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Research Project: Advancing Molecular Pest Management, Diagnostics, and Eradication of Fruit Flies and Invasive Species

Location: Tropical Pest Genetics and Molecular Biology Research Unit

2025 Annual Report

Objectives
Objective 1: Increase the effectiveness of sterile insect techniques for pest management including the development of next generation methods to achieve sterility, advances in mass insect rearing, and new combinations of techniques for cost-effective suppression and eradication of tephritids. Sub-objective 1A: Improvement of tephritid strains by characterizing strain domestication and colony infusions by quantifying the genetic and phenotypic effects and changes in microbial communities. Sub-objective 1B: Appraisal of Sterile Insect Technique strains for efficacy and efficiency. Objective 2: Identify pathways and risk factors for invasive tropical pest introduction, improve pest surveillance and detection methods, and analyze pest population dynamics at multiple levels to increase the protection of agriculture in Hawaii and the U.S. mainland. Sub-objective 2A: Identify attractant for female oriental fruit fly using host fruit volatiles associated with oviposition. Sub-objective 2B: Develop tools for pathway analysis of invasive Bactrocera and other tropical pests to improve bio-surveillance methods. Sub-objective 2C: Evaluate improvements to Male Annihilation Technique under low prevalence scenarios via changes in application density and pattern.

Approach
Research Goal 1A: Quantify the effect of cycling rearing temperatures, colony infusion protocols, and domestication on fly quality as determined by previously established performance metrics (flight ability, locomotor activity, adult longevity, time to sexual maturity, and fecundity) and microbial community diversity. Research Goal 1B: Evaluate current methods and develop standardized protocols for appraising the efficacy of mass-reared sterile flies in suppressing wild populations that can be used as a standard to determine if a new strain is able to be adopted. Hypothesis 2A: Host fruit odor based female attractant attracts more oviposition-ready females than odor from torula yeast. Hypothesis 2B: Genome-wide population genomics across the geographic range of emerging Bactrocera species, along with other tropical pests, will allow development of SNP-based source estimation along with other tools that can be applied to detection surveys, and improve the understanding of pathways of these invasive pests and improved control. Hypothesis 2C: An application density of half of the standard for male annihilation technique (currently 600 spots per square mile) will be at least as effective at killing male B. dorsalis.

Progress Report
This research project focused on utilizing molecular, genetic, and suborganismal approaches to improve technologies to manage tropical fruit flies and other pest species, as well as applications of biocontrol and microbial tools for pest. This is the final report for the project 2040-22430-028-000D, “Advancing Molecular Pest Management, Diagnostics, and Eradication of Fruit Flies and Invasive Species”, which will be succeeded by a new project titled “Expanding Molecular Pest Management in Tropical Fruit Flies and Other Invasive Species”. In support of Sub-objective 1A, research concluded on the strain-specific genomic characterization of tephritid species maintained in colony across USDA. The genetic basis of the black pupae phenotype across Tephritidae was completed. Utilizing state-of-the-art long read sequencing, a genome assembly of a single male individual Ceratitis capitata of the Sterile Insect Technique (SIT) strain released by the California Department of Food and Agriculture (CDFA) has been completed as well as a genome assembly of the Zeugodacus cucurbitae translocated strain (T1). In both of these assemblies, the translocation causing sex sorting and the structure of the sex chromosomes was completed which are new to science. The Bactrocera tryoni Ourimbah white pupae genetic sexing strain created by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) was sequenced and assembled in a gapless assembly, the translocation was characterized. This represents the first CRISPR-based fully functional genetic sexing system. A male and female Bactrocera oleae were sequenced and assembled, the male assembly is gapless and the primary symbiont for the species that enables its utilization of unripe olives was assembled into one contig and two circular plasmids. The foundational knowledge on the genetic basis of pupal color as well as translocation breakpoints and structure of the sex chromosomes has been utilized broadly by researchers internationally, including in the development of novel genetic sexing systems in tephritid fruit flies and other Diptera using next generation genetic biocontrol methods. In parallel with the advances in the foundational genomics of the genetic sexing systems, work has been completed to characterize fruit fly microbiomes in the rearing environments associated with mass rearing for SIT. One study evaluated how age and inoculation procedures impact gut bacterial establishment in the Mediterranean fruit fly. This is especially pertinent, as the methods detailed in that study allow for manipulation of reared flies from multiple sources, including those mass-reared and irradiated by the CDFA. Across the duration of the project, ARS researchers have characterized the microbiome dynamics of laboratory- and mass-reared fruit flies. Their results indicate that the gut microbiome is dynamic, and multiple species and strains are present within these systems. However, the adult microbiome is prone to manipulation and replacement with strains from wild fruit flies. Compared to wild populations, the microbiomes of reared fruit flies are considerably different, although they undergo changes through the development of the fruit fly. These results have been disseminated to USDA-Animal and Plant Health Inspection Service (APHIS) stakeholders. Progress on Sub-objective 1B was impacted by travel restrictions related to the COVID-19 pandemic. Despite this, ARS scientists completed high-throughput sequencing of wild-infused fly lines for oriental fruit fly and melon Fly, which provide important information for developing new genetic sexing strains (GSS) for SIT. Additionally, a small cage study was completed and published in 2022 on mating competitiveness of an existing GSS with specific testing of varying overflooding ratios and the presence of mass-reared females. Economic analysis is ongoing with colleagues from USDA-APHIS. Under Sub-objective 2B, ARS researchers developed numerous resources and tools for pathway analysis and bio-surveillance methods for invasive Bactrocera and other tropical pests. To accomplish this, they completed population genomics work with Bactrocera frauenfeldi, A. fraterculus, B. albistrigata, and now Bactrocera tryoni from South Asia, Southeast Asia, Australia, and the Pacific Islands utilizing whole-genome sequencing and mapping to identify and employ single-nucleotide polymorphism (SNP) variants for establishment of population level markers. These variants were used collectively to create a phylogeny to determine their taxonomic relationships. The creation of this phylogeny was followed with an analysis of the whole SNP dataset to identify a subset that resulted in the same phylogenetic relationship as the whole SNP dataset. Applying to the frauenfeldi/albistrigata complex and combining with classical morphological-based taxonomy, the ARS researchers have support to synonymize this complex and develop strong population level markers for the now synonymized fraunfeldi complex (which would have been considered a distinct species before). In addition to Bactrocera species, extensive advances in structure and characterization of the Anastrepha fraterculus species complex in South and Central America have been made, facilitated by long read sequencing of members across the complex and populations. The outcomes of these projects have led to the current utilization of whole genome sequencing for the analysis of outbreak populations of B. dorsalis and A. ludens in cooperation with USDA-APHIS, as well as in support of resolution of the systematics and taxonomy in combination with morphological characters, which has allowed for the release of the Adult Bactrocera fruit fly ID key. In support of Sub-objective 2C, experiments to test the effectiveness of lower application densities of male annihilation technique (MAT) spots (lure + insecticide) in Southern California and Florida via mark-release-recapture (MRR) were fully successful. These inter-agency efforts led by ARS researchers from Hilo, Hawaii, with USDA-APHIS and Florida Department of Agriculture and CDFA colleagues supported a rule change in Florida, California, and in the USDA-APHIS guidelines, with the number of spots for MAT against B. dorsalis has been decreased from 230 to 110 per km2, leading to significant cost, material, and labor savings while simultaneously increasing the effectiveness of MAT. Additional MRR experiments on a species of fruit fly that responds to a different male lure, cuelure, were completed and indicate that reducing the application density below the manufacturer’s recommended level improves effectiveness. Follow-up experiments and computer modeling of trap application patterns were completed and published in a two-part paper; these results can be integrated with MAT in programs responding to incursions by invasive tropical tephritid fruit flies. Finally, fruit fly tracking via harmonic-radar tagged flies was conducted by ARS researchers in Hilo, Hawaii, in the field with and without MAT, with results indicating that saturation of attractant lowers performance of MAT at higher densities.

Accomplishments
1. If you’re thinkin’ about my pupae, it matters if it’s black or it’s white. Fruit flies in the family Tephritidae cause damages to specialty crops, threatening billions of dollars of production and market access of American produce. The Sterile Insect Technique (SIT) is a powerful tool for preventing the establishment of fruit flies in the mainland United States. Releasing sterile insects prevents reproduction in wild populations, and its efficacy is improved when only sterile males are released. This requires techniques for separating males from females prior to emergence as adult flies. To support the development of SIT strains for male-only releases, ARS researchers in Hilo, Hawaii, in collaboration with scientists across several international institutions identified the genetic basis of two different pupal color polymorphisms, black and white, and recreated the phenotypes using gene editing techniques in six distinct fruit fly species across the four most destructive fruit fly genera that threaten American agriculture. In one species, this mutation was used to create a functioning genetic sexing system using pupal color sex separation. Knowledge of the genetic basis and the ability to genetically manipulate traits that can be used to sort and separate male and female fruit flies will be valuable in creating new SIT strains to protect American specialty crops.

2. Release of comprehensive fruit fly ID tools for the highly important Bactrocera pest species group. Bactrocera is a genus of pest fruit flies from Asia that is one of the greatest threats for introduction into the mainland United States and could have major impacts on specialty crop markets. ARS researchers in Hilo, Hawaii, in cooperative research between USDA-ARS, University of Hawaii Manoa, and USDA-APHIS, have developed and curated robust collections of Bactrocera flies, representing one of the most complete collections worldwide. Through utilization of integrated taxonomy and genomics, the systematics has been defined and resolved. The outcome of this effort is the release of the Adult Bactrocera fruit fly ID tool as a lucid key (https://keys.lucidcentral.org/search/adult-bactrocera-fruit-fly-id/), which integrates high quality illustrations and morphological descriptions to allow for rapid morphological identification of adult flies following the resolved taxonomy. This tool is utilized by USDA identifiers as well as other stakeholder groups to allow for rapid and accurate identification of flies, greatly enhancing control efforts for this invasive species.

U.S. DEPARTMENT OF AGRICULTURE

Tropical Pest Genetics and Molecular Biology Research Unit: Hilo, HI