Metatranscriptomic Analysis Reveals an Imbalance of Hepatopancreatic Flora of Chinese Mitten Crab Eriocheir sinensis with Hepatopancreatic Necrosis Disease

Abstract

Hepatopancreas necrosis disease (HPND) of the Chinese mitten crab Eriocheir sinensis causes huge economic loss in China. However, the pathogenic factors and pathogenesis are still a matter of dissension. To search for potential pathogens, the hepatopancreatic flora of diseased crabs with mild symptoms, diseased crabs with severe symptoms, and crabs without visible symptoms were investigated using metatranscriptomics sequencing. The prevalence of Absidia glauca and Candidatus Synechococcus spongiarum decreased, whereas the prevalence of Spiroplasma eriocheiris increased in the hepatopancreatic flora of crabs with HPND. Homologous sequences of 34 viral species and 4 Microsporidian species were found in the crab hepatopancreas without any significant differences between crabs with and without HPND. Moreover, DEGs in the hepatopancreatic flora between crabs with severe symptoms and without visible symptoms were enriched in the ribosome, retinol metabolism, metabolism of xenobiotics by cytochrome P450, drug metabolism-cytochrome P450, biosynthesis of unsaturated fatty acids, and other glycan degradation. Moreover, the relative abundance of functions of DEDs in the hepatopancreatic flora changed with the pathogenesis process. These results suggested that imbalance of hepatopancreatic flora was associated with crab HPND. The identified DEGs were perhaps involved in the pathological mechanism of HPND; nonetheless, HPND did not occur due to virus or microsporidia infection.

Keywords: Eriocheir sinensis; hepatopancreas necrosis disease; hepatopancreatic flora; metatranscriptomics sequencing.

Figure 1

The common clinical signs of crabs with HPND. The body weights were about 100–150 g. (A) hepatopancreas of healthy crab, the hepatopancreas golden and plump; (B) hepatopancreas of crab with mild signs, the hepatopancreas are yellow and do not degenerate significantly; (C) the hepatopancreas turms pale yellow and begins to degeneration; (D) the hepatopancreas turns white and gradually atrophies; (E) the hepatopancreas erodes; (F) the hepatopancreas disappears.

Figure 2

Top 10 taxon in relative abundance at different taxonomic levels. (A), (B), (C), (D), (E), and (F) represented Phyla, Classes, Orders, Families, Genera, and Species, respectively.

Figure 3

Clustering of relative abundance at the genus level. The horizontal axis represented sample information; vertical axis represented species information; the cluster tree on the left side was taxon clustering tree; the color intensity in the square grid represented the bacterial relative abundance, which is named as z-value and generated by the relative abundance of the genus in each line after normalization treatment.

Figure 4

Volcano plot for gene expression among different samples. (A) Diseased crabs with mild signs VS. Healthy crabs; (B) Diseased crabs with severe signs VS. Healthy crabs; (C) Diseased crabs with severe signs VS. Diseased crabs with mild signs.

Figure 5

Venn diagram of DEGs identified by metatranscriptomic sequencing. (A) Venn diagrams of all DEGs among Diseased crabs with mild signs and Healthy crabs, Diseased crabs with severe signs and Healthy crabs, and Diseased crabs with severe signs and Diseased crabs with mild signs; (B) Venn diagrams of up-regulated DEGs among Diseased crabs with mild signs and Healthy crabs, Diseased crabs with severe signs and Healthy crabs, and Diseased crabs with severe signs and Diseased crabs with mild signs; (C) Venn diagram of down-regulated DEGs among Diseased crabs with mild signs and Healthy crabs, Diseased crabs with severe signs and Healthy crabs, and Diseased crabs with severe signs and Diseased crabs with mild signs.

Figure 6

GO enrichment analysis of DEGs identified in the hepatopancreatic flora. (A) diseased crabs with mild signs vs. healthy crabs; (B) diseased crabs with severe signs vs. healthy crabs; (C) diseased crabs with severe signs vs. diseased crabs with mild signs. BP, CC, and MF represented biological processes, cellular components and molecular functions, respectively.

Figure 7

14 enriched genes to retinol metabolism in diseased crabs with severe signs vs. healthy crabs were downregulated. The green boxes represent the downregulated genes.

Figure 8

Functional abundances of different samples at eggNOG, KEGG, and CaZy levels. (A) KEGG; (B) eggNOG; (C) CaZy.