Theobroma cacao is affected by viruses on every continent where the crop is cultivated, with the most well-known ones belonging to the Badnavirus genus. One of these, cacao mild mosaic virus (CaMMV), is present in the Americas, and is transmitted by several species of Pseudococcidae (mealybugs). To determine which species are associated with virus-affected cacao plants in North America, and to assess their potential as vectors, mealybugs (n = 166) were collected from infected trees in Florida, and identified using COI, ITS2, and 28S markers. The species present were Pseudococcus jackbeardsleyi (38%; n = 63), Maconellicoccus hirsutus (34.3%; n = 57), Pseudococcus comstocki (15.7%; n = 26), and Ferrisia virgata (12%; n = 20). Virus acquisition was assessed by testing mealybug DNA (0.8 ng) using a nested PCR that amplified a 500 bp fragment of the movement protein–coat protein region of CaMMV. Virus sequences were obtained from 34.6 to 43.1% of the insects tested; however, acquisition did not differ among species, X2 (3, N = 166) = 0.56, p < 0.91. This study identified two new mealybug species, P. jackbeardsleyi and M. hirsutus, as potential vectors of CaMMV. This information is essential for understanding the infection cycle of CaMMV and developing effective management strategies.

Theobroma cacao is affected by viruses on every continent where the crop is cultivated, with the best-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 different biological control organisms. 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 informative for Pseudococcus comstocki (n=4) and Maconellicoccus hirsutus (n=8), but only CO1 provided unambiguous identification for Pseudococcus jackbeardsleyi (n=11). Primer pair D2F/D2R is not recommended for mealybug identification, as it frequently yielded sequences of Anagyrus sp., an Encyrtid parasitoid wasp commonly used for biocontrol. This study describes molecular diagnostic protocols for identifying cacao-associated mealybugs and detecting the presence of certain parasitoids. This information is essential for selecting the most effective interventions as part of an integrated pest management program.