Transcriptome analysis of liver, gill and intestine in rainbow trout (Oncorhynchus mykiss) symptomatically or asymptomatically infected with Vibrio anguillarum.

Rainbow trout (Oncorhynchus mykiss), an important economic cold-water fish worldwide, is severely threatened by viruses and bacteria in the farming industry. The vibriosis outbreak has caused a significant setback to aquaculture. Vibrio anguillarum, one of the common disease-causing vibriosis associated with severe lethal vibriosis in aquaculture, infects fish mainly by adsorption and invasion of the skin, gills, lateral line and intestine. To investigate the defense mechanism of rainbow trout against the pathogen after infection with Vibrio anguillarum, trout were intraperitoneally injected by Vibrio anguillarum and divided into symptomatic group (SG) and asymptomatic group (AG) according to the phenotype. RNA-Seq technology was used to evaluate the transcriptional signatures of liver, gill and intestine of trout injected with Vibrio anguillarum (SG and AG) and corresponding control groups (CG(A) and CG(B)). The GO and KEGG enrichment analyses were used to investigate the mechanisms underlying the differences in susceptibility to Vibrio anguillarum. Results showed that in SG, immunomodulatory genes in the cytokine network were activated and tissue function-related genes were down-regulated, while apoptosis mechanisms were activated. However, AG responded to Vibrio anguillarum infection by activating complement related immune defenses, while metabolism and function related genes were up-regulated. Conclusively, a rapid and effective immune and inflammatory response can successfully defend Vibrio anguillarum infection. However, a sustained inflammatory response can lead to tissue and organ damage and cause death. Our results may provide a theoretical basis for breeding rainbow trout for disease resistance.

Identification and characterization of mkk genes and their expression profiles in rainbow trout (Oncorhynchus mykiss) symptomatically or asymptomatically infected with Vibrio anguillarum.

Mitogen-activated protein kinase kinases (MKKs) are intermediate kinases of mitogen-activated protein kinases (MAPKs) signaling pathways. MKKs are activated by mitogen-activated protein kinase kinase kinase (MKKK) and then the activated MKKs trigger the activation of downstream MAPKs. MAPK signaling pathways play an important role in regulating immune functions including apoptosis and inflammation. However, studies on identification and characterization of mkk repertoire in rainbow trout (Oncorhynchus mykiss) are still limited. Trout experienced 4 rounds (4R) of whole genome duplication (WGD), thus exhibiting increased paralogs of mkks with potentially functional diversity. In this study, we identified 17 mkk genes in trout and the following bacterial challenge (Vibrio anguillarum) studies showed functional diversity of different mkk subtypes. Vibrio anguillarum infection resulted in significantly up-regulated mkk2 subtypes in spleen and liver, and mkk4b3 in spleen, suggesting immunomodulation was regulated by activation of ERK, p38 and JNK pathways. Compared to other mkk subtypes, mkk6s were down-regulated in symptomatic group, rather than asymptomatic group. The organisms present negative feedback on MAPK activation, thus reducing extra damage to cells. We observed down-regulated mkk6s with up-regulated genes (dusp1 & dusp2) involved in negative feedback of MAPK activation. Based on these results, we might propose the distinct expression patterns of genes associated with MAPK pathways resulted in different phenotypes and symptoms of trout in response to bacterial challenge.

Pleiotropic Role of Rainbow Trout CXCRs in Response to Disease and Environment: Insights from Transcriptional Signatures and Structure Analysis.

Chemokines are cytokines with chemoattractant capacities that exert their physiological functions through the binding of chemokine receptors. Thus, chemokine and receptor complexes exert important roles in regulating development and homeostasis during routine immune surveillance and inflammation. Compared to mammals, the physiology and structure of chemokine receptors in fish have not been systematically studied. Furthermore, the salmonid-specific whole genome duplication has significantly increased the number of functional paralogs of chemokine receptors. In this context, in the current study, trout exhibited 17 cxcr genes, including 12 newly identified and 5 previously identified receptors. Interestingly, gene expression of brain cxcr1 and cxcr4, kidney cxcr3 and cxcr4, and spleen cxcr3, cxcr4, and cxcr5 subtypes were altered by bacterial infection, whereas brain cxcr1, kidney cxcr1 and cxcr7, and liver cxcr2, cxcr3, and cxcr4 subtypes were changed in response to environmental changes. Based on protein structures predicted by ColabFold, the conserved amino acids in binding pockets between trout CXCR4.1 subtypes and human CXCR4 were also analyzed. Our study is valuable from a comparative point of view, providing new insights into the identification and physiology of salmonid chemokine receptors.

Identification, characterization, and transcription of serotonin receptors in rainbow trout (Oncorhynchus mykiss) in response to bacterial infection and salinity changes.

Serotonergic system is involved in the regulation of physiological functions and behavioral traits including cognition, memory, aggression, stress coping, appetite and immunomodulation. Serotonin exerts its functions via binding distinct serotonin receptors which are classified into 7 groups. Salmonid exhibits expanded functional gene copies due to salmonid-specific whole genome duplication. However, serotonin receptor (htr) repertoire is not fully identified in rainbow trout (Oncorhynchus mykiss). In this study, we identified 39 htr genes, including 14 htr1, 4 htr2, 4 htr2 like, 3 htr3, 4 htr4, 2 htr5, 2 htr6, and 6 htr7 subtypes. We investigated physiological functions of serotonin receptors in response to bacterial pathogens exposure and salinity changes. We showed htr1, htr2, htr4 and htr7 subtypes were associated with immunomodulation in response to Vibrio anguillarum or Aeromonas salmonicida infection. Saltwater (salinity of 15) transfer significantly altered htr1, htr2, htr4, and htr7 subtypes, suggesting trout Htr was associated with osmoregulation. We further showed residues interacted with inverse agonist (methiothepin) and serotonin analogue (5-Carboxamidotryptamine) were conserved between trout and human, suggesting exogenous ligands targeting human HTRs might have a role in aquaculture. This study showed duplicated trout Htrs might be physiologically neofunctionalized and potentially exhibit pleiotropic effects in regulating immunomodulation and osmoregulation.

Identification and characterization of caspases genes in rainbow trout (Oncorhynchus mykiss) and their expression profiles after Aeromonas salmonicida and Vibrio anguillarum infection.

Caspases are highly conserved cysteine-dependent aspartyl-specific proteases that play an important role in regulating cell death and inflammation. However, the caspase genes have not been systematically studied in rainbow trout (Oncorhynchus mykiss). Rainbow trout experienced 4 rounds (4R) of genome duplication in the evolutionary history. Thereby an increased numbers of paralogs are observed in trout, probably with more complicated gene functions. We identified 18 caspase genes in rainbow trout, including two inflammatory caspases (casp1a, casp1b), six apoptosis executioner caspases (casp3, casp3a1, casp3a2, casp3b, casp6, and casp7), nine apoptosis initiator caspases (casp2a, casp2b, casp8, casp9a, casp9b, casp10a, casp10b, casp20a, and casp20b) and one uncategorized caspase gene (casp17). To investigate the potentially physiological functions of caspase genes, we challenged the rainbow trout with Aeromonas salmonicida (A. salmonicida) and Vibrio anguillarum (V. anguillarum). Results showed that the CASP3-regulated intrinsic apoptosis was activated after A. salmonicida infection, while the CASP8 and CASP6-regulated extrinsic apoptosis exerted the greatest effect on trout challenged with V. anguillarum. In response to V. anguillarum infection, the data of RNA-Seq further showed the casp8 was tightly integrated with the significantly enriched Gene Ontology terms and functional pathways, including apoptosis regulation, pathogen detection and immunomodulation. Our study provides a foundation for the physiological functions and regulatory network of the caspase genes in teleosts.

Transcriptional Profiles of Genes Related to Stress and Immune Response in Rainbow Trout (Oncorhynchus mykiss) Symptomatically or Asymptomatically Infected With Vibrio anguillarum.

Rainbow trout (Oncorhynchus mykiss) is one of the most common aquaculture fish species worldwide. Vibriosis disease outbreaks cause significant setbacks to aquaculture. The stress and immune responses are bidirectionally modulated in response to the health challenges. Therefore, an investigation into the regulatory mechanisms of the stress and immune responses in trout is invaluable for identifying potential vibriosis treatments. We investigated the transcriptional profiles of genes associated with stress and trout immune functions after Vibrio anguillarum infection. We compared the control trout (CT, 0.9% saline injection), asymptomatic trout (AT, surviving trout with minor or no symptoms after bacteria injection), and symptomatic trout (ST, moribund trout with severe symptoms after bacteria injection). Our results showed activated immunomodulatory genes in the cytokine network and downregulated glucocorticoid and mineralocorticoid receptors in both AT and ST, indicating activation of the proinflammatory cytokine cascade as a common response in AT and ST. Moreover, the AT specifically activated the complement- and TNF-associated immune defenses in response to V. anguillarum infection. However, the complement and coagulation cascades, as well as steroid hormone homeostasis in ST, were disturbed by V. anguillarum. Our studies provide new insights toward understanding regulatory mechanisms in stress and immune functions in response to diseases.