A molecular identification approach for five species of mealybug (Hemiptera: Pseudococcidae) on citrus fruit exported from South Africa

Within South Africa, millions of rands in revenue are generated annually from the export of fruit produce. Citrus fruits are exported mostly to the U.S.A., South Korea and China. These countries have strict biosecurity laws in place which prohibit the import of specific problem insects, including several species of mealybug (Hemiptera: Pseudococcidae). Although the adult females for most species of mealybug are routinely identified through morphological keys, eggs and nymphs (crawlers) are more problematic. As such, export consignments are regularly refused based on the presence of unidentifiable mealybug nymphs or eggs. The aim of this paper is to report an easy, user-friendly molecular laboratory technique to accurately identify mealybug eggs and crawlers to species level. We amplified and sequenced a 749 bp portion of the mitochondrial DNA cytochrome oxidase subunit one (COI) gene. Phylogenetic trees indicated that the five species included here (Planococcus citri (Risso), Paracoccus burnerae (Brain), Pseudococcus longispinus (Targioni-Tozzetti), Ps. calceolariae (Maskell) and Ps. viburni (Signoret)) are reciprocally monophyletic and follow the 10× rule where sequence divergences separating species are an order of magnitude larger than divergences within species. As such, DNA can be extracted from eggs, crawlers or adults; analysed and compared with reference data, and unequivocally assigned to one of the five species.