Identification of four secretory phospholipase A2s in a lepidopteran insect, Acrolepiopsis sapporensis, and their functional association with cellular immune responses

Abstract

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 A₂ (PLA₂) provides an initial substrate, arachidonic acid (AA), for subsequent eicosanoid biosynthesis.

Results: This study identified four different secretory PLA₂ (As-PLA₂A-As-PLA₂D) genes encoded in the Asian onion moth, Acrolepiopsis sapporensis. A phylogenetic analysis indicated that As-PLA₂A and As-PLA₂D are clustered with Group III PLA₂s while As-PLA₂B and As-PLA₂C are clustered with Group XII and Group X PLA₂s, respectively. Expression levels of these PLA₂ genes increased along with larval development, especially in the fat body. A bacterial immune challenge upregulated the basal expression levels of the four PLA₂ genes, which resulted in significant increases of the PLA₂ enzyme activity. The enzyme activity was susceptible to a calcium chelator or reducing agent, suggesting Ca²⁺ dependency and disulfide linkage required for the catalytic activities of the secretory type of PLA₂s. In addition, the PLA₂ activity was also susceptible to bromophenacyl bromide (BPB), a specific inhibitor to sPLA₂, but not to intracellular PLA₂ 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 PLA₂(s) responsible for the immunity, individual RNA interference (RNAi) treatments specific to each of the four PLA₂s were performed. Injection of gene-specific double-stranded RNAs caused significant reductions in the transcript level in all four PLA₂s. In all four PLA₂s, the RNAi treatments prevented the cellular immune response even after the immune challenge.

Conclusion: This study reports four secretory PLA₂s encoded in A. sapporensis and their function in mediating cellular immunity.

Keywords: Acrolepiopsis sapporensis; PLA2; RNAi; eicosanoid; immunity; nodulation.