RNA-Seq Analysis of the Growth Hormone Transgenic Female Triploid Atlantic Salmon (Salmo salar) Hepatic Transcriptome Reveals Broad Temperature-Mediated Effects on Metabolism and Other Biological Processes.

This study examined the impact of rearing temperature (10.5, 13.5 or 16.5°C) on the hepatic transcriptome of AquAdvantage Salmon (growth hormone transgenic female triploid Atlantic salmon) at an average weight of 800 g. Six stranded PE libraries were Illumina-sequenced from each temperature group, resulting in an average of over 100 M raw reads per individual fish. RNA-sequencing (RNA-seq) results showed the greatest difference in the number of differentially expressed transcripts (1750 DETs), as revealed by both DESeq2 and edgeR (q < 0.05; fold-change > |1.5|), was between the 10.5 and 16.5°C temperature groups. In contrast, 172 and 52 DETs were found in the 10.5 vs. 13.5°C and the 13.5 vs. 16.5°C comparisons, respectively. Considering the DETs between the 10.5 and 16.5°C groups, 282 enriched gene ontology (GO) terms were identified (q < 0.05), including "response to stress", "immune system process", "lipid metabolic process", "oxidation-reduction process", and "cholesterol metabolic process", suggesting elevated temperature elicited broad effects on multiple biological systems. Pathway analysis using ClueGO showed additional impacts on amino acid and lipid metabolism. There was a significant positive correlation between RNA-seq and real-time quantitative polymerase chain reaction (RT-qPCR) results for 8 of 9 metabolic-related transcripts tested. RT-qPCR results also correlated to changes in fillet tissue composition previously reported in these salmon (e.g., methionine and lysine concentrations positively correlated with hsp90ab1 transcript expression), suggesting that rearing temperature played a significant role in mediating metabolic/biosynthetic pathways of AquAdvantage Salmon. Many transcripts related to lipid/fatty acid metabolism (e.g., elovl2, fabpi, hacd2, mgll, s27a2, thrsp) were downregulated at 16.5°C compared to both other temperature groups. Additionally, enrichment of stress-, apoptosis- and catabolism-relevant GO terms at 16.5°C suggests that this temperature may not be ideal for commercial production when using freshwater recirculating aquaculture systems (RAS). This study relates phenotypic responses to transcript-specific findings and therefore aids in the determination of an optimal rearing temperature for AquAdvantage Salmon. With approval to grow and sell AquAdvantage Salmon in the United States and Canada, the novel insights provided by this research can help industry expansion by promoting optimal physiological performance and health.

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.

Comparative Genomics Analysis of Vibrio anguillarum Isolated from Lumpfish (Cyclopterus lumpus) in Newfoundland Reveal Novel Chromosomal Organizations.

Vibrio anguillarum is a Gram-negative marine pathogen causative agent of vibriosis in a wide range of hosts, including invertebrates and teleosts. Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to control sea lice (Lepeophtheirus salmonis) infestations in the Atlantic salmon (Salmo salar) aquaculture industry. V. anguillarum is one of the most frequent bacterial pathogens affecting lumpfish. Here, we described the phenotype and genomic characteristics of V. anguillarum strain J360 isolated from infected cultured lumpfish in Newfoundland, Canada. Koch's postulates determined in naïve lumpfish showed lethal acute vibriosis in lumpfish. The V. anguillarum J360 genome was shown to be composed of two chromosomes and two plasmids with a total genome size of 4.56 Mb with 44.85% G + C content. Phylogenetic and comparative analyses showed that V. anguillarum J360 is closely related to V. anguillarum strain VIB43, isolated in Scotland, with a 99.8% genome identity. Differences in the genomic organization were identified and associated with insertion sequence elements (ISs). Additionally, V. anguillarum J360 does not possess a pJM1-like plasmid, typically present in virulent isolates from the Pacific Ocean, suggesting that acquisition of this extrachromosomal element and the virulence of V. anguillarum J360 or other Atlantic isolates could increase.

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 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.

Optimization and field use of a bioassay to monitor sea lice Lepeophtheirus salmonis sensitivity to emamectin benzoate.

A bioassay for sea lice Lepeophtheirus salmonis sensitivity towards emamectin benzoate (EMB) was validated for field use. A probit regression model with natural responsiveness was used for the number of affected (moribund or dead) sea lice in bioassays involving different concentrations of EMB. Bioassay optimization included an evaluation of the inter-rater reliability of sea lice responsiveness to EMB and an evaluation of gender-related differences in susceptibility. Adoption of a set of bioassay response criteria improved the concordance (evaluated using the concordance correlation coefficient) between raters' assessments and the model estimation of EC50 values (the 'effective concentration' leading to a response of 50% of the lice not prone to natural response). An evaluation of gender-related differences in EMB susceptibility indicated that preadult stage female sea lice exhibited a significantly larger sensitivity towards EMB in 12 of 19 bioassays compared to preadult males. In order to evaluate sea lice sensitivity to EMB in eastern Canada, the intensive salmon farming area in the Bay of Fundy in southwestern New Brunswick was divided into 4 distinct regions based on industry health management practices and hydrographics. A total of 38 bioassays were completed from 2002 to 2005 using populations of preadult stage sea lice collected from Atlantic salmon Salmo salar farms within the 4 described regions. There was no significant overall effect of region or year on EC50 values; however, analysis of variance indicated a significant effect of time of year on EC50 values in 2002 and a potential effect in 2004 to 2005. Although the range of EC50 values obtained in this 3 yr study did not appear sufficient to affect current clinical success in the control of sea lice, the results suggest a seasonal- or temperature-associated variation in sensitivity to EMB. This will need to be considered if changes in EMB efficacy occur in the future.