No guts, no glory! Uncovering cryptic parasitoid-host relationships in the Euwallacea fornicatus species complex through molecular gut content analysis

In recent years, biological control efforts have focused on the discovery, identification, and mass-rearing of natural enemies associated with the Euwallacea fornicatus species complex, in particular, the polyphagous shothole borer (PSHB), E. fornicatus sensu stricto. PSHB is an invasive ambrosia beetle that has caused significant damage in numerous native and ornamental trees, including agricultural crops, in southern California and other regions worldwide. Multi-year collection of parasitoids emerging from native shothole borer-infested wood, lead to the identification of several potential natural enemies, of which, one commonly encountered species, and six less common species, belonged to the flat wasp family, Bethylidae. In the PSHB system, the acts of parasitism and parasitoid emergence are impossible to observe directly since they occur within the beetle gallery. Thus, in an attempt to confirm the parasitic link between PSHB and each parasitoid, we designed a probe-based qPCR assay to detect remnants of PSHB DNA in the gut of the parasitoids. Detection limits of the assay were determined using 10-fold serial dilutions of PSHB DNA in that of a “surrogate” parasitoid species, Tamarixia radiata, a parasitoid of the Asian citrus psyllid. Among the parasitoids that emerged from the native infested logs, only two species consistently showed evidence of having consumed PSHB, namely the Braconids, Eucosmophorus sp. and Cosmophorus sp. None of the bethylids consistently tested positive, not even a Phymastichus sp. that, through laboratory rearing, has already been shown to parasitize PSHB. Reasons for this discrepancy and the implications of our findings are discussed.