Fruit fly (Diptera: Tephritidae) species identification: a rapid molecular diagnostic technique for quarantine application

Abstract

AbstractNew Zealand is currently the only major fruit producing country in the world that is free of economically important fruit flies. As part of the effort to maintain this status, there is a need to supplement quarantine decision-making procedures with a means of rapidly identifying immature life stage infestations to the species level. Here we describe a molecular method that achieves this, using simple restriction patterns of ribosomal DNA (rDNA) as diagnostic markers. The 18S and 18S plus internal transcribed spacer (ITS) regions were amplified from larval DNA by the polymerase chain reaction (PCR). Nineteen species, spanning four genera (including five subgenera ofBactrocera) were analysed. Restriction analysis of the 18S PCR product provided poor resolution, even at the generic level. Digestion of the 18S + ITS PCR product, however, generated thirteen diagnostic haplotypes as defined by the composite restriction patterns fromRsaI,Sau3aHaeIII andAluI. No variation was detected at these restriction sites within or between populations. Twenty two restriction enzymes have been screened, but diagnostic RFLPs have yet to been found for six out of the tenBactrocera (Bactrocera)species;B. passiflorae(Froggatt) cannot be distinguished fromB. facialis(Coquillet), norB. kirki(Froggatt) fromB. trilineola(Froggatt) orB. neohumeralis(Hardy) fromB. tryoni(Froggatt). Geographic origin could assist in distinguishing the first four species, but the latter pair are very closely related with overlapping origins, hosts and adult morphology. All six species, however, are considered high risk with respect to their likely establishment in New Zealand. Therefore diagnosis based on this molecular technique would support the same quarantine decision. We consider this method could be useful as a diagnostic technique and discuss directions for further development.