A single multiplex PCR reaction for distinguishing strains of Queensland fruit fly Bactrocera tryoni (Diptera: Tephritidae) Yizhou Chen, 1 Bernard C Dominiak 2 * and Brendon A O’Rourke 1 1 Elizabeth Macarthur Agricultural Institute, New South Wales Department of Primary Industries, PMB 8, Camden, NSW 2570, Australia. 2 New South Wales Department of Primary Industries, Locked Bag 21, Orange, NSW 2800, Australia. Abstract The sterile insect technique (SIT) has been used to suppress or eradicate fruit flies. It is critical to be able to identify sterile and wild flies so that informed decisions can be made during eradication activities. The current dye marking approach can be flawed on a small number of occasions, and a genetic method is needed to test suspect misidentified samples. As a proof of concept, a single multiplex PCR with nine microsatellite markers was used to study the genetic structure of Queensland fruit flies Bactrocera tryoni (Froggatt) in 11 locations in southern New South Wales. Cluster analysis demonstrated that one cluster was exclusive to the sterile mass-reared flies. A second distinct cluster was exclusive for one site in a wetter cooler area. The other sites were admixture of two main clusters. These nine microsatellite markers could be used to distinguish laboratory-reared flies from field flies. The mass-reared flies would need to be reanalysed after each introduction of wildness. Key words fruit fly freedom, incursion, sterile insect technique, trade. INTRODUCTION The Queensland fruit fly (Qfly) Bactrocera tryoni is one of the most serious pests of Australian horticulture as it attacks a broad range of fruit crops and many vegetables (Drew et al. 1978; Hancock et al. 2000). Qfly was originally distributed in coastal Queensland and possibly northern New South Wales, but it is now widely established in Australia’s east coast and far inland (Meats 1981; Gilchrist et al. 2006). The past and present distribution of Qfly was reviewed by Dominiak and Daniels (2012). The total export value for Australia’s top 25 commodities that are fruit fly hosts has been estimated at $432 million. Additionally, more than $1 billion worth of products traded domestically are vulnerable to this pest. As a result, Qfly poses a major threat to national and interna- tional market access for horticultural commodities produced in eastern Australia. It was estimated in a national stocktake that $128.7 million would be spent on fruit fly surveillance and management activities in Australia from July 2003 to June 2008 (Plant Health Australia 2009). A Fruit Fly Exclusion Zone (FFEZ) was established in 1994 covering important horticultural production areas in south- eastern Australia, encompassing irrigated agricultural land in New South Wales, South Australia and Victoria. The FFEZ was managed by the Tri-State Fruit Fly Committee (Yonow & Sutherst 1998). The Risk Reduction Zone (RRZ) was a buffer zone about 80 km wide that surrounds the FFEZ. Qfly populations in the RRZ were managed and frequently eradicated, depending on the proximity and risk certain locations posed to the FFEZ. The FFEZ did experience incursions, and subsequently, tempo- rary establishment and some outbreaks of wild Qfly occurred in most years. Gilchrist et al. (2006) claimed that most Qfly incursions into the FFEZ originated from the RRZ. Following the declaration of an outbreak (Dominiak et al. 2011), a quarantine area of 15 km radius was imposed, and all produce within this 707 sq km had to be treated before it was traded with a fruit fly sensi- tive market (Dominiak 2012). Area freedom was reinstated after certain periods of nil detection after the last wild Qfly was trapped. The period varied depending on the export market. However, following the wettest 2 year period on record in 2010–2011 (Webb 2012), eradication became technically unfeasible and economically unsustainable, and the legislation underpinning the NSW portion of the FFEZ was withdrawn in July 2013 (Dominiak et al. 2015). However, prior to the closure of the FFEZ, and following the introduction and temporary establishment of Qfly in these other- wise pest-free production areas, incursions were eradicated using a range of management strategies. These techniques included bait sprays, cover sprays, ground sprays, male annihilation tech- nique, fruit destruction and sterile insect technique (SIT) (Dominiak & Ekman 2013). SIT is an effective, species-specific and environmentally friendly method for controlling pest popu- lations (Knipling 1955) and has been widely used for control of Mediterranean fruit fly Ceratitis capitata (Wiedemann; Diptera: Tephritidae) and other insect species such as Anastrepha ludens (Loew) (Lux et al. 2002; Barry et al. 2003; Toledo et al. 2004). Typically in the FFEZ, an eradication program consisted of a 12 week chemical program (minimum period) and sometimes followed by a release of sterile fruit flies, depending on the time of eradication and location. Qfly SIT programs usually released sterile flies for 10 weeks to eradicate the remnants of the wild fly population. These flies were dyed as it is vitally important for identification services to be able to accurately differentiate wild and sterile flies. The incorrect identification of an undyed sterile Qfly as a wild fly (false positive) could cause the extension of the suspension period of an outbreak in an otherwise fruit fly-free *bernie.dominiak@dpi.nsw.gov.au © 2016 Australian Entomological Society doi: 10.1111/aen.12190 Austral Entomology (2016) ••, ••–••