Identification of a novel toll gene (Shtoll3) from the freshwater crab Sinopotamon henanense and its expression pattern changes in response to cadmium followed by Aeromonas hydrophila infection

Abstract

Toll signaling is essential for expression of immune genes which are important for defense against bacterial, fungal and viral infections in invertebrates. Although several toll genes have been identified in the crustaceans, none of them has been investigated in freshwater crab Sinopotamon henanense. Moreover, the effect of cadmium (Cd) on toll gene expression has never been examined on the freshwater crabs which live in the sediments and are prone to heavy metal bioaccumulation. Our transcriptomic analysis of hepatopancreas tissue reveals that toll3 gene expression has been decreased when treated with Cd. In this study, we cloned one toll gene (hereby designated Shtoll3) from the crab. The full-length cDNA of Shtoll3 was 4488 bp, with an ORF of 3693 bp encoding a putative protein of 1230 amino acids, a 5′-untranslated region of 414 bp and a 3′-untranslated region of 781 bp. Phylogenetic analysis showed that ShToll3 was clustered into the group of DmToll8. The tissue distribution results showed that Shtoll3 was expressed widely in different tissues, with the highest in gills, and the lowest in hemocytes. Shtoll3 expression was down-regulated only in midguts after Aeromonas hydrophila infection. With Cd presence, Shtoll3 expression in response to A. hydrophila were up-regulated in midguts and gills, which was further confirmed by western blotting analysis. Moreover, the mRNA level of two antimicrobial peptides (AMPs) crustin and c-lys, which possibly responded to Cd and A. hydrophila stimulation through Shtoll3, were analysised.Thus, we conclude that Cd changes the susceptibility of Shtoll3 to A. hydrophila infection in gills and midguts. This suggest that Shtoll3 may contribute to the innate immune defense of S. henanense to A. hydrophila and Cd can modify the immune function in epithelium.