The immune function of a novel crustin with an atypical WAP domain in regulating intestinal microbiota homeostasis in Litopenaeus vannamei

Crustins are a family of antimicrobial peptides (AMP) with multiple functions, including antimicrobial activity, capability of protease inhibition, phagocytosis promotion, and wound healing in crustaceans. Till present, several members of crustins have been identified and their activities were studied. However, there are still less investigations on how they play functions in vivo. Here, we identified a novel crustin with an atypical WAP domain, LvCrustin Ⅰ-1, which is mainly distributed in tissues, including intestine, gill, epidermis and stomach of the shrimp Litopenaeus vannamei. The expression level of LvCrustin Ⅰ-1 was significantly up-regulated at 3 h, 6 h, 12 h, and 24 h after Vibrio parahaemolyticus infection. Knockdown of LvCrustin Ⅰ-1 with dsRNA resulted in a significant increase of the bacteria number in hepatopancreas of shrimp upon V. parahaemolyticus infection, showing that LvCrustin Ⅰ-1 participated in pathogen infection process. Recombinant LvCrustin Ⅰ-1 protein showed microorganism-binding activity rather than antibacterial activity against tested bacteria. Furthermore, significant difference existed between the intestinal microbiota in shrimp before and after LvCrustin Ⅰ-1 knockdown based on the result of alpha and NMDS analyses. Knockdown of LvCrustin Ⅰ-1 increased the proportion of Demequina, Nautella, Propionibacterium, Anaerospora and decreased the proportion of Bacteroidia and Vibrio. These data suggest that LvCrustin Ⅰ-1 might perform its immunological function through modulation of the intestinal microbiota homeostasis rather than direct inhibition of bacterial growth in shrimp.