Dual-target detection using simultaneous amplification of PCR in clarifying interaction between Opiinae species (Hymenoptera: Braconidae) associated with Bactrocera spp. (Diptera: Tephritidae) infesting several crops

Simultaneous polymerase chain reaction (PCR) by using multiplex PCR was conducted to clarify the interaction between Opiinae associated with Bactrocera carambolae, B. papayae, and B cucurbitae. We sequenced and characterized a dual-band target detected on Opiinae DNA fragments by using two combination pairs of universal primers on two molecular markers, namely cytochrome oxidase subunit I (COI) and cytochrome b. Additionally, each Bactrocera species was identified by amplifying the COI region. Sequence data obtained from multiplex PCR seemed very effective in confirming species-level distinction that has been shown in tree topology. We conducted phylogenetic analyses prior to clarification of the interaction among three taxa levels. Interestingly, the sequences obtained from the simultaneous PCR successfully differentiated between six closely related Opiinae species in three genera, which could potentially be mass-reared as biocontrol agents of bactroceran fruit flies that infest several species of fruit. We discovered, proved, and added molecular data to clarify the interaction between Opiinae parasitoids, their host (Bactrocera spp.), and associated plants species. Psyttalia fletcheri parasitizing Bactrocera cucurbitae, which infests the ridge gourd fruit, has been added as a new record from Malaysia. This information would be extremely useful in taxonomic identification of species as part of an effective method in biological control program of the targeted fruit fly pests and their associated crops.