MicroRNAs (miRNAs) are key regulators in fish immune responses. However, no study has previously characterized the impact of polyriboinosinic polyribocytidylic acid (pIC) and formalin-killed typical Aeromonas salmonicida (ASAL) on miRNA expression in Atlantic salmon fed a commercial diet with and without immunostimulant CpG. To this end, first, we performed small RNA deep sequencing and qPCR analyses to identify and confirm pIC- and/or ASAL-responsive miRNAs in the head kidney of salmon fed a control diet. DESeq2 analyses identified 12 and 18 miRNAs differentially expressed in pIC and ASAL groups, respectively, compared to the controls. Fifteen of these miRNAs were studied by qPCR; nine remained significant by qPCR. Five miRNAs (miR-27d-1-2-5p, miR-29b-2-5p, miR-146a-5p, miR-146a-1-2-3p, miR-221-5p) were shown by qPCR to be significantly induced by both pIC and ASAL. Second, the effect of CpG-containing functional feed on miRNA expression was investigated by qPCR. In pre-injection samples, 6 of 15 miRNAs (e.g., miR-181a-5-3p, miR-462a-3p, miR-722-3p) had significantly lower expression in fish fed CpG diet than control diet. In contrast, several miRNAs (e.g., miR-146a-1-2-3p, miR-192a-5p, miR-194a-5p) in the PBS- and ASAL-injected groups had significantly higher expression in CpG-fed fish. Multivariate statistical analyses confirmed that the CpG diet had a greater impact on miRNA expression in ASAL-injected compared with pIC-injected fish. This study identified immune-relevant miRNA biomarkers that will be valuable in the development of diets to combat infectious diseases of salmon.

MicroRNAs (miRNAs) are important post-transcriptional gene expression regulators. Here, 448 different miRNA genes, including 17 novel miRNAs, encoding for 589 mature Atlantic salmon miRNAs were identified after sequencing 111 samples (fry, pathogen challenged fry, various developmental and adult tissues). This increased the reference miRNAome with almost one hundred genes. Prior to isomiR characterization (mature miRNA variants), the proportion of erroneous sequence variants (ESVs) arising in the analysis pipeline was assessed. The ESVs were biased towards 5’ and 3’ end of reads in unexpectedly high proportions indicating that measurements of ESVs rather than Phred score should be used to avoid misinterpreting ESVs as isomiRs. Forty-three isomiRs were subsequently discovered. The biological effect of the isomiRs measured as increases in target diversity was small (<3%). Five miRNA genes showed allelic variation that had a large impact on target gene diversity if present in the seed. Twenty-one miRNAs were ubiquitously expressed while 31 miRNAs showed predominant expression in one or few tissues, indicating housekeeping or tissue specific functions, respectively. The miR-10 family, known to target Hox genes, were highly expressed in the developmental stages. The proportion of miR-430 family members, participating in maternal RNA clearance, was high at the earliest developmental stage.

Macrophages are among the first cells to respond to infection and disease. While microRNAs (miRNAs) are involved in the process of monocyte-to-macrophage differentiation in mammals, less is known in teleost fish. Here, Atlantic salmon head kidney leukocytes (HKLs) were used to study the expression of miRNAs in response to in vitro culture. The morphological analysis of cultures showed predominantly monocyte-like cells on Day 1 and macrophage-like cells on Day 5, suggesting that the HKLs had differentiated from monocytes to macrophages. Day 5 HKLs also contained a higher percentage of phagocytic cells. Small RNA sequencing and qPCR analysis were applied to examine the miRNA diversity and expression. There were 370 known mature Atlantic salmon miRNAs in HKLs. Twenty-two miRNAs (15 families) were downregulated while 44 miRNAs (25 families) were upregulated on Day 5 vs. Day 1. Mammalian orthologs of many of the differentially expressed (DE) miRNAs are known to regulate macrophage activation and differentiation, while the teleost-specific miR-2188, miR-462 and miR-731 were also DE and are associated with immune responses in fish. In silico predictions identified several putative target genes of qPCR-validated miRNAs associated with vertebrate macrophage differentiation. This study identified Atlantic salmon miRNAs likely to influence macrophage differentiation, providing important knowledge for future functional studies.