sPLA2 behaves like a prophylactic agent and mediates cellular and humoral immune responses in Plutella xylostella

Most immune effectors are inducible to microbial pathogen infection while some are already present to act as prophylactic immunity against as yet unseen infection. This study identified secretory phospholipase A(2) (sPLA(2)) as a prophylactic factor in diamondback moth, Plutella xylostella. Western blotting using a polyclonal antibody raised against other lepidopteran sPLA(2) reacted specifically with similar to 25 kDa protein, which was present at approximately 0.4 mM in the plasma of naive larvae. Interrogation of P. xylostella transcriptomes revealed an open-reading frame for sPLA(2) (Px-sPLA(2)), exhibiting high homology with other Group III sPLA(2)s. Px-sPLA(2) was expressed in all developmental stages. In the larval stage, bacterial challenge induced its expression in hemocytes and fat body but not in gut or epidermis. RNA interference (RNAi) suppressed Px-sPLA(2) messenger RNA level and sPLA(2) activity in plasma. An inhibition zone assay showed that Px-sPLA(2) exhibited antibacterial activities against different species, because specific RNAi knockdown impaired the activity. The RNAi treatment also suppressed the cellular immune response assessed by hemocyte nodule formation and humoral immune response assessed by antimicrobial peptide gene expression. Finally, benzylideneacetone (BZA, a specific sPLA(2) inhibitor) treatment inhibited plasma sPLA(2) activity of naive larvae in a dose-dependent manner. An addition of BZA significantly increased the bacterial virulence of an entomopathogen, Bacillus thuringiensis. These results suggest that Px-sPLA(2) is an immune-associated factor of P. xylostella and its relatively high level of concentration in the plasma of naive larvae strongly suggests its role as a prophylactic factor in defending against pathogens at early infection stages.