A non-destructive, fast, inexpensive, non-toxic chelating resin-based DNA extraction protocol for insect voucher specimens and associated microbiomes.

Identifying a DNA extraction method that yields high quantity and quality DNA is a crucial component of molecular ecological studies; and the best suited method can vary greatly depending on research priorities. Here, we propose a nondestructive extraction method for insect museum vouchers aimed at analyzing gut-associated microbiomes. The leafhopper Euscelidius variegatus (Kirschbaum) (Hemiptera: Cicadellidae) associated with the bacterial plant pathogen Flavescence dorée phytoplasma, a member of the genus 'Candidatus Phytoplasma' (Mollicutes: Acholeplasmataceae), was used as an experimental model. We developed and refined a resin-based DNA extraction protocol by testing the effects of prelysis bleaching and postlysis proteinase K inactivation on DNA quality and yield. We found that bleaching did not compromise the integrity of insect and associated bacterial DNA and that excluding the inactivation of proteinase K did not interfere with quantitative polymerase chain reaction analysis. Based on our findings, we recommend a DNA extraction protocol for insect voucher specimens and associated microbiomes that includes a prelysis bleaching step to chemically degrade external contaminants without proteinase K inactivation, thereby reducing processing time. Our refined protocol resulted in a high DNA yield, which we successfully analyzed using quantitative polymerase chain reaction analysis and other downstream molecular applications, including targeted high-throughput sequencing.