Detection of environmental DNA of the Japanese orange fly, Bactrocera tsuneonis (Diptera: Tephritidae), from immature mandarin orange fruits

Early detection of insect pest outbreaks is critical for effective pest control. However, this is challenging, especially for pest species such as the Japanese orange fly, Bactrocera tsuneonis (Miyake) (Diptera: Tephritidae), whose larvae grow inside fruits. B. tsuneonis is a univoltine citrus pest, with females laying their eggs directly into the fruits during summer, and larvae develop inside the fruits until pupation in early winter. To monitor B. tsuneonis outbreaks, we developed and validated methods for the detection of environmental DNA (eDNA) in immature fruits collected during summer using quantitative real-time PCR. eDNA samples were prepared from individual fruits with oviposition pinholes left by gravid females during ovipositor probing and oviposition. By immersing the fruits in distilled water for 1 h to extract eDNA, B. tsuneonis eDNA was detected in 20% of the fruits. In addition, B. tsuneonis eDNA was detected in a fruit without oviposition pinholes, albeit at a low rate, suggesting that residual eDNA on fruit surfaces during mating and searching for oviposition sites can be detected. While there remains a possibility of cross-contamination between infected and uninfected fruits due to the lack of negative controls during eDNA extraction, we conducted preliminary field examination in several orchards where B. tsuneonis was either visually observed or not. B. tsuneonis eDNA was detected even at a location where adults were not observed visually, but were captured using plastic-tub traps, indicating a low population density. Thus, our method has practical applicability in various areas, regardless of B. tsuneonis population density.