Effect of Dietary Tryptophan on Growth, Intestinal Microbiota, and Intestinal Gene Expression in an Improved Triploid Crucian Carp

Abstract

Tryptophan (Trp) has received increasing attention in the maintenance of intestinal function. In this study, improved triploid crucian carp (ITCC) fed diets containing 6.35 g kg^-1 Trp had higher average daily gain (ADG) and improved villus height (VH) and crypt depth (CD) in the intestine compared to the control group. To elucidate the potential mechanisms, we used RNA sequencing (RNA-seq) to investigate changes in the intestinal transcriptome and 16S rRNA gene sequencing to measure the intestinal microbiota in response to 6.35 g kg^-1 Trp feeding in ITCC. Dietary Trp altered intestinal gene expression involved in nutrient transport and metabolism. Differentially expressed transcripts (DETs) were highly enriched in key pathways containing protein digestion and absorption and the AMPK signaling pathway. 16S rRNA sequencing showed that 6.35 g kg^-1 Trp significantly increased the abundance of the genus Cetobacterium, and the Firmicutes/Bacteroidetes ratio at the phylum level (P < 0.05). In addition, bacterial richness indices (Simpson index) significantly increased (P < 0.05) community evenness in response to 6.35 g kg^-1 Trp. In conclusion, appropriate dietary Trp improves the growth performance, and influences the intestinal flora of ITCC. This study might be helpful to guide the supply of dietary exogenous Trp in ITCC breeding.

Keywords: 16S rRNA; improved triploid crucian carp; intestinal flora; transcriptome; tryptophan.

Figure 1

Effects of dietary tryptophan on the growth performance of ITCC. (A) Average final weight of ITCC. (B) The average daily gain of ITCC. The Trp concentrations in the five experimental diets were determined to be 1.85 (control), 3.35, 4.85, 6.35, and 7.85 g kg⁻¹ Trp diet (basal diets supplemented with 0, 1.5, 3.0, 4.5, and 6.0 g kg⁻¹ Trp).

Figure 2

Cluster analysis of DETs by the FPKM value. The X-axis indicates the samples in the different groups. The sample on the left is from the 6.35 Trp group, and the sample on the right is from the 1.85 Trp group. The Y-axis is the gene cluster across the 1.85 and 6.35 Trp groups. Color from red to blue, indicated that the log10 (FPKM+1) values were from large to small, red color indicates high expression level and blue color indicates low expression level.

Figure 3

The enriched GO terms of the DETs. The X-axis indicates the number of DETs for each GO term; the y-axis corresponds to the GO terms. *represent the significantly enriched terms (p < 0.05).

Figure 4

PPI network of differentially expressed genes in the group fed 6.35 g kg⁻¹ Trp diets compared with the control group and two significant modules identified among the PPI network using the molecular complex detection method with a score of >13. Blue nodes represent the upregulated genes; green nodes represent the downregulated genes; (A) PPI network of differentially expressed genes in the group fed 6.35 g kg⁻¹ Trp diets compared with the control group; (B) module 1, MCODE score = 23; (C) protein-protein interaction network of 15 hub genes.

Figure 5

Comparison of the gene expression levels of RNA-seq with real-time PCR. The right axis represents the expression levels determined by RNA-seq in FPKM units, and the left axis represents gene expression levels determined by real-time PCR. Bars represent the mean (±SE) of three samples. The black column indicates the FPKM value; the grey column indicates the real-time PCR using β-actin as a reference gene. Data represent relative mRNA expression of (A) Erp27, (B) CPA5, (C) CPA1, (D) CTRL, (E) LOC109066737, (F) ssbp4, (G) NEU3, and (H) Per2 determined by quantitative real-time PCR.

Figure 6

Alpha diversity of the gut microbiota in control group and 6.35Trp group. (A) Shannon's diversity index. (B) Chao1. (C) Observed-species. (D) Simpson. **p < 0.01 vs. control group by Tukey's post-hoc test.

Figure 7

Principal coordinate analysis (PCoA) of the microbial community composition of the control group and 6.35 Trp group. Horizontal coordinates indicate one principal component, vertical coordinates indicate another principal component, and percentages indicate the value of the principal component's contribution to sample differences; each point in the graph represents a sample, and samples from the same group are represented using the same color. Blue represents the 6.35 Trp group, and red represents the 1.85 Trp group.

Figure 8

Linear discriminant analysis coupled with effect size (LEfSe) of the control group and 6.35 Trp group. The LDA value distribution histogram shows species with an LDA score greater than the set value (set to 4 by default), and the biomarkers with statistically significant differences between groups. The prefixes “p,” “c,” “o,” “f,” “g,” “s,” and “t” represent the annotated level of phylum, class, order, family, genus, species, and strain. Green represents the 6.35 Trp group, and red represents the 1.85 Trp group.