Universal Mitochondrial Multi-Locus Sequence Analysis (mtMLSA) to Characterise Populations of Unanticipated Plant Pest Biosecurity Detections

Simple Summary Agricultural and environmental sustainability requires effective biosecurity responses that prevent the establishment or spread of exotic insect pests. Understanding where new detections may have come from or if recurrent detections are connected contributes to this. Suitable population genetic markers use relatively rapidly evolving gene regions which render the PCR method species-specific at best. Because resource limitations mean these are pre-emptively developed for the highest risk species, populations of other exotic pests are unable to be characterised at the time. Here we have developed a generic method that is useful across species within the same taxonomic Order, including where there is little or no prior knowledge of their gene sequences. Markers are formed by concomitant sequencing of four gene regions. Sequence concatenation was shown to retrieve higher resolution signatures than standard DNA barcoding. The method is encouragingly universal, as illustrated across species in ten fly and 11 moth superfamilies. Although as-yet untested in a biosecurity situation, this relatively low-tech, off-the-shelf method makes a proactive contribution to the toolbox of quarantine agencies at the time of detection without the need for impromptu species-specific research and development. Biosecurity responses to post-border exotic pest detections are more effective with knowledge of where the species may have originated from or if recurrent detections are connected. Population genetic markers for this are typically species-specific and not available in advance for any but the highest risk species, leaving other less anticipated species difficult to assess at the time. Here, new degenerate PCR primer sets are designed for within the Lepidoptera and Diptera for the 3 ' COI, ND3, ND6, and 3 ' plus 5 ' 16S gene regions. These are shown to be universal at the ordinal level amongst species of 14 and 15 families across 10 and 11 dipteran and lepidopteran superfamilies, respectively. Sequencing the ND3 amplicons as an example of all the loci confirmed detection of population-level variation. This supported finding multiple population haplotypes from the publicly available sequences. Concatenation of the sequences also confirmed that higher population resolution is achieved than for the individual genes. Although as-yet untested in a biosecurity situation, this method is a relatively simple, off-the-shelf means to characterise populations. This makes a proactive contribution to the toolbox of quarantine agencies at the time of detection without the need for unprepared species-specific research and development.