Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge

We propose that expression of four genes encoding secretory phospholipases A2 (sPLA2) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis of which depends on PLA2-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA2 activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA2 and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA2s. The recombinant sPLA2s were inhibited by sPLA2 inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA2 genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA2s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA2s showed the presence of the sPLA2 enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA2 genes that mediate nodulation reactions strongly supports our hypothesis that sPLA2s are central enzymes in insect cellular immune reactions.