Protocol for spatial sampling and monitoring infection disease dynamics and pathogen spread in social ambrosia beetle colonies

Here, we present a protocol for investigating spatial sampling, infection, and pathogen spread in ambrosia beetle colonies to explore infectious disease dynamics in social organisms. We describe steps for rearing Xyleborus affinis colonies, preparing and performing fungal ( Metarhizium ) infection assays, and examining vertical and horizontal disease transmission. We then detail procedures for spatial sampling within beetle colonies for adult beetle and progeny distribution, microbial commensal and/or pathogen distribution, and for microscopic imaging of colonies and the infection process, including cryo-sectioning. For complete details on the use and execution of this protocol, please refer to Masoudi et al. 1 • Laboratory rearing of ambrosia beetles in sawdust-based artificial medium • Immersion-based fungal infection of beetles using entomopathogenic conidia • Vertical and horizontal transmission assays in plates and colony nest habitats • Spatial CFU quantification and fluorescence imaging of infection dynamics Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for investigating spatial sampling, infection, and pathogen spread in ambrosia beetle colonies to explore infectious disease dynamics in social organisms. We describe steps for rearing Xyleborus affinis colonies, preparing and performing fungal ( Metarhizium ) infection assays, and examining vertical and horizontal disease transmission. We then detail procedures for spatial sampling within beetle colonies for adult beetle and progeny distribution, microbial commensal and/or pathogen distribution, and microscopic imaging of colonies and infection, including cryo-sectioning.