Mining Lepeophtheirus salmonis RNA-Seq data for qPCR reference genes and their application in Caligus elongatus

Lepeophtheirus salmonis and Caligus elongatus are two parasitic copepod species posing a significant threat to salmonid aquaculture. Consequently, several gene expression studies are executed each year to gain new knowledge and treatment strategies. Though, to enable accurate gene expression measurements by quantitative real time PCR, stable reference genes are needed. Previous studies have mainly focused on a few genes selected based on their function as housekeeping genes, as these are often stably expressed in various cells and tissues. In the present study, however, RNA-sequencing data from 127 L. salmonis samples from different life stages and diverse environmental conditions were used to identify new candidate reference genes displaying low variation. From this, six genes were selected, and the stability validated by qPCR on samples from different life stages. Since neither a genome nor comprehensive RNA sequencing data are available for C. elongatus, homologous genes to those identified for L. salmonis were identified within a C. elongatus transcriptome assembly and validated by qPCR in different life stages. Overall, the genes eukaryotic translation initiation factor 1A (EIF1A) and serine/threonine-protein phosphatase 1 (PP1) displayed the highest stability in L. salmonis, while the combination of PP1 and ribosomal protein S13 (RPS13) was found to have the highest stability in C. elongatus. These genes are well-suited reference genes for qPCR applications which allow for accurate normalization of target genes.