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

Insect species complexes challenge entomologists in a variety of ways ranging from quarantine protection to pest management. Billbugs (Coleoptera: Curculionidae: Sphenophorus spp. Schönherr) represent one such species complex that has been problematic from a pest management perspective. These grass-feeding weevils reduce the aesthetic and functional qualities of turfgrass. Sixty-four species of billbugs are native to North America, and at least ten are associated with damage to turfgrass. Billbug species are sympatric in distribution and their species composition and seasonal biology varies regionally. Since their management relies heavily on proper choice of insecticide active ingredients and timing of insecticide applications that target specific life stages, understanding billbug seasonal biology underpins the development of efficient management programs. However, billbug seasonal biology investigations are currently hindered by our inability to identify the damaging larval stage to species level. DNA barcoding, which involves the use of short DNA sequences that are unique for each species, represents one potential tool that can aid these efforts. By combining DNA-based species identification with morphometric measures capable of serving as a proxy of larval development, it may be possible to gain a more holistic understanding of billbug seasonal biology. In this study, we developed a DNA barcoding reference library using cytochrome oxidase subunit 1 (COI) sequences from morphologically identified adult billbugs collected across Indiana, Missouri, Arizona, and Utah. Next, we applied our reference library for comparison and identification of unknown larval specimens collected across the growing season in Utah and Indiana. We then used a combination of DNA barcoding and larval head capsule diameters acquired from samples collected across a short span of the growing season to produce larval phenology maps. Adult billbug COI sequences varied within species, but the variation was not shaped by geography, indicating that this locus itself could resolve larval species identity. Overlaid with head capsule diameter data from specimens collected across the growing season, a better understanding of billbug species composition and seasonal biology emerged. This knowledge will provide researchers with the tools necessary to fill critical gaps in our understanding of billbug biology thereby improving turfgrass pest management. Using this approach researchers will be able to support efforts to provide growers with the information necessary to develop more prescriptive, location-based management programs and reduce the ecological footprint of turfgrass pest management