Molecular cloning, characterization and expression analysis of two apoptosis genes, caspase and nm23, involved in the antibacterial response in Chinese mitten crab, Eriocheir sinensis

Apoptosis is a central regulatory feature of the immune system, and the most common form of death among immunological cells. However, the function of apoptosis, within the innate immune system of invertebrates, remains largely unknown. For this reason, we investigated the immune functionality of two apoptosis genes, caspase and nm23, in the Chinese mitten crab (Eriocheir sinensis), which is a commercially important and disease vulnerable aquaculture species. The entire length caspase and nm23 cDNA genes were cloned using PCR, based on an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The caspase cDNA contained an 1119 bp open reading frame that encoded a putative 372 amino acid protein, while nm23 cDNA contained a 456 bp open reading frame that encoded a putative 151 amino acid protein. Comparison, with other reported invertebrate and vertebrate sequences, revealed the presence of conserved enzyme active sites that were common among caspase and nm23 superfamilies. In brief, caspase and nm23 mRNA expression in E. sinensis were (a) both detected in all tissues, including the hemocytes, heart, hepatopancreas, gill, stomach, muscle, intestine, brain and eyestalk, and (b) responsive in hemocytes, gill and hepatopancreas to a Vibrio anguillarum immuno-challenge all appeared sharp increase. Collectively, the data presented here demonstrate the successful isolation of caspase and nm23 apoptosis genes from the Chinese mitten crab, and their role in the innate immune system of an invertebrate.

Research highlights
► Crab ES-Casp belongs to long prodomain caspases, with DED or CARD domains being absent.
► Possible role of ES-Nm23 in apoptosis signaling as it is in the same phylogenetic group as human NM23-H1 and NM23-H2.
► Caspase expression is immunoresponsive and may possess immune functionality in crabs.
► Caspase and Nm23 are involved in defense response against bacterial infection in crabs.