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

Abstract

New 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 of Bactrocera) 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 from Rsal, Sau3a Haelll and Alul. 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 ten Bactrocera (Bactrocera) species; B. passiflorae (Froggatt) cannot be distinguished from B. facialis (Coquillet), nor B. kirki (Froggatt) from B. trilineola (Froggatt) or B. neohumeralis (Hardy) from B. tnjoni (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.

The complete nucleotide sequence of the mitochondrial genome of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae)

“…Focusing on species discrimination for import/export controls and general biosecurity, most available tests are based on PCR-RFLP assays targeting sequences from the nuclear ribosomal cluster (Armstrong et al, 2000) and mitochondrial CR, rrnS and rrnL, but none of these tests are currently capable of discriminating between all the key species, and attempts are being undertaken do develop more sensitive and efficient tests, i.e. taking advantage of barcoding data (Armstrong and Ball, 2005) or in the form of a micro-array biochip (Frey and Pfunder, 2006).…”

Mitotic and polytene chromosomes analysis of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)

“…Several autosomal recessive mutations were described for B. dorsalis s.s., belonging to five linkage groups [reviewed in McCombs and Saul (1992)]. Efforts have also been made in developing molecular markers for species identification (Armstrong et al 1997;Armstrong and Cameron 2000;Nakahara 2001, 2002;Nakahara et al 2001;Naeole and Haymer 2003) or for the analysis of the genetic structure of natural populations (Aketarawong et al 2007;Chen and Ye 2008;Nakahara et al 2008).…”

Identification of forensically important Chrysomya (Diptera: Calliphoridae) species using the second ribosomal internal transcribed spacer (ITS2)

“…The complete internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) has been found to be a reliable marker for PCR-RFLP identification of many species, including economically important fruit flies [7] and more recently, morphologically similar dipteran larvae of forensic importance [8]. These findings suggest that the ITS regions might be sufficiently variable to construct unique identification markers for a number of carrion-breeding blowflies, including Australian members of the genus Chrysomya.…”