DNA barcoding is a diagnostic molecular method that is routinely applied in Australian quarantine and biosecurity laboratories to identify morphologically problematic specimens and confirm new fruit fly incursions.
The Australian Department of Agriculture and Water Resources also regularly uses it to screen immature stages (e.g. eggs) collected from fruit to exclude the non-tephritid exotic pest Drosophila suzukii (A. Broadley pers. comm.).
DNA barcoding of an unknown insect specimen involves obtaining a DNA sequence of a specific region of the genome, and then comparing it with a database of sequences from positively identified reference specimens. On this site, we present PCR protocols and primer details for the standard COI barcode, along with POP4, RPA2, DDOSTs2, and EIF3L. See PCR protocols.
There are currently many reference DNA barcoding sequences available. On this site, verified reference data sets for COI, POP4, RPA2, DDOSTs2, and EIF3L are provided for download and use.
Additionally, the Barcode of Life Data Systems (BOLD) (accessed Oct 2017) holds 860 x Tephritidae, including 197 x Bactrocera ‘species with barcodes’.
This also includes data from NCBI GenBank and of regional studies covering a broad range of endemic or intercepted tephritid species e.g. 60 species from Oceania (Armstrong and Ball 2005), 153 species from Africa (Virgilio et al. 2012), 135 species from Europe (Smit et al. 2013).
There are a number of potential issues that should be taken into consideration when applying a DNA barcoding approach to fruit fly species identification:
- Many studies have not used the same gene region rendering the data unusable (Boykin et al. 2012)
- Some commonly applied combinations of ‘universal’ polymerase chain reaction (PCR) primers (e.g. LCO/HCO) may amplify a nuclear copy (a numt pseudogene) of the COI barcode region in some fruit flies (Blacket et al. 2012). The development of dacine-specific COI primers appears to overcome this issue (Krosch et al. unpublished).
- The presence of fruit fly species complexes can limit precise species identification (e.g. Blacket et al. 2012, Jiang et al. 2014), enabling identification only to the complex level (e.g. B. tryoni complex).
- The confidence of assigning an unknown sequence to a reference species can be problematic if all relevant potential taxa are not able to be included in the reference dataset (e.g. Virgilio et al. 2012). The degree of identification uncertainty is dependent upon a number of factors, including the accuracy of the taxonomic reconstruction (Lou and Goulding 2010), how recently the sister species diverged (e.g. van Velzen et al. 2012), and the geographic scale of reference samples (Bergsten et al. 2012).
Traditionally, DNA barcoding has been the most frequently utilised molecular tool to assist in species identification.
The DNA barcode refers to the mitochondrial gene for cytochrome oxidase subunit I (COI), however several new diagnostic loci have been explored and their ability to separate similar species pairs is outlined in Molecular diagnostics available to distinguish similar species.
Additionally, DNA barcoding data can be used for other applications in a biosecurity context; for example, tracing maternal lineages and dispersal (Blacket et al. 2017) and inferring possible source populations (Barr et al. 2014).
Download summary of Molecular diagnostics available to distinguish similar species