Project description:A total of 55 individuals were analysed: 15 migratory brown trout (Salmo trutta) individuals from the Redon river, 15 sedentary brown trout (S. trutta) individuals from the Redon river, 15 sedentary brown trout (S. trutta) individuals from the Chevenne river, and 10 Atlantic salmon (S. salar) individuals of a hatchery strain. For each individual, RNA was isolated twice from different parts of the same tissue, independently reverse transcribed into Cy3-labeled cDNA and then probed on two different slides, which leads to total of 110 single slide experiments.
Project description:The proliferative darkening syndrome (PDS) is an annually recurring disease that causes species-specific die-off of brown trout (Salmo trutta fario) with a mortality rate of near 100 % in pre-alpine rivers of central Europe. So far the etiology and causation of this disease is still unclear. The objective of this study was to identify the cause of PDS using a next-generation technology detection pipeline. Following the hypothesis that PDS is caused by an infectious agent, brown trout specimens were exposed to water from a heavily affected pre-alpine river with annual occurrence of the disease. Specimens were sampled over the entire time period from potential infection through death. Transcriptomic analysis (microarray) and RT-qPCR of brown trout liver tissue evidenced strong gene expression response of immune-associated genes. Messenger RNA of specimens with synchronous immune expression profiles were ultra-deep sequenced using next-generation sequencing technology (NGS). Bioinformatic processing of generated reads and gap-filling Sanger re-sequencing of the identified pathogen genome revealed strong evidence that a piscine-related reovirus is the causative organism of PDS. The identified pathogen is phylogenetically closely related to the family of piscine reoviruses (PRV) which are considered as the causation of different fish diseases in Atlantic and Pacific salmonid species such as Salmo salar and Onchorhynchus kisutch. This study also highlights that the approach of first screening immune responses along a timeline in order to identify synchronously affected stages in different specimens which subsequently were ultra-deep sequenced is an effective approach in pathogen detection. In particular, the identification of specimens with synchronous molecular immune response patterns combined with NGS sequencing and gap-filling re-sequencing resulted in the successful pathogen detection of PDS.
Project description:contemporary natural selection in brown trout (Salmo trutta) at transcriptome level - QuantSeq 3' RNA-Seq forward raw sequence reads
