Transcriptome and 16S rRNA Analyses Reveal That Hypoxic Stress Affects the Antioxidant Capacity of Largemouth Bass ( Micropterus salmoides), Resulting in Intestinal Tissue Damage and Structural Changes in Microflora


doi: 10.3390/antiox12010001.

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Zhuo Song et al.


Antioxidants (Basel).


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Abstract

Dissolved oxygen (DO) is a key factor affecting the health of aquatic organisms in an intensive aquaculture environment. In this study, largemouth bass (Micropterus salmoides) were subjected to acute hypoxic stress for 96 h (DO: 1.00 mg/L) followed by recovery under sufficient DO conditions (DO: 7.50 mg/L) for 96 h. Serum biochemical indices, intestinal histomorphology, the transcriptome, and intestinal microbiota were compared between hypoxia-treated fish and those in a control group. The results showed that hypoxia caused oxidative stress, exfoliation of the intestinal villus epithelium and villus rupture, and increased cell apoptosis. Transcriptome analyses revealed that antioxidant-, inflammation-, and apoptosis-related pathways were activated, and that the MAPK signaling pathway played an important role under hypoxic stress. In addition, 16S rRNA sequencing analyses revealed that hypoxic stress significantly decreased bacterial richness and identified the dominant phyla (Proteobacteria, Firmicutes) and genera (Mycoplasma, unclassified Enterobacterales, Cetobacterium) involved in the intestinal inflammatory response of largemouth bass. Pearson’s correlation analyses showed that differentially expressed genes in the MAPK signaling pathway were significantly correlated with some microflora. The results of this study will help to develop strategies to reduce damage caused by hypoxic stress in aquacultured fish.


Keywords:

16S rDNA; Micropterus salmoides; dissolved oxygen; intestine; transcriptome.

Conflict of interest statement

The authors declare no conflict of interest.

Figures


Figure 1



Figure 1

Linear interpolation graph for 96h-LH50 (n = 20).


Figure 2



Figure 2

Serum biochemical indices of largemouth bass under 96 h of hypoxic stress and recovery for 96 h (n = 12). (A): Glucose concentration. (B): Lactic acid concentration. (C): Malondialdehyde (MDA) content. (D): Superoxide dismutase (SOD) activity. (E): Catalase (CAT) activity. Different letters above bars indicate significant differences among sampling times. Asterisks indicate significant difference between control group and hypoxia treated group at each time point.


Figure 3



Figure 3

Intestinal morphology of largemouth bass under hypoxic stress for 0 h (A), 6 h (B), 24 h (C), 96 h (D) and 96 h of recovery under sufficient DO conditions (E) (n = 12). Solid arrow: intestinal epithelial injury; dotted arrow: ruptured intestinal villus.


Figure 4



Figure 4

Effects of hypoxic stress (0 h (A), 6 h (B), 24 h (C), 96 h (D)) and reoxygenation (96 h (E)) on intestinal cell apoptosis in largemouth bass (n = 12). White arrows indicate apoptotic cells.


Figure 5



Figure 5

(A): Volcanic diagram of differentially expressed genes (DEGs) in Ctrl vs. Hyp groups (n = 3). (B): Volcanic map of DEGs in Hyp vs. Rec groups (n = 3). (C): Volcanic map of DEGs in Ctrl vs. Rec groups (n = 3). Grey dots represent genes with no significant difference in transcript levels between groups; red dots and blue dots represent significantly upregulated and downregulated DEGs, respectively. (D): Transcriptome analysis of DEGs quantity and expression (n = 3). (E): Venn diagram showing number of DEGs between and among groups (n = 3).


Figure 6



Figure 6

KEGG pathway enrichment analysis of DEGs in the intestine of largemouth bass under acute hypoxic stress (n = 3).


Figure 7



Figure 7

qRT-PCR analysis of differentially expressed genes in Ctrl group, Hyp group, and Rec group (n = 12). Different letters indicate significant differences in gene transcript levels among groups.


Figure 8



Figure 8

Intestinal alpha diversity of largemouth bass under hypoxic stress. (A): Observed operational taxonomic units (OTUs) index (alpha) (n = 10). (B): Shannon’s index (alpha) (n = 10), (C): Simpson’s index (alpha) (n = 10). (D): Chao1 index (alpha), * and ** indicate significant differences in abundance among different groups (Kruskal–Wallis test, *: p < 0.05, **: p < 0.01) (n = 10). (E): Relative abundance of main taxa of intestinal microflora in Ctrl group, Hyp group, and Rec group (ad) (n = 10). Each bar represents relative abundance in each sample, and shows 27 most abundant taxa.


Figure 9



Figure 9

(A): Taxa shown in histogram were determined to differ significantly in abundance among Ctrl, Hyp, and Rec groups by Kruskal–Wallis test ((p < 0.05, LDA score > 4, (a): Ctrl vs. Hyp; (b): Hyp vs. Rec; (c): Ctrl vs. Rec) (n = 10). (B): Abundance of major bacteria (ad) in Ctrl, Str, and Rec groups (*, p < 0.05; **, p < 0.01) (n = 10).


Figure 10



Figure 10

(A): Venn diagram showing number of OTUs shared between and among Ctrl, Hyp, and Rec groups (n = 10). (B): Abundance ratio of intestinal microflora in Ctrl, Hyp, and Rec groups with predicted level 2 functions (n = 10).


Figure 11



Figure 11

Correlation between DEGs and intestinal microflora at the genus level (*, p < 0.05; **, p < 0.01).

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