Project : USDA ARS

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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

2023 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 third year of project number 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 project number 2040-22430-026-000D are now respectively Objectives 1 and 2 in this newly established project plan. In fiscal year 2023 (FY23), evaluation of microbial diversity and contributions of the gut microbiome to fly colony production continued in support of Sub-objective 1A. To build upon work from the previous fiscal year, bacterial and yeast isolates have been collected from healthy and diseased fruit flies and are currently being identified by 16S-rRNA gene sequencing. Evaluation of bacterial establishment at different timepoints has been assessed, and reintroduction of cultured isolates is achievable at early periods after fly emergence. Initial experiments evaluating the impact of diet and microbiota on fly performance have begun. Studies to compare how different rearing environments impact fly microbiomes are ongoing. Additionally, population genetic evaluation of genetic sexing strain (GSS) and wild type melon fly in colony have been initiated also to support Sub-objective 1A. This was accomplished using whole-genome sequencing to identify loci under selection as a result of domestication. Results show large regions of high-differentiation as result of the initial selection event and the maintenance in colony. To appraise Sterile Insect Technique (SIT) strains for efficacy and efficiency, scientists in Hilo, Hawaii, have implemented mesocosm-scale stable populations of tephritid fruit flies in large (21.6 m3) outdoor field enclosures in support of Sub-objective 1B. These small populations are ideal for testing suppression of actual fruit fly populations via releases of sterile males produced by irradiation or via GSS lines. The system is currently being used to test suppression of oriental fruit fly populations via standard irradiation-based SIT. To facilitate the identification of a female Oriental fruit fly (OFF) attractant using host fruit volatiles associated with oviposition in support of Sub-objective 2A, fruit juices were tested on mated OFF females for attraction using laboratory bioassays. Of the fruit-based attractants tested, guava juice, mango, and Surinam cherry were identified as the most attractive to mated OFF females. A combination of gas chromatography-electroantennographic detection (GC-EAD) and gas chromatography-mass-spectrometry (GC-MS) revealed 16, 27, and 11 volatile chemicals that are EAD-active (i.e. detected by antennae) from guava juice, mango, and Suriman cherry, respectively. Three different blends (16-, 27-, and 11-component blends) were composed based on the ratio of those volatiles released from different fruit sources. All three blends were tested in bioassays and were demonstrated to be more attractive than the control. The most attractive of these blends, the 16-component blend, was tested in the field, and was similarly attractive to female OFF flies as the original material (guava juice). The development of tools for pathway analysis of invasive Bactrocera and other tropical pests to improve bio-surveillance methods was pursued in support of Sub-objective 2B. To accomplish this, the species Bactrocera frauenfeldi, B. fraterculus, B. albistrigata, B. kirki, and B. psidii from South Asia, Southeast Asia and the Pacific Islands were sequenced for whole-genome sequencing and mapped to the B. dorsalis genome to identify single-nucleotide polymorphism (SNP) variants. 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. This resulted in a significantly minimized computation time while yielding an accurate taxonomic assessment which can also be used to identify novel species. These methods will be applied to other fruit fly species complexes that are of economic importance. To evaluate improvements to the Male Annihilation Technique (MAT) in support of Sub-objective 2C, mark-release-recapture experiments with sterile OFF were completed in Florida this year by ARS scientists from Hilo, Hawaii, and other Federal and State partners. In combination with previous results from California and Hawaii, this information was used by a USDA-APHIS expert panel to reduce the recommended application density of (MAT) against oriental fruit fly by one half. Related work on parametrizing a movement model has seen a breakthrough: ARS researchers in Hilo, Hawaii, plus collaborators have been able to track individual tagged fruit flies in an open field using harmonic radar technology. This has enabled the partial parametrization of random correlated walk models, an improvement over a simple diffusion-based approach. Testing of different trapping patterns has been conducted via simulation by ARS scientists in Hilo, Hawaii, in collaboration with USDA-APHIS and North Carolina State University researchers, with results published in a peer reviewed journal.

Accomplishments
1. Reduced application density for male annihilation technique (MAT) against oriental fruit fly. As a result of ARS research conducted by scientists in Hilo, Hawaii, USDA-APHIS has revised their guidelines on application density of MAT against oriental fruit fly to half the previous value as of January 2023, as have agencies overseas (e.g., an Australian areawide project in Malaysia). 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. Software tool for processing accurate genome assembly. Genome sequencing and assembly represents a large component of research programs in the Life Sciences. From disease research, agriculture research, and to conservation, genome assembly is the foundation of both exploratory and hypothesis-based studies, and highly accurate genome assemblies are necessary to do downstream analyses. ARS researchers at Hilo, Hawai'i, have developed a useful and widely adopted software program (HiFiAdapterFilt) that facilitates genome assembly by removing erroneous sequencing artifacts to prevent their integration into genome assemblies. HiFiAdapterFilt has been used and cited in high-impact studies such as the Human Pan-genome and provides a valuable tool that facilitates accurate genome assembly.

U.S. DEPARTMENT OF AGRICULTURE

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