Activation of immune-associated phospholipase A2 is functionally linked to Toll/Imd signal pathways in the red flour beetle, Tribolium castaneum

Bacterial challenge enhances phospholipase A2 (PLA2) activity, which in turn induces biosynthesis of various eicosanoids that mediate non-self recognition signal to immune effectors in insects. But, there is little information on how PLA2 activity is controlled after the non-self recognition. A recent genome analysis of the red flour beetle, Tribolium castaneum, has annotated both Toll and Imd signal pathways that are presumably considered to specifically respond to different microbial infections to express specific antimicrobial peptides (AMPs). This study set up a hypothesis that PLA2 activation is linked to Toll and Imd pathways in T. castaneum. Bacterial challenge to the larvae of T. castaneum induced expressions of Toll and Imd genes. Different AMP genes were induced in larvae infected with Gram-positive or -negative bacteria. RNA interference using double-stranded RNAs (dsRNAs) specific to different Toll and Imd genes showed differential inhibition of these AMP expressions, indicating that the Toll and Imd pathways play critical roles in the production of AMPs by specifically responding to various bacterial challenges in T. castaneum. These Toll and Imd immune signals are also linked to the activation of PLA2 in T. castaneum. Activation of PLA2 was significantly induced in response to bacterial challenge, but was inhibited by dsRNAs specific to different Toll and Imd genes. The activation of PLA2 via Toll and Imd pathways could be explained by induction of PLA2 gene expression because the dsRNA treatments of Toll and Imd genes suppressed the gene expression of PLA2 in response to bacterial challenge. The functional links were further supported by an immunofluorescence assay of PLA2 intracellular translocation. Upon bacterial challenge, hemocytes from control larvae showed intracellular translocation of their PLA2s near to cell membrane, but hemocytes from larvae treated with dsRNAs of the Toll and Imd genes did not show the translocation, at which the PLA2s appeared to be evenly spread in the cytoplasm. These results suggest that recognition of bacterial challenge initiates Toll and Imd pathways in T. castaneum, which subsequently induces the activation of immune-associated PLA2s as well as gene expression of various AMPs.