Environmental DNA assays for Laricobius beetles (Coleoptera: Derodontidae), biocontrol agents of the hemlock woolly adelgid in North America

Abstract The hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), is an invasive pest causing significant ecological and economic damage to certain hemlock tree (Tsuga (Endlicher) Carrière, Pinales:Pinaceae) species. In response to this invasive threat, biological control strategies have been implemented, introducing natural predators such as Laricobius nigrinus Fender (Coleoptera: Derodontidae) and, more recently, Laricobius osakensis Montgomery and Shiyake (Coleoptera: Derodontidae), as specialist predators against A. tsugae. However, the genetic and morphological similarities between L. osakensis and both L. nigrinus and the native beetle, Laricobius rubidus LeConte (Coleoptera: Derodontidae), pose challenges in their identification. Effective monitoring of released predators is integral to evaluating the success of biological control measures. Environmental DNA (eDNA) holds potential for various detection applications, including species monitoring. In this study, we developed specific primers and probes targeting the mitochondrial cytochrome oxidase 1 gene sequences, achieving high specificity despite their 95% sequence similarity. With an optimal annealing temperature of 60 °C, our tools effectively differentiated L. osakensis from the other 2 beetles and demonstrated eDNA detection sensitivity down to 2 copies/µl. This research underscores the potential of precise molecular tools for advancing biological control and biodiversity assessment against invasive threats like A. tsugae.