Application of PCR-RFLP of COI Gene for Identification of Life Stages of Bactrocera dorsalis and Ceratitis anonae Infesting Citrus in Southeastern Nigeria

The exotic Bactrocera dorsalis and the native Ceratitis anonae infest citrus and constitute a threat to citrus production in Nigeria. The immature stages of the two species are difficult to differentiate morphologically and share the same fruit in the study area. A 658 bp portion of the COI gene was amplified and sequenced in 88 life stages of the species using LCO1490/HCO2198 primers. The enzymes RsaI, Sau3AI and Hsp92II were used to construct banding patterns distinguishing life stages of B. dorsalis from C. anonae. DNA barcoding confirmed the species to be B. invadens syn. n. B. dorsalis and C. anonae. Rsa I produced bands of 322 and 336 bp in B. dorsalis and 201 and 457 bp in C. anonae. Sau3AI produced bands of 103, 144 and 364 bp in B. dorsalis and 164 and 471 bp in C. anonae. Hsp92II produced fragment lengths of 137 and 521 bp in B. dorsalis and 110, 231 and 290 bp in C. anonae. The identification method was confirmed by DNA barcoding and molecular phylogenetic analysis. All the life stages within a species produced the same banding patterns with the enzymes. This molecular method will be applied at quarantine checkpoints for interception of the invasive B. dorsalis. It will also be crucial for early detection of infestation and in creating and maintaining fruit fly exclusion zones by implementation of appropriate management programmes for B. dorsalis and C. anonae infesting citrus in Nigeria.