DNA Barcodes for thrips species and development of multiplex real-time PCR assay for Frankliniella occidentalis Pergande, Frankliniella panamensis Hood, Thrips palmi Karny and Thrips tabaci Lindeman (Thysanoptera: Thripidae)

ABSTRACTThrips (Order Thysanoptera) species are agriculturally important pests and vectors of plant disease. These tiny insects are commonly found in all life stages on imported commodities at the New Zealand border. Morphological identification of thrips is able to be performed on adults, but the identification keys for immature stages are inadequate and so DNA barcoding is regularly used for their identification. Here, we have generated cytochrome oxidase I (COI) DNA barcode data for 29 thrips species from 124 individuals, focusing on Frankliniella occidentalis, a dominant species intercepted at New Zealand border, followed by F. panamensis, Thrips palmi and T. tabaci. In addition, a multiplex real-time PCR assay has been developed to target the four thrips species. This assay is intended to facilitate the identification of quarantine interceptions with greater accuracy and faster diagnostic turnaround times, regardless of the developmental stages of the thrips. The developed assay demonstrated a high level of specificity for all the four target species and could detect as few as 10 copies/µL of the target DNA. Linear responses and high correlation coefficients between the amount of DNA and Cq values for each species were also achieved. The method was successfully tested on single egg, larva and adult samples and proved to be applicable for all life stages of the four species. Overall, this study has shown that the developed assay is an effective biosecurity tool for rapid and reliable identification of the target thrips species.KEYWORDS: Thripidaebiosecurityquarantine pestsborder detection AcknowledgementsThis work was part of the project (11 Int 03) funded by Operational Research programme from the Ministry for Primary Industries (MPI), New Zealand. We would like to thank all the Entomology staff at Plant Health and Environment Laboratory (PHEL), MPI for identifying the species, keeping the specimens, and performing the DNA extraction for this research. Our thanks go to Dr David Waite (PHEL, MPI) for conducting the internal review of the manuscript and his valuable suggestions. Great thanks also go to the editor and two anonymous reviewers for their constructive comments and edits to the manuscript, which has improved it significantly.Disclosure statementNo potential conflict of interest was reported by the author(s).Author contributionLK, DG, DL and AS conceived research.LK and DG secured funding.AS and DL designed the assay.AS, AT, YC and DL conducted all the experiments, including optimisation, specificity and sensitivity and blind panel testing of the assay.DG acquired thrips specimens and conducted morphological identification of the specimens.DL analysed the data and generated the figures.DL and AS wrote the draft manuscript. All authors read, edited and approved the manuscript.Data accessibility statementThe data used for this study were submitted into Zenodo, see xxx10.5281/zenodo.5748573