White spot syndrome virus impact on the expression of immune genes and gut microbiome of black tiger shrimp Penaeus monodon

Abstract

The gut microbiome plays an essential role in the immune system of invertebrates and vertebrates. Pre and pro-biotics could enhance the shrimp immune system by increasing the phenoloxidase (PO), prophenoloxidase (ProPO), and superoxide dismutase activities. During viral infection, the host immune system alteration could influence the gut microbiome composition and probably lead to other pathogenic infections. Since the JAK/STAT pathway is involved in white spot syndrome virus (WSSV) infection, we investigated the intestine immune genes of STAT-silenced shrimp. During WSSV infection, expression levels of PmVago1, PmDoral, and PmSpätzle in PmSTAT-silenced shrimp were higher than normal. In addition, the transcription levels of antimicrobial peptides, including crustinPm1, crustinPm7, and PmPEN3, were higher in WSSV-challenged PmSTAT-silenced shrimp than the WSSV-infected normal shrimp. Meanwhile, PmSTAT silencing suppressed PmProPO1, PmProPO2, and PmPPAE1 expressions during WSSV infection. The microbiota from four shrimp tested groups (control group, WSSV-infected, PmSTAT-silenced, and PmSTAT-silenced infected by WSSV) was significantly different, with decreasing richness and diversity due to WSSV infection. The relative abundance of Bacteroidetes, Actinobacteria, and Planctomycetes was reduced in WSSV-challenged shrimp. However, at the species level, P. damselae, a pathogen to human and marine animals, significantly increased in WSSV-challenged shrimp. In constrast, Shewanella algae, a shrimp probiotic, was decreased in WSSV groups. In addition, the microbiota structure between control and PmSTAT-silenced shrimp was significantly different, suggesting the importance of STAT to maintain the homeostasis interaction with the microbiota.

Figure 1

(A) PmSTAT and IE1 transcription levels in shrimp intestine at 24 h post-WSSV injection. PmSTAT was suppressed by injecting PmSTAT dsRNA 10 µg per shrimp’s gram, then 16 h later, followed by the same amount with the first injection. The control group was injected with PBS instead PmSTAT dsRNA. WSSV was injected at 24 h after the 2nd PmSTAT dsRNA injection. Shrimp intestines were collected at 24 h post-infection. In addition, we investigated the transcription levels of immune-related genes upon PmSTAT silencing during WSSV infection. (B) Transcription levels of interferon-like gene (PmVago 1, 4, and 5). (C) Expression levels of immune-related genes, including JAK/STAT pathway (PmDOME and PmJAK), Toll pathway (MyD88, Castus, Dorsal, and Spätzle), IMD pathway (Relish) and inhibitor of kappa B kinase (IKKβ, IKKε1, and IKKε2). Moreover, Transcription levels of immune genes were also observed. (D) Transcription levels of antimicrobial peptide and (E) phenol oxidase cascade.

Figure 2

(A) Intestinal bacterial composition in PBS, PmSTAT-silenced, WSSV-challenged, and PmSTAT-silenced combined WSSV challenged on phylum level. The most abundant genera (B) and species level (C). The relative abundance of potential probiotic, Shewanella algae (D).

Figure 3

(A) Principal coordinate analysis (PCoA) based on unweighted UniFrac distance and (B) unweighted UPGMA clustering. The knSTAT refers to PmSTAT silencing shrimp.