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Abstract The immunomodulatory effects of omega-3 and omega-6 fatty acids are a crucial subject of investigation for sustainable fish aquaculture, as fish oil is increasingly replaced by terrestrial vegetable oils in aquafeeds. Unlike previous research focusing on fish oil replacement with vegetable alternatives, our study explored how the omega-6 to omega-3 polyunsaturated fatty acid (PUFA) ratio in low-fish oil aquafeeds influences Atlantic salmon's antiviral and antibacterial immune responses. Atlantic salmon were fed aquafeeds rich in soy oil (high in omega-6) or linseed oil (high in omega-3) for 12 weeks and then challenged with bacterial (formalin-killed Aeromonas salmonicida) or viral-like (polyriboinosinic polyribocytidylic acid) antigens. The head kidneys of salmon fed high dietary omega-3 levels exhibited a more anti-inflammatory fatty acid profile and a restrained induction of pro-inflammatory and neutrophil-related genes during the immune challenges. The high-omega-3 diet also promoted a higher expression of genes associated with the interferon-mediated signaling pathway, potentially enhancing antiviral immunity. This research highlights the capacity of vegetable oils with different omega-6 to omega-3 PUFA ratios to modulate specific components of fish immune responses, offering insights for future research on the intricate lipid nutrition-immunity interplay and the development of novel sustainable low-fish oil clinical aquaculture feeds.

Due to the nature of open-pen farming, salmon are exposed to numerous pathogens shared by other farms and their wild counterparts. Industry must manage these outbreaks through vaccination, oral or bath treatments, and more recently through functional feed administration. Globally, the most important pathogens of salmon are sea lice (Lepeophtheirus salmonis, and Caligus rogercresseyi) not only due to their direct impacts on the host, but indirectly by enhancing host susceptibility to co-infection. This study aims to characterize molecular responses during a co-infection of L. salmonis and a salmon orthomyxovirus (infectious salmon anemia virus; ISAv) under administration of four functional feed diets: a control feed with a low 0.3 % EPA/DHA + high-ω6 (Ctrl), an EPA/DHA enriched pro-inflammatory diet 1 % EPA/DHA + high-ω6; FA + I), an EPA/DHA enriched anti-inflammatory diet (1 % EPA/DHA+high-ω3; FA-I), and a low EPA/DHA feed (0.3 %) with an immunostimulant added (0.3 % EPA/DHA + high-ω6 + immunostimulant (IS); Ctrl + IS). Atlantic salmon (40 fish per tank; 8 tanks per feed) were acclimated to one of the four experimental diets. Sea lice copepodids were introduced to all experimental tanks and 10 fish sampled from each tank at each time point (prior to infection [−3], and 11, 33, 47 days post infection). A high virulence ISAv isolate (ISAV-HPR4) was intraperitoneally injected into donor fish 6 days prior to their transfer into the experimental tanks for cohabitation (ca. 10–15 % of tank density; 4 tanks per feed group) to achieve peak shedding rates at time of stocking. Fatty acid enriched (FA + I and FA-I) diets had a significant impact on sea lice abundance on fish infected with lice only. Gene expression profiles measured by reverse transcriptase-qPCR showed significant upregulation in several antiviral genes (irf7b and mxb) associated with the interferon system in all but the fish fed the immunostimulating diet. An increase in transcript levels (irf7b, isg15a, mmp-9, mxb) accounted for high lice and high viral loads in diets FA + I and Ctrl + IS. As feeds successful at reducing lice were the least successful in survival of co-infected fish, there appears to be a trade-off for better anti-parasitic responses, which is enhanced through dietary supplementation. This will require further research to ensure careful selection of feed combinations and expected co-infection challenges.

We investigated the immunomodulatory effect of varying levels of dietary ω6/ω3 fatty acids (FA) on Atlantic salmon (Salmo salar) antibacterial response. Two groups were fed either high-18:3ω3 or high-18:2ω6 FA diets for 8 weeks, and a third group was fed for 4 weeks on the high-18:2ω6 diet followed by 4 weeks on the high-18:3ω3 diet and termed “switched-diet”. Following the second 4 weeks of feeding (i.e., at 8 weeks), head kidney tissues from all groups were sampled for FA analysis. Fish were then intraperitoneally injected with either a formalin-killed Renibacterium salmoninarum bacterin (5 × 107 cells mL−1) or phosphate-buffered saline (PBS control), and head kidney tissues for gene expression analysis were sampled at 24 h post-injection. FA analysis showed that the head kidney profile reflected the dietary FA, especially for C18 FAs. The qPCR analyses of twenty-three genes showed that both the high-ω6 and high-ω3 groups had significant bacterin-dependent induction of some transcripts involved in lipid metabolism (ch25ha and lipe), pathogen recognition (clec12b and tlr5), and immune effectors (znrf1 and cish). In contrast, these transcripts did not significantly respond to the bacterin in the “switched-diet” group. Concurrently, biomarkers encoding proteins with putative roles in biotic inflammatory response (tnfrsf6b) and dendritic cell maturation (ccl13) were upregulated, and a chemokine receptor (cxcr1) was downregulated with the bacterin injection regardless of the experimental diets. On the other hand, an inflammatory regulator biomarker, bcl3, was only significantly upregulated in the high-ω3 fed group, and a C-type lectin family member (clec3a) was only significantly downregulated in the switched-diet group with the bacterin injection (compared with diet-matched PBS-injected controls). Transcript fold-change (FC: bacterin/PBS) showed that tlr5 was significantly over 2-fold higher in the high-18:2ω6 diet group compared with other diet groups. FC and FA associations highlighted the role of DGLA (20:3ω6; anti-inflammatory) and/or EPA (20:5ω3; anti-inflammatory) vs. ARA (20:4ω6; pro-inflammatory) as representative of the anti-inflammatory/pro-inflammatory balance between eicosanoid precursors. Also, the correlations revealed associations of FA proportions (% total FA) and FA ratios with several eicosanoid and immune receptor biomarkers (e.g., DGLA/ARA significant positive correlation with pgds, 5loxa, 5loxb, tlr5, and cxcr1). In summary, dietary FA profiles and/or regimens modulated the expression of some immune-relevant genes in Atlantic salmon injected with R. salmoninarum bacterin. The modulation of Atlantic salmon responses to bacterial pathogens and their associated antigens using high-ω6/high-ω3 diets warrants further investigation.

Gill damage represents a significant challenge in the teleost fish aquaculture industry globally, due to the gill’s involvement in several vital functions and direct contact with the surrounding environment. To examine the local and systemic effects accompanying gill damage (which is likely to negatively affect gill function) of Atlantic salmon, we performed a field sampling to collect gill and liver tissue after several environmental insults (e.g., harmful algal blooms). Before sampling, gills were visually inspected and gill damage was scored; gill scores were assigned from pristine [gill score 0 (GS0)] to severely damaged gills (GS3). Using a 44K salmonid microarray platform, we aimed to compare the transcriptomes of pristine and moderately damaged (i.e., GS2) gill tissue. Rank Products analysis (5% percentage of false-positives) identified 254 and 34 upregulated and downregulated probes, respectively, in GS2 compared with GS0. Differentially expressed probes represented genes associated with functions including gill remodeling, wound healing, and stress and immune responses. We performed gill and liver qPCR for all four gill damage scores using microarray-identified and other damage-associated biomarker genes. Transcripts related to wound healing (e.g., neb and klhl41b) were significantly upregulated in GS2 compared with GS0 in the gills. Also, transcripts associated with immune and stress-relevant pathways were dysregulated (e.g., downregulation of snaclec 1-like and upregulation of igkv3) in GS2 compared with GS0 gills. The livers of salmon with moderate gill damage (i.e., GS2) showed significant upregulation of transcripts related to wound healing (i.e., chtop), apoptosis (e.g., bnip3l), blood coagulation (e.g., f2 and serpind1b), transcription regulation (i.e., pparg), and stress-responses (e.g., cyp3a27) compared with livers of GS0 fish. We performed principal component analysis (PCA) using transcript levels for gill and liver separately. The gill PCA showed that PC1 significantly separated GS2 from all other gill scores. The genes contributing most to this separation were pgam2, des, neb, tnnt2, and myom1. The liver PCA showed that PC1 significantly separated GS2 from GS0; levels of hsp70, cyp3a27, pparg, chtop, and serpind1b were the highest contributors to this separation. Also, hepatic acute phase biomarkers (e.g., serpind1b and f2) were positively correlated to each other and to gill damage. Gill damage-responsive biomarker genes and associated qPCR assays arising from this study will be valuable in future research aimed at developing therapeutic diets to improve farmed salmon welfare.

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.

Moraxella catarrhalis causes significant health problems, including 15-20% of otitis media cases in children and ~10% of respiratory infections in adults with chronic obstructive pulmonary disease. The lack of an efficacious vaccine, the rapid emergence of antibiotic resistance in clinical isolates, and high carriage rates reported in children are cause for concern. In addition, the effectiveness of conjugate vaccines at reducing the incidence of otitis media caused by Streptococcus pneumoniae and nontypeable Haemophilus influenzae suggest that M. catarrhalis infections may become even more prevalent. Hence, M. catarrhalis is an important and emerging cause of infectious disease for which the development of a vaccine is highly desirable. Studying the pathogenesis of M. catarrhalis and the testing of vaccine candidates have both been hindered by the lack of an animal model that mimics human colonization and infection. To address this, we intranasally infected chinchilla with M. catarrhalis to investigate colonization and examine the efficacy of a protein-based vaccine. The data reveal that infected chinchillas produce antibodies against antigens known to be major targets of the immune response in humans, thus establishing immune parallels between chinchillas and humans during M. catarrhalis infection. Our data also demonstrate that a mutant lacking expression of the adherence proteins MhaB1 and MhaB2 is impaired in its ability to colonize the chinchilla nasopharynx, and that immunization with a polypeptide shared by MhaB1 and MhaB2 elicits antibodies interfering with colonization. These findings underscore the importance of adherence proteins in colonization and emphasize the relevance of the chinchilla model to study M. catarrhalis-host interactions.