Molecular Identification of Mealybug Species (Hemiptera: Pseudococcidae) Affecting <em>Theobroma cacao</em> for Improved Pest Management

Theobroma cacao is affected by viruses on every continent where the crop is cultivated (North America, South America, Asia, and Africa), with the most well-known ones belonging to the Badnavirus genus. Badnaviruses are transmitted by several species of Pseudococcidae, a large, taxonomically diverse group of insects collectively known as mealybugs. Effective management of mealybugs depends on accurate identification of species present, as even closely related species have distinct life cycles and are vulnerable to chemical control at different times. To develop a molecular identification strategy, mealybugs were collected from four randomly selected T. cacao trees in a greenhouse in Florida, and DNA was extracted from individual specimens. This study compares the usefulness of the COI, ITS2, and 28S markers using the primer pairs (MFCO1/MRCO1, ITS2-M-F/ITS2-M-R, D10F/D10R, and D2F/D2R) to identify mealybugs associated with cacao plants in North America. All markers were equally informative for Pseudococcus comstocki (n=4) and Maconelicoccus hirsutus (n=8), but only CO1 provided unambiguous identification for Pseudococcus jackbeardsleyi (n=11). However, primer pair D2F/D2R is not recommended for mealybug identification. In addition to poor amplification, many of the sequences obtained were Anagyrus sp., wasp-like parasites frequently used for biocontrol. This study describes molecular diagnostic protocols for identifying cacao-infecting mealybugs and estimating the prevalence of certain parasitoids. This information is essential for selecting the most effective interventions as part of an integrated pest management program.