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 Tropical Crop and Commodity Protection Research: Hilo, HI
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                        Acebes-Doria, Angelita
                                    Cha, Dong
                                    Follett, Peter
                                    Johnson, Melissa
                                    Liang, Peishih
                                    Manoukis, Nicholas
                                    Sun, Xiuxiu
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ARS Home Pacific West Area Hilo, Hawaii Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center Tropical Crop and Commodity Protection Research Research





- Manoukis, Nicholas
- Johnson, Melissa
- Cha, Dong
- 2023
- 2022
- 2021
- Publications
- Crop Protection & Quarantine (304)

Research Project: Development of New and Improved Surveillance, Detection, Control, and Management Technologies for Fruit Flies and Invasive Pests of Tropical and Subtropical Crops **Location:
                            Tropical Crop and Commodity Protection Research** 2021 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: Enhance or develop new technologies for the biological control of tephritids and other tropical pests by developing new methods for testing for host specificity, improved mass rearing techniques, enhanced understanding of the fundamental biology of parasitism and insect pathology, and the integration of biological control agent ecology into management techniques. 
Sub-objective 2A: Investigate cues driving host specificity in braconid parasitoids of fruit flies in order to improve the safety and acceptability of biological control programs using these wasps.
Sub-objective 2B: Explore the genomic basis for host preference and the role of associated viruses in host suitability of tephritid parasitoids.
Objective 3: 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 3A: Identify attractant for female oriental fruit fly using host fruit volatiles associated with oviposition.
Sub-objective 3B: Develop tools for pathway analysis of invasive Bactrocera and other tropical pests to improve bio-surveillance methods.
Sub-objective 3C: Evaluate improvements to Male Annihilation Technique under low prevalence scenarios via changes in application density and pattern.
Objective 4: Develop new methods for invasive pest control including reduced-risk insecticides, new practices for insecticide resistance management, and new components and programs for IPM for tephritids and other tropical plant pests of quarantine significance for Hawaii and the U.S. mainland to promote the unimpeded movement of fruit and vegetable exports. 
Sub-objective 4A: Investigate the molecular, physiological, or behavioral basis of evolving resistance to chemical and biological control of tephritids and other tropical pests.
Sub-objective 4B: Validate the effectiveness of coffee berry borer pest control techniques in the context of a comprehensive IPM system to enable economically viable control. 
Sub-objective 4C: Develop baseline biological assessments, survey, monitoring, and control tools based on behavioral interventions and other methods for established and emerging insect pests of tropical agriculture (e.g. the Queensland longhorn beetle, Acalolepta aesthetica and the little fire ant, Wasmannia auropunctata). **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: Visual cues, particularly color and shape, are drivers of host specificity in parasitoids used in classical and augmentative biological control programs against tephritid pests (Psyttalia, Fopius, and Dichasmomorpha). 
Hypothesis 2B: Across braconid parasitoid species which parasitize tephritids, novel mechanisms for overcoming hosts defenses have developed, which play a role in a species host specificity and host range.
Hypothesis 3A: Host fruit odor based female attractant attracts more oviposition-ready females than odor from torula yeast.
Hypothesis 3B: 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 3C: 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. 
Research Goal 4A: Determine the extent to which wild melon fly have become resistant to insecticides and devise strategies for insecticide rotation and resistance monitoring. Prescribe a standardized test for resistance for use by collaborators at other research centers in geographic locations where flies are established.
Research Goal 4B: To determine the optimal combination of control measures for CBB management in Hawaii, add new techniques, and deliver a “smart agriculture” app.
Research Goal 4C: Develop trapping systems, behavior modification systems, and genetic assays for emerging and established invasive species that attack tropical crops and commodities in the Pacific basin. **Progress Report**
                                

This research project focuses on foundational and applied research towards improving control and detection of fruit fly pests and other tropical pest species. This report covers the first year of the new project 2040-22430-027-00D, “Development of New and Improved Surveillance, Detection, Control, and Management Technologies for Fruit Flies and Invasive Pests of Tropical and Sub-tropical Crops”, which replaces the former project 2040-22430-026-00D, “Detection, Control and Area-wide Management of Fruit Flies and Other Quarantine Pests of Tropical/Subtropical Crops”.  Overall, this project supports current fruit fly eradication and exclusion programs throughout the United States, developing improvements to treatments, lures, trapping, and detection.

For Objective 1, we attempted to begin colony infusion experiments, which rely on sourcing wild flies from various locations across the state of Hawaii, including field sites on Hawaii Island as well as Kauai coffee fields. Due to COVID-19, travel to collect wild flies was significantly hampered, and despite attempts to collect flies locally near the lab as allowed by COVID restrictions, sufficient numbers of flies could not be collected to perform the replicated experiments as described in the project plan. Non-replicated infusions were performed for melon fly and oriental fruit fly for use in experiments requiring recently infused flies, and quality control data on these flies were collected, but ideally, it would be best to target replicated infusions as described in the project plan in year two as field work and travel is allowed. Also, due to the need to reduce rearing operations to minimal levels, a detailed assessment of quality metrics of genetic sexing lines was not performed. However, experiments were planned, and new materials gathered, such as flight mills and recording equipment. As well, methods for establishing stable populations of fruit flies in large outdoor field enclosures have been developed and wo;; be tested in the second year of the project plan. 
 
For biocontrol experiments in Objective 2, vision cue tests have focused on the ovo-parasitoid Fopius arisanus this year. The method had to be modified from the one used with larval parasitoid, but data have now been collected on about 50% of the required trials for this species, meeting the goal for the first year of the project plan. Additionally, in olive fruit fly, populations at the Lalamilo test site near Waimea are being monitored to determine if this species is parasitized in Hawaii, and if so, if it is from already established natural enemies, or if new parasitoids were introduced during the introduction of the olive fly. For Sub-objective 2B, all species of parasitoids have been collected through our own rearing or from collaborators and these have  been cryogenically stored for analysis. For F. arisanus, F. ceratitivorous, Psyttalia fletcherii, and Dichasmomorpha longicaudata, library preparation and sequencing has been completed and assemblies are being finalized to complete reference genomes for these samples. Additionally, in D. longicaudata, tissue specific collections have been performed and RNA is being prepared for sequencing to investigate multitrophic interactions between host fly species, wasp, and wasp associated virus. 
 
In support of Sub-objective 3A, fruits that were more attractive to mated female oriental fruit flies (OFF) than torula yeast bait were selected as original materials to develop synthetic chemical lure targeting mated female OFF. Currently, traps baited with torula yeast are being used to monitor invading OFF females. However, torula yeast baited traps are minimally effective, short-lived, and not user friendly. In two-choice laboratory bioassays, traps baited with 5g of a host fruit (guava, guava juice, mango, Surinam cherry, orange, or white sapote) captured significantly greater numbers of mated female OFF (13-15 day old) than traps baited with 5g of torula yeast. In subsequent multiple-choice tests comparing the attractiveness of torula yeast and the six host fruits, traps baited with guava juice, mango and Surinam cherry captured greatest numbers of mated female flies. Genome wide population genetic analysis in B. correcta and B. zonata has been initiated through generation of genome-wide single nucleotide polymorphism (SNP) datasets from sample locations in South and Southeast Asia from collections maintained at the University of Hawaii Insect Museum. Analyses of this data was completed using reference-free methods. High quality reference genomes are still ongoing for those two species, but specimens have been identified. For other pests, such as the coconut rhinoceros beetle, fruit piercing moth, oriental fruit fly, and Mexican fruit fly reference genomes have been completed in the first year of this project following the methods outlined in Sub-objective 2B. For Sub-objective 3C, the first round of mark-release-recapture experiments were not performed, but methods to effectively sterilize B. dorsalis using new RadSource X-Ray irradiation equipment was developed and tested in Hilo. Additionally, ongoing communication with the California Department of Agriculture on the mechanism and methods to allow release of flies in Southern California has been ongoing and approval to release flies was granted pending travel approvals and timing of releases. 

In support of Sub-objective 4A, Spinosad resistant lines from three source populations of melon fly, Zeugodacus cucurbitae, were established in Hilo, Hawaii, and further selected for increased resistance. Together with susceptible colony flies, these lines are now available to provide Spinosad susceptible and resistant melon flies (respectively), to determine the genetic basis of insecticide resistance. Following one additional generation and an insecticide treatment of susceptible and resistant flies, genetic analysis will be conducted to identify the genes and gene products associated with resistance. Cooperations have been developed to survey for Spinosad resistance on Hawaii, Maui, and Oahu for both B. dorsalis and Z. cucurbitae starting in July of 2021. To help develop plans for managing resistance, alternative management approaches are being surveyed, including testing new insecticide products in collaboration with industry, and we have been working on evaluating colored kaolin products for use in the field to deter oviposition as part of an effort to develop a comprehensive Integrated Pest Management (IPM) program for tephritid management. 

In support of Sub-objective 4B, research was initiated to investigate the flight behavior of coffee berry borer (CBB) across three sites on Hawaii Island. These sites represent three different growing regions on Hawaii Island (Kona, Ka’u and Hilo) at low and mid-elevations. This study will continue for two years and the results will be used to optimize the timing and height of Beauveria bassiana sprays. Information on CBB flight behavior will also be used to design studies for physical exclusion of CBB using border crops. Research was also initiated in 2021 to quantify minimum levels of post-harvest ground and tree sanitation needed for successful CBB management. The timing of CBB emergence and infestation of the new season’s crop is currently being followed until harvest. “Best Beans”, the CBB control decision support and coffee quality application (app), is in the testing phase with growers on Hawaii island. Multiple features are available in the current version of the app, including a “no-count” trap estimation method, two methods for monitoring infestation in trees, a heatmap showing localized infestations in a field, field reports with infestation percentage, graphs showing infestation and flight activity over time, and management recommendations based on data submitted by the user. With the October 2020 detection of Coffee Leaf Rust (CLR, Hemileiavastatrix) in Hawaii, we have also added a feature that will allow growers to monitor CLR in their fields. 

In support of Sub-objective 4C, research was initiated in December 2020 to develop survey, monitoring and control methods for Coffee Leaf Rust (CLR). Monthly surveys of CLR incidence are being conducted at 25 commercial coffee farms in the Kona coffee-growing district of Hawaii Island, where the disease has been centralized since its detection. The combination of data on CLR incidence and severity, weather, spore movement, plant phenology, and agronomic practices will be used to develop predictive disease models and IPM guidelines that can be used to manage CLR in Hawaii. Also, in support of Sub-objective 4C, properties in East Hawaii Island infested with Queensland longhorned beetles, Acaloleptaaesthetica, were identified and a trapping study was deployed to assess the effectiveness of commercial longhorned beetle lures. The results show that commercial lures are not effective and species-specific work is necessary to develop an early detection and rapid response strategy. Additionally, a genetic sequencing diagnostic assay was developed to differentiate species’ beetle larval samples on Hawaii Island. This assay was then used to develop a molecular presence/absence assay to determine the presence or absence of A. aesthetica from insect frass. **Accomplishments**
                                

1. Bioinformatic software for identifying and removing adapter sequences from PacBio circular consensus sequences. Pacific Biosciences High-Fidelity (HiFi) circular consensus sequencing (CCS) is currently the standard for whole-genome sequencing. Unfortunately, the current circular consensus basecaller that is part of the sequencing pipeline fails to remove a small portion of adapter sequences from the (CCS) reads. ARS researchers in Hilo, Hawaii, wrote bioinformatic software called HiFiAdapterFilt to identify adapter sequences from CCS reads and remove them from the CCS read pool to produce assembly-ready reads. This bioinformatic tool has been adopted by large genome sequencing consortiums such as the Darwin Tree of Life Project and Vertebrate Genome Project to prepare their CCS reads for assembly. This tool has greatly benefitted the genome sequencing community with as many as many as 20 downloads per day when it was first released. 2. Developed a genetic assay for detecting an invasive beetle from wood frass. With obscure species such as wood boring or longhorned beetles which live most of their immature lives in wood, it is difficult to identify larvae to species due to inadequate larval identification keys. Because they are obscured from visual inspection, beetle larvae can quickly invade a new environment and thus it is necessary to have methods to identify beetle species at the larval stage or from non-destructive sampling methods. The Queensland longhorned beetle is an invasive species on Hawaii island with a broad host range. To rapidly identify host species in the absence of larval tissue, ARS scientists in Hilo, Hawaii, developed a genetic assay to detect the presence of beetle DNA from frass material. This assay is currently being used to identify new hosts and the geographic range of this species in the Hawaiian Islands. **Review Publications**
                            *Manoukis, N. 2021. Drivers of mosquito mating. Science. 371(6527):340-341. https://doi.org/10.1126/science.abf7917.*
                                *Knope, M.L., Funk, V.A., Johnson, M.A., Wagner, W.L., Datlof, E.M., Johnson, G., Crawford, D., Bonafacino, J.M., Morden, C.W., Lorence, D.H., Wood, K.R., Meyer, J., Carlquist, S. 2020. Dispersal and adaptive radiation of Bidens (Compositae) across the remote archipelagoes of Polynesia. Journal of Systematics and Evolution. 58(6):805-822. https://doi.org/10.1111/jse.12704.*
                                *Johnson, M., Manoukis, N. 2020. Abundance of Coffee Berry Borer in feral, abandoned, and managed coffee on Hawaii Island. Journal of Applied Entomology. 144(10):920-928. https://doi.org/10.1111/jen.12804.*
                                *Manoukis, N., Carvalho, L.A. 2020. Flight burst duration as an indicator of flight ability and physical fitness in two species of Tephritid fruit flies. Journal of Insect Science. 20(5). Article 11. https://doi.org/10.1093/jisesa/ieaa095.*
                                *Paranhos, B.A., Poncio, S., Morelli, R., Nava, D.E., Nogueria De Sa, L., Manoukis, N. 2020. Non-target effects of the exotic generalist parasitoid wasp Fopius arisanus (Sonan) estimated via competition assays against Doryctobracon areolatus (Szepligeti) on both native and exotic fruit fly hosts. Biocontrol. 66:83-96. https://doi.org/10.1007/s10526-020-10057-4.*
                                *Johnson, M., Ruiz-Diaz, C.P., Manoukis, N., Rodrigues, J.C. 2020. Coffee berry borer (Hypothenemus hampei), a global pest of coffee: perspectives from historical and recent invasions, and future priorities. Insects. 11(12). Article 882. https://doi.org/10.3390/insects11120882.*
                                *Feugere, L., Gibson, G., Manoukis, N., Roux, O. 2021. Mosquito sound communication: are male swarms loud enough to attract females?. Journal of the Royal Society Interface. 18(177). Article 20210121. https://doi.org/10.1098/rsif.2021.0121.*
                                *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.*
                                *Ward, C.M., Aumann, R.A., Whitehead, M.A., Nikolouli, K., Leveque, G., Gouvi, G., Fung, E., Reiling, S.J., Djambazian, H., Hughes, M.A., Whiteford, S., Caceres-Barrios, C., Nguyen, T.N., Choo, A., Crisp, P., Sim, S.B., Geib, S.M., Marec, F., Hacker, I., Ragoussis, J., Darby, A.C., Bourtzis, K., Baxter, S.W., Schetelig, M.F. 2021. White pupae genes in the Tephritids Ceratitis capitata, Bactrocera dorsalis and Zeugodacus cucurbitae: a story of parallel mutations. Nature Biotechnology. 12. Article 491. https://doi.org/10.1038/s41467-020-20680-5.*


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