Molecular mechanism research of crayfish prx 6 gene participating in defending against E. ictaluri infection

Objective Procambarus clarkii was utilized as the research subject to investigate the role of the prx 6 gene in the antimicrobial innate immune response mechanism. This study aimed to provide novel strategies for disease prevention and control in crayfish aquaculture.

Methods Prx 6 double-stranded RNA (dsRNA) was prepared by an in vitro transcription method and subsequently injected into crayfish blood sinusoids using sterile syringes. The interference effect was evaluated through qRT-PCR. To confirm whether prx 6 gene was involved in the process of crayfish antimicrobial innate immunity, the relative expression of prx 6 in haemocytes, hepatopancreas, gills, and intestine tissues after challenging with E. ictaluri was examined. In RNAi assay, crayfish survival rates were determined post E. ictaluri challenge. And changes of antimicrobial peptide genes expression levels were analyzed by qRT-PCR.

Results The results of qRT-PCR analysis showed the up-regulated expression levels of prx 6 gene in various crayfish tissues post stimulation with E. ictaluri revealed the role in crayfish innate immune mediating function against bacterial pathogens. To further investigate the regulating function, dsRNA targeting Prx 6 molecule was prepared in vitro and injected into crayfish, and the relative expression levels of prx 6 in hepatopancreas were detected. The results showed that its expression level was obviously suppressed. In RNAi assay, there was a significant decrease in crayfish survival rates post E. ictaluri injection, comparing with the dsGFP control group. This result revealed the critical role of crayfish prx 6 in defending against bacterial infections. Additionally, some antimicrobial peptides genes were detected post E. ictaluri challenge in RNAi assay. The corresponding results showed that crayfish endogenous antimicrobial peptide genes (ALF 4, ALF 1, crustin 1, crustin 2, lys, and lectin 2) were significantly suppressed, comparing with the dsGFP group. We speculated that prx 6 likely influenced crayfish resistance to pathogens by modulating antimicrobial peptide genes expression, which in turn resulted in the decrease of survival rates.

Conclusion It was hypothesized that prx 6 might participate in the process of the innate immune responses of crayfish against E. ictaluri by regulating the expression of antimicrobial peptides genes.