DNA Barcodes for Fruit Fly Species from Pacific Islands and Development of Multiplex Real-Time PCR Assay for Bactrocera facialis, B. passiflorae, B. kirki and B. distincta (Tephritidae: Diptera)

Polyphagous fruit fly (Diptera: Tephritidae) pests from the Pacific Islands pose a biosecurity risk to New Zealand, a country free from pest fruit flies. Among them, Bactrocera facialis, B. passiflorae, B. kirki, and B. distincta are sympatric species commonly intercepted at immature stages at the border. However, current mitochondrial cytochrome oxidase I (COI) barcode data lack sufficient variation for a confident identification of the above four species. To address this, we generated COI barcode data for 403 fruit fly individuals including these four species and an additional 17 related fruit fly species. A phylogenetic analysis of the COI sequences of B. facialis revealed two genetically distinct populations, one closely related to B. passiflorae. Complete mitochondrial genomes were explored, identifying minor fixed differences in Cytochrome b (CYTB), NADH dehydrogenase 2 (ND2), and ATP synthase membrane subunit 6 (ATP6) genes. Based on sequence data for COI, ND2, and ATP6 genes, a multiplex real-time PCR assay has been developed and validated for the four target species. Each assay demonstrated high specificity, with no cross-reactions, and sensitivity as low as 10 copies/μL of the target DNA. This study shows that the developed assays enable the rapid and reliable identification of the target fruit fly species, supporting global biosecurity efforts.