Simple and Rapid PCR-RFLP based species identification in Drosophila suzukii and Drosophila immigrans larvae

Wild fruit fly larvae can be distinguished at the family level such as Drosophilidae and Tephritidae larvae are based on size or spiracles arrangement, but such visual distinction is more intricate between Drosophilid species. In the present study, to identify the Drosophila immigrans and Drosophila suzukii species at the larval stage, we employed a simple, rapid PCR-RFLP experiment. Wild suspected larvae from Drosophila immigrans and Drosophila suzukii species were collected from 3 different geographical regions. DNA was isolated and a PCR-RFLP profile was developed using the XmnI restriction enzyme. Sequencing of the mitochondrial COI gene from larvae was performed to evaluate the species. PCR-RFLP analysis from Drosophila suzukii wild larvae generated three distinct bands with 120bp, 210bp and 250bp. Similarly, Drosophila immigrans generated 2 distinct bands with 270bp and 310bp. Restriction digestion products from both the larvae and adult flies were the same, hence the accuracy of the PCR-RFLP profile developed from both suspected wild larvae and adult flies was 100%. Sequencing analysis confirms that wild field collected larvae belong to Drosophila suzukii and Drosophila immigrans. Present study clearly shows that PCR‐RFLP is a fast, simple, inexpensive and accurate molecular method and it can be successfully used for species identification at the early stages of the life cycle of Drosophila species.