Population Genomics, Diagnostic Strain Determination, and SIT Characterization for the Mexican Fruit Fly Anastrepha ludens

Title Population genomics, diagnostic strain determination, and SIT characterization for the Mexican fruit fly Anastrepha ludens Abstract The following is a proposal for the Plant Protection Act 2019. The primary purpose of this agreement is to develop and improve molecular identification and diagnostic capacity for the Mexican fruit fly, Anastrepha ludens. Anastrepha ludens is a destructive agricultural pest across its range of Central America and Mexico. Although recorded from >30 species of host fruit, it poses a significant threat to U.S. agriculture due to proximity to citrus production in south Texas and California. Control of A. ludens in these regions relies on extensive surveillance and area- wide management, including use of Sterile Insect Technique (SIT). When A. ludens is intercepted via routine trapping networks in the U.S., two main regulatory determinations are of utmost importance: 1) delimiting wild, fertile flies from more numerous SIT flies; and 2) predicting the geographic origin of wild flies. Molecular diagnostic tools would greatly increase our capacity to make these determinations, and this agreement will create such diagnostic tools. Specifically, we will develop sets of diagnostic markers for three purposes: 1) SIT strain determination for A. ludens rearing lines used in the U.S. and abroad; 2) genetic characterization of mass-reared SIT strains (assessing inbreeding, genetic diversity, heterozygosity, etc.), and 3) improved geographic source determination for trapped wild flies, focusing on northern and eastern Mexico adjacent to the U.S. border. To accomplish these goals, we will make use of existing specimen collections and use double-digest Restriction-site Associated DNA sequencing (ddRAD) to assemble large genome-wide population genetic datasets. From these population genomic resources, we can then identify highly informative sets of markers for particular diagnostic tasks. By utilizing existing collections and established molecular protocols in which we are experts, we will be able to avoid the main anticipated obstacles facing these types of projects (sampling and marker validation). Our research team has extensive expertise in tephritid population genetics and developing molecular diagnostic tools (e.g. Dupuis et al. 2018 Biol Inv 20:1211-1228), which includes the first range-wide population genomic assessment of A. ludens (Dupuis et al. 2019 Evol App 12:1641-1660). With extensive collaborations across the tephritid research community, this project has a high likelihood of success. Specific objectives: The goal of this agreement is to develop and improve molecular identification and diagnostic capacity for Anastrepha ludens. We have three specific objectives, all of which will use large population genomic datasets (thousands of genetic markers for hundreds to thousands of individuals) as their foundation. Our objectives are: 1) Develop and demonstrate a set of molecular diagnostic markers for identifying and delimiting the SIT rearing line strains used in the U.S. and abroad. 2) Develop and demonstrate a set of molecular diagnostic markers for genetic characterization of mass-reared SIT strains. These markers will be used to quantitatively measure population genetic statistics such as inbreeding, genetic diversity, and heterozygosity. 3) Improve geographic source determination for trapped wild flies by sequencing additional individuals from northern and eastern Mexico adjacent to the U.S. border. Keywords: population genomics, Diptera, Tephritidae, Anastrepha, recurrently invading pests, molecular diagnostics, pathway analysis