Loss of bmp15 function in the seasonal spawner Atlantic salmon results in ovulatory failure.

Bone morphogenetic protein 15 (BMP15) is an oocyte-specific growth factor important for successful female reproduction in mammals. While mutations in BMP15/Bmp15 cause ovulatory deficiency and/or infertility in certain mammalian species, loss of bmp15 in zebrafish, a continuous spawner and the only bmp15 knockout model in fish to date, results in complete arrest of follicle development and later female-to-male sex reversal, preventing to examine effects on ovulation/fertilization. Here, we used Atlantic salmon, a seasonal spawner, and generated bmp15 mutants to investigate ovarian development and fertility. Histological and morphometric analyses revealed that in biallelic frameshift (bmp15 fs/fs) mutant ovaries, folliculogenesis started earlier, resulting in an advanced development compared to wild-type (WT) controls, accompanied by a weaker expression of the (early) oocyte-specific factor figla. This precocious ovarian development was followed in bmp15 fs/fs females by enhanced follicle atresia during vitellogenic stages. Although genes involved in steroid synthesis and signaling (star, cyp11b, cyp17a1 and esr1) were dramatically higher in late vitellogenic bmp15 fs/fs mutant ovaries, estradiol-17ß plasma levels were lower than in WT counterparts, potentially reflecting compensatory changes at the level of ovarian gene expression. At spawning, bmp15 fs/fs females displayed lower gonado-somatic index values and reduced oocyte diameter, and the majority (71.4%), showed mature non-ovulating ovaries with a high degree of atresia. The remaining (28.6%) females spawned eggs but they either could not be fertilized or, upon fertilization, showed severe malformations and embryonic mortality. Our results show that Bmp15 is required for proper follicle recruitment and growth and later ovulatory success in Atlantic salmon, providing an alternative candidate target to induce sterility in farmed salmon. Moreover, since loss of bmp15 in salmon, in contrast to zebrafish, does not result in female-to-male sex change, this is the first mutant model in fish allowing further investigations on Bmp15-mediated functions in the ovulatory period.

Keratinocytes drive the epithelial hyperplasia key to sea lice resistance in coho salmon.

BACKGROUND: Salmonid 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. RESULTS: We 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. CONCLUSIONS: Our 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.

Endocrine and Transcriptome Changes Associated with Testicular Growth and Differentiation in Atlantic Salmon (Salmo salar L.).

Sexual maturation of Atlantic salmon males is marked by dramatic endocrine changes and rapid growth of the testes, resulting in an increase in the gonad somatic index (GSI). We examined the association of gonadal growth with serum sex steroids, as well as pituitary and testicular gene expression levels, which were assessed with a DNA oligonucleotide microarray. The testes transcriptome was stable in males with a GSI < 0.08% despite the large difference between the smallest and the largest gonads. Fish with a GSI ≥ 0.23% had 7-17 times higher serum levels of five male steroids and a 2-fold increase in progesterone, without a change in cortisol and related steroids. The pituitary transcriptome showed an upregulation of the hormone-coding genes that control reproduction and behavior, and structural rearrangement was indicated by the genes involved in synaptic transmission and the differentiation of neurons. The observed changes in the abundance of testicular transcripts were caused by the regulation of transcription and/or disproportional growth, with a greater increase in the germinative compartment. As these factors could not be separated, the transcriptome results are presented as higher or lower specific activities (HSA and LSA). LSA was observed in 4268 genes, including many genes involved in various immune responses and developmental processes. LSA also included genes with roles in female reproduction, germinal cell maintenance and gonad development, responses to endocrine and neural regulation, and the biosynthesis of sex steroids. Two functional groups prevailed among HSA: structure and activity of the cilia (95 genes) and meiosis (34 genes). The puberty of A. salmon testis is marked by the predominance of spermatogenesis, which displaces other processes; masculinization; and the weakening of external regulation. Results confirmed the known roles of many genes involved in reproduction and pointed to uncharacterized genes that deserve attention as possible regulators of sexual maturation.

Cellular heterogeneity in red and melanized focal muscle changes in farmed Atlantic salmon (Salmo salar) visualized by spatial transcriptomics.

Spatial transcriptomics is a technique that provides insight into gene expression profiles in tissue sections while retaining structural information. We have employed this method to study the pathological conditions related to red and melanized focal changes in farmed Atlantic salmon (Salmo salar). Our findings support a model where similar molecular mechanisms are involved in both red and melanized filet discolorations and genes associated with several relevant pathways show distinct expression patterns in both sample types. Interestingly, there appears to be significant cellular heterogeneity in the foci investigated when looking at gene expression patterns. Some of the genes that show differential spatial expression are involved in cellular processes such as hypoxia and immune responses, providing new insight into the nature of muscle melanization in Atlantic salmon.

Variation in phenotypic plasticity across age-at-maturity genotypes in wild Atlantic salmon.

Evolution of phenotypic plasticity requires genotype-environment interaction. The discovery of two large-effect loci in the vgll3 and six6 genomic regions associated with the number of years the Atlantic salmon spend feeding at sea before maturation (sea age), provides a unique opportunity to study evolutionary potential of phenotypic plasticity. Using data on 1246 Atlantic salmon caught in the River Surna in Norway, we show that variation in mean sea age among years (smolt cohorts 2013-2018) is influenced by genotype frequencies as well as interaction effects between genotype and year. Genotype-year interactions suggest that genotypes may differ in their response to environmental variation across years, implying genetic variation in phenotypic plasticity. Our results also imply that plasticity in sea age will evolve as an indirect response to selection on mean sea age due to a shared genetic basis. Furthermore, we demonstrate differences between years in the additive and dominance functional genetic effects of vgll3 and six6 on sea age, suggesting that evolutionary responses will vary across environments. Considering the importance of age at maturity for survival and reproduction, genotype-environment interactions likely play an important role in local adaptation and population demography in Atlantic salmon.

Overruled by nature: A plastic response to environmental change disconnects a gene and its trait.

In Atlantic salmon, age at maturation is a life history trait governed by a sex-specific trade-off between reproductive success and survival. Following environmental changes across large areas of the Northeast Atlantic, many populations currently display smaller size at age and higher age at maturation. However, whether these changes reflect rapid evolution or plasticity is unknown. Approximately 1500 historical and contemporary salmon from the river Etne in Western Norway, genotyped at 50,000 SNPs, revealed three loci associated with age at maturation. These included vgll3 and six6 which collectively explained 36%-50% of the age at maturation variation in the 1983-1984 period. These two loci also displayed sex-specific epistasis, as the effect of six6 was only detected in males bearing two copies of the late maturation allele for vgll3. Strikingly, despite allelic frequencies at vgll3 remaining unchanged, the combined influence of these genes was nearly absent in all samples from 2013 to 2016, and genome-wide heritability strongly declined between the two time-points. The difference in age at maturation between males and females was upheld in the population despite the loss of effect from the candidate loci, which strongly points towards additional causative mechanisms resolving the sexual conflict. Finally, because admixture with farmed escaped salmon was excluded as the origin of the observed disconnection between gene(s) and maturation age, we conclude that the environmental changes observed in the North Atlantic during the past decades have led to bypassing of the influence of vgll3 and six6 on maturation through growth-driven plasticity.

Ontogenetic variation in the marine foraging of Atlantic salmon functionally links genomic diversity with a major life history polymorphism.

The ecological role of heritable phenotypic variation in free-living populations remains largely unknown. Knowledge of the genetic basis of functional ecological processes can link genomic and phenotypic diversity, providing insight into polymorphism evolution and how populations respond to environmental changes. By quantifying the marine diet of Atlantic salmon, we assessed how foraging behaviour changes along the ontogeny, and in relation to genetic variation in two loci with major effects on age at maturity (six6 and vgll3). We used a two-component, zero-inflated negative binomial model to simultaneously quantify foraging frequency and foraging outcome, separately for fish and crustaceans diets. We found that older salmon forage for both prey types more actively (as evidenced by increased foraging frequency), but with a decreased efficiency (as evidenced by fewer prey in the diet), suggesting an age-dependent shift in foraging dynamics. The vgll3 locus was linked to age-dependent changes in foraging behaviour: Younger salmon with vgll3LL (the genotype associated with late maturation) tended to forage crustaceans more often than those with vgll3EE (the genotype associated with early maturation), whereas the pattern was reversed in older salmon. Vgll3 LL genotype was also linked to a marginal increase in fish acquisition, especially in younger salmon, while six6 was not a factor explaining the diet variation. Our results suggest a functional role for marine feeding behaviour linking genomic diversity at vgll3 with age at maturity among salmon, with potential age-dependent trade-offs maintaining the genetic variation. A shared genetic basis between dietary ecology and age at maturity likely subjects Atlantic salmon populations to evolution induced by bottom-up changes in marine productivity.

Gene co-expression patterns in Atlantic salmon adipose tissue provide a molecular link among seasonal changes, energy balance and age at maturity.

Sexual maturation in many fishes requires a major physiological change that involves a rapid transition between energy storage and usage. In Atlantic salmon, this transition for the initiation of maturation is tightly controlled by seasonality and requires a high-energy status. Lipid metabolism is at the heart of this transition since lipids are the main energy storing molecules. The balance between lipogenesis (lipid accumulation) and lipolysis (lipid use) determines energy status transitions. A genomic region containing a transcription co-factor of the Hippo pathway, vgll3, is the main determinant of maturation timing in Atlantic salmon. Interestingly, vgll3 acts as an inhibitor of adipogenesis in mice and its genotypes are potentially associated with seasonal heterochrony in lipid storage and usage in juvenile Atlantic salmon. Here, we explored changes in expression of more than 300 genes directly involved in the processes of adipogenesis, lipogenesis and lipolysis, as well as the Hippo pathway in the adipose tissue of immature and mature Atlantic salmon with distinct vgll3 genotypes. We found molecular evidence consistent with a scenario in which immature males with different vgll3 genotypes exhibit contrasting seasonal dynamics in their lipid profiles. We also identified components of the Hippo signalling pathway as potential major drivers of vgll3 genotype-specific differences in adipose tissue gene expression. This study demonstrates the importance of adipose gene expression patterns for directly linking environmental changes with energy balance and age at maturity through genetic factors bridging lipid metabolism, seasonality and sexual maturation.

Habitat modulates population-level responses of freshwater salmon growth to a century of change in climate and competition.

The impacts of climate change are widespread and threaten natural systems globally. Yet, within regions, heterogeneous physical landscapes can differentially filter climate, leading to local response diversity. For example, it is possible that while freshwater lakes are sensitive to climate change, they may exhibit a diversity of thermal responses owing to their unique morphology, which in turn can differentially affect the growth and survival of vulnerable biota such as fishes. In particular, salmonids are cold-water fishes with complex life histories shaped by diverse freshwater habitats that are sensitive to warming temperatures. Here we examine the influence of habitat on the growth of sockeye salmon (Oncorhynchus nerka) in nursery lakes of Canada's Skeena River watershed over a century of change in regional temperature and intraspecific competition. We found that freshwater growth has generally increased over the last century. While growth tended to be higher in years with relatively higher summer air temperatures (a proxy for lake temperature), long-term increases in growth appear largely influenced by reduced competition. However, habitat played an important role in modulating the effect of high temperature. Specifically, growth was positively associated with rising temperatures in relatively deep (>50 m) nursery lakes, whereas warmer temperatures were not associated with a change in growth for fish among shallow lakes. The influence of temperature on growth also was modulated by glacier extent whereby the growth of fish from lakes situated in watersheds with little (i.e., <5%) glacier cover increased with rising temperatures, but decreased with rising temperatures for fish in lakes within more glaciated watersheds. Maintaining the integrity of an array of freshwater habitats-and the processes that generate and maintain them-will help foster a diverse climate-response portfolio for important fish species, which in turn can ensure that salmon watersheds are resilient to future environmental change.

Pacific salmon in the Canadian Arctic highlight a range-expansion pathway for sub-Arctic fishes.

Rapid climate change is altering Arctic ecosystems at unprecedented rates. These changes in the physical environment may open new corridors for species range expansions, with substantial implications for subsistence-dependent communities and sensitive ecosystems. Over the past 20 years, rising incidental harvest of Pacific salmon by subsistence fishers has been monitored across a widening range spanning multiple land claim jurisdictions in Arctic Canada. In this study, we connect Indigenous and scientific knowledges to explore potential oceanographic mechanisms facilitating this ongoing northward expansion of Pacific salmon into the western Canadian Arctic. A regression analysis was used to reveal and characterize a two-part mechanism related to thermal and sea-ice conditions in the Chukchi and Beaufort seas that explains nearly all of the variation in the relative abundance of salmon observed within this region. The results indicate that warmer late-spring temperatures in a Chukchi Sea watch-zone and persistent, suitable summer thermal conditions in a Beaufort Sea watch-zone together create a range-expansion corridor and are associated with higher salmon occurrences in subsistence harvests. Furthermore, there is a body of knowledge to suggest that these conditions, and consequently the presence and abundance of Pacific salmon, will become more persistent in the coming decades. Our collaborative approach positions us to document, explore, and explain mechanisms driving changes in fish biodiversity that have the potential to, or are already affecting, Indigenous rights-holders in a rapidly warming Arctic.

[68Ga]Pentixafor PET/CT for staging and prognostic assessment of newly diagnosed multiple myeloma: comparison to [18F]FDG PET/CT.

PURPOSE: To evaluate the prognostic performance of [68Ga]Pentixafor PET/CT at baseline for staging of patients with newly diagnosed multiple myeloma (MM) and to compare it with [18F]FDG PET/CT and the Revised-International Staging System (R-ISS). METHODS: Patients who underwent [68Ga]Pentixafor and [18F]FDG PET/CT imaging were retrospectively included. Patient staging was performed according to the Durie-Salmon PLUS staging system based on [68Ga]Pentixafor PET/CT and [18F]FDG PET/CT images, and the R-ISS. Progression-free survival (PFS) at patient follow-up was estimated using the Kaplan-Meier estimator and compared using the log-rank test. Area under the receiver operating characteristic curve (AUC) was calculated to assess predictive performance. RESULTS: Fifty-five MM patients were evaluated. Compared with [18F]FDG PET, [68Ga]Pentixafor PET detected 25 patients as the same stage, while 26 patients were upstaged and 4 patients were downstaged (P = 0.001). After considering the low-dose CT data, there was no statistically significant difference in the number of patients classified in each stage using [68Ga]Pentixafor PET/CT and [18F]FDG PET/CT (P = 0.091). [68Ga]Pentixafor PET/CT-based staging discriminated PFS outcomes in patients with different disease stages (stage I vs. stage II, stage I vs. stage III, and stage II vs. stage III; all P < 0.05), whereas for [18F]FDG PET/CT, there was only a difference in median PFS between stage I and III (P = 0.021). When staged by R-ISS, the median PFS for stage III was significantly lower than that for stage I and II (P = 0.008 and 0.035, respectively). When predicting 2-year PFS based on staging, the AUC of [68Ga]Pentixafor PET/CT was significantly higher than that of [68Ga]Pentixafor PET (0.923 vs. 0.821, P = 0.002), [18F]FDG PET (0.923 vs. 0.752 P = 0.002), and R-ISS (0.923 vs. 0.776, P = 0.005). CONCLUSIONS: [68Ga]Pentixafor PET/CT-based staging possesses substantial potential to predict disease progression in newly diagnosed MM patients.

Odor exposure during imprinting periods increases odorant-specific sensitivity and receptor gene expression in coho salmon (Oncorhynchus kisutch).

Pacific salmon are well known for their homing migrations; juvenile salmon learn odors associated with their natal streams prior to seaward migration, and then use these retained odor memories to guide them back from oceanic feeding grounds to their river of origin to spawn several years later. This memory formation, termed olfactory imprinting, involves at least in part, sensitization of the peripheral olfactory epithelium to specific odorants. We hypothesized that this change in peripheral sensitivity is due to exposure-dependent increases in the expression of odorant receptor (OR) proteins that are activated by specific odorants experienced during imprinting. To test this hypothesis, we exposed juvenile coho salmon, Oncorhynchus kisutch Walbaum, to the basic amino acid odorant L-arginine during the parr-smolt transformation (PST), when imprinting occurs, and assessed sensitivity of the olfactory epithelium to this and other odorants. We then identified the coho salmon orthologue of a basic amino acid odorant receptor (BAAR) and determined the mRNA expression levels of this receptor and other transcripts representing different classes of OR families. Exposure to L-arginine during the PST resulted in increased sensitivity to that odorant and a specific increase in BAAR mRNA expression in the olfactory epithelium relative to other ORs. These results suggest that specific increases in ORs activated during imprinting may be an important component of home stream memory formation and this phenomenon may ultimately be useful as a marker of successful imprinting to assess management strategies and hatchery practices that may influence straying in salmon.

Coronary circulation enhances the aerobic performance of wild Pacific salmon.

Female Pacific salmon often experience higher mortality than males during their once-in-a-lifetime up-river spawning migration, particularly when exposed to secondary stressors (e.g. high temperatures). However, the underlying mechanisms remain unknown. One hypothesis is that female Pacific salmon hearts are more oxygen-limited than those of males and are less able to supply oxygen to the body's tissues during this demanding migration. Notably, female hearts have higher coronary blood flow, which could indicate a greater reliance on this oxygen source. Oxygen limitations can develop from naturally occurring coronary blockages (i.e. coronary arteriosclerosis) found in mature salmon hearts. If female hearts rely more heavily on coronary blood flow but experience similar arteriosclerosis levels as males, they will have disproportionately impaired aerobic performance. To test this hypothesis, we measured resting (RMR) and maximum metabolic rate (MMR), aerobic scope (AS) and acute upper thermal tolerance in coho salmon (Oncorhynchus kisutch) with an intact or artificially blocked coronary oxygen supply. We also assessed venous blood oxygen and chemistry (cortisol, ions and metabolite concentrations) at different time intervals during recovery from exhaustive exercise. We found that coronary blockage impaired MMR, AS and the partial pressure of oxygen in venous blood (PvO2) during exercise recovery but did not differ between sexes. Coronary ligation lowered acute upper thermal tolerance by 1.1°C. Although we did not find evidence of enhanced female reliance on coronary supply, our findings highlight the importance of coronary blood supply for mature wild salmon, where migration success may be linked to cardiac performance, particularly during warm water conditions.

Flavobacterium facile sp. nov., isolated from water system of Atlantic salmon (Salmo salar) fry cultured in Chile.

Strain T-12T, an orange, Gram-stain-negative, non-motile, rod-shaped strain, was isolated in November 2013 from water samples collected from an Atlantic salmon (Salmo salar) fry culturing system at a fish farm in Chile. Phylogenetic analysis based on 16S rRNA sequences (1394 bp) revealed that strain T-12T belonged to the genus Flavobacterium, showing close relationships to Flavobacterium bernardetii F-372T (99.48 %) and Flavobacterium terrigena DS-20T (98.50 %). The genome size of strain T-12T was 3.28 Mb, with a G+C content of 31.1 mol%. Genome comparisons aligned strain T-12T with Flavobacterium bernardetii F-372T (GCA_011305415) and Flavobacterium terrigena DSM 17934T (GCA_900108955). The highest digital DNA-DNA hybridization (dDDH) values were 42.6 % with F. bernardetii F-372T (GCA_011305415) and 33.9 % with F. terrigena DSM 17934T (GCA_900108955). Pairwise average nucleotide identity (ANI) calculations were below the species cutoff, with the best results with F. bernardetii F-372T being: ANIb, 90.33 %; ANIm, 91.85 %; and TETRA, 0.997 %. These dDDH and ANI results confirm that strain T-12T represents a new species. The major fatty acids were iso-C15 : 0 and C15 : 1ω6с. Detected polar lipids included phospholipids (n=2), aminophospholipid (n=1), aminolipid (n=1) and unidentified lipids (n=2). The predominant respiratory quinone was menaquinone MK7 (80 %) followed by MK-6 (20 %). Phenotypic, chemotaxonomic, and genomic data support the classification of strain T-12T (=CECT 30410T=RGM 3222T) as representing a novel species of Flavobacterium, for which the name Flavobacterium facile sp. nov. is proposed.

Flavobacterium psychraquaticum sp. nov., isolated from water system of Atlantic salmon (Salmo salar) smolts cultured in Chile.

Strain LB-N7T, a novel Gram-negative, orange, translucent, gliding, rod-shaped bacterium, was isolated from water samples collected from an open system of Atlantic salmon (Salmo salar) smolts in a fish farm in Chile during a flavobacterial infection outbreak in 2015. Phylogenetic analysis based on 16S rRNA sequences (1337 bp) revealed that strain LB-N7T belongs to the genus Flavobacterium and is closely related to the type strains Flavobacterium ardleyense A2-1T (98.8 %) and Flavobacterium cucumis R2A45-3T (96.75 %). The genome size of strain LB-N7T was 2.93 Mb with a DNA G+C content 32.6 mol%. Genome comparisons grouped strain LB-N7T with Flavobacterium cheniae NJ-26T, Flavobacterium odoriferum HXWNR29T, Flavobacterium lacisediminis TH16-21T and Flavobacterium celericrescens TWA-26T. The calculated digital DNA-DNA hybridization values between strain LB-N7T and the closest related Flavobacterium strains were 23.3 % and the average nucleotide identity values ranged from 71.52 to 79.39 %. Menaquinone MK-6 was the predominant respiratory quinone, followed by MK-7. The major fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The primary polar lipids detected included nine unidentified lipids, two amounts of aminopospholipid and phospholipids, and a smaller amount of aminolipid. Phenotypic, genomic, and chemotaxonomic data suggest that strain LB-N7T (=CECT 30406T=RGM 3221T) represents as a novel bacterial species, for which the name Flavobacterium psychraquaticum sp. nov. is proposed.

A comprehensive transcriptional body map of Atlantic salmon unveils the vital role of the intestine in the immune system and highlights functional specialization within its compartments.

The intestine is a barrier organ that plays an important role in the immune system of Atlantic salmon. The immune functions are distributed among the diffuse gut lymphoid tissue containing diverse immune cells, and other cell types. Comparison of intestinal transcriptomes with those of other organs and tissues offers an opportunity to elucidate the specific roles of the intestine and its relationship with other parts of the body. In this work, a meta-analysis was performed on a large volume of data obtained using a genome-wide DNA oligonucleotide microarray. The intestine ranks third by the expression level of immune genes after the spleen and head kidney. The activity of antigen presentation and innate antiviral immunity is higher in the intestine than in any other tissue. By comparing transcriptome profiles, intestine shows the greatest similarity with the gill, head kidney, spleen, epidermis, and olfactory rosette (descending order), which emphasizes the integrity of the peripheral mucosal system and its strong connections with the major lymphoid organs. T cells-specific genes dominate among the genes co-expressed in these tissues. The transcription signature of CD8+ (86 genes, r > 0.9) includes a master gene of immune tolerance foxp3 and other negative regulators. Different segments of the intestine were compared in a separate experiment, in which expression gradients along the intestine were found across several functional groups of genes. The expression of luminal and intracellular (lysosome) proteases is markedly higher in pyloric caeca and distal intestine respectively. Steroid metabolism and cytochromes P450 are highly expressed in pyloric caeca and mid intestine while the distal intestine harbors genes related to vitamin and iron metabolism. The expression of genes for antigen presenting proteins and immunoglobulins shows a gradual increase towards the distal intestine.

Current vaccination strategy against Piscirickettsia salmonis in Chile based only on the EM-90 genogroup shows incomplete cross-protection for the LF-89 genogroup.

Piscirickettsia salmonis, the primary bacterial disease in Chilean salmon farming, necessitates a constant refinement of control strategies. This study hypothesized that the current vaccination strategy for SRS control in the Chilean Atlantic salmon aquaculture industry, which has been in place since 2017 (ALPHA JECT® 5.1 plus LiVac®), solely relies on vaccines formulated with the EM-90 genogroup of P. salmonis (PS-EM-90), triggering a partial cross-immunity response in fish infected with the LF-89 genogroup (PS-LF-89). Relative Percent Survival (RPS) and cell-mediated immune (CMI) response were evaluated in Atlantic salmon post-smolts vaccinated with the standard vaccination strategy but challenged with both PS-EM-90 and PS-LF-89, in addition to other vaccination strategies considering primo vaccination and booster with other commercial vaccines and the possible enhancing effects of the combination with a natural immunomodulator (PAQ-Xtract®) administered orally. The intraperitoneal (I.P.) challenge was performed after 2395 degree-days (DD) after the start of the immunostimulant delivery, 1905 DD after the primo vaccination, and 1455 DD after the booster vaccination. Unvaccinated fish showed 73.6 and 41.7% mortality when challenged with PS-EM-90 and PS-LF-89, respectively. Fish infected with PS-LF-89 died significantly faster (21 days post-infection, dpi) than fish challenged with PS-EM-90 (28 dpi) (p = 0.0043) and had a higher probability of death (0.4626) than fish challenged with PS-EM-90. RPS had a significant positive correlation with the PS-EM-90 load of the P. salmonis genogroup (r = 0.540, p < 0.01) but not with the PS-LF-89 load (r = 0.155, p > 0.05). This demonstrated that the immunization strategies were more effective in lowering PS-EM-90 loads, resulting in higher survival rates in fish challenged with PS-EM-90. The current industry vaccination strategy recorded a 100% RPS when fish were challenged with PS-EM-90, but the RPS dropped significantly to 77% when fish were challenged with PS-LF-89, meaning that the strategy did not show complete cross-protection. But after adding PAQ-Xtract®, the RPS improved from 77% to 92% in fish that were vaccinated with the standard method but then challenged with PS-LF-89. The most effective vaccination strategy was based on LiVac® as primo vaccination and ALPHA JECT® 5.1 plus LiVac® as booster vaccination, with or without PAQ-Xtract®, in both PS-EM-90 (100%) and PS-LF-89 (96%) challenged fish. The serum concentration of anti-P. salmonis IgM did not show a correlation with the protection of immunization strategies expressed in survival. Low serum IL-12 and high serum IFNγ concentrations showed a correlation with higher bacterial loads and lower survival. Aggregate analysis showed a significant correlation between higher numbers of CD8+ cells in the head-kidney, higher fish survival, and a lower bacterial load. The immunization strategies were safe for fish and induced only mild microscopic lesions in the gut. Taken together, our results help to better understand the biological interaction between P. salmonis and post-smolt vaccinated Atlantic salmon to deepen the knowledge on vaccine-induced protection, CMI immune response, and cross-immunity applied to improve the current immunization strategy for SRS control in the Chilean salmon industry.

Environmental influence on the Atlantic salmon transcriptome and methylome during sea lice infestations.

The fish's immune response is affected by different factors, including a wide range of environmental conditions that can also disrupt or promote changes in the host-pathogen interactions. How environmental conditions modulate the salmon genome during parasitism is poorly understood here. This study aimed to explore the environmental influence on the Salmo salar transcriptome and methylome infected with the sea louse Caligus rogercresseyi. Atlantic salmon were experimentally infected with lice at two temperatures (8 and 16 °C) and salinity conditions (32 and 26PSU). Fish tissues were collected from the infected Atlantic salmon for reduced representation bisulfite sequencing (RRBS) and whole transcriptome sequencing (RNA-seq) analysis. The parasitic load was highly divergent in the evaluated environmental conditions, where the lowest lice abundance was observed in fish infected at 8 °C/26PSU. Notably, transcriptome profile differences were statistically associated with the number of alternative splicing events in fish exposed to low temperature/salinity conditions. Furthermore, the temperature significantly affected the methylation level, where high values of differential methylation regions were observed at 16 °C. Also, the association between expression levels of spliced transcripts and their methylation levels was determined, revealing significant correlations with Ferroptosis and TLR KEEG pathways. This study supports the relevance of the environmental conditions during host-parasite interactions in marine ecosystems. The discovery of alternative splicing transcripts associated with DMRs is also discussed as a novel player in fish biology.

Mucosal barrier status in Atlantic salmon fed rapeseed oil and Schizochytrium oil partly or fully replacing fish oil through winter depression.

The study was designed to investigate the effects of replacing fish oil by algal oil and rapeseed oil on histomorphology indices of the intestine, skin and gill, mucosal barrier status and immune-related genes of mucin and antimicrobial peptide (AMP) genes in Atlantic salmon (Salmo salar). For these purposes, Atlantic salmon smolts were fed three different diets. The first was a control diet containing fish oil but no Schizochytrium oil. In the second diet, almost 50 % of the fish oil was replaced with algal oil, and in the third diet, fish oil was replaced entirely with algal oil. The algal oil contained mostly docosahexaenoic acid (DHA) and some eicosapentaenoic acid (EPA). The study lasted for 49 days in freshwater (FW), after which some fish from each diet group were transferred to seawater (SW) for a 48-h challenge test at 33 ppt to test their ability to tolerate high salinity. Samples of skin, gills, and mid intestine [both distal (DI) and anterior (AI) portions of the mid intestine] were collected after the feeding trial in FW and after the SW-challenge test to assess the effects of the diets on the structure and immune functions of the mucosal surfaces. The results showed that the 50 % VMO (Veramaris® algal oil) dietary group had improved intestinal, skin, and gill structures. Principal component analysis (PCA) of the histomorphological parameters demonstrated a significant effect of the algal oil on the intestine, skin, and gills. In particular, the mucosal barrier function of the intestine, skin, and gills was enhanced in the VMO 50 % dietary group after the SW challenge, as evidenced by increased mucous cell density. Immunolabelling of heat shock protein 70 (HSP70) in the intestine (both DI and AI) revealed downregulation of the protein expression in the 50 % VMO group and a corresponding upregulation in the 100 % VMO group compared to 0 % VMO. The reactivity of HSP70 in the epithelial cells was higher after the SW challenge compared to the FW phase. Immune-related genes related to mucosal defense, such as mucin genes [muc2, muc5ac1 (DI), muc5ac1 (AI), muc5ac2, muc5b (skin), and muc5ac1 (gills)], and antimicrobial peptide genes [def3 (DI), def3 (AI), and cath1 (skin)] were significantly upregulated in the 50 % VMO group. PCA of gene expression demonstrated the positive influences on gene regulation in the 50 % VMO dietary group. In conclusion, this study demonstrated the positive effect of substituting 50 % of fish oil with algal oil in the diets of Atlantic salmon. The findings of histomorphometry, mucosal mapping, immunohistochemistry, and immune-related genes connected to mucosal responses all support this conclusion.

Impact of freshwater rearing history on Atlantic salmon gill response to viral stimulation post seawater transfer.

Land-based recirculating aquaculture systems (RAS) have risen in prevalence in recent years for Atlantic salmon production, enabling intensive production which allows increased growth and environmental control, but also having the potential for reducing water use and eutrophication. The Atlantic salmon has an anadromous life history with juvenile stages in freshwater (FW) and on-growing in seawater (SW), enabled by a transformational process known as smoltification. The timing of smoltification and transfer of smolts from FW to SW is critical under commercial production with high mortalities during this period. The impact of FW rearing system on immune function following seawater transfer (SWT) is not well understood. In this study parr were raised in either RAS or a traditional open-LOCH system until smolting and then transferred to a common marine environment. Two-weeks post-SWT fish were immune stimulated with a viral mimic (poly I:C) for 24 h to assess the ability to mount an antiviral immune response, assessed by whole transcriptome analysis of gill tissue, an important immune organ in fish. We show that unstimulated smolts reared in the LOCH had higher immune gene expression than those reared in RAS as determined by functional analysis. However, following stimulation, smolts reared in the RAS mounted a greater magnitude of response with a suite of immune genes displaying higher fold induction of transcription compared to LOCH reared smolts. We suggest RAS smolts have a lower steady state immune-associated transcriptome likely due to an unvarying environment, in terms of environmental factors and lack of exposure to pathogens, which shows a compensatory mechanism following stimulation allowing immune 'catch-up' with those reared in the LOCH. Alternatively, the RAS fish are experiencing an excessive response to the immune stimulation.