Fruit flies of the family Tephritidae especially genus Bactrocera pose a serious threat to fruits and vegetables throughout the world including Pakistan. To investigate the diversity and distribution of Bactrocera species, flies were collected in field through pheromone traps and also through emergence from infested fruit samples in the lab. Pheromone traps were installed at 43 locations across 18 districts of the country and in the same season infested fruits were collected from nine districts. At first a total of 12,215 flies were identified morphologically using the taxonomic keys from David and Ramani 2011 and Zubair et al. 2019 and subsequently confirmed by amplification and sequencing of the mitochondrial cytochrome c oxidase I (mt-COI) gene for DNA barcoding of selected samples. Results reveal occurrence of six Bactrocera species in Pakistan; Bactrocera dorsalis, Bactrocera zonata, Bactrocera correcta, Bactrocera cucurbitae, Bactrocera tau, Bactrocera scutellaris, and one species of Dacus ciliatus during May to September 2023. Our study also provides the first molecular confirmation of Bactrocera scutellaris and Dacus ciliatus in Pakistan using mt-COI barcoding, validating previous morphological reports. The findings from our study have significant implications for integrated pest management (IPM), as the development of effective single or combined management strategies for specific pests or pest complexes requires accurate and reliable species identification tools. Without accurate identification and an understanding of where these species are located, the resources and efforts invested in managing these flies could be wasted. Overall, our study highlights both the diversity of fruit fly species in Pakistan and provides the first molecular verification of Bactrocera scutellaris and Dacus ciliatus, offering a reliable foundation for targeted IPM interventions.
