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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

2024 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
The purpose of this research project is to develop new and improved surveillance, detection, control, and management technologies for fruit flies and invasive pests of tropical and subtropical crops. This report summarizes the fourth year of project 2040-22430-028-000D, “Advancing Molecular Pest Management, Diagnostics, and Eradication of Fruit Flies and Invasive Species". Due to changes in research unit structure, Objectives 1 and 3 in the previous project (2040-22430-026-000-D) are now respectively Objectives 1 and 2 in the current plan. In fiscal year 2024, to support Sub-objective 1A, ARS researchers in Hilo, Hawaii, conducted three experiments to characterize fruit fly microbial communities. One study evaluated the developmental changes of the gut microbiota in following adult emergence in two fruit fly species (Mediterranean fruit fly and melon fly) from wild and reared populations and identified rapid repopulation of the gut microbiota following insect emergence. Another experiment evaluating the impact of host fruit on the female oriental fruit fly microbiome has also been published. A longitudinal study on evaluating irradiated mass reared fruit fly microbiomes has also been performed in flies reared by the California Department of Agriculture (CDFA), where fruit fly performance metrics were compared alongside gut microbiome surveys. Fly metrics were adversely affected by gamma irradiation sterilization of males compared to control males, but there were no adverse effects observed in the gut microbiome. These results have been disseminated to CDFA and APHIS cooperators. New microbial profiling techniques have been demonstrated using full length 16S sequencing using Pacific Biosciences Revio HiFi sequencing, which has dramatically increased the throughput and specificity of 16S profiling in the lab. Two additional conceptual manuscripts were published in support of this objective. For Sub-objective 1B, and to appraise Sterile Insect Technique (SIT) strains for efficacy and efficiency, ARS scientists completed high-throughput sequencing of wild-infused fly lines for Oriental fruit fly and Melon Fly. These datasets are currently being analyzed and being used to assess the ability to infuse lines and improve fitness of SIT reared genetic sexing strains for field suppression. Supporting Sub-objective 2A, ARS researchers continue to identify key attractant components among the 16-component blend that was similarly attractive to female oriental fruit flies compared to the original source material (guava juice). The researchers have been focusing on identifying key chemical attractants that can still maintain attractiveness at high release rates, which helps to make the lure better at attracting female oriental fruit flies in the field. Their data also suggests that the guava juice-based attractant blend is more effective at monitoring oviposition-ready female oriental fruit flies than the current standard female oriental fruit fly trap baited with torula yeast. Under Sub-objective 2B, ARS researchers continued to develop new tools for pathway analysis and bio-surveillance methods for invasive Bactrocera and other tropical pests. To accomplish this, they continued to 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 use of sequencing by binding technology on the Element Biosciences Aviti instrument substantially lowered the cost of data generation, while increasing the accuracy of the data almost an order of magnitude. 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). For Sub-objective 2C, a manuscript describing results of mark-release-recapture (MRR) experiments conducted with sterile oriental fruit fly in California and Florida by ARS scientists with USDA-APHIS and State Department of Agriculture colleagues was submitted, reviewed, and published. This has supported a rule change in Florida, where the number of spots for male annihilation technique will be decreased from 230 to 110 per km2. MRR experiments in Hawaii are nearing conclusion (data analysis) investigating the same question of application density for a Cuelure-responding fly, Zeugodacus cucurbitae. A study on tracking Queensland fruit fly in Australia using harmonic radar (HR) technology has been published, a first in the world for this species. ARS scientists in Hilo, Hawaii, have completed a small trial using HR to track individual melon flies in the field where male annihilation (Cuelure) has been applied at varying densities. Also, in support of Sub-objective 2C, two manuscripts on a concentric ring design (“core and perimeter”) approach to delimitation of an invading population of insects have been completed. The first reports on field experiments (MRR) conducted in Hawaii with Mediterranean fruit fly, the second is a theoretical examination of the “core and perimeter” approach. These data will allow parametrization of improved movement models for fruit flies. Simplified and improved modeling of insect traps has been completed and is in the process of being disseminated.

Accomplishments
1. Reduced application density for male annihilation technique (MAT). As a result of research conducted by ARS scientists in Hilo, Hawaii, the California Department of Food and Agriculture (CDFA) and the Florida Department of Agriculture and Consumer Services (FDACS) have followed USDA-Animal and Plant Health Inspection Service (APHIS) and revised their guidelines on application density of male annihilation technique (MAT) against oriental fruit fly to half the previous value. A reduction in application density will result in large cost savings for the material used, labor needed to apply over invasion areas, and environmental consequences of the insecticide used in conjunction with the attractant in MAT. For a high-detection year in California such as 2015, the change is estimated to result in $380,000 in direct savings.

2. A bioinformatic pipeline for assembling and analyzing insect genomes. The cost of sequencing and assembling insect genomes de novo has decreased significantly with the availability of new sequencing technologies. The USDA, ARS has made significant investments in the latest generation of sequencing technologies, and ARS researchers in Hilo, Hawaii, have developed a bioinformatic pipeline for producing contiguous, complete, and chromosome-scale genome assemblies to support USDA-ARS genome sequencing efforts and developed it within a portable container to allow easy implementation on any compute infrastructure. This published workflow has been applied to insects across the breadth of Arthropoda and provides a valuable resource for assembling genomes of non-model taxa. Applications of the resulting genome assemblies include gene annotation resources that can be applied to crop protection and pollinator health.

U.S. DEPARTMENT OF AGRICULTURE

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