Identification of four secretory phospholipase A(2)s in a lepidopteran insect, Acrolepiopsis sapporensis, and their functional association with cellular immune responses

BACKGROUND: Eicosanoids are a group of the oxygenated C20 polyunsaturated fatty acids and play crucial roles in mediating various insect physiological processes. Catalytic activity of phospholipase A(2) (PLA(2)) provides an initial substrate, arachidonic acid (AA), for subsequent eicosanoid biosynthesis. RESULTS: This study identified four different secretory PLA(2) (As-PLA(2)A–As-PLA(2)D) genes encoded in the Asian onion moth, Acrolepiopsis sapporensis. A phylogenetic analysis indicated that As-PLA(2)A and As-PLA(2)D are clustered with Group III PLA(2)s while As-PLA(2)B and As-PLA(2)C are clustered with Group XII and Group X PLA(2)s, respectively. Expression levels of these PLA(2) genes increased along with larval development, especially in the fat body. A bacterial immune challenge upregulated the basal expression levels of the four PLA(2) genes, which resulted in significant increases of the PLA(2) enzyme activity. The enzyme activity was susceptible to a calcium chelator or reducing agent, suggesting Ca(2+) dependency and disulfide linkage required for the catalytic activities of the secretory type of PLA(2)s. In addition, the PLA(2) activity was also susceptible to bromophenacyl bromide (BPB), a specific inhibitor to sPLA(2), but not to intracellular PLA(2) inhibitors. An addition of BPB to the immune challenge significantly prevented hemocyte-spreading behavior of A. sapporensis. BPB treatment also suppressed a cellular immune response measured by hemocyte nodule formation. However, the immunosuppression was significantly rescued by the AA addition. To determine the PLA(2)(s) responsible for the immunity, individual RNA interference (RNAi) treatments specific to each of the four PLA(2)s were performed. Injection of gene-specific double-stranded RNAs caused significant reductions in the transcript level in all four PLA(2)s. In all four PLA(2)s, the RNAi treatments prevented the cellular immune response even after the immune challenge. CONCLUSION: This study reports four secretory PLA(2)s encoded in A. sapporensis and their function in mediating cellular immunity.