Functional analysis of subterranean termite antifungal peptides using RNA interference

Social insects face high risks of infection and disease transmission, and strong selective pressures have influenced the evolution of adaptations to cope with disease. The entomopathogenic fungus Metarhizium anisopliae, which can evade the host immune system after cuticular penetration, is ubiquitous in soil occupied by the eastern subterranean termite Reticulitermes flavipes, and has likely influenced the evolution of immune defenses in these termites. The humoral immune response in insects involves pattern recognition proteins (PRPs) that recognize pathogens and initiate immune pathways leading to the production of antimicrobial peptides (AMPs).  Termite Gram-negative bacteria binding proteins (GNBPs), a class of PRPs which exhibit antifungal b(1,3)-glucanase activity, are secreted from the salivary glands with termicins (AMPs) and are spread over the cuticle during mutual grooming. These molecules may act synergistically to defend against fungal pathogens. A selective sweep in termicins in Reticulitermes suggests that positive selection has changed the amino acid composition of the peptide, and that it may be a central point in the evolutionary arms race between termites and their pathogens. We are characterizing the functional roles of GNBPs and termicins in R. flavipes by knocking down gene expression with RNA interference.  Initial results from survival experiments show that termites fed termicin-specific double stranded RNA are more susceptible to infection by M. anisopliae relative to controls. This suggests that termicins are critical in defending against infection by M. anisopliae, and the externalization of these peptides may prevent cuticular penetration and initial infection.