetic study of the new species. Infected tissues were fixed in buffered formalin for pathological investigations. RESULTS: The fully developed eggs of H. persica sp. nov. are distinguished from those previously described from this host on the basis of their measurements (size, 54-68 × 31-43 µm; polar plugs, 6.4-9.7 × 8.4-12 µm; shell thickness, 3.5-6.1 µm) and a delicate but ornate uterine layer (UL) covering the entire eggshell including the polar plugs. Histopathological examination revealed a fibro-granulomatous inflammation in the ovary and the serosal layer of the stomach of infected fish. Maximum-likelihood (ML) phylogenetic analysis recovered a sister relationship between the new species of marine origin and Huffmanela species previously collected from freshwater hosts. CONCLUSIONS: The present study is the first to report the molecular characterization and phylogenetic position of a teleost-associated marine species of the genus Huffmanela. A comprehensive list of nominal and innominate populations of Huffmanela is also provided.

Basal and immune-responsive transcript expression of two Atlantic salmon interferon regulatory factor 2 (irf2) paralogues.

Atlantic salmon (Salmo salar) is one of the most economically important aquaculture species globally. However, disease has become a prevalent threat to this industry. A thorough understanding of the genes and molecular pathways involved in the immune responses of Atlantic salmon is imperative for selective breeding of disease-resistant broodstock, as well as developing new diets and vaccines to mitigate the impact of disease. Members of the interferon regulatory factor (IRF) family of transcription factors play roles in the induction of interferons and other cytokines involved in host immune responses to intracellular and parasitic pathogens. IRF family members also play diverse roles in other biological processes, such as stress response, reproduction and development. The current study focused on one member of the IRF family: interferon regulatory factor 2 (irf2). As previously shown, due to the genome duplication that occurred ∼80 million years ago in the salmonid lineage, there are two irf2 paralogues in the Atlantic salmon genome. In silico analyses at the cDNA and deduced amino acid levels were conducted followed by phylogenetic tree construction with IRF2 amino acid sequences from various ray-finned fishes, cartilaginous fish and tetrapods. qPCR was then used to analyze paralogue-specific irf2 constitutive expression across 17 adult tissues, as well as responses to the viral mimic pIC (i.e., synthetic double-stranded RNA analog) in cultured macrophage-like cells (in vitro) and to infection with the Gram-negative bacterium Moritella viscosa in skin samples (in vivo). The qPCR studies showed sex- and paralogue-specific differences in expression across tissues. For example, expression of both paralogues was higher in ovary than in testes; expression (considering both sexes together) was highest for irf2-1 in gonad and for irf2-2 in hindgut. Both irf2 paralogues were responsive to pIC stimulation, but varied in their induction level, with irf2-1 having an overall stronger response than irf2-2. Only one paralogue, irf2-2, was significantly responsive to M. viscosa infection. Differences in irf2-1 and irf2-2 transcript expression levels constitutively across tissues, and in response to pIC and M. viscosa, may suggest neo- or subfunctionalization of the duplicated genes. This novel information expands current knowledge and provides insight into how genome duplication events may impact host regulation of important immune markers.

Applying genetic technologies to combat infectious diseases in aquaculture.

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.

Interacting Effects of Sea Louse (Lepeophtheirus salmonis) Infection and Formalin-Killed Aeromonas salmonicida on Atlantic Salmon Skin Transcriptome.

Lepeophtheirus salmonis (sea lice) and bacterial co-infection threatens wild and farmed Atlantic salmon performance and welfare. In the present study, pre-adult L. salmonis-infected and non-infected salmon were intraperitoneally injected with either formalin-killed Aeromonas salmonicida bacterin (ASAL) or phosphate-buffered saline (PBS). Dorsal skin samples from each injection/infection group (PBS/no lice, PBS/lice, ASAL/no lice, and ASAL/lice) were collected at 24 h post-injection and used for transcriptome profiling using a 44K salmonid microarray platform. Microarray results showed no clear inflammation gene expression signatures and revealed extensive gene repression effects by pre-adult lice (2,189 down and 345 up-regulated probes) in the PBS-injected salmon (PBS/lice vs. PBS/no lice), which involved basic cellular (e.g., RNA and protein metabolism) processes. Lice repressive effects were not observed within the group of ASAL-injected salmon (ASAL/lice vs. ASAL/no lice); on the contrary, the observed skin transcriptome changes -albeit of lesser magnitude (82 up and 1 down-regulated probes)- suggested the activation in key immune and wound healing processes (e.g., neutrophil degranulation, keratinocyte differentiation). The molecular skin response to ASAL was more intense in the lice-infected (ASAL/lice vs. PBS/lice; 272 up and 11 down-regulated probes) than in the non-infected fish (ASAL/no lice vs. PBS/no lice; 27 up-regulated probes). Regardless of lice infection, the skin's response to ASAL was characterized by the putative activation of both antibacterial and wound healing pathways. The transcriptomic changes prompted by ASAL+lice co-stimulation (ASAL/lice vs. PBS/no lice; 1878 up and 3120 down-regulated probes) confirmed partial mitigation of lice repressive effects on fundamental cellular processes and the activation of pathways involved in innate (e.g., neutrophil degranulation) and adaptive immunity (e.g., antibody formation), as well as endothelial cell migration. The qPCR analyses evidenced immune-relevant genes co-stimulated by ASAL and lice in an additive (e.g., mbl2b, bcl6) and synergistic (e.g., hampa, il4r) manner. These results provided insight on the physiological response of the skin of L. salmonis-infected salmon 24 h after ASAL stimulation, which revealed immunostimulatory properties by the bacterin with potential applications in anti-lice treatments for aquaculture. As a simulated co-infection model, the present study also serves as a source of candidate gene biomarkers for sea lice and bacterial co-infection.

Global gene expression responses of Atlantic salmon skin to Moritella viscosa.

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.

Expression of immune markers of white sturgeon (Acipenser transmontanus) during Veronaea botryosa infection at different temperatures.

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-ß (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.

Analysis of the white sturgeon (Acipenser transmontanus) immune response during immunostimulation and Veronaea botryosa infection.

Systemic phaeohyphomycosis caused by Veronaea botryosa is regarded as an important emerging mycotic disease of sturgeon aquaculture. However, no vaccines or treatments are currently available. The effects of dietary ß-glucan supplementation on resistance to V. botryosa infection was examined in controlled challenges by exposing immunostimulated and control fish to ~7.25 × 105 fungal spores/fish via intra-muscular injection. Six weeks post-challenge, cumulative mortality was determined and antibodies to acute phase-proteins (APP) were used to quantify the conserved APP peptides in the serum of challenged and control fish using Western blot. Transcript levels for all tested pro-inflammatory cytokines, APP, and regulatory cytokines in the spleen were similar amongst treatments at the end of the three-week feeding period. However, significantly higher survival occurred in fingerlings fed 0.3% ß-glucans compared to non-immunostimulated fish groups (p < 0.05) six weeks post-challenge. A strong proinflammatory response was detected in exposed treatment groups, and greater survival at 6 weeks was associated with higher transcript abundance of Il-17 in fish fed ß-glucans. Findings support the important role of this cytokine in response to fungal infection.

Estimating the dispersal of Lepeophtheirus salmonis sea lice within and among Atlantic salmon sites of the Bay of Fundy, New Brunswick.

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 < .001). This study concludes that sea lice burdens within Atlantic salmon farms in the Bay of Fundy, NB, are affected by within site management and could be improved by synchronizing treatments between sites.

Transcriptome Analysis of Atlantic Salmon (Salmo salar) Skin in Response to Sea Lice and Infectious Salmon Anemia Virus Co-Infection Under Different Experimental Functional Diets.

Sea lice (Lepeophtheirus salmonis) are ectoparasitic copepods that cause significant economic loss in marine salmoniculture. In commercial salmon farms, infestation with sea lice can enhance susceptibility to other significant pathogens, such as the highly contagious infectious salmon anemia virus (ISAv). In this study, transcriptomic analysis was used to evaluate the impact of four experimental functional feeds (i.e. 0.3% EPA/DHA+high-ω6, 0.3% EPA/DHA+high-ω6+immunostimulant (IS), 1% EPA/DHA+high-ω6, and 1% EPA/DHA+high-ω3) on Atlantic salmon (Salmo salar) during a single infection with sea lice (L. salmonis) and a co-infection with sea lice and ISAv. The overall objectives were to compare the transcriptomic profiles of skin between lice infection alone with co-infection groups and assess differences in gene expression response among animals with different experimental diets. Atlantic salmon smolts were challenged with L. salmonis following a 28-day feeding trial. Fish were then challenged with ISAv at 18 days post-sea lice infection (dpi), and maintained on individual diets, to establish a co-infection model. Skin tissues sampled at 33 dpi were subjected to RNA-seq analysis. The co-infection's overall survival rates were between 37%-50%, while no mortality was observed in the single infection with lice. With regard to the infection status, 756 and 1303 consensus differentially expressed genes (DEGs) among the four diets were identified in "lice infection vs. pre-infection" and "co-infection vs. pre-infection" groups, respectively, that were shared between the four experimental diets. The co-infection groups (co-infection vs. pre-infection) included up-regulated genes associated with glycolysis, the interferon pathway, complement cascade activity, and heat shock protein family, while the down-regulated genes were related to antigen presentation and processing, T-cell activation, collagen formation, and extracellular matrix. Pathway enrichment analysis conducted between infected groups (lice infection vs. co-infection) resulted in several immune-related significant GO terms and pathways unique to this group, such as "autophagosome", "cytosolic DNA-sensing pathway" and "response to type I interferons". Understanding how experimental functional feeds can impact the host response and the trajectory of co-infections will be an essential step in identifying efficacious intervention strategies that account for the complexities of disease in open cage culture.

Hydrogen peroxide treatment and its impacts on Lepeophtheirus salmonis originating from the Bay of Fundy, Canada.

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.

Increasing temperatures accentuate negative fitness consequences of a marine parasite.

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.

Identification of critical enzymes in the salmon louse chitin synthesis pathway as revealed by RNA interference-mediated abrogation of infectivity.

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.

Salmon immunological defence and interplay with the modulatory capabilities of its ectoparasite Lepeophtheirus salmonis.

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.

Transcriptomic Profiling in Fins of Atlantic Salmon Parasitized with Sea Lice: Evidence for an Early Imbalance Between Chalimus-Induced Immunomodulation and the Host's Defense Response.

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 < 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 < 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's fins. The comparison between ATT and ADJ sites demonstrated the upregulation of a suite of immune-relevant transcripts, evidencing the salmon'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.

Impact of co-infection with Lepeophtheirus salmonis and Moritella viscosa on inflammatory and immune responses of Atlantic salmon (Salmo salar).

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.

Impact of rearing temperature on the innate antiviral immune response of growth hormone transgenic female triploid Atlantic salmon (Salmo salar).

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.