Cloning and functional analysis of Rac1 in Crassostrea hongkongensis reveals its role in immune defense against Vibrio harveyi

Abstract

Crassostrea hongkongensis is an economically important marine bivalve species in the South China Sea, but its aquaculture industry is frequently threatened by Vibrio harveyi infections, leading to mass mortality events. Rac1 plays crucial roles in immune regulation, yet its function in C. hongkongensis remains poorly understood. This study systematically investigated the immunoregulatory mechanisms of Rac1 gene in C. hongkongensis against V. harveyi infection. Our results demonstrated that V. harveyi infection significantly increased ROS and MDA levels (P < 0.05), while markedly reducing the activities of SOD and CAT (P < 0.05), indicating the induction of oxidative stress. Notably, the activities of immune-related enzymes, including LZM, PO, ACP, and MPO, were significantly upregulated (P < 0.05), confirming the activation of multiple immune defense systems against pathogen invasion. Through molecular cloning, we obtained the ORF of Rac1, 579 bp, encoding 192 amino acids from the C. hongkongensis. ChRac1 exhibits high conservationat, suggesting functional similarity to its homologs in other species. ChRac1 was detected in multiple tissues of C. hongkongensis via RT-qPCR, with the highest expression in the hepatopancreas. Following immune challenge with Poly(I:C), LPS, or V. harveyi, ChRac1 expression in the hepatopancreas showed significant induction at 12 h post-treatment (P < 0.05). RNA interference-mediated silencing of ChRac1 significantly downregulated the expression of the downstream gene PI3Kα at 12 h post-infection (P < 0.05). Concurrently, the activities of antioxidant enzymes SOD and CAT were aberrantly elevated (P < 0.05), while the enzymatic activities of PO, ACP, and MPO were markedly suppressed (P < 0.05). These findings demonstrate that ChRac1 plays a pivotal role in innate immunity by regulating both antioxidant systems and immune enzyme activities. This study provides novel insights into the immune mechanisms of bivalves and establishes an important theoretical foundation for healthy aquaculture and disease-resistant breeding of C. hongkongensis.