Research Project: Detection, Control and Area-wide Management of Fruit Flies and Other Quarantine Pests of Tropical/Subtropical Crops

Location: Tropical Crop and Commodity Protection Research

2020 Annual Report

Objectives
The goals of our project plan are to conduct foundational research to create the knowledge base necessary to develop innovative control methods and IPM strategies, and to conduct applied research to produce information and products that improve pest control in tropical agriculture. The four major objectives of our program are: Objective 1: Model pest invasion pathways, and investigate the genomics/genetics, physiology/behavior, population dynamics, biology/ecology, and natural enemies of tropical and subtropical fruit flies and other invasive pests to develop technologies to control (contain, suppress, and eradicate) these pests in Hawaii and the Pacific, the U.S. mainland, and elsewhere. 1A: Build an analysis of emerging tephritid fruit fly genomes, including linkage mapping, uniform and consistent gene structural and functional annotation, and comparative genomic analysis. 1B: Conduct surveys on coffee berry borer (CBB) distribution and abundance on Hawaii Island to provide a baseline for a predictive model that integrates GIS, pest insect population dynamics, host plant phenology, weather data and grower practices to drive area-wide management of CBB on Hawaii Island. Objective 2: Determine physiological, genetic, and biological factors limiting the effectiveness of the sterile insect technique (SIT) and natural enemies in control and eradication of fruit flies and other tropical plant pests of quarantine significance. 2A: Improve the effectiveness of mass reared fruit flies for SIT by quantifying the impact of colony infusion on incorporating wild genetics into the SIT colony, and correlating fly performance with genomic markers. 2B: Investigate parasitoid-fruit fly host interactions from the molecular to the field level. Objective 3: To increase export of tropical fruits and vegetables, improve attractants and trapping systems for surveillance and detection, and develop lures, baits, and reduced risk pesticides for area-wide IPM of fruit flies and/or other tropical plant pests of quarantine significance. 3A: Evaluation of C. capitata, B.dorsalis and B. cucurbitae captures in traps baited with solid trimedlure (TML), methyl eugenol (ME) and raspberry ketone (RK) male lure and insecticide dispensers weathered in Hawaii and California. 3B: Evaluation of mixtures, weathering and chemical degradation of SPLAT-spinosad ME and cue-lure (C-L) for fruit fly control under Hawaii and California conditions. 3C: Evaluation of a new attractant system for detection, monitoring and control of the sweetpotato vine borer, a pest of quarantine significance in Hawaii. Objective 4: Provide baseline information for development of low prevalence and/or pest-free zones, for implementation in Hawaii and the U.S. mainland, to promote or allow unimpeded movement of fruit and vegetable exports. 4A: Create area of low pest prevalence (ALPP) as an independent measure of systems approaches for melon fly. 4B: Utilize models to evaluate the sensitivity of trapping grids for detection and control of insect pests such as tephritid fruit flies. 4C. Effectiveness of foliar and bait sprays against C. capitata, B.dorsalis, B. cucurbitae and B. latifrons.

Approach
Hypothesis 1A: Tephritid genomes have a core set of genes that are related to their proliferation as pests world-wide. If we have trouble generating crosses from a particular species or have issues generating genomic DNA, other species could be sequenced. Approach 1B: Collect baseline data on distribution and abundance of Coffee Berry Borer and associated environmental and climatic data. Then use GIS techniques to produce a region-wide assessment of infestation and economic impact. If this does not work, surveys can be replaced with grower-collected data. Approach 2A: Combine genetic, proteomic and phenotypic data into a synthetic analysis. If portions experiments fail or cannot be integrated, publish portions independently. Approach 2B: Examine tephritid host-parasitoid biology across levels of biological organization to allow integration of foundational knowledge benefiting classical biological control of tephritids. If international releases are impeded by regulatory issues, field work in Hawaii will be done. Hypothesis 3A: Solid male lure wafers with solid insecticidal tape are just as effective, but more convenient and safer to handle than current liquid lure-insecticide formulations used for fruit fly detection programs. If data are inconclusive, chemical analyses of weathered dispensers from trials will at least provide “use pattern” and formulation data for future trials. Hypothesis 3B: Generic combination SPLAT-MAT-spinosad-methyl eugenol/cue-lure mixture will perform as well as Min-U-Gel with naled and ME and C-L separately for fruit fly control/eradication. If the sprayable mixtures are too expensive, recommend addition of small amounts of cue-lure or raspberry ketone to STATIC-spinosad-ME as part of a tank mix. Hypothesis 3C: A binary male attractant system identified with sweetpotato vine borer populations in Vietnam will provide significantly greater male catch in Hawaii populations than male catch in traps baited with an initially identified single compound lure. In cases where low populations are encountered, trials will be shifted to other fields or other time of year where higher populations are present. Hypothesis 4A: Mass trapping using a plant-odor lure, male lure and protein baits can create areas of low pest prevalence (ALPP) in commercial crops and reduce the risk of a mating pair in a consignment when combined with a second measure such as a (less than 99.9986% effective) quarantine treatment. Alternatively, investigate the effects of other independent measures such as poor host status and quarantine treatments. Approach 4B: Develop a biologically-based mathematical model of tephritid traps in a landscape that allows formal quantification of trap network sensitivity. Hypothesis 4C: Current preharvest foliar insecticides being used in IPM systems against other fruit and vegetable pests (e.g., spotted wing drosophila and Asian citrus psyllid) in California and Florida are sufficient to meet quarantine requirements for fruit flies when introduced into Florida and California. Alternatives are to use current practices of either malathion protein bait or GF-120 sprays.

Progress Report
This is the final report for project 2040-22430-026-00-D, which has been replaced by our new project, 2040-22430-027-00D. In support of Sub-objective 1A, foundational genomic resources for tephritid fruit flies were developed, including submission of Tephritid genomes into National Center for Biotechnology Information (NCBI) and i5k databases, and further, five publications on improved assemblies, such as a draft assembly of the Mexican fruit fly (Anastrepha ludens) were written. Diagnostic tools for the melon fly and Mexican fruit fly have been developed to identify source/strain of detected flies. These tools are now being tested and implemented at the USDA, Animal and Plant Health Inspection Service/Plant Protection and Quarantine (USDA-APHIS PPQ) in addition to being applied to new samples as they are collected. Through this project, scientists in Hilo, Hawaii, have made substantial progress in the process of insect genome sequencing, including implementation of single insect assemblies using the PacBio Sequel system, and the methods developed are being applied across many pest species as a component of the Ag100Pest initiative at ARS. Successful examples of this are present in Tephritids, as well as in other pest systems, such as the Spotted-lanternfly assembly. A related project with the University of Hawaii includes a curated collection of Bactrocera specimens and DNA samples, with taxonomic and genomic information digitized. The collection now includes specimens from South Asia, Philippines and South East Asia, from which samples have been lacking. These samples have been used to develop new species level diagnostic methods, as well as source estimation methods across many species of tephritid flies in collaboration with USDA-APHIS Plant Protection Quarantine. For Sub-objective 1B, three years of detailed data collection on Coffee Berry Borer (CBB) and coffee agroecosystems (2016, 2017, and 2018 seasons) were successfully conducted including the Kona and Kau coffee growing regions on the Big Island, Oahu, and Puerto Rico. Comprehensive geo-referenced data collected using an electronic system include monitoring of CBB populations, plant phenology, management practices and weather conditions. On Hawaii island alone, the effort encompassed 1,406 individual site visits across 21 sites, with 6,512 monitoring traps serviced, 18,223 branches checked for infestation level and almost 43,000 berries were manually dissected. This dataset has been invaluable to research and management of CBB. Researchers in Hilo, Hawaii, led collection, analysis, and publication on CBB research since 2015, with key data on the distribution and abundance of this pest that include: information on coffee phenology, reservoirs of CBB in the field between seasons, CBB development, effects of unmanaged and feral trees, optimization of sampling, and remote sensing (and GIS geographic information systems) detection of coffee plants. An important product has been the development of an app to provide decision support to coffee growers based on these data, which includes a decision model that responds to data collected by the grower with the app. Data collection was optimized based on experience from the extensive 3-year dataset collected. In support of Sub-objective 2A, through utilization of genomic and proteomic approaches, novel interactions between parasitoid wasps, their hosts, and wasp associated viruses were discovered, including the rapid integration of a virus genome into its parasitoid host, a novel finding. With collaborators at California State University Monterey Bay, ARS scientists in Hilo, Hawaii, have effectuated shipments of the oriental fruit fly parasitoid Fopius arisanus to Senegal, West Africa (Crop Protection Directorate, Ministry of Agriculture). Several years of shipments supporting the efforts of the ministry were completed to establish this biological control agent in Senegal. A similar project in Brazil has been halted, because the regulatory bodies in that country have not given clearance for release of F. arisanus. In support of Sub-objective 2B, two genetic sexing line colony infusion studies, the melon fly T1 white pupae line, and the Vienna 8 medfly, have been completed to evaluate the impact of infusion towards improving fitness traits in the flies. Using a combination of functional genomics, genetics, and CRISPR/Cas9 technology, with collaborators the genetic basis of the white pupal trait in three species of Tephritids were characterized and these phenotypes were recreated in a wild type line. This leads the way for rapid development of Sterile Insect Technique strains in new species through the use of sex-specific pupal color sorting. A new method for quantifying individual fruit fly flight ability and overall condition (“Flight Burst Duration”, “FBD”) was developed, described, and validated. In order to further the goal of releasing the parasitoid Fopius arisanus (Sonan) in the field in Brazil, researchers in Hilo, Hawaii, collaborated with scientists at the Brazilian Agricultural Research Corporation (EMBRAPA) on specificity testing with native host flies and in competition with a key native parasitoid wasp. Progress on Sub-objective 3A included the binary male attractant system (with Type 1 and Type 2 components), which identified with sweet potato vine borer populations in Vietnam and has been shown to similarly provide significantly greater male catch in Hawaii populations compared to male catch using only the Type 1 compound. These outcomes have led to greater than 10-fold increase in male catch compared to prior lures. These lures are also productive over time, with little drop-off in male sweet potato vine borer catch over the course of a three-month weathering period. Overall, results to date document greatly enhanced trap catch effectiveness of a Type 1 + Type 2 lure combination, compared to catch baited with the Type 1 lure only, with good persistence of the effectiveness of the binary attractant system over time. Supporting Sub-objective 3B, for Integrated Pest Management (IPM) of tephritid fruit flies, field weathering trials of solid trimedlure (TML), methyl eugenol (ME), and raspberry keytone (RK) lure wafers with the insecticide dichlorovinyl dimethyl phosphate (DDVP) are complete. By deploying combination lure traps, money is saved in operational programs that were previously using single lure traps, making exclusion programs more efficient. Additionally, combination male annihilation techniques (SPLAT-MAT-spinosad-ME/C-L mixture [Specialized Pheromone and Lure Application Technology – Male Annihilation Technique – Spinosad – Methyl Eugenol/Cuelure]) were completed for similar situations where control or eradication of more than one invading Bactrocera fruit fly species was required. In support of Sub-objective 4A, results of experimental field studies (“mark release-recapture”) testing the effectiveness of mass trapping have been completed, together with field estimates of oriental fruit fly survival. ARS scientists in Hilo, Hawaii, have completed and published mark-release-recapture experiments with oriental fruit fly in Hawaii indicating that a lower application density of SPLAT-MAT is more effective than the currently used rate; these results hold promise for reducing product application during incursions in California and elsewhere. A model was developed and released to quantify trap network sensitivity to incursions of invasive pests. This was parameterized via extensive “Mark- Release-Recapture" (MRR) field experiments, as well as estimates of field survivorship for the oriental fruit fly. Field experiments included Mediterranean fruit fly, oriental fruit fly, melon fly (all in Hawaii), plus Queensland fruit fly (Australia). Detailed trap behavioral research with Mediterranean fruit fly was also completed and reported. The trap model (“TrapGrid”) was incorporated into the Agent-Based Simulation “MED-FOES” (“MEDiterranean Fruit fly Outbreak and Eradication Simulation) as of version 0.6. MED-FOES simulated outbreaks of the Mediterranean fruit fly in “free” areas and assists with setting biologically meaningful and effective quarantine lengths. The TrapGrid model has been adapted is by USDA-APHIS-PPQ-CPHST (Raleigh, NC), to develop new guidelines on delimitation trapping for tephritids and other invasive pest insects, and has most recently been used by this group to analyze delimitation approaches to a lepidopeteran (European grape vine moth), a coleopteran (Japanese beetle), and a mollusc (Giant African land snails). Research results supporting Sub-objective 4B on foliar chemicals currently being used to control Asian citrus psyllid (ACP) were completed for potential use in a systems approach for fruit flies during quarantines. Often, treatments that farmers are already using for ACP are likely effective on tephritids, but this project tested their efficacy directly. Studies in Hawaii included a comparison of the effects of 19 foliar pesticides to the current standards (Malathion protein bait or GF-120 Naturalyte Fruit Fly Bait), for the uninterrupted movement of commercial citrus or cherries in the event of future fruit fly quarantines in growing areas of California. Additionally, the impact of foliar sprays on beneficial hymenopterous insects was determined. Dimethoate, Malathion, Mustang, Assail, Warrior, Actara, and Lorsban consistently produced high fruit fly mortality compared to the standards. However, many of these foliar insecticides also produced high mortality on natural enemies compared to GF-120 Naturalyte Fruit Fly Bait. Our results support development of a systems approach for relief from fruit fly quarantines, allowing farmers already applying established treatments for these other pests to still be allowed to harvest and market their products; however, detrimental effects on natural enemies should be recognized.

Accomplishments
1. Trap model is key to biologically relevant and effective trap network design. Surveillance and detection trap network design is a difficult task for action agencies dealing with invasive insects, and decision on size, density, and duration are often based on qualitative expert assessments or experience with other species. TrapGrid, a model developed by researchers in Hilo, Hawaii, provides quantitative estimates of capture probability over time; when combined with field-estimated measurements of trap attraction, this tool provides actionable information on which to base trap network design. This model has been adapted by USDA-APHIS to develop new guidelines for delimitation trapping for a wide range of agricultural pests including flies, moths, beetles, and a snail. New trapping network designs are currently being evaluated by APHIS using this model that will increase detection probability while minimizing the required number of traps.

2. Coffee Berry Borer (CBB) management app developed. Using three years of monitoring experience in 21 sites on Hawaii island plus over a dozen more on Oahu and Puerto Rico, researchers in Hilo, Hawaii, developed a scientifically defensible minimal data collection set and implemented a mobile application (“App”) that allows growers to monitor their fields for CBB and provides them with guidance on its management. The app allows for 1) spatial CBB distribution in the field to guide sprays, 2) “no count” trap catch estimation, 3) beetle position assessment, 4) management logging, and 5) weather data analysis. Together, these are used to provide guidance to the grower. A technology transfer agreement is currently being developed with a private company that is producing weather sensor networks in Hawaii to allow further development and public availability of the app.

3. Established reporting tool and experimental colony of the Queensland Longhorned Beetle (QLB) using artificial diet. An experimental colony of Queensland Longhorned Beetle, a new invasive pest in Hawaii, was established at the USDA-ARS in Hilo, Hawaii, using wild-collected individuals from infested wood. The individuals are reared on an artificial beetle diet formulated for Asian Longhorned Beetles and optimized for QLB. This colony is currently being used to perform biological assessments such as susceptibility to biopesticides, fungal biocontrol agents, nematode biocontrol agents, and phenologic assessments. In addition, a QLB reporting tool was established online and distributed to residents of Hawaii Island. Nearly one hundred reports have been submitted by the public over the course of three months which includes two reports in Hilo which previously had no reported beetle activity.

4. Determined genetic basis of white pupae in four tephritid species. The gene causing white pupae in the Mediterranean fruit fly, the oriental fruit fly, the Queensland fruit fly, and the melon fly was identified and empirically validated using CRISPR-Cas9 mediated targeted mutagenesis. Using a combination of bioinformatic analysis (whole-genome and transcriptome) and selected introgressions between B. dorsalis and B. tryoni to introgress the white pupae phenotype from the B. dorsalis DTWP strain in a B. tryoni genetic background, the white pupae gene was identified to be a putative metabolite transport protein. Transcripts of this gene in individuals with the white pupae phenotype either produce a non-functional protein or contain a premature stop codon in the exonic region which results in a truncated protein. The simplicity of this mutation and ease in recreating with CRISPR technology opens the door for straightforward genetic sexing technologies across pest Tephritid species.

Review Publications
Szyniszewska, A., Collier, T.C., Manoukis, N., Hastings, J.M., Bigsby, K., Kriticos, D., Leppla, N.C. 2020. CLIMEX and MEDFOES models for predicting the variability in growth potential and persistence of the Mediterranean fruit fly (Diptera: Tephritidae) populations. Annals of the Entomological Society of America. 113(2):114-124. https://doi.org/10.1093/aesa/saz065.
Kingan, S.B., Urban, J., Lambert, C.C., Baybayan, P., Childers, A.K., Coates, B.S., Scheffler, B.E., Hackett, K.J., Korlack, J., Geib, S.M. 2019. A high-quality genome assembly from a single, field-collected Spotted Lanternfly (Lycorma delicatula) using the PacBio Sequel II System. Gigascience. 8(10):1-10. https://doi.org/10.1093/gigascience/giz122.
Hamilton, L.J., Hollingsworth, R.G., Sabado-Halpern, M., Manoukis, N., Follett, P.A., Johnson, M. 2019. Coffee berry borer (Hypothenemus hampei) (Coleoptera: Curculionidae) development across an elevational gradient on Hawai‘i Island: applying laboratory degree-day predictions to natural field populations. PLoS One. 14(7). https://doi.org/10.1371/journal.pone.0218321.
Koch, J.B., Dupuis, J.R., Jardeleza, M., Ouedraogo, N., Geib, S.M., Follett, P.A., Price, D.K. 2020. Evidence for evolutionary change in invasive populations of Drosophila suzukii in Hawai‘i. Biological Invasions. 22:1753-1770. https://doi.org/10.1007/s10530-020-02217-5.
Garzon, I., Geib, S.M., Bremer, F., Barr, N. 2020. Implementing low-cost, high accuracy DNA barcoding from single molecule sequencing to screen larval tephritid fruit fly intercepted at ports of entry. Annals of the Entomological Society of America. 113(4):288-297. https://doi.org/10.1093/aesa/saz071.
Rubinoff, D., Reil, J., Osborne, K., Gregory, C., Geib, S.M., Dupuis, J. 2020. Phylogenomics of an endangered beetle reveals challenges and opportunities for broader conservation planning in a rapidly disappearing desert ecosystem. Biodiversity and Conservation Journal. 29:2185-2200. https://doi.org/10.1007/s10531-020-01968-w.
Hood, G.R., Powell, T.H., Doellmam, M.M., Sim, S.B., Glover, M., Yee, W.L., Goughnour, R.B., Mattsson, M., Schwarz, D., Feder, J.L. 2020. Rapid and repeatable host plant shifts drive reproductive isolation following a human-mediated introduction. Journal of Molecular Evolution. 74(1):156-168. https://doi.org/10.1111/evo.13882.
Bellinger, M.R., Paudel, R., Starnes, S., Kambic, L., Kantar, M., Wolfgruber, T., Lamour, K., Geib, S.M., Sim, S.B., Miyasaka, S., Helmkampf, M., Shintaku, M. 2020. Taro genome assembly and linkage map reveal QTLs for resistance to Taro Leaf Blight. G3, Genes/Genomes/Genetics. 10(8):2763-2775. https://doi.org/10.1534/g3.120.401367.
Johnson, M.A., Fortna, S., Manoukis, N. 2020. Evaluation of exclusion netting for Coffee Berry Borer (Hypothenemus hampei) management. Crop Protection Journal. 11(6). https://doi.org/10.3390/insects11060364.
Helmkampf, M., Bellinger, R., Geib, S.M., Sim, S.B., Takabayashi, M. 2019. Draft genome of the rice coral Montipora capitata obtained from linked-read sequencing. Genome Biology and Evolution. 11(7):2045-2054. https://doi.org/10.1093/gbe/evz135.
Johnson, M.A., Fortna, S., Hollingsworth, R., Manoukis, N. 2019. Postharvest population reservoirs of Coffee Berry Borer (Coleoptera: Curculionidae) on Hawai‘i Island. Journal of Economic Entomology. 112(6):2833-2841. https://doi.org/10.1093/jee/toz219.