Amoebic Gill Disease (AGD), caused by the protozoan extracellular parasite Paramoeba perurans (P. perurans) is a disease affecting Atlantic salmon (Salmo salar). This study investigated the gill transcriptomic profile of pre-clinical AGD using RNA-sequencing (RNA-seq) technology. RNA-seq libraries generated at 0, 4, 7, 14 and 16 days post infection (dpi) identified 19,251 differentially expressed genes (DEGs) of which 56.2% were up-regulated. DEGs mapped to 224 Gene Ontology (GO) terms including 140 biological processes (BP), 45 cellular components (CC), and 39 molecular functions (MF). A total of 27 reference pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and 15 Reactome gene sets were identified. The RNA-seq data was validated using real-time, quantitative PCR (qPCR). A host immune response though the activation of complement and the acute phase genes was evident at 7 dpi, with a concurrent immune suppression involving cytokine signalling, notably in interleukins, interferon regulatory factors and tumour necrosis factor-alpha (tnf-α) genes. Down-regulated gene expression with involvement in receptor signalling pathways (NOD-like, Toll-like and RIG-1) were also identified. The results of this study support the theory that P. perurans can evade immune surveillance during the initial stages of gill colonisation through interference of signal transduction pathways.

RNA G-quadruplexes (rG4) have recently emerged as major regulatory elements in both mRNA 21 and non-coding RNA. To investigate the biological roles of the rG4 structures, chemists have 22 developed a variety of highly specific and potent ligands. All these ligands bind to the rG4 by 23 staking on their top, and the specificity of binding is demonstrated in comparison to other 24 structures such as duplex or three-way junctions. It remains unclear whether rG4-ligands 25 merely stabilize fully formed rG4 structures, or if they actively participate in the folding of the 26 rG4 structure through their association with an unfolded RNA sequence. In order to access 27 the innate steps of ligand-rG4 association and mechanisms, robust in vitro techniques, 28 including FRET, electrophoretic mobility shift assay and reverse transcriptase stalling assays, 29 were used to examine the capacity of five well-known G4 ligands to induce rG4 structures 30 derived from either long non-coding RNAs of from synthetic RNAs. It was found that both 31 PhenDC3 and PDS induce rG4 formation in unfolded single RNA strands. This discovery has 32 important implications for the interpretation of RNA-seq experiments. Overall, in vitro data 33 that can assist biochemists in selecting the optimal G4-ligands for their RNA cellular 34 experiments are presented, while also considering the effects induced by these ligands of the 35 rG4.

Amoebic Gill Disease (AGD), caused by the protozoan extracellular parasite Paramoeba perurans, is a disease affecting Atlantic salmon (Salmo salar) aquaculture. Many studies to date have investigated the pathogenesis of ADG focusing on the host immune response in the gill after the appearance of clinical symptoms. This study investigated the gill transcriptomic profile of pre-clinical AGD using RNA-sequencing (RNA-seq) technology. RNA-seq libraries generated at 4, 7, 14 and 16 days post inoculation (dpi) identified 29,357 Differentially Expressed Genes (DEGs). RNA-seq data was validated using real-time, quantitative PCR (qPCR) analysis of 10 selected immune genes. DEGs mapped to 224 Gene Ontology (GO) terms, 27 reference pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and 15 Reactome Gene Sets. Immune suppression was evident at 7 dpi, prior to there being any evidence of ADG on the gill, involving signalling pathways for interleukins, Nod-like receptors, B-cell and T-cell receptors, and the differentiation of Th1/Th2 and Th17 cells. The results of this study suggest a mechanism for how N. perurans circumvents the host immune response to establish a successful infection, and could potentially lead to the development of novel strategies for AGD mitigation or prevention in aquaculture.