Methanotroph (Methylococcus capsulatus) bacteria meal, influences the intestinal microbiome, and improves intestinal barrier function and immunity in hybrid grouper (Epinephelus fuscointestinetatus ♀ × E. lanceolatus ♂)

Abstract

The hybrid grouper (Epinephelus fuscointestinetatus ♀ × E. lanceolatus ♂) is an important aquaculture marine species in China. The current study was designed to test the effects of methanotroph (Methylococcus capsulatus) bacteria meal (MBM) on hybrid grouper growth and intestinal health outcomes. Five iso-nitrogenous and iso-lipidic diets were formulated, comprising a control diet (MBM0) with 400 g/kg fish meal (FM) as the main protein source, and four experimental diets, in which FM was replaced at 5% (MBM2), 10% (MBM4), 20% (MBM8) and 30% (MBM12) with 20, 40, 80 and 120 g/kg of MBM, respectively. The 450 healthy juvenile hybrid grouper (initial weight 28.84 ± 0.05 g) were randomly allocated into five groups, with three replicates in each group and 30 fish in each replicate. They were fed for eight weeks prior to being sampled for transcriptome and microbiome research. The final body weight (FBW, P < 0.001) and weight gain rate (WGR, P < 0.001) were obviously enhanced in MBM2 and MBM8 groups compared to MBM0 group, and the specific growth rate (SGR) in the MBM2 and MBM8 groups were significantly enhanced compared to MBM0 group (P < 0.001). The condition factor (CF) in MBM4 and MBM8 groups were considerably reduced than that in MBM0 group (P = 0.008). The hepatosomatic index (HSI) in MBM8 and MBM12 groups were obviously enhanced than that in the other groups (P < 0.001). In the intestinal microflora analysis, the number of operational taxonomic units (OTUs) exhibited a significant linear increase with increasing MBM replacement ratio (P = 0.015). Concurrently, the richness of bacterial families and genera showed significant linear enrichment (P = 0.023 and P = 0.028, respectively). There was a decrease in the relative abundance of the potentially pathogenic genera Photobacterium and Vibrio in the MBM-fed groups compared with the control group. The study has effectively shown that MBM may significantly enhance the health and growth of hybrid groupers by partially replacing FM in diets at the required amount of 3.70%, as determined by WGR regression analysis.

Keywords: Intestinal microflora; Juvenile hybrid grouper; Methanotroph bacteria meal; Transcriptome analysis.

Fig. 1

The relationship between weight gain rate (WGR) and methanotroph (M. capsulatus) bacteria meal (MBM) replacement level in juvenile hybrid grouper for 8 weeks. FM = fish meal.

Fig. 2

Venn diagram of the operational taxonomic units (OTUs) in hybrid grouper. The experimental diets consisted of a control group (MBM0) and four treatment groups supplemented with varying levels of methanotroph (M. capsulatus) bacteria meal (MBM): 2% (MBM2), 4% (MBM4), 8% (MBM8), and 12% (MBM12), respectively.

Fig. 3

Relative abundance of the intestinal microbiome of hybrid groupers. (A) Relative abundance at phyla level. (B) Relative abundance at class level. (C) Relative abundance at genera level. The experimental diets consisted of a control group (MBM0) and four treatment groups supplemented with varying levels of methanotroph (M. capsulatus) bacteria meal (MBM): 2% (MBM2), 4% (MBM4), 8% (MBM8), and 12% (MBM12, respectively.

Fig. 4

Characteristic taxa distribution of the intestinal microflora of hybrid grouper. (A) Facultatively anaerobic. (B) Gram negative. (C) Potentially pathogenic. (D) Stress tolerant. (E) Aerobic. (F) Gram positive. (G) Contain mobile elements. (H) Biofilm formers. (I) Anaerobic. The experimental diets consisted of a control group (MBM0) and four treatment groups supplemented with varying levels of methanotroph (M. capsulatus) bacteria meal (MBM): 2% (MBM2), 4% (MBM4), 8% (MBM8), and 12% (MBM12), respectively.

Fig. 5

Functional pathways enrichment at A class in KEGG of the hybrid grouper's intestinal microflora. The hybrid groupers were fed either control diet (MBM0) or diets containing 12% methanotroph (M. capsulatus) bacteria meal (MBM12).

Fig. 6

Volcano plot of distribution of differentially expressed genes (DEGs) in hybrid groupers between MBM12 and MBM0 groups. The hybrid groupers were fed either control diet (MBM0) or the diet containing 12% methanotroph (M. capsulatus) bacteria meal (MBM12). FC = fold change; Nodiff = no difference.

Fig. 7

Functional enrichment of differentially expressed genes (DEGs) by GO enrichment analysis for GO term classification at level 1 and level 2 (A), biological processes, molecular functions, and cellular components (B).

Fig. 8

KEGG pathways classification and enrichment analysis of the different expression genes (DEGs) of hybrid grouper fed MBM12 diets. (A) KEGG pathways classification at class A and class B. (B) The top twenty pathways identified by KEGG enrichment. The hybrid groupers were fed the diet containing 12% methanotroph (M. capsulatus) bacteria meal (MBM12).

Fig. 9

Heat map of 25 differentially expressed genes (DEGs) in hybrid groupers. The hybrid groupers were fed either control diet (MBM0) or diets containing 12% methanotroph (M. capsulatus) bacteria meal (MBM12). The red and green boxes in the figure represent the relative expression levels of genes, with red indicating increased expression and green indicating decreased expression.

Fig. 10

Pearson correlation heatmap of relative abundance of intestinal microbiota and expression of differentially expressed genes (DEGs) in hybrid groupers fed in diets containing 12% methanotroph (M. capsulatus) bacteria meal (MBM12). ∗∗ Denote extremely significant correlation (P < 0.01), and ∗ denote significant correlation (P < 0.05).