First Molecular Verification of the Two-Spot Cotton Leafhopper Amrasca biguttula (Hemiptera: Cicadellidae) in the United States

This report contains the first molecular record of the two-spot cotton leafhopper, Amrasca biguttula (Ishida) (Hemiptera: Cicadellidae), in the United States. Nymphs of multiple instars and adult specimens were collected from a cotton (Gossypium hirsutum) field in Macon County, Alabama, in August 2025. While distinct paired dark spots were observed on the forewings of adult specimens, this trait was inconsistently present on nymphal wing pads. Cytochrome oxidase I (COI) DNA barcoding confirmed the specimen identity. The United States sequence shared > 99% identity with Asian A. biguttula references, and phylogenetic analysis placed it within the A. biguttula clade with 100% posterior probability support. Although this pest was previously reported in 2023 from Puerto Rico based solely on morphological traits, our findings provide the first DNA-confirmed evidence of its presence in the United States. Given its well-documented role in damaging cotton across Asia and Africa, this report underscores the urgent need for monitoring and development of management strategies in United States cotton-growing regions.