Cycloheximide-ProducingStreptomycesAssociated withXyleborinus saxeseniiandXyleborus affinisFungus-Farming Ambrosia Beetles

AbstractSymbiotic microbes help a myriad of insects acquire nutrients. Recent work suggests that insects also frequently associate with actinobacterial symbionts that produce molecules to help defend against parasites and predators. Here we explore a potential association between Actinobacteria and two species of fungus-farming ambrosia beetles,Xyleborinus saxeseniiandXyleborus affinis. We isolated and identified actinobacterial and fungal symbionts from laboratory reared nests, and characterized small molecules produced by the putative actinobacterial symbionts. One 16S rRNA phylotype ofStreptomyces(XylebKG-1) was abundantly and consistently isolated from the nests and adults ofX. saxeseniiandX. affinisnests. In addition toRaffaelea sulphurea, the symbiont thatX. saxeseniicultivates, we also repeatedly isolated a strain ofNectriasp. that is an antagonist of this mutualism. Inhibition bioassays betweenS. griseusXylebKG-1 and the fungal symbionts fromX. saxeseniirevealed strong inhibitory activity of the actinobacterium towards the fungal antagonistNectriasp. but not the fungal mutualistR. sulphurea. Bioassay guided HPLC fractionation ofS. griseusXylebKG-1 culture extracts, followed by NMR and mass spectrometry identified cycloheximide as the compound responsible for the observed growth inhibition. A biosynthetic gene cluster putatively encoding cycloheximide was also identified inS. griseusXylebKG-1. The consistent isolation of a single 16S phylotype ofStreptomycesfrom two species of ambrosia beetles, and our finding that a representative isolate of this phylotype produces cycloheximide, which inhibits a parasite of the system but not the cultivated fungus, suggests that these actinobacteria may play defensive roles within these systems.