Transcriptome analysis and immune gene expression of channel catfish (Ictalurus punctatus) fed diets with inclusion of frass from black soldier fly larvae

Abstract

The larval waste, exoskeleton shedding, and leftover feed components of the black soldier fly and its larvae make up the by-product known as frass. In this study, we subjected channel catfish (Ictalurus punctatus) to a 10-week feeding trial to assess how different dietary amounts of frass inclusion would affect both systemic and mucosal tissue gene expression, especially in regard to growth and immune-related genes. Fish were divided in quadruplicate aquaria, and five experimental diets comprising 0, 50, 100, 200, and 300 g of frass per kilogram of feed were fed twice daily. At the end of the trial, liver, head kidney, gill, and intestine samples were collected for gene expression analyses. First, liver and intestine samples from fish fed with a no frass inclusion diet (control), low-frass (50 g/kg) inclusion diet, or a high-frass (300 g/kg) inclusion diet were subjected to Illumina RNA sequencing to determine global differential gene expression among diet groups. Differentially expressed genes (DEGs) included the upregulation of growth-related genes such as glucose-6-phosphatase and myostatin, as well as innate immune receptors and effector molecules such as toll-like receptor 5, apolipoprotein A1, C-type lectin, and lysozyme. Based on the initial screenings of low/high frass using RNA sequencing, a more thorough evaluation of immune gene expression of all tissues sampled, and all levels of frass inclusion, was further conducted. Using targeted quantitative PCR panels for both innate and adaptive immune genes from channel catfish, differential expression of genes was identified, which included innate receptors (TLR1, TLR5, TLR9, and TLR20A), proinflammatory cytokines (IL-1β type a, IL-1β type b, IL-17, IFN-γ, and TNFα), chemokines (CFC3 and CFD), and hepcidin in both systemic (liver and head kidney) and mucosal (gill and intestine) tissues. Overall, frass from black soldier fly larvae inclusion in formulated diets was found to alter global gene expression and activate innate and adaptive immunity in channel catfish, which has the potential to support disease resistance in this species in addition to demonstrated growth benefits.

Keywords: RNA-seq; adaptive immunity; alternative diets; channel catfish; feed additives; frass; innate immunity; metabolism.

FIGURE 1

Number of significant DEGs identified between the control (0 g/kg) and the low-frass (50 g/kg) and high-frass (300 g/kg) supplemented diets. The downregulated DEGs (white bars) and upregulated DEGs (gray bars) are shown for the intestine and liver (p-adj <0.05; fold change >2).

FIGURE 2

Venn diagram showing unique DEGs among the low-frass (50 g/kg) and high-frass (300 g/kg) supplemented diets in the intestine and liver.

FIGURE 3

Relative expression of toll-like receptors: TLR1 (A), TLR 3 (B), TLR5 (C), TLR 9 (D), TLR20A (E), and TLR 21 (F) in the head kidney, liver, gills, and intestine of channel catfish fed with frass-supplemented diets (50–300 g/kg). Data are presented as mean ± SE, and multiple reference genes were used to normalize with the target gene (n = 4). TLR gene expression among the frass diets (50–300 g/kg) was significantly upregulated when compared to the control (0 g/kg). Different alphabets indicate the significant differences between frass-supplemented diets (p < 0.05).

FIGURE 4

Relative expression of proinflammatory cytokines: IL-1β type a (A), IL-1β type b (B), IL-17 (C), TNFα (D), and IFN-γ (E), and antimicrobial peptide and hepcidin (F) in the head kidney, liver, gills, and intestine of channel catfish fed with frass-supplemented diets (50–300 g/kg). Data are presented as mean ± SE, and multiple reference genes were used to normalize with the target gene (n = 4). Gene expression among the frass diets (50–300 g/kg) was significantly upregulated when compared to the control (0 g/kg). Different alphabets indicate the significant differences between frass-supplemented diets (p < 0.05).

FIGURE 5

Relative expression of complementary factor C3 (A) and complement factor D (B) in the head kidney, liver, gills, and intestine of channel catfish fed with frass-supplemented diets (50–300 g/kg). Data are presented as mean ± SE, and multiple reference genes were used to normalize with the target gene (n = 4). Different alphabets indicate the significant differences between frass-supplemented diets (p < 0.05).

FIGURE 6

Relative expression of adaptive immunity genes: IgM (A), CD4 (B), β2M (C), and MHC class 2 (D) in the head kidney, liver, gills, and intestine of channel catfish fed with frass-supplemented diets (50–300 g/kg). Data are presented as mean ± SE, and multiple reference genes were used to normalize with the target gene (n = 4). Different alphabets indicate the significant differences between frass-supplemented diets (p < 0.05).