The effects of insulin-like growth factor-I (IGF-I), insulin, and leucine on protein turnover and pathways that regulate proteolytic gene expression and protein polyubiquitination were investigated in primary cultures of 4-day-old rainbow trout myocytes. Supplementing media with 100 nM IGF-I increased protein synthesis by 13% ( P < 0.05) and decreased protein degradation by 14% ( P < 0.05). Treatment with 1 μM insulin increased protein synthesis by 13% ( P < 0.05) and decreased protein degradation by 17% ( P < 0.05). Supplementing media containing 0.6 mM leucine with an additional 2.5 mM leucine did not increase protein synthesis rates but reduced rates of protein degradation by 8% ( P < 0.05). IGF-I (1 nM–100 nM) and insulin (1 nM-1 μM) independently reduced the abundance of ubiquitin ligase mRNA in a dose-dependent manner, with maximal reductions of ∼70% for muscle atrophy F-box (Fbx) 32, 40% for Fbx25, and 25% for muscle RING finger-1 (MuRF1, P < 0.05). IGF-I and insulin stimulated phosphorylation of FOXO1 and FOXO4 ( P < 0.05), which was inhibited by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, and decreased the abundance of polyubiquitinated proteins by 10–20% ( P < 0.05). Supplementing media with leucine reduced Fbx32 expression by 25% ( P < 0.05) but did not affect Fbx25 nor MuRF1 transcript abundance. Serum deprivation decreased rates of protein synthesis by 60% ( P < 0.05), increased protein degradation by 40% ( P < 0.05), and increased expression of all ubiquitin ligases. These data suggest that, similar to mammals, the inhibitory effects of IGF-I and insulin on proteolysis occur via P I3-kinase/protein kinase B signaling and are partially responsible for the ability of these compounds to promote protein accretion.

Background: Diverse microbial communities colonizing the intestine of fish contribute to their growth, digestion, nutrition, and immune function. We hypothesized that fecal samples representing the gut microbiota of rainbow trout could be associated with differential growth rates observed in fish breeding programs. If true, harnessing the functionality of this microbiota can improve the profitability of aquaculture. The first objective of this study was to test this hypothesis if gut microbiota is associated with fish growth rate (body weight). Four full-sibling families were stocked in the same tank and fed an identical diet. Two fast-growing and two slow-growing fish were selected from each family for 16S rRNA microbiota profiling. Microbiota diversity varies with different DNA extraction methods. The second objective of this study was to compare the effects of five commonly used DNA extraction methods on the microbiota profiling and to determine the most appropriate extraction method for this study. These methods were Promega-Maxwell, Phenol-chloroform, MO-BIO, Qiagen-Blood/Tissue, and Qiagen-Stool. Methods were compared according to DNA integrity, cost, feasibility and inter-sample variation based on non-metric multidimensional scaling ordination (nMDS) clusters. Results: Differences in DNA extraction methods resulted in significant variation in the identification of bacteria that compose the gut microbiota. Promega-Maxwell had the lowest inter-sample variation and was therefore used for the subsequent analyses. Beta diversity of the bacterial communities showed significant variation between breeding families but not between the fast- and slow-growing fish. However, an indicator analysis determined that cellulose, amylose degrading and amino acid fermenting bacteria (Clostridium, Leptotrichia, and Peptostreptococcus) are indicator taxa of the fast-growing fish. In contrary, pathogenic bacteria (Corynebacterium and Paeniclostridium) were identified as indicator taxa for the slow-growing fish. Conclusion: DNA extraction methodology should be carefully considered for accurate profiling of the gut microbiota. Although the microbiota was not significantly different between the fast- and slow-growing fish groups, some bacterial taxa with functional implications were indicative of fish growth rate. Further studies are warranted to explore how bacteria are transmitted and potential usage of the indicator bacteria of fast-growing fish for development of probiotics that may improve fish health and growth.

In salmonids, the majority of circulating insulin-like growth factor-I (IGF-I) is bound to IGF binding proteins (IGFBP), with IGFBP-2b being the most abundant in circulation. We used CRISPR/Cas9 methodology to disrupt expression of a functional IGFBP-2b protein by co-targeting for gene editing IGFBP-2b1 and IGFBP-2b2 subtypes, which represent salmonid-specific gene duplicates. Twenty-four rainbow trout were produced with mutations in the IGFBP-2b1 and IGFBP-2b2 genes. Mutant fish exhibited between 8–100% and 2–83% gene disruption for IGFBP-2b1 and IGFBP-2b2, respectively, with a positive correlation (P < 0.001) in gene mutation rate between individual fish. Analysis of IGFBP-2b protein indicated reductions in plasma IGFBP-2b abundance to between 0.04–0.96-fold of control levels. Plasma IGF-I, body weight, and fork length were reduced in mutants at 8 and 10 months post-hatch, which supports that IGFBP-2b is significant for carrying IGF-I. Despite reduced plasma IGF-I and IGFBP-2b in mutants, growth retardation in mutants was less severe between 10 and 12 months post-hatch (P < 0.05), suggesting a compensatory growth response occurred. These findings indicate that gene editing using CRISPR/Cas9 and ligand blotting is a feasible approach for characterizing protein-level functions of duplicated IGFBP genes in salmonids and is useful to unravel IGF-related endocrine mechanisms.

Growth rates in fish are largely dependent on genetic and environmental factors, of which the latter can be highly variable throughout development. For this reason, muscle growth in fish is particularly dynamic as muscle structure and function can be altered by environmental conditions, a concept referred to as muscle plasticity. Myogenic regulatory factors (MRFs) like Myogenin, MyoD, and Pax7 control the myogenic mechanisms regulating quiescent muscle cell maintenance, proliferation, and differentiation, critical processes central for muscle plasticity. This review focuses on recent advancements in molecular mechanisms involving microRNAs (miRNAs) and DNA methylation that regulate the expression and activity of MRFs in fish. Findings provide overwhelming support that these mechanisms are significant regulators of muscle plasticity, particularly in response to environmental factors like temperature and nutritional challenges. Genetic variation in DNA methylation and miRNA expression also correlate with variation in body weight and growth, suggesting that genetic markers related to these mechanisms may be useful for genomic selection strategies. Collectively, this knowledge improves the understanding of mechanisms regulating muscle plasticity and can contribute to the development of husbandry and breeding strategies that improve growth performance and the ability of the fish to respond to environmental challenges.

This study determined the effect of genetic variation, feed deprivation, and insulin-like growth factor-I (IGF-I) on weight loss, plasma IGF-I and growth hormone, and indexes of protein degradation in eight full-sibling families of rainbow trout. After 2 wk of feed deprivation, fish treated with IGF-I lost 16% less ( P < 0.05) wet weight than untreated fish. Feed deprivation increased growth hormone ( P < 0.05) and decreased IGF-I ( P < 0.05), but hormone levels were not altered by IGF-I. Plasma 3-methylhistidine concentrations were not affected by IGF-I but were decreased after 2 wk ( P < 0.05) and increased after 4 wk ( P < 0.05) of feed deprivation. In white muscle, transcript abundance of genes in the ubiquitin-proteasome, lysosomal, and calpain- and caspase-dependent pathways were affected by feed deprivation ( P < 0.05). IGF-I prevented the feed deprivation-induced upregulation of MAFbx (F-box) and cathepsin transcripts and reduced abundance of proteasomal mRNAs ( P < 0.05), suggesting that reduction of protein degradation via these pathways may be partially responsible for the IGF-I-induced reduction of weight loss. Family variations in gene expression, IGF-I concentrations, and weight loss during fasting suggest genetic variation in the fasting response, with considerable impact on regulation of proteolytic pathways. These data indicate that nutrient availability, IGF-I, and genetic variation affect weight loss, in part through alterations of proteolytic pathways in rainbow trout, and that regulation of genes within these pathways is coordinated in a way that supports a similar physiological response.