Identification and differentiation of the cryptic cucurbit fruit fly species Dacus frontalis, Dacus ciliatus and Bactrocera cucurbitae (Diptera: Tephritidae) using PCR-RFLP for quarantine applications

Tephritid fruit flies attacking cucurbits are major agricultural pests that cause severe damage to their host plants, and are listed as quarantine pests in many countries. The classical morphological identification of Tephritid species is an arduous task due to presence of cryptic species complexes, as in those attacking cucurbits, and due to the reduced number of species-specific larval morphological characters. A quick and reliable species differentiation method is required to identify and prevent the establishment of invasive species in new territories. For this purpose, in this study, we developed a PCR-RFLP methodology to differentiate between three cucurbits Tephritid fruit fly species, namely Dacus frontalis, Dacus ciliatus and Bactrocera cucurbitae, which are hard to differentiate by morphological characters. The PCR-RFLP method is based on the mitochondrial COI gene barcoding region, which was successfully amplified in specimens from the three species. Sequence analysis revealed that the restriction enzyme RsaI clearly separated -among the three target species either at the larval or adult stage. Hence, this method can be used to improve decision-making procedures at quarantine checkpoints, especially when only immature stages are present in each quarantined commodity, avoiding time-consuming rearing until adult emergence for morphological identification.