Optimized Field Collection and Gut Dissection Workflows for Microbiome Studies of the Citrus Root Weevil, Diaprepes abbreviatus.

Careful dissection of insect gut tissues is essential for microbiome studies to ensure accurate characterization of internal microbial communities and preservation of DNA integrity. Because insect-associated microbiomes are highly sensitive to contamination, effective removal of external microbes prior to dissection is critical to minimize bias in downstream analyses. While ethanol- and bleach-based surface sterilization methods are commonly used, standardized workflows integrating field collection, sterilization, and dissection remain limited. Here, we present a step-by-step protocol for the field collection, surface sterilization, and dissection of gut tissues from the agricultural pest Diaprepes abbreviatus (Coleoptera: Curculionidae), optimized for genomic DNA extraction and microbiome analyses. Using wild-caught specimens, this workflow incorporates a rigorous surface sterilization and dissection strategy that minimizes external contamination while preserving biologically relevant microbial signatures and DNA integrity for downstream microbiome analyses. The protocol provides a standardized framework for insect gut microbiome studies and can be broadly adapted to other wild-caught insect species requiring careful collection, disinfection, and sterile dissection prior to molecular analysis. The protocol integrates field collection and laboratory processing steps into a streamlined workflow that minimizes contamination while preserving tissue integrity for downstream applications. Key features • Designed for wild-caught Diaprepes abbreviatus collected directly from agricultural host trees, this protocol can also be adapted for other insect species. • Integrates field collection, surface sterilization, and sterile gut dissection into a single workflow to minimize contamination. • Sequential ethanol and diluted bleach treatment effectively removes external microbes prior to dissection. • Enables isolation of intact gut tissues suitable for high-quality DNA extraction and downstream microbiome sequencing.