RESUMO
Piscirickettsiosis is the most important bacterial disease in the Chilean salmon industry, which has borne major economic losses due to failure to control it. Cells use extracellular vesicles (EVs) as an inter-cellular communicators to deliver several factors (e.g., microRNAs) that may regulate the responses of other cells. However, there is limited knowledge about the identification and characterization of EV-miRNAs in salmonids or the effect of infections on these. In this study, Illumina sequencing technology was used to identify Coho salmon plasma EV-miRNAs upon Piscirickettsia salmonis infection at four different time points. A total of 118 novels and 188 known EV-miRNAs, including key immune teleost miRNAs families (e.g., miR-146, miR-122), were identified. A total of 245 EV-miRNAs were detected as differentially expressed (FDR < 5%) in terms of control, with a clear down-regulation pattern throughout the disease. KEGG enrichment results of EV-miRNAs target genes showed that they were grouped mainly in cellular, stress, inflammation and immune responses. Therefore, it is hypothesized that P. salmonis could potentially benefit from unbalanced modulation response of Coho salmon EV-miRNAs in order to promote a hyper-inflammatory and compromised immune response through the suppression of different key immune host miRNAs during the course of the infection, as indicated by the results of this study.
Assuntos
Doenças dos Peixes/microbiologia , MicroRNAs/metabolismo , Oncorhynchus kisutch/metabolismo , Infecções por Piscirickettsiaceae/imunologia , Animais , Vesículas Extracelulares/metabolismo , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica , Inflamação , Oncorhynchus kisutch/genética , Oncorhynchus kisutch/imunologia , Piscirickettsia/fisiologiaRESUMO
The increasing capacity of transcriptomic analysis by high throughput sequencing has highlighted the presence of a large proportion of transcripts that do not encode proteins. In particular, long non-coding RNAs (lncRNAs) are sequences with low coding potential and conservation among species. Moreover, cumulative evidence has revealed important roles in post-transcriptional gene modulation in several taxa. In fish, the role of lncRNAs has been scarcely studied and even less so during the immune response against sea lice. In the present study we mined for lncRNAs in Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynkus kisutch), which are affected by the sea louse Caligus rogercresseyi, evaluating the degree of sequence conservation between these two fish species and their putative roles during the infection process. Herein, Atlantic and Coho salmon were infected with 35 lice/fish and evaluated after 7 and 14 days post-infestation (dpi). For RNA sequencing, samples from skin and head kidney were collected. A total of 5658/4140 and 3678/2123 lncRNAs were identified in uninfected/infected Atlantic and Coho salmon transcriptomes, respectively. Species-specific transcription patterns were observed in exclusive lncRNAs according to the tissue analyzed. Furthermore, neighbor gene GO enrichment analysis of the top 100 highly regulated lncRNAs in Atlantic salmon showed that lncRNAs were localized near genes related to the immune response. On the other hand, in Coho salmon the highly regulated lncRNAs were localized near genes involved in tissue repair processes. This study revealed high regulation of lncRNAs closely localized to immune and tissue repair-related genes in Atlantic and Coho salmon, respectively, suggesting putative roles for lncRNAs in salmon against sea lice infestation.
Assuntos
Doenças dos Peixes/genética , Imunidade/genética , Infestações por Piolhos/genética , Oncorhynchus kisutch/genética , RNA Longo não Codificante/genética , Salmo salar/genética , Transcriptoma , Animais , Copépodes/imunologia , Copépodes/fisiologia , Doenças dos Peixes/imunologia , Perfilação da Expressão Gênica , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Parasita , Infestações por Piolhos/imunologia , Infestações por Piolhos/parasitologia , Oncorhynchus kisutch/imunologia , Oncorhynchus kisutch/parasitologia , Salmo salar/imunologia , Salmo salar/parasitologia , Especificidade da Espécie , Cicatrização/genéticaRESUMO
The genes of the major histocompatibility complex (MHC) are amongst the most variable in vertebrates and represent some of the best candidates to study processes of adaptive evolution. However, despite the number of studies available, most of the information on the structure and function of these genes come from studies in mammals and birds in which the MHC class I and II genes are tightly linked and class II alpha exhibits low variability in many cases. Teleost fishes are among the most primitive vertebrates with MHC and represent good organisms for the study of MHC evolution because their class I and class II loci are not physically linked, allowing for independent evolution of both classes of genes. We have compared the diversity and molecular mechanisms of evolution of classical MH class II alpha and class II beta loci in farm populations of three salmonid species: Oncorhynchus kisutch, Oncorhynchus mykiss and Salmo salar. We found single classical class II loci and high polymorphism at both class II alpha and beta genes in the three species. Mechanisms of evolution were common for both class II genes, with recombination and point mutation involved in generating diversity and positive selection acting on the peptide-binding residues. These results suggest that the maintenance of variability at the class IIalpha gene could be a mechanism to increase diversity in the MHC class II in salmonids in order to compensate for the expression of one single classical locus and to respond to a wider array of parasites.
Assuntos
Evolução Molecular , Genes MHC da Classe II , Salmonidae/genética , Salmonidae/imunologia , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , Primers do DNA/genética , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Variação Genética , Antígenos de Histocompatibilidade Classe II/genética , Modelos Genéticos , Dados de Sequência Molecular , Oncorhynchus kisutch/genética , Oncorhynchus kisutch/imunologia , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/imunologia , Recombinação Genética , Salmo salar/genética , Salmo salar/imunologia , Salmonidae/classificação , Seleção Genética , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
We have used the expression library immunization technology to study the protection of Coho salmon Oncorhynchus kisutch to the infection with Piscirickettsia salmonis. Purified DNA from this bacterium was sonicated and the fragments were cloned in the expression vector pCMV-Bios. Two libraries were obtained containing 22,000 and 28,000 colonies and corresponding to approximately 8 and 10 times the genome of the pathogen, respectively. On average, the size of the inserts ranged between 300 and 1,000 bp. The plasmid DNA isolated from one of these libraries was purified and 20 micrograms were injected intramuscularly into 60 fish followed by a second dose of 10 micrograms applied 40 days later. As control, fish were injected with the same amount of DNA of the vector pCMV-Bios without insert. The titer of IgM anti-P. salmonis of vaccinated fish, evaluated 60 days post-injection, was significantly higher than that of the control group injected with the vector alone. Moreover, this response was specific against P. salmonis antigens, since no cross reaction was detected with Renibacterium salmoninarum and Yersinia ruckeri. The vaccinated and control fish were challenged 60 days after the second dose of DNA with 2.5 x 10(7) P. salmonis corresponding to 7.5 times the LD50. At 30 days post-challenge, 100% mortality was obtained with the control fish while 20% of the vaccinated animals survived. All surviving fish exhibited a lower bacterial load in the kidney than control fish. The expression library was also tested in Balb/c mice and it was found that the humoral immune response was specific to P. salmonis and it was dependent on the amount of DNA injected.