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, <i>Amrasca biguttula</i> (Ishida) (Hemiptera: Cicadellidae), in the United States. Nymphs of multiple instars and adult specimens were collected from a cotton (<i>Gossypium hirsutum</i>) 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. <i>Cytochrome oxidase I</i> (COI) DNA barcoding confirmed the specimen identity. The United States sequence shared > 99% identity with Asian <i>A. biguttula</i> references, and phylogenetic analysis placed it within the <i>A. biguttula</i> 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.