Integrative functional analyses using rainbow trout selected for tolerance to plant diets reveal nutrigenomic signatures for soy utilization without the concurrence of enteritis

Abstract

Finding suitable alternative protein sources for diets of carnivorous fish species remains a major concern for sustainable aquaculture. Through genetic selection, we created a strain of rainbow trout that outperforms parental lines in utilizing an all-plant protein diet and does not develop enteritis in the distal intestine, as is typical with salmonids on long-term plant protein-based feeds. By incorporating this strain into functional analyses, we set out to determine which genes are critical to plant protein utilization in the absence of gut inflammation. After a 12-week feeding trial with our selected strain and a control trout strain fed either a fishmeal-based diet or an all-plant protein diet, high-throughput RNA sequencing was completed on both liver and muscle tissues. Differential gene expression analyses, weighted correlation network analyses and further functional characterization were performed. A strain-by-diet design revealed differential expression ranging from a few dozen to over one thousand genes among the various comparisons and tissues. Major gene ontology groups identified between comparisons included those encompassing central, intermediary and foreign molecule metabolism, associated biosynthetic pathways as well as immunity. A systems approach indicated that genes involved in purine metabolism were highly perturbed. Systems analysis among the tissues tested further suggests the interplay between selection for growth, dietary utilization and protein tolerance may also have implications for nonspecific immunity. By combining data from differential gene expression and co-expression networks using selected trout, along with ontology and pathway analyses, a set of 63 candidate genes for plant diet tolerance was found. Risk loci in human inflammatory bowel diseases were also found in our datasets, indicating rainbow trout selected for plant-diet tolerance may have added utility as a potential biomedical model.

Fig 1. Experimental design.

Fig 2. Bar chart of histopathological scoring.

The Y-axis shows cumulative score, where higher scoring equals increase in severity. The X-axis indicates fish strain (HC or ARS-KO) along with treatment (PM or FM diet). The chart key and color are indicative of scoring variables: Mucosal fold fusion (MF), lamina propria width and cellularity (LP), sub-epithelial mucosa width and cellularity (SM), total inflammatory cell number (TIC), number of eosinophilic granulocytes (EG), degree of enterocyte supranuclear vacuolization (SNV) and number of goblet cells (GC).

Fig 3. Photomicrographs of distal intestines at 400x magnification.

(a). A representative image of selected fish reared for 12-weeks on plant protein based feed, similar to selected and non-selected fish reared on the fishmeal based feed (b). A representative image of non-selected fish reared for 12-weeks on the plant protein based feed. Intracytoplasmic supranuclear vacuoles (asterisk), intraepithelial lymphocytes (arrowhead) and mucous cell hyperplasia (arrow) are so indicated. Scale is depicted by horizontal bar.

Fig 4. Venn diagram of the number of significant (p-adj < 0.05; FPKM > 1) differentially expressed genes.

Fig 5. Weighted correlation network analysis among plant-diet tolerant selected fish fed either the FM diet (n = 10) or a 100% plant-protein replacement PM diet (n = 10), using RNA-seq data from liver tissues.