Validation of reference genes for RT-qPCR analysis of growth hormone receptor and growth hormone expression in the tench (Tinca tinca) fed substituting poultry meal for fish meal

Abstract

The tench (Tinca tinca L., 1758) is a promising freshwater species in which numerous studies have been conducted recently to develop a dedicated feed for optimized growth rate. The aim of this study was to assess the stability of seven candidate reference genes, including 18S ribosomal RNA (18S rRNA), β-actin (actb), α-tubulin (tuba), ribosomal protein l8 (rpl8), 40S ribosomal protein s11 (rps11), glyceraldehyde-3-phosphate dehydrogenase (gapdh) and elongation factor 1 alpha (ef1a), to select the best calibrator for nutrigenomic studies that would improve our understanding and illustrate the molecular basis of nutrient-related gene expression in response to feed mixes in the tench. An additional aim was to verify the hypothesis that the functioning of the somatotropic axis in the tench (growth hormone receptor, ghr, and growth hormone, gh) depends on dietary treatment. The analysis was based on five tissue samples (liver, spleen, gut, heart and muscle) collected from adult tench females fed with 0% and 100% replacement of fish meal (FM) with poultry by-product meal (PBM). The study demonstrated that the most stable genes in most tissues were rpl8 and rps11, with gapdh showing the greatest stability, along with rpl8, in the liver. Moreover, a comparison of relative transcript abundance between the tissues revealed that ghr demonstrated the highest expression in the liver, while gh demonstrated the highest expression in the spleen. Based on the selected reference genes, it was determined that the feed did not affect the level of expression of ghr and gh in most tissues except the spleen (ghr). The study also involved a phylogenetic analysis based on the amino acid sequences of Rpl8, Rps11 and Ghr, and it was confirmed that the tench is the most closely related with Asian cyprinids (i.e. Megalobrama amblycephala). The study identified suitable reference genes for RT-qPCR in the tench, and the effect of two feed variants on the somatotropic axis was determined. The study opens the way for further nutrigenomic studies aimed to develop a feed best matched for the needs of the tench and optimizing its growth. Statement of relevanceThe effect of feed intake on gene expression in fish species is of particular interest of aquaculture sector which seeks for alternative protein sources, but also new species to adapt for intensive culture.The study demonstrated that the most stable genes in most tissues were rpl8 and rps11, with gapdh showing the greatest stability, along with rpl8, in the liver.Comparison of relative transcript abundance between tissues revealed that ghr demonstrated the highest expression in the liver, while gh demonstrated the highest expression in the spleen.Presented work characterizes genes with stable expression across various tench tissue types and identifies suitable reference genes for RTq-PCR for nutrient-related gene expression studies.Presented study was performed for tench for the first time and provides information on influence of poultry by-product meal on the tench organism.Information on tench genetic studies, especially combined with dietary treatments are sparse, therefore presented results address this gap and will bring attention of Aquaculture readers.