Characterizing Billbug (Sphenophorus spp.) Seasonal Biology Using DNA Barcodes and a Simple Morphometric Analysis

Simple Summary Billbugs (Sphenophorus spp.) are a group of grass-feeding weevils considered to be one of the most important and widespread insect pests of turfgrass. However, our limited understanding of regional variation in billbug species composition and inability to identify the damaging larval stage to species level, has hindered our ability to resolve the seasonal biology of many billbug species and constrained development of effective management approaches. In this study, we developed a robust DNA barcoding approach for identification of morphologically cryptic billbug larvae. Using this molecular tool combined with larval head capsule measurements we characterized regional variation in billbug species and developed larval phenology charts. Our approach provides researchers with the molecular tools necessary to fill critical gaps in our understanding of billbug seasonal biology and will facilitate the development of improved turfgrass pest management programs. Billbugs (Sphenophorus spp.) are a complex of grass-feeding weevil species that reduce the aesthetic and functional qualities of turfgrass. Effective billbug monitoring and management programs rely on a clear understanding of their seasonal biology. However, our limited understanding of regional variation in the species compositions and seasonal biology of billbugs, stemming primarily from our inability to identify the damaging larval stage to species level, has hindered efforts to articulate efficient IPM strategies to growers. We used a combination of DNA barcoding methods and morphometric measures to begin filling critical gaps in our understanding of the seasonal biology of the billbug species complex across a broad geographic range. First, we developed a DNA barcoding reference library using cytochrome oxidase subunit 1 (COI) sequences from morphologically identified adult billbugs collected across Indiana, Missouri, Utah and Arizona. Next, we used our reference library for comparison and identification of unknown larval specimens collected across the growing season in Utah and Indiana. Finally, we combined our DNA barcoding approach with larval head capsule diameter, a proxy for developmental instar, to develop larval phenology charts. Adult COI sequences varied among billbug species, but variation was not influenced by geography, indicating that this locus alone was useful for resolving larval species identity. Overlaid with head capsule diameter data from specimens collected across the growing season, a better visualization of billbug species composition and seasonal biology emerged. This approach will provide researchers with the tools necessary to fill critical gaps in our understanding of billbug biology and facilitate the development of turfgrass pest management programs.