Effect of Purslane (Portulaca oleracea L.) on Intestinal Morphology, Digestion Activity and Microbiome of Chinese Pond Turtle (Mauremys reevesii) during Aeromonas hydrophila Infection

Abstract

Large-scale mortality due to Aeromonas hydrophila (A. hydrophila) infection has considerably decreased the yield of the Chinese pond turtle (Mauremys reevesii). Purslane is a naturally active substance with a wide range of pharmacological functions, but its antibacterial effect on Chinese pond turtles infected by A. hydrophila infection is still unknown. In this study, we investigated the effect of purslane on intestinal morphology, digestion activity, and microbiome of Chinese pond turtles during A. hydrophila infection. The results showed that purslane promoted epidermal neogenesis of the limbs and increased the survival and feeding rates of Chinese pond turtles during A. hydrophila infection. Histopathological observation and enzyme activity assay indicated that purslane improved the intestinal morphology and digestive enzyme (α-amylase, lipase and pepsin) activities of Chinese pond turtle during A. hydrophila infection. Microbiome analysis revealed that purslane increased the diversity of intestinal microbiota with a significant decrease in the proportion of potentially pathogenic bacteria (such as Citrobacter freundii, Eimeria praecox, and Salmonella enterica) and an increase in the abundance of probiotics (such as uncultured Lactobacillus). In conclusion, our study uncovers that purslane improves intestinal health to protect Chinese pond turtles against A. hydrophila infection.

Keywords: A. hydrophila; Chinese pond turtle; intestine; microbiome; purslane.

Figure 1

Effect of purslane on symptoms of the body surface, survival, and feeding rates of Chinese pond turtles during A. hydrophila infection. (A–C) Example of symptoms on the body surface of the limbs. The pink circle represents the exposed muscle layer and epidermal ulceration, the green circle indicates the neonatal black epidermis. (D) The cumulative survival rate of each group. Survival rate (%) = (currently surviving turtles/total turtles) × 100. (E) Heat map of the feeding rate of each group. Feeding rate (%) = (1 − average residual feed/average feeding amount) × 100.

Figure 2

Effect of purslane on intestinal structure and digestive enzyme activities of Chinese pond turtles during A. hydrophila infection. (A–F) Histopathological changes in the control (A,D), infection (B,E) and purslane (C,F) groups of Chinese pond turtle’s intestine. The red bidirectional arrow shows villus height and the yellow bidirectional arrow indicates crypt depth. Red unidirectional arrow means shed intestinal mucosa and the orange unidirectional arrow represents newly generated intestinal mucosa. (G,H) Villus height and crypt depth in different groups. (I) Intestinal health status (organ index) of Chinese pond turtles in different groups. (J–L) The activity of α-Amylase, lipase, and pepsin in the Chinese pond turtle’s intestine. One-way ANOVA plus Bonferroni post-tests, * p < 0.05.

Figure 3

OTU differential analysis of control, infection, and purslane groups. (A) Histogram (above) and Venn diagram (below) of OTUs number in different groups. (B) OTU ternary analysis of different groups in the Chinese pond turtle intestines. The three corners represent the control, infection, and purslane samples, and the color of solid circles in the figure represents the annotation of OTUs at the phyla level, and the size of the circles stands for the average relative abundance (%) at the phyla level.

Figure 4

Species annotation and evaluation. (A) Rank-Abundance curve at the OTU level. (B) Sobs index at the OTU level. (C,D) Shannon and Simpson index at the OTU level. (E,F) Ace and Chao index at the OTU level. * p < 0.05 indicates a significant difference between the two groups, one-way ANOVA plus Bonferroni post-tests.

Figure 5

Hierarchical clustering trees of Chinese pond turtle intestines at the family level. Clustering analysis based on Bray–Curtis dissimilarity.

Figure 6

Comparison of microbial community structures among control, infection, and purslane groups. (A) Sunburst plots at the phyla level and pie charts of the most abundant genus. (B) Species composition of each sample.

Figure 7

Comparison of microbial community differences between control or purslane and Infection groups. The chart lists the top 15 abundant species in control, infection, and purslane samples, and data are presented as means ± standard deviation. Wilcoxon rank sum test was applied to determine if differences between groups were significant (p < 0.05). The communities with red and green fonts indicate potential pathogens and probiotics with significant changes in abundance, respectively.