Validation of the Mitochondrial Genome of Planococcus citri and Molecular Tools for Species-Level Identification of Citrus-Associated Mealybugs in South Africa

Abstract

Citrus production in South Africa is affected by seven mealybug (Hemiptera: Coccomorpha: Pseudococcidae) species, with Planococcus citri (Risso) being the most prevalent and the pest status of the other six species being generally minor or even negligible. Infestations can reduce fruit production and quality due to sooty mould development. Accurate species-level identification of mealybugs is valuable for effective agricultural pest management and in some cases may be of phytosanitary relevance. Traditional morphological identification is challenging. Slide mounted adult female specimens are usually required for accurate morphological identification, a process that is time consuming and requires specialist skills. Molecular diagnostics targeting mitochondrial genes, particularly the cytochrome c oxidase subunit I (COI), have become valuable rapid tools for reliable species discrimination in most insect groups. The use of molecular techniques in combination with morphological assessments provides stronger support for accurate species identification. This study reports the first validated mitochondrial genome of P. citri, an important citrus pest, alongside partial mitochondrial sequences for the six other mealybug species previously reported on citrus and two other species that are not associated with citrus in South Africa. Using high-throughput and Sanger sequencing combined with manual curation, a high-confidence P. citri mitogenome was assembled while draft mitogenomes were generated for the other species. Two complementary molecular assays were developed and validated. These consisted of a set of multiplex PCRs and restriction enzyme digest assays that enable rapid, same-day differentiation of the nine mealybug species. The multiplex PCR assays provide sensitive and specific detection, while the restriction digest assays act as a confirmatory tool and fail-safe mechanism, allowing early identification of discrepancies. In one of the species, Nipaecoccus viridis, greater genetic diversity was uncovered, suggesting cryptic speciation. This study underscores the complexities of unarmoured scale insect mitogenomics, including repetitive regions, low gene conservation and scarce reference sequences while providing a robust and efficient molecular framework that enhances accurate mealybug identification.