Mitigation of Invasive Pest Threats to U.S. Subtropical Agriculture

Goals / Objectives
1. Identify semiochemicals that mediate the behavior and physiology of exotic insect pests and develop semiochemical-based tools that lead to products for pest detection, behavioral disruption, and surveillance. [NP 304, Component 4, Problem Statement 4A] 1.A. Document behavioral responses and olfactory chemoreception to semiochemicals that have potential application in pest monitoring and control systems. 1.B. Identify and quantify insect semiochemicals; develop improved systems to collect, analyze and identify semiochemicals. 1.C. Develop synthetic and natural product lures based on host location and/or feeding cues that are formulated for effective use in IPM programs. 2. Evaluate â¿¿next generationâ¿¿ technologies to advance methods for fruit fly detection and eradication, such as lasers, sonic methods, or nano-technologies. [NP 304, Component 4, Problem Statement 4A] 2.A. Compare available remote trapping systems that can automatically count and identify trapped insects; optimize automated detection systems for adult fruit fly pests. 2.B. Evaluate imaging techniques and non-destructive chemical analysis methods for detection of larval infestation within host fruit. 3. Evaluate essential oils for natural insect pest toxicants and elucidate the physiological mechanisms underlying resistance to conventional pesticides. [NP 304, Component 4, Problem Statement 4A] 3.A. Determine the molecular/physiological basis for development of pesticide resistance in adult fruit flies. 3.B. Identify plant essential oils and plant extracts that have insecticidal, repellent, or oviposition deterrent properties against adult fruit flies and other target pests. 3.C. Determine the specific chemical components responsible for the toxic/repellent effects of essential oils and plant extracts. 4. Integrate tools, technologies and management strategies to reduce the threat of pest establishment and mitigate the impact of exotic pest incursions. [NP 304, Component 4, Problem Statement 4A] 4.A. Develop new approaches to assess efficacy of lures, repellents, and control practices based on spatial statistics and contour analysis of trap captures in the field. 4.B. Develop improved IPM protocols for tephritid fruit flies, ambrosia beetles, and other target pests by integrating semiochemical-based technologies with other management options, such as biological control, judicious alternating use of insecticides to manage pesticide resistance, sanitation and other cultural practices.

Project Methods
Research will consist of field and laboratory experiments to investigate aspects of basic biology, physiology and chemical ecology that can be exploited to develop integrated pest management approaches for invasive insects that impact subtropical agriculture. Target species will include pests that threaten to invade or have recently established in south Florida, including fruit flies in the family Tephritidae (Oriental fruit fly, Medfly, and Anastrepha species) and ambrosia beetles that vector fungal pathogens (redbay ambrosia beetle and Euwallacea shot-hole borers). Strategies will include (1) identifying semiochemicals from natural product sources that can alter insect behavior, such as attractants, repellents, and oviposition deterrents; (2) developing new detection and delimitation tools that will include formulated lures, discrete attract-and-kill bait stations, next generation technologies like smart traps for automated pest surveillance, and non-destructive imaging techniques for detection of hidden infestation in agricultural commodities; (3) improving pesticide resistance management by identification of pesticide modes of action and physiological responses of fruit flies to toxicants; (4) evaluating plant essential oils as sources of natural toxicants (biopesticides) with less environmental impact; and (5) developing new approaches to assess efficacy of lures, repellents, and control practices based on spatial statistics and contour analysis of trap captures in the field.

Progress 10/01/23 to 09/30/24

Outputs
PROGRESS REPORT Objectives (from AD-416): 1. Identify semiochemicals that mediate the behavior and physiology of exotic insect pests and develop semiochemical-based tools that lead to products for pest detection, behavioral disruption, and surveillance. [NP 304, Component 4, Problem Statement 4A] 1.A. Document behavioral responses and olfactory chemoreception to semiochemicals that have potential application in pest monitoring and control systems. 1.B. Identify and quantify insect semiochemicals; develop improved systems to collect, analyze and identify semiochemicals. 1.C. Develop synthetic and natural product lures based on host location and/or feeding cues that are formulated for effective use in IPM programs. 2. Evaluate ⿿next generation⿿ technologies to advance methods for fruit fly detection and eradication, such as lasers, sonic methods, or nano- technologies. [NP 304, Component 4, Problem Statement 4A] 2.A. Compare available remote trapping systems that can automatically count and identify trapped insects; optimize automated detection systems for adult fruit fly pests. 2.B. Evaluate imaging techniques and non-destructive chemical analysis methods for detection of larval infestation within host fruit. 3. Evaluate essential oils for natural insect pest toxicants and elucidate the physiological mechanisms underlying resistance to conventional pesticides. [NP 304, Component 4, Problem Statement 4A] 3.A. Determine the molecular/physiological basis for development of pesticide resistance in adult fruit flies. 3.B. Identify plant essential oils and plant extracts that have insecticidal, repellent, or oviposition deterrent properties against adult fruit flies and other target pests. 3.C. Determine the specific chemical components responsible for the toxic/ repellent effects of essential oils and plant extracts. 4. Integrate tools, technologies and management strategies to reduce the threat of pest establishment and mitigate the impact of exotic pest incursions. [NP 304, Component 4, Problem Statement 4A] 4.A. Develop new approaches to assess efficacy of lures, repellents, and control practices based on spatial statistics and contour analysis of trap captures in the field. 4.B. Develop improved IPM protocols for tephritid fruit flies, ambrosia beetles, and other target pests by integrating semiochemical-based technologies with other management options, such as biological control, judicious alternating use of insecticides to manage pesticide resistance, sanitation and other cultural practices. Approach (from AD-416): Research will consist of field and laboratory experiments to investigate aspects of basic biology, physiology and chemical ecology that can be exploited to develop integrated pest management approaches for invasive insects that impact subtropical agriculture. Target species will include pests that threaten to invade or have recently established in south Florida, including fruit flies in the family Tephritidae (Oriental fruit fly, Medfly, and Anastrepha species) and ambrosia beetles that vector fungal pathogens (redbay ambrosia beetle and Euwallacea shot-hole borers). Strategies will include (1) identifying semiochemicals from natural product sources that can alter insect behavior, such as attractants, repellents, and oviposition deterrents; (2) developing new detection and delimitation tools that will include formulated lures, discrete attract- and-kill bait stations, next generation technologies like smart traps for automated pest surveillance, and non-destructive imaging techniques for detection of hidden infestation in agricultural commodities; (3) improving pesticide resistance management by identification of pesticide modes of action and physiological responses of fruit flies to toxicants; (4) evaluating plant essential oils as sources of natural toxicants (biopesticides) with less environmental impact; and (5) developing new approaches to assess efficacy of lures, repellents, and control practices based on spatial statistics and contour analysis of trap captures in the field. Mediterranean fruit fly (medfly), Ceratitis capitata, is a serious pest of fruit crops globally and is a quarantine pest in the USA. Current management of this pest relies heavily on the male-specific attractant trimedlure for monitoring its populations. However, due to cost and availability there is urgent need for new male attractants. ARS scientists in Miami, Florida have previously identified tea tree oil (TTO) as a strong male medfly attractant and have developed a thin-layer chromatography (TLC) method for separating TTO into five distinct fractions. Through gas chromatography coupled with electroantennographic detection (GC-EAD) and electroantennography (EAG) studies, 10 compounds (terpinolene, a-terpinene, ¿-terpinene, p-cymene, (+)-terpinen-4-ol, (-)- terpinene-4-ol, a-phellendrene, myrcene, (+)-limonene, and (-)-limonene) have been identified as potential medfly attractants from fractionated TTO. Short-range attraction assays in the laboratory identified 7 of these compounds as attractive to male medfly. In collaboration with ARS scientists in Hilo, Hawaii, ongoing work are investigating the long-range attraction of these 7 compounds in semi-field cage trials to further identify new male medfly attractants. Caribbean fruit fly (caribfly), Anastrepha suspensa, is a major pest of guava and a quarantine pest in citrus growing regions of the USA. Due to large natural caribfly populations and their resistance to chemical insecticides, guava growers rely on mesh coverings to protect developing fruits from fly infestations. However, this management strategy is not ideal due to labor cost and environmental impact. To develop alternative control strategies, scientists at the ARS in Miami, Florida, and Hilo, Hawaii , are investigating the use of components from coconut free fatty acids (CFFA) as oviposition repellents/deterrents. In laboratory trials, wax substrates treated with the full CFFA blend, and 4 single components, significantly reduced female caribfly oviposition compared to untreated wax substrates. Ongoing field trials are validating these compounds on reducing caribfly infestation on intact guava fruit. Currently, regulatory agencies monitor caribfly and the Mexican fruit fly (mexfly), Anastrepha ludens, a major quarantine pest of citrus, utilizing traps baited with food-based protein baits. These protein baits must be changed frequently and attract many non-target insects which requires frequent servicing from technicians in the field. Thus, species-specific lures are needed for attracting and monitoring caribfly and mexfly populations. In collaboration with APHIS scientists in Buzzards Bay, MA, ARS scientists in Miami, FL, are investigating the pheromone production of male caribfly and mexfly as potential female attractants for both species. A few key compounds have been identified and currently laboratory experiments are ongoing to evaluate the attractant attributes to the two species. The giant African snail (GAS), Achatina fulica, is native to Africa, and it was first detected in South Florida in September 2011 and has been found in Florida on numerous occasions. It spread fast and caused potential economic harm in U.S. agriculture and also transmit lethal disease to human. In collaboration with APHIS, ARS scientists in Miami, Florida initiated research to determine the key components in GAS volatile emission that can be potentially used as training aids for dogs. Volatile emissions from available invasive gastropods species Asian tramp snail (Bradybaena similaris), a serious pest of gardens, crops, and vegetables.in South Florida, were collected at different life stages. Results showed that some chemical similarities with GAS, which significantly facilitate the GAS studies. This is an ongoing study. Progress was made on Objective 2 as follows: Current surveillance networks for pest fruit flies rely on many static traps baited with synthetic lures that require regular servicing to sort, count, and identify insect captures. Development of automated ⿿smart traps⿿ could reduce program costs and optimize detection systems. Scientists at ARS in Miami, FL, have developed a prototype automated trap for tephritid flies that photographs the captures and transmits images using Wi-Fi technology to the cloud for processing. However, the development and application of automatic identification algorithms will require large-scale data collection. Currently, more than 1,000 tephritid fruit fly images have been collected by ARS scientists for trainings of algorithm models and image analysis. The development of AI-driven algorithm systems was initiated with collaborators in ARS Hilo, Hawaii, and University of Texas (NACA). The current inspection of incoming fruit at US ports of entry for quarantine fruit fly pests is achieved by manually cutting open a small sample (= 2%) of fruit and searching for larvae. Scientists at ARS in Miami, Florida, used a Cyranose 320 (C-320) electronic sensor as a high- throughput screening method for fruit fly infestation of guava. Based on GC-MS results, five chemicals were selected which tentatively increase considerably on infested fruit. The C-320 was trained to identify cis-3- hexen-1-ol, ethyl hexanoate, cis-3-hexenyl acetate, hexyl acetate, and hydrocinnamyl acetate, respectively, as key compounds responding to fruit fly infestation. Currently, the detection sensitivity is also under evaluation and protocols for improving high throughput detection was under development using C-320. This is ongoing study. Progress was made on Objective 3 as follows: To improve the isolation of plant extracted EO components, high- performance thin-layer chromatography (HPTLC), an efficient, rapid, and convenient tool with low costs, was used for isolation of TTO components that attract male medflies. ARS scientists in Miami, Florida, explored the utilization of HPTLC fingerprinting in identifying components in TTO and four other Melaleuca spp. EOs. Variations were observed in the profile of bands and peak intensity of TTO and Melaleuca spp. The results showed that HPTLC can be used as a stand-alone fingerprinting platform for TTO and other Melaleuca EO, which facilitated the isolation process. Recently, a growing number of plant essential oils (EOs) have shown insecticidal effect and were promising environmentally sound alternatives to conventional pesticides for control of tephritid fruit flies. ARS scientists in Miami, Florida, continue to investigate EOs from Apiaceae, Asteraceae, Cupressaceae, Fabaceae, and Lamiaceae for toxicity against adult Caribbean fruit fly (CFF). Of the EOs tested, Cabreuva wood (CwEO) from Fabaceae showed the highest level of toxicity and thus the most promising candidate as a biorational insecticide. The constituents of CwEO were further identified by GC-MS to be oxygenated sesquiterpenes (E)-nerolidol, ¿-bisabolol, epi-¿-bisabolol, and (2E,6Z) farnesol. Additional studies are needed to determine the specific chemical components that confer toxicity to CFF. To understand the development of insecticide resistance in tephritid flies, ARS scientists in Miami, Florida, continues to detect and document resistance levels of caribfly in Miami-Dade County, Florida. From March to June 2024, wild caribflies were collected and assayed as done in 2023 to evaluate resistance development to methomyl, and resistance ratio was < 9 which showed mild resistance development. Cross resistance of the resistant strains (F11) was also tested, and results showed that little or no cross resistance were discovered among tested pesticides, including Spinosad, naled, cyfluthrin, and methomyl. Synergists of PBO, DEM, and DEF were also evaluated to identify resistance mechanism and results indicated PBO and DEM both played important roles in inhibiting resistance development in insecticides with different classes. Alternative control strategies will be needed to prevent further resistance development. Progress was made on Objective 4 as follows: To understand the spatial distribution of ambrosia beetle in avocado groves, ARS scientists in Miami Florida monitored the abundance of ambrosia beetle in avocado groves that constituted mapping population of avocado germplasm in SHRS at Miami FL, to understand the association among ambrosia beetle species, avocado varieties, and laurel wilt disease Harringtonia lauricola (LW). Traps baited with ethanol and a-copaene lures were deployed in avocado groves around healthy and LW infested trees to monitor the abundance of ambrosia beetle species and to better understand the interactions among the tritrophic level. In addition, anecdotal observations suggest that avocado trees colonized by Trichoderma sp. are less impacted by LW, ARS scientists are investigating the volatile production profiles of avocado trees that appear to be colonized by unknown Trichoderma sp. and uncolonized trees. This research will have better understanding how Trichoderma sp. affecting the LW disease in avocado trees. To develop novel biological control agents for invasive pest management in subtropical agriculture, in collaboration with Guizhou University (China) and University of Florida, ARS scientist in Miami, Florida, investigated several natural enemies species including Neoseiulus mites, two parasitoid species, Aphelinus varipes and Lysiphlebia japonica, and entomopathogenic nematode species (EPNs) on their efficiency in controlling various insect pests like sugarcane aphid, western flower thrips and HBW. Results from lab, greenhouse, and field trials indicated that all tested natural enemy⿿s species were effective in controlling their pest respectively, which expanded the application of biological control as a component for integrated pest management for subtropical agricultural pests. These studies provided useful guidance on system approach by incorporating biological control agents for invasive pest management in US Subtropical agriculture. Artificial Intelligence (AI)/Machine Learning (ML) The project involved developing an unmanned/automated insect trap to facilitate the detection, monitoring, and management of invasive pest species in subtropical agriculture in USA. The automated trap includes an image-capturing system to collect images of captured insect, and an image- analysis system to identify the species through machine learning algorithms. ACCOMPLISHMENTS 01 Identified new toxicity against Caribbean fruit fly. Natural compound structures are good sources of new insecticides in integrated pest management strategies. Chalcones are naturally occurring compounds that are aromatic enones and play important roles in agricultural and public health insect pests. In collaboration with scientists from Marmara University, Turkey, ARS scientists in Miami, Florida designed and synthesized 14 new chalcone derivatives, that were tested in this study for their toxicities against female CFF. Adult topical bioassays showed that derivative 2 (1-(4-cinnamoylphenyl)-3-(p-tolyl)urea) exhibited the highest toxicity against CFFs. This work revealed some lead structures are critical for further design of efficient chemicals for tephritid fruit fly management. 02 Identified structure-related activity in kairomone as an alternative to TML. Trimedlure (TML) is a powerful attractant used extensively throughout the world for detecting and monitoring populations of the Mediterranean fruit fly, Ceratitis capitata. However, the cost and limited availability of TML have prompted for the research on alternative attractants. ARS scientists in Miami, Florida investigated thymol and carvacrol, along with their propyl, butyl, benzyl, and octyl ethers for potential attraction of C. capitata as alternative to TML. Bioassay results showed thymol and carvacrol, along with their propyl, butyl, benzyl, and octyl ethers captured more male C. capitata. Furthermore, thymol benzyl and octyl ethers, and carvacrol benzyl ether elicited significantly greater antennal responses in C. capitata. This study revealed some key structural features responsible for attraction, which will benefit future researchers in developing alternative lures for C. capitata. 03 Improved analytical protocol for chemical analysis using HPTLC. Previous bioassays revealed that male Medflies are attracted to tea tree oil (TTO), an essential oil (EO) from Melaleuca alternifolia. Since TTO is available from multiple manufacturers, there is considerable variation in its chemical composition. Therefore, ARS scientists in Miami, Florida developed an analytical tool using high performance thin-layer chromatography (HPTLC) to evaluate variations in chemical composition of TTO from different sources. HPTLC protocols characterized variation of several TTOs on the planar chromatography without the need of time-consuming depletion strategies for the GC-MS system. This analytical protocol provides a quick and inexpensive screening tool to assess the variability present among EO samples. 04 Oviposition repellents for the melon fly. Melon fly, Zeugodacus cucurbitae, is a serious pest of many important fruit crops worldwide and a quarantine pest in the USA. ARS scientists in Miami, Florida and Hilo, Hawaii investigated the potential use of coconut free fatty acid (CFFA) components as oviposition repellents for this pest. In the laboratory, a 5-component blend of CFFA components (caprylic, capric, oleic, stearic, and linoleic acids) significantly reduced Z. cucurbitae oviposition on pumpkin agar. Over a 24-hour period female flies made 74. 3% fewer visits, spent 69.8% less time, and oviposited 89.3% fewer eggs on pumpkin agar treated with the 5-component blend compared to untreated pumpkin agar. Since all five compounds are registered food additives and generally regarded as safe, this 5-component blend has potential for use in behavioral control strategies against Z. cucurbitae. 05 Identified the most effective trap and commercial lure for the hibiscus bud weevil. The hibiscus bud weevil (HBW), Anthonomous testaceosquamosus, is an invasive weevil and major pest of tropical hibiscus, Hibiscus rosa-sinensis, in south Florida. No research has been done to identify the best trapping systems for monitoring HBW in hibiscus nurseries. In collaboration with scientists from the University of Florida, ARS scientists at Miami, Florida investigated the efficiency of commercially available traps and lures used for other weevil pests for their ability to capture and attract HBW. Laboratory and semi-field tests identified yellow sticky traps as the most effective trap for HBW and the cranberry weevil lure as the most attractive lure for HBW. Growers are now able to utilize this trap/lure combination for monitoring HBW in their hibiscus nurseries to develop treatment strategies for the pest.

Impacts
(N/A)

Publications

Shelly, T.E., Cloonan, K.R. 2023. Male lures and the detection of tephritid fruit flies: assessing the relationships between lure amount and release rate and trap captures of invasive pest species. Crop Protection. 176.Article 106504. https://doi.org/10.1016/j.cropro.2023.106504.
Vargas, G., Velazquez-Hernandez, Y., Greene, D.A., Yang, X., Revynthi, A.M. 2024. Entomopathogenic nematodes to control the hibiscus bud weevil Anthonomus testaceosquamosus (Coleoptera: Curculionidae), above ground and on soil surface. Biological Control. 69:91⿿101. https://doi.org/10.1007/ s10526-024-10242-9.
Ataide, L.M., Vargas, G., Velazquez-Hernandez, Y., Arauz, I.R., Villamarin, P., Canon, M.A., Yang, X., Riley, S.S., Revynthi, A.M. 2024. Efficacy of conventional and biorational insecticides against the invasive pest thrips parvispinus (thysanoptera: thripidae) under containment conditions. Insects. 15(1):48. https://doi.org/10.3390/insects15010048.
Ataide, L.M., Greene, D.A., Cloonan, K.R., Gill, M.A., Vargas, G., Tabanca, N., Arauz, I.R., Valezquez-Hernandez, Y., Revynthi, A.M. 2024. Exploring market-available commercial pheromone lures and traps for controlling the hibiscus bud weevil, anthonomus testaceosquamosus (coleoptera: curculionidae). Journal of Economic Entomology. https://doi.org/10.1093/ jee/toae105.
Demir, S., Karaalp, C., Tabanca, N., Bernier, U.R., Linthicum, K. 2023. Evaluation of the repellent activity of 13 Achillea L. species from Turkiye against the Virus vector Aedes aegypti (L.) Mosquitoes. Kafkas Universitesi Veteriner Fakultesi Dergisi. 29(1):33-40. https://doi.org/10. 9775/kvfd.2022.28409.
Huang, Y., Yang, X., Bai, Q., Zang, L., Tang, L., Singh, S. 2024. Performance of the two parasitoid species, Aphelinus varipes and Lysiphlebia japonica against sugarcane aphid, Melanaphis sacchari. Biological Control. https://doi.org/10.1016/j.biocontrol.2024.105532.
Sampson, B.J., Tabanca, N., Werle, C.T., Stringer, S.J., Wedge, D.E., Moraes, R., Dehgan, B. 2022. Insecticidal activity of jatropha extracts against the azalea lace bug, stephanitis pyrioides (Hemiptera: Tingidae). Journal of Economic Entomology. 116(1):192⿿201. https://doi.org/10.1093/ jee/toac187.
Vargas, G., Greene, A., Velazquez-Hernandez, Y., Yang, X., Kendra, P.E., Revynthi, A.M. 2023. A prophylactic application of systemic insecticides contributes to the management of the hibiscus bud weevil anthonomus testaceosquamosus Linell (Coleoptera: Curculionidae). Agriculture. 13(10) :1879. https://doi.org/10.3390/agriculture13101879.
Zhang, Y., Zang, L., Guo, L., Singh, S., Wu, S., Yang, X., Tang, L. 2024. Neoseiulus mites as biological control agents against Megalurothrips usitatus (Thysanoptera: Thripidae) and Frankliniella intonsa (Thysanoptera: Thripidae) on cowpea crop: laboratory to field. Journal of Economic Entomology. https://doi.org/10.1093/jee/toae118.

Progress 10/01/22 to 09/30/23

Outputs
PROGRESS REPORT Objectives (from AD-416): 1. Identify semiochemicals that mediate the behavior and physiology of exotic insect pests and develop semiochemical-based tools that lead to products for pest detection, behavioral disruption, and surveillance. [NP 304, Component 4, Problem Statement 4A] 1.A. Document behavioral responses and olfactory chemoreception to semiochemicals that have potential application in pest monitoring and control systems. 1.B. Identify and quantify insect semiochemicals; develop improved systems to collect, analyze and identify semiochemicals. 1.C. Develop synthetic and natural product lures based on host location and/or feeding cues that are formulated for effective use in IPM programs. 2. Evaluate ⿿next generation⿿ technologies to advance methods for fruit fly detection and eradication, such as lasers, sonic methods, or nano- technologies. [NP 304, Component 4, Problem Statement 4A] 2.A. Compare available remote trapping systems that can automatically count and identify trapped insects; optimize automated detection systems for adult fruit fly pests. 2.B. Evaluate imaging techniques and non-destructive chemical analysis methods for detection of larval infestation within host fruit. 3. Evaluate essential oils for natural insect pest toxicants and elucidate the physiological mechanisms underlying resistance to conventional pesticides. [NP 304, Component 4, Problem Statement 4A] 3.A. Determine the molecular/physiological basis for development of pesticide resistance in adult fruit flies. 3.B. Identify plant essential oils and plant extracts that have insecticidal, repellent, or oviposition deterrent properties against adult fruit flies and other target pests. 3.C. Determine the specific chemical components responsible for the toxic/ repellent effects of essential oils and plant extracts. 4. Integrate tools, technologies and management strategies to reduce the threat of pest establishment and mitigate the impact of exotic pest incursions. [NP 304, Component 4, Problem Statement 4A] 4.A. Develop new approaches to assess efficacy of lures, repellents, and control practices based on spatial statistics and contour analysis of trap captures in the field. 4.B. Develop improved IPM protocols for tephritid fruit flies, ambrosia beetles, and other target pests by integrating semiochemical-based technologies with other management options, such as biological control, judicious alternating use of insecticides to manage pesticide resistance, sanitation and other cultural practices. Approach (from AD-416): Research will consist of field and laboratory experiments to investigate aspects of basic biology, physiology and chemical ecology that can be exploited to develop integrated pest management approaches for invasive insects that impact subtropical agriculture. Target species will include pests that threaten to invade or have recently established in south Florida, including fruit flies in the family Tephritidae (Oriental fruit fly, Medfly, and Anastrepha species) and ambrosia beetles that vector fungal pathogens (redbay ambrosia beetle and Euwallacea shot-hole borers). Strategies will include (1) identifying semiochemicals from natural product sources that can alter insect behavior, such as attractants, repellents, and oviposition deterrents; (2) developing new detection and delimitation tools that will include formulated lures, discrete attract- and-kill bait stations, next generation technologies like smart traps for automated pest surveillance, and non-destructive imaging techniques for detection of hidden infestation in agricultural commodities; (3) improving pesticide resistance management by identification of pesticide modes of action and physiological responses of fruit flies to toxicants; (4) evaluating plant essential oils as sources of natural toxicants (biopesticides) with less environmental impact; and (5) developing new approaches to assess efficacy of lures, repellents, and control practices based on spatial statistics and contour analysis of trap captures in the field. Mediterranean fruit fly, or medfly Ceratitis capitata, is a worldwide pest of fruit crops. Trimedlure is currently the only commercialized attractant used by regulatory agencies world-wide to monitor medfly populations. To develop alternative attractants, ARS scientist in Miami, Florida, developed a thin-layer chromatography assay for separating and assaying fractions of tea tree oil, a known medfly attractant, to develop alternatives. Using gas chromatography coupled with electroantennographic detection and gas chromatography coupled with mass spectrometry, five distinct fractions from tea tree oils were identified and four monoterpenes from fraction 1, and two alcohols from fraction 3 have been identified as attractive to male medflies. Behavioral assays indicated that the two alcohols were more attractive than tea tree oils, and the addition of one monoterpene to the alcohols increased attraction compared to either alcohol alone. These studies are useful for the development of low-cost alternative attractants for medfly management. Caribbean fruit fly, or caribfly, Anastrepha suspensa, is a production pest of guava and other specialty fruits in Florida. Traps baited with food-based lures are used to monitor caribfly populations in fruit fly free zones. As one of the effective means for fruit fly management, oviposition repellents may offer growers alternative control tools with lower cost for control of this pest. ARS scientists in Miami, Florida, in collaboration with ARS scientists in Hilo, Hawaii, are investigating the use of coconut oil derived free fatty acids as caribfly oviposition repellents. Preliminary behavioral assays in the laboratory indicate that the complete fatty acid blend reduces caribfly oviposition for 14 days, and ongoing work is investigating the deterrent effects of six individual fatty acid constituents. This is an ongoing study. The invasive lychee erinose mite, Aceria litchi, is a microscopic and serious pest of lychee fruit trees in Florida. Currently, little is known about the chemical ecology of lychee mites. In collaboration with the University of Florida, ARS scientists in Miami, Florida investigated potential host-based attractants or host defensive compounds induced by mite infestation. Qualitative comparison of the headspace profiles of uninfested and infested lychee plants revealed that six volatile organic compounds nonanal, decanal, limonene, sabinene, ÿ-caryophyllene, and ar- curcumene were present in samples of infested lychee plants, and the amounts varied from infested plants. Behavioral bioassays showed that the chemical concentration significantly influenced mite attraction, and provide useful information on developing attractive lures for lychee mite management. The hibiscus bud weevil, Anthonomus testaceosquamosus, is an invasive pest attacking flower buds of hibiscus (Hibiscus rosa-sinensis) in South Florida. The volatile organic compounds released by hibiscus plants are responsible for the attraction of the hibiscus bud weevil, and different color cultivars show variation in attraction to this weevil. In collaboration with the University of Florida, ARS scientists in Miami, Florida, investigated volatile compounds from the leaves, buds, and open flowers of different cultivars. In red-flowering cultivars, saturated and unsaturated fatty alcohols and aldehydes were the primary compounds identified in open flower and bud mixtures of alkanes, primary alcohols, aldehydes, and fatty acids were typically highest in leaves. Identification of volatiles from cultivars with pink and yellow flowers are ongoing. The giant African snail, Achatina fulica, is native to Africa and a quarantine pest in southern Florida. The snail has posed significant threat to agricultural production as well as transmitting diseases to human. In collaboration with APHIS, ARS scientists in Miami, Florida, developed a methodology to determine the key components that are emitted by snail for detection by dogs. To identify biomarkers of giant African snail, volatile emissions from four invasive gastropods species (Macrochlamys indica, Bulimulus guadalupensis, Zachrysia provisoria, and Parmarion martensi) were analyzed and the methodology was developed for African snail biomarker analysis. Using this methodology, odor profiles among African snail, Z. provisoria, and M. indica, was varied but shared similarity. This study is currently ongoing. Current surveillance networks for pest fruit flies rely on many static traps baited with synthetic lures that require regular servicing to sort, count, and identify insect captures. Development of automated ⿿smart traps⿿ could reduce program costs and optimize detection systems. Scientists at ARS in Miami, Florida, have developed a prototype automated trap for tephritid flies that photographs the captures and transmits images using Wi-Fi technology to the cloud for processing. However, the development and application of automatic identification algorithms will require large-scale data collection. Collaborations are being sought with scientist in ARS Hilo, Hawaii, researchers in China (NFCA), and a company to facilitate development of automated traps. The current inspection of incoming fruit at US ports of entry for quarantine fruit fly pests is achieved by manually cutting open a small sample (= 2%) of fruit and searching for larvae. Consequently, there is a need for more sensitive, high-throughput screening methods. Scientists at ARS in Miami, Fl, are evaluating imaging techniques and non-destructive chemical analysis methods for detection of larval infestation. Initial experiments using the Cyranose Electronic Nose (eNose) on infested guava identified four VOCs. Further experiments will be conducted to optimize the testing system.Progress was made on Objective 3 as follows: Development of insecticide resistance in tephritid flies is an ongoing challenge. ARS scientists in Miami, Florida, continues to detect and document resistance levels of caribfly in Miami-Dade County, FL. From March to May 2023, wild caribflies were collected and assayed as done in 2022 to determine the median lethal dosage of methomyl to detect the insecticide resistance level. This was then compared to baseline susceptibility to determine the resistance ratio, which was <8 for both males and females. Results indicated that caribfly populations are beginning to develop low levels of resistance to carbamate insecticides. ARS will continue to monitor changes in caribfly resistance levels over time; however, to prevent further resistance development, alternative control strategies will be required. To improve the isolation of plant extracted EO components, high- performance thin-layer chromatography, an efficient, rapid, and convenient tool with low costs, were used for isolation of tea tree oil components that attract male medflies. ARS scientists in Miami, Florida, explored the utilization of high-performance thin-layer chromatography fingerprinting in identifying components in tea tree oils and four other Melaleuca spp. essential oils. Variations were observed in the profile of bands and peak intensity of tea tree oils and Melaleuca spp. The results showed that high-performance thin-layer chromatography can be used as a stand-alone fingerprinting platform for tea tree oils and other Melaleuca essential oils, which facilitated the isolation process. Current management of tephritid fruit flies typically relies on bait systems that incorporate attractants with insecticides. Extensive use of synthetic insecticides has led to an increase of pesticide resistance of these pests. To develop alternative biopesticide, ARS researchers in Miami, Florida, identified plant EO, from three chamomile species, German chamomile (Matricaria chamomilla), Roman chamomile (Chamaemelum nobile), and Chinese chamomile (Chrysanthemum x morifolium) and evaluated their toxicity against caribflies. Results showed that all essential oils showed some mortality against caribfly adults, with the highest toxicity from C. x morifolium oil. The monoterpene compounds (borneol and bornyl acetate) are high in C. x morifolium oil which may contribute to their toxicity. Research is currently ongoing to further identify the active components of C. x morifolium oil against caribflies. Alternative environmentally safe products for control of tephritid fruit flies are urgently needed. Phthalimides are found in both natural and synthetic products with a myriad of biological activities, including toxicity against insects. In collaboration with Marmara University, Turkey, ARS scientists in Miami, Florida, investigated a series of 13 phthalimide derivatives to determine the toxicities against female caribflies. Bioassays showed that three phthalimide derivatives (4a, 4c, and 4d) exhibited potent insecticidal activity against caribflies with median lethal dose values ranging from 0.70 to 1.91 µg/fly. The physicochemical properties of these phthalimides demonstrated that higher lipophilicity tended to show stronger insecticidal activity. This study showed that phthalimides demonstrated potential for development of new biopesticides for control of caribflies. To evaluate efficacy of cultural practices, scientists from University of Florida, in collaboration with ARS, investigated the effect of avocado canopy cover on ambrosia beetle abundance. Monitoring for 12 months indicated that groves with full canopy cover exhibited the highest number of beetles and the lowest light intensity. The opposite was found for topworked and newly planted groves. In addition, solar radiation had a significant effect on beetle dispersal flight. The results indicate that thinning canopy cover, which increases light intensity, suppresses ambrosia beetle abundance in commercial groves, thereby reducing the spread of laurel wilt disease. Artificial Intelligence (AI)/Machine Learning (ML) The project involved developing an unmanned/automated insect trap to facilitate the detection, monitoring, and management of invasive pest species in subtropical agriculture in USA. The automated trap includes an image-capturing system to collect images of captured insect, and an image- analysis system to identify the species through machine learning algorithms. ACCOMPLISHMENTS 01 Repellents for pest ambrosia beetles. Redbay ambrosia beetle and tea shot-hole borer are vectors of laurel wilt and Fusarium dieback, respectively, two fungal diseases of avocado, woody ornamentals, and native forest trees. Incorporating a repellent into pest management programs may reduce the incidence of these diseases. ARS scientists from Miami, Florida, identified piperitone as a new beetle repellent and compared its efficacy to two other repellents, verbenone and a- farnesene. Beetle captures in traps baited with lures were compared to those containing lures plus a repellent (a push-pull design). Results showed that farnesene was ineffective; however, piperitone and verbenone were equally effective, reducing captures by 50-70% for 10-12 weeks. Since piperitone is less expensive than verbenone, the standard beetle repellent. This study identifies an economical alternative for management of the two beetles with push-pull design in commercial avocado groves. 02 Identification of host kairomones for lychee erinose mite. The invasive lychee erinose mite (LEM, Aceria litchii) is a serious pest of lychee fruit trees in Florida. This tiny mite lives mainly on the underside of the leaves, preventing flowering and fruit production. Currently, little is known about the chemical ecology of LEM and no known attractants or repellents exist. In collaboration with the University of Florida, ARS scientists in Miami, Florida, investigated potential host-based attractants or host defensive compounds induced by LEM infestation. Qualitative comparison of the headspace profiles of uninfested and infested lychee plants revealed that six volatile organic compounds (VOCs) nonanal, decanal, limonene, sabinene, ÿ- caryophyllene and ar-curcumene were present in almost all samples of uninfested and infested lychee plants. Behavioral (olfactometer) bioassays with these six VOCs demonstrated that with these six VOCs, the chemical concentration significantly influenced LEM attraction, which was typically the most attractive at lower concentrations. The findings of this study provide useful information on the development of a field lure that can be used in pest management programs for LEM. 03 Effect of entomopathogenic fungus Metarhizium anisopliae on morphological changes of insect. Biological control using the entomopathogenic fungus M. anisopliae has shown potential for management of various insect pests, including tephritid fruit flies. Destruxin A (DA) is a mycotoxin isolated from M. anisopliae, but the mechanism of toxicity against insects remains unknown. To better understand the mode of action of DA, scientists from Guangdong Academy of Agricultural Science (China) and USDA-ARS in Miami, Florida, used histopathological methods to investigate the effect of DA on target cells and tissues of the silkworm Bombyx mori. At low doses (i.e., 0. 01µg/g), the hemocytes were the most sensitive to DA. At higher doses (i.e., > 0.1µg/g), the muscle cells, fat body, and Malpighian tubules showed morphological changes 24 h after treatment. This study elucidated the target sites in B. mori that responded to DA treatment, indicating that the defensive hemocytes were the first cells damaged. The results of this study will help develop mycopesticides and novel immunosuppressants for improved management of tephritid fruit fly pupae. 04 Identified new plant based toxicants against the Caribbean fruit fly. Tephritid fruit flies are among the most serious agricultural pests worldwide. Current management typically relies on bait sprays or bait stations that incorporate insecticides, but concerns have been raised regarding the adverse effects on the environment and increased insecticide resistance of the pest. To develop environmentally friendly alternative biopesticides, ARS researchers in Miami, Florida, evaluated plant EOs extracted from three species of chamomile plants, commonly used in therapeutic applications, German chamomile (GC, Matricaria chamomilla), Roman chamomile (RC, Chamaemelum nobile) and Chinese chamomile (CC, Chrysanthemum x morifolium). Results showed that CCEO demonstrated the most significant insecticidal activity against caribfly female adults. This study discovered that CCEO is a promising source of potential alternative pesticides for CFF. 05 Discovered novel toxicants against the Caribbean fruit fly. Natural products with insecticidal effects against insect pest have the potential as alternatives to synthetic insecticides for pest control. Phthalimides (isoindoline-1,3-diones, and their N-substituted analogs), are found in natural products, can be synthesized in the laboratory, and have demonstrated biological activities including insecticidal attributes. In collaboration with Marmara University, Turkey, ARS scientists in Miami, Florida, investigated 13 phthalimide derivatives for their toxicities against female caribflies. Results showed that three phthalimide derivatives (4a, 4c, and 4d) exhibited potent insecticidal activity against caribflies with LD50 values ranging from 0.70 to 1.91 ��g/fly. The physicochemical properties of these phthalimides demonstrated that higher lipophilicity tended to show good insecticidal activity. This study revealed key structural features responsible for the insecticidal activity and can be promising for the development of new biopesticides. 06 Vibrational communication identified in the spotted lanternfly. The spotted lanternfly Lycorma delicatula, is a polyphagous insect pest that invaded the United States in 2014, in Berks County, Pennsylvania, and has since spread to several northeastern states threatening the agriculture production of many crops. ARS scientists from Miami, Florida, in collaboration with scientists from USDA-APHIS, demonstrated that adult and fourth-instar L. delicatula are attracted to broadcasts of 60-Hz acoustic/vibrational stimuli in the laboratory. This finding expanded the current knowledge on a wider frequency that is attractive to L. delicatula. This study suggests that a vibroacoustic trap may be developed for management of L. delicatula in the field.

Impacts
(N/A)

Publications

Rohde, B.B., Cooperband, M.F., Canlas, I., Mankin, R.W. 2022. Evidence of receptivity to vibroacoustic stimuli in the spotted lanternfly lycorma delicatula (Hemiptera: Fulgoridae). Journal of Economic Entomology. 115(6) :2116-2120. https://doi.org/10.1093/jee/toac167.
Panthi, B., Cloonan, K.R., Rodriguez-Saona, C., Kirkpatrick, D.M., Short, B.D., Aflitto, N.C., Andrews, H., Ballman, E., Beal, D.J., Beers, E.H. 2022. Using red panel traps to detect spotted-wing drosophila and its infestation in us berry and cherry crops. Journal of Economic Entomology. 115:(6), 1995-2003. https://doi.org/10.1093/jee/toac134.
Ali, A., Tabanca, N., Raman, V., Avonto, C., Yang, X., Demirci, B., Chittiboyina, A.G., Khan, I.A. 2023. Chemical composition of essential oils from German, Roman, and Chinese chamomile flowers and their biological activities against three economically important insects. Records of Natural Products. 17(4): 595-614. https://doi.org/10.25135/rnp. 378.2211.2627.
Kendra, P.E., Larissa, G., Tabanca, N., Montgomery, W.S., Schnell, E.Q., Deyrup, M.A., Cloonan, K.R. 2023. Risk assessment of Hass avocado and Mexican Lauraceae for attack by redbay ambrosia beetle (Coleoptera: Curculionidae: Scolytinae). Agricultural and Forest Entomology. 25(2): 285- 302. https://doi.org/10.1111/afe.12551.
Cloonan, K.R., Montgomery, W.S., Narvaez, T.I., Carrillo, D., Kendra, P.E. 2022. Community of bark and ambrosia beetles (coleoptera: curculionidae: scolytinae and platypodinae) in agricultural and forest ecosystems with Laurel Wilt. Insects. 13(11):971. https://doi.org/10.3390/insects13110971.
Menocal, O., Cruz, L.F., Kendra, P.E., Berto, M., Carrillo, D. 2023. Flexibility in the ambrosia symbiosis of Xyleborus bispinatus. Frontiers in Microbiology. 14:1110474. https://doi.org/10.3389/fmicb.2023.1110474.
Tok, F., Yang, X., Tabanca, N., Kocyigit-Kaymakciogl, B. 2023. Synthesis of phthalimide derivatives and their insecticidal activity against caribbean fruit fly, Anastrepha suspensa (Loew). Topic Issue: Frontiers in Chemical Ecology. Biomolecules EISSN 2218-273X. 13(2): 361. https://doi. org/10.3390/biom13020361.
Kendra, P.E., Montgomery, W.S., Tabanca, N., Schnell, E.Q., Vazquez, A., Menocal, O., Carrillo, D., Cloonan, K.R. 2023. Piperitone (p-menth-1-en-3- one): A new repellent for tea shot hole borer (Coleoptera: Curculionidae) in Florida avocado groves. Biomolecules. 13(4): 656. https://doi.org/10. 3390/biom13040656.
Vázquez, A., Tabanca, N., Kendra, P.E. 2023. HPTLC analysis and chemical composition of selected melaleuca essential oils. Molecules. 28(9):3925. https://doi.org/10.3390/molecules28093925.
Vázquez, A., K. R. Cloonan, B. B. Rhode, M. A. Gill, L. K. Mosser, J. H. Crane, D. Carrillo, and P. E. Kendra. 2022. Attraction and longevity of 2- and 3-component food cone lures for the Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae). J. Econ. Entomol. 115(4): 1231-1239.
Romero, P., L.A. Ibarra-Juárez, D. Carrillo, J.A. Guerrero-Analco, P.E. Kendra, A.L. Kiel-Martínez, and L. Guillén. 2022. Electroantennographic responses of wild and laboratory-reared females of Xyleborus affinis Eichhoff and Xyleborus ferrugineus (Fabricius) (Coleoptera: Curculionidae: Scolytinae) to ethanol and bark volatiles of three host-plant species. Topic Issue: Frontiers in Chemical Ecology. Insects 13(7): 655.
Niogret, J., Kendra, P.E., Ekaynati, A., Zhang, A., Marelli, J., Tabanca, N., Epsky, N.D. 2022. Development of a kairomone-based attractant as a monitoring tool for the cocoa pod borer, Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillariidae). Insects 13(9): 813.
Roh, G., Kendra, P.E., Zhu, J.J., Roda, A., Loeb, G.M., Tay, J., Cha, D.H. 2023. Coconut oil derived five-component synthetic oviposition deterrent for oriental fruit fly, Bactrocera dorsalis. Pest Management Science. https://doi.org/10.1002/ps.7584.
Greene, D.A., Yang, X., Velazquez-Hernandez, Y., Vargas, G., Kendra, P.E., Mannion, C., Revynthi, A.M. 2023. Lethal and sublethal effects of contact insecticides and horticultural oils on the hibiscus bud weevil, anthonomus testaceosquamosus linell (Coeloptera: Curculionidae). Insects. 14(6):544. https://doi.org/10.3390/insects14060544.
Ataide, L.M., Tabanca, N., Canon, M.A., Schnell, E.Q., Narvaez, T.I., Cloonan, K.R., Kendra, P.E., Carrillo, D. 2023. Volatile characterization of lychee plant tissues (Litchi chinensis) and the effect of key compounds on the behavior of lychee erinose mite (Aceria litchii). Biomolecules EISSN 2218-273X. 13:933. https://doi.org/10.3390/biom13060933.
Cloonan, K.R., Montgomery, W.S., Narvaez, T.I., Kendra, P.E. 2023. A new repellent for redbay ambrosia beetle (Coleoptera: Curculionidae: Scolytinae), a primary vector of the mycopathogen that causes laurel wilt. Plants. 12(13):2406. https://doi.org/10.3390/plants12132406.
Thomas, G., Rusman, Q., Morrison III, W.R., Magalhaes, D.M., Dowell, J.A., Ngumbi, E., Osei-Owusu, J., Kansman, J., Gaffke, A.M., Jayanthi, K., Kim, S., Tabanca, N. 2023. Deciphering plant-insect-microorganism signals for sustainable crop production. Biomolecules EISSN 2218-273X. 13(6):997. https://doi.org/10.3390/biom13060997.
Ying, F., Hu, L., Li, Z., Yang, X., Kendra, P.E., Hu, Q. 2023. Effects of destruxin a on hemocytes of the domestic silkworm, bombyx mori. Frontiers in Microbiology. 14 : Article 1210647. https://doi.org/10.3389/fmicb.2023. 1210647.

Progress 10/01/21 to 09/30/22

Outputs
PROGRESS REPORT Objectives (from AD-416): 1. Identify semiochemicals that mediate the behavior and physiology of exotic insect pests and develop semiochemical-based tools that lead to products for pest detection, behavioral disruption, and surveillance. [NP 304, Component 4, Problem Statement 4A] 1.A. Document behavioral responses and olfactory chemoreception to semiochemicals that have potential application in pest monitoring and control systems. 1.B. Identify and quantify insect semiochemicals; develop improved systems to collect, analyze and identify semiochemicals. 1.C. Develop synthetic and natural product lures based on host location and/or feeding cues that are formulated for effective use in IPM programs. 2. Evaluate ⿿next generation⿿ technologies to advance methods for fruit fly detection and eradication, such as lasers, sonic methods, or nano- technologies. [NP 304, Component 4, Problem Statement 4A] 2.A. Compare available remote trapping systems that can automatically count and identify trapped insects; optimize automated detection systems for adult fruit fly pests. 2.B. Evaluate imaging techniques and non-destructive chemical analysis methods for detection of larval infestation within host fruit. 3. Evaluate essential oils for natural insect pest toxicants and elucidate the physiological mechanisms underlying resistance to conventional pesticides. [NP 304, Component 4, Problem Statement 4A] 3.A. Determine the molecular/physiological basis for development of pesticide resistance in adult fruit flies. 3.B. Identify plant essential oils and plant extracts that have insecticidal, repellent, or oviposition deterrent properties against adult fruit flies and other target pests. 3.C. Determine the specific chemical components responsible for the toxic/ repellent effects of essential oils and plant extracts. 4. Integrate tools, technologies and management strategies to reduce the threat of pest establishment and mitigate the impact of exotic pest incursions. [NP 304, Component 4, Problem Statement 4A] 4.A. Develop new approaches to assess efficacy of lures, repellents, and control practices based on spatial statistics and contour analysis of trap captures in the field. 4.B. Develop improved IPM protocols for tephritid fruit flies, ambrosia beetles, and other target pests by integrating semiochemical-based technologies with other management options, such as biological control, judicious alternating use of insecticides to manage pesticide resistance, sanitation and other cultural practices. Approach (from AD-416): Research will consist of field and laboratory experiments to investigate aspects of basic biology, physiology and chemical ecology that can be exploited to develop integrated pest management approaches for invasive insects that impact subtropical agriculture. Target species will include pests that threaten to invade or have recently established in south Florida, including fruit flies in the family Tephritidae (Oriental fruit fly, Medfly, and Anastrepha species) and ambrosia beetles that vector fungal pathogens (redbay ambrosia beetle and Euwallacea shot-hole borers). Strategies will include (1) identifying semiochemicals from natural product sources that can alter insect behavior, such as attractants, repellents, and oviposition deterrents; (2) developing new detection and delimitation tools that will include formulated lures, discrete attract- and-kill bait stations, next generation technologies like smart traps for automated pest surveillance, and non-destructive imaging techniques for detection of hidden infestation in agricultural commodities; (3) improving pesticide resistance management by identification of pesticide modes of action and physiological responses of fruit flies to toxicants; (4) evaluating plant essential oils as sources of natural toxicants (biopesticides) with less environmental impact; and (5) developing new approaches to assess efficacy of lures, repellents, and control practices based on spatial statistics and contour analysis of trap captures in the field. Oriental fruit fly (OFF) is a destructive pest of tropical fruit crops worldwide. Current control programs rely on surveillance of male OFF, which is less effective for mitigating pest impact (caused by females). Under an Interagency Agreement with APHIS, ARS scientists in Miami, Florida, and Hilo, Hawaii, investigated the relationship between OFF olfactory preferences and oviposition. In lab bioassays using mated females, some preferred protein (torula yeast, the standard female lure) however, some preferred host fruit (guava volatiles). Females attracted to fruit odors laid 2.4x more eggs than females attracted to protein. Results suggest that host-based lures are better for trapping oviposition- ready female OFF. Parallel tests with Caribbean fruit fly, another pest of guava, are in progress to determine potential utility of host odors for management of this pest. Mediterranean fruit fly (medfly) is another major pest of fruit production globally. Trimedlure, a synthetic male attractant, is used to monitor medfly populations. However, due to cost and availability concerns, APHIS recently identified a need for new male attractants. ARS researchers in Miami, Florida, evaluated a series of essential oils (EOs), including leaf and fruit EO of Juniperus foetidissima and leaf EO of Origanum vulgare and Monarda didyma. Lab bioassays indicated that components of these EOs were attractive to medflies. Chemical analyses indicated the major constituents of J. foetidissima EO were a- and ÿ- thujone, and the O. vulgare and M. didyma EOs were rich in carvacrol and thymol, respectively. Tests are ongoing to determine the bioactivity of these novel compounds and their potential applications as alternative medfly lures. The collection, analysis and accurate identification of bioactive chemicals is a fundamental requirement for developing semiochemical-based tools for pest management. We previously identified tea tree oil (TTO) as an EO highly attractive to male medfly. Separation of TTO by thin layer chromatography (TLC) generated five major fractions, two of which were attractive. However, identification of specific attractants has been challenging since TTO is a complex mixture of terpenoids and TTOs vary based on the manufacturer. ARS-Miami has recently developed high performance TLC (HPTLC) protocols to evaluate variations in EOs. HPTLC provides a rapid screening method that improves separation, resolution, and isolation of the individual chemicals in TTO, facilitating identification of alternative attractants for medfly. Lychee erinose mite (LEM), recently established in Florida, is a high priority pest with potential to devastate the state⿿s lychee production. This microscopic mite attacks new leaves and flower buds, thereby preventing fruit development. No attractants are known for LEM; therefore, ARS researchers in Miami, Florida, in collaboration with the University of Florida (UF), initiated research to identify attractive host kairomones. Chemical analyses indicated that LEM infestation induces significant changes in host emissions, and lab bioassays demonstrated preferential attraction of LEM to particular host tissues. Research is in progress to identify the attractants used for host location, and to determine their application in monitoring and control systems for LEM. Hibiscus bud weevil (HBW) is emerging as a major pest in Florida. Females oviposit in flower buds, larvae feed within the buds, and buds drop prior to flowering. Lures are critically needed for early HBW detection. ARS researchers in Miami, Florida, in collaboration with UF, conducted two lines of research to identify potential attractants: host volatiles and species-specific pheromones. Volatile collections and chemical analyses found 42 compounds (primarily long-chain fatty acids) emitted from hibiscus plants and 4 male-produced aggregation pheromone components. Lab bioassays and electrophysiology studies (to evaluate attraction and olfaction, respectively) are ongoing to identify the most promising candidates for development of HBW lures. The giant African snail (GAS) is an invasive pest that causes feeding damage to a variety of agricultural, ornamental, and native plants. In recent years, GAS has established in Florida, prompting an extensive eradication program. In collaboration with APHIS-PPQ, ARS researchers in Miami, Florida, and Hilo, Hawaii, are conducting research to determine if signature volatiles are emitted from GAS mucus trails. If so, these chemical signals could provide the basis for trained canine detection. Initial research documented the odor profile emitted from GAS. Current research is identifying biomarkers from other land gastropods in Florida to determine which volatiles are unique to GAS and potentially diagnostic of pest GAS populations. This project recently received funding through the USDA Plant Protection Act Section 7721. Surveillance networks for pest fruit flies rely on a large number of static traps baited with synthetic lures. These traps require regular servicing to sort, count, and identify insect captures. Development of automated ⿿smart traps⿿ could reduce program costs and optimize detection systems. ARS researchers in Miami, Florida, have developed a prototype trap entrance camera that is undergoing small-scale trials to determine its potential for image collection. However, the development and application of automatic identification algorithms will require large- scale data collection. Collaborations are being sought with researchers in China (Chinese Academy of Science, Beijing) and a private company that has assembled 15,000 images of tephritid fruit flies; access to these datasets would greatly facilitate development of automated traps. At U.S. ports of entry, inspectors check incoming produce shipments for fruit fly infestation by manually cutting open a small sample (= 2%) of fruit and searching for larvae. Consequently, there is a need for more sensitive, high-throughput screening methods. ARS researchers in Miami, Florida, are evaluating imaging techniques and non-destructive chemical analysis methods for detection of larval infestation. Initial experiments using an electronic sensor prototype, the Cyranose Electronic Nose (Sensigent Inc.), found that this system could differentiate chemicals produced by infested and damaged fruit. Experiments are ongoing to determine infestation thresholds required for consistent detection by either gas chromatography or electronic means. Caribbean fruit fly (CFF) is a quarantine pest of citrus and a production pest of guava in Florida. As with other pests, development of insecticide resistance is an ongoing challenge. ARS researchers in Miami, Florida, initiated a long-term project to document resistance levels of CFF in Miami-Dade County, FL. Initial work established a susceptible lab colony in 2020 and determined baseline susceptibility to methomyl. In 2022, wild CFF were assayed to determine the median lethal dosage (LD50) of methomyl, which was compared to baseline susceptibility to determine the resistance ratio. Results indicated that CFF populations are beginning to develop low levels of resistance to carbamate insecticides. CFF resistance levels will be monitored over time; however, to prevent further resistance, alternative control strategies will be required. In recent years, many essential oils (EOs) have been shown to possess insecticidal properties. These biopesticides provide environmentally sound alternatives to conventional pesticides. We evaluated 9 EOs extracted from plants in the Cupressaceae, Myrtaceae, Zygophyllaceae, Lamiaceae, Asteraceae, and Apiaceae for toxicity against adult CFF. Of the EOs tested, 6 were effective toxicants, particularly 3 from species of chamomile (Asteraceae): German, Roman, and Chinese chamomile. EO of Chinese chamomile, rich in oxygenated sesquiterpenes, showed the highest level of toxicity and thus the most promise for development as a biorational insecticide. Additional research is needed to determine the specific chemical components that confer toxicity to CFF. With the impending loss of methyl bromide, alternative fumigation strategies are needed for protection and disinfestation of postharvest commodities. ARS researchers in Miami, Florida, and Hilo, Hawaii, in collaboration with APHIS, developed fumigation protocols using ethyl formate (EF) to control OFF and CFF. We also evaluated combination treatments using EF with anisole (a volatile plant oil) for control of CFF and false spider mite. Results showed that addition of anisole improved efficacy of EF fumigation; the combination achieved complete control of both pests at lower concentrations than fumigation with either fumigant alone. Further testing will optimize EF and anisole concentrations to achieve synergistic fumigation protocols for postharvest pests. Laurel wilt (LW), a lethal disease of avocado and other U.S. laurel trees, is caused by a fungal symbiont of redbay ambrosia beetle. However, this pathogen has since transferred to at least 9 other beetle species, contributing to the loss of ~200,000 avocado trees in Florida. Sanitation practices (e.g., pruning, stumping, removal of infected trees) are used by growers to reduce the incidence of LW. To evaluate efficacy of cultural practices, scientists from UF, in collaboration with ARS researchers in Miami, Florida, investigated the effect of avocado canopy cover on ambrosia beetle abundance. Monitoring for 12 months indicated that groves with full canopy cover exhibited the highest number of beetles and the lowest light intensity. The opposite was found for topworked and newly planted groves. In addition, solar radiation had a significant effect on beetle dispersal flight. The results indicate that thinning canopy cover, which increases light intensity, suppresses ambrosia beetle abundance in commercial groves, thereby reducing the spread of LW. ACCOMPLISHMENTS 01 Repellents for pest ambrosia beetles. Redbay ambrosia beetle and tea shot-hole borer are vectors of laurel wilt and Fusarium dieback, respectively, two fungal diseases of avocado, woody ornamentals and native forest trees. Incorporating a repellent into pest management programs may reduce the incidence of these diseases. ARS researchers in Miami, Florida, identified piperitone as a new beetle repellent and compared its efficacy to two other repellents, verbenone and a- farnesene. Beetle captures in traps baited with lures were compared to captures in traps containing lures plus a repellent (a push-pull design) . Farnesene was ineffective; however, piperitone and verbenone were equally effective, reducing captures by 50-70% for 10-12 weeks. Since piperitone is less expensive than verbenone, the standard beetle repellent, these studies identify an economical alternative for management of both pests in commercial avocado groves. 02 Risk assessment of Hass avocado and Mexican laurels for attack by redbay ambrosia beetle. Redbay ambrosia beetle vectors the pathogen that causes laurel wilt, a lethal disease of avocado and other U.S. trees in the laurel family. First detected in Georgia in 2002, the wood- boring pest has since spread to 12 southeastern states. With time, it will likely enter Mexico, threatening native forest species and the Mexican avocado industry based on the Hass cultivar. In advance of an incursion, ARS researchers in Miami, Florida, and the Instituto de Ecología (Veracruz, Mexico) conducted research to assess the potential risk. Freshly cut wood from Hass avocado and 8 native laurels (collected from Veracruz) was shipped to Florida to determine the beetle⿿s boring preferences in lab bioassays and relative attraction in field tests. Results indicated that Hass avocado, Persea schiedeana, and Ocotea sp. are highly attractive species susceptible to beetle attack. These findings emphasize the need for effective, early detection systems for redbay ambrosia beetle to prevent severe economic and ecological impact of laurel wilt in Mexico. 03 Functional roles of tea shot-hole borer symbionts. Tea shot-hole borer (TSHB) vectors a fungus that causes Fusarium dieback, a destructive disease of avocado trees. Current lures contain quercivorol, an attractant produced by the beetle⿿s fungal symbionts. The lure contains a mixture of compounds, so the exact attractant is unknown. The beetle also carries multiple symbionts, and the function of each symbiont is unknown. ARS researchers in Miami, Florida, in collaboration with the University of Florida, conducted research with pure cultures of six symbionts to address these questions. A study of the fungal volatile emissions revealed that trans-p-menth-2-en-ol and limonene are beetle attractants. Another study identified which symbionts provide nutrition for beetle development, and which ones were most pathogenic to Florida avocado trees. This information will direct future research in development of improved management programs for TSHB and Fusarium dieback disease. 04 Nitric oxide (NO) fumigation for postharvest pest control. Postharvest pest control needs safe and effective alternative treatments due to the phase out of methyl bromide. NO is a newly discovered fumigant that has been demonstrated efficacious against a variety of insect pests and safe to fumigated products. ARS researchers in Miami, Florida, and Salinas, California, in collaboration with Kansas State University, developed fumigation protocols using NO to control ham mites, Tyrophagus putrescentiae, on ham meat under ultra-low oxygen conditions. The results showed that complete control was achieved for all mite life stages; eggs were the most tolerant stage, but 100% mortality was obtained after 48 and 24 h treatments at 0.5 and 1.0% NO concentrations, respectively. NO fumigation, a safe and effective treatment, provides a potential alternative to methyl bromide fumigation for postharvest pest control. 05 Insecticide resistance mechanisms of Diamondback moth. The diamondback moth (DBM), Plutella xylostella, is a destructive pest of cruciferous crops. Due to extensive use of chemical controls, DBM has evolved resistance to diamide insecticides, including chlorantraniliprole. To better understand the resistance mechanisms of DBM, ARS researchers in Miami, Florida, in collaboration with the Guangdong Academy of Agricultural Science (Guangdong, China) used a comparative transcriptomic approach to analyze the genes associated with insecticide resistance. The study identified 21 metabolism-related genes associated with resistance development to chlorantraniliprole and Bacillus thuringiensis insecticides. This research provides a better understanding of DBM resistance mechanisms, facilitating design of new insecticide combinations for improved pest management.

Impacts
(N/A)

Publications

Shabbir, M.N., Yang, X., Yin, F., Batool, R., Kendra, P.E., Li, Z. 2021. Bacillus thuringiensis and Chlorantraniliprole Trigger the Expression of Detoxification-Related Genes in the Larval Midgut of Plutella xylostella. Frontiers in Physiology. 12:780255. https://doi.org/10.3389/fphys.2021. 780255.
Revynthi, A.M., Cruz, L.F., Cannon, M.A., Crane, J.A., Kendra, P.E., Mannion, C., Carrillo, D. 2022. Evaluation of abamectin as a potential chemical control for the lychee erinose mite (Acari: eriophyidae), a new invasive pest in Florida. Florida Entomologist. 105(1):1-5. https://doi. org/10.1653/024.105.0101.
Kurtca, M., Tumen, I., Keskin, H., Tabanca, N., Yang, X., Demirci, B., Kendra, P.E. 2021. Chemical composition of essential oils from leaves and fruits of Juniperus foetidissima and their attractancy and toxicity to two economically important tephritid fruit fly species, Ceratitis capitata and Anastrepha suspensa. Molecules. 26(24):7504. https://doi.org/10.3390/ molecules26247504.
Yang, X., Liu, Y., Singh, R., Phillips, T.W. 2022. Nitric oxide fumigation for control of ham mite, Tyrophagus putrescentiae (Sarcoptiformes: Acaridae). Journal of Economic Entomology. 115(2):501-507. https://doi.org/ 10.1093/jee/toac014.
Revynthi, A.M., Velazquez Hernandez, Y., Canon, M.A., Greene, D.A., Vargas, G., Kendra, P.E., Mannion, C.M. 2021. Biology of Anthonomus testaceosquamosus (Coleoptera: Curculionidae): a new pest of tropical hibiscus. Insects. 13(1):13. https://doi.org/10.3390/insects13010013.
Kendra, P.E., Cruz, L.F., Tabanca, N., Menocal, O., Schnell, E.Q., Carrillo, D. 2022. Volatile emissions and relative attraction of the fungal symbionts of tea shot hole borer (coleoptera: curculionidae). Biomolecules EISSN 2218-273X. 12(1):97. https://doi.org/10.3390/ biom12010097.
Menocal, O., Kendra, P.E., Padilla, A., Chagas, P.C., Chagas, E.A., Crane, J.H., Carrillo, D. 2022. Influence of canopy cover and meteorological factors on the abundance of bark and ambrosia beetles (coleoptera: curculionidae) in avocado orchards affected by laurel wilt. Agronomy. 12(3) :547. https://doi.org/10.3390/agronomy12030547.
Yusufoglu, H.S., Alqarni, M.H., Salkini, M.A., Tabanca, N., Demirci, B., Kendra, P.E. 2021. Chemical composition of essential oils of Pulicaria species growing in Saudi Arabia and activity for Mediterranean fruit fly, Ceratitis capitata. Phytochemistry Letters. 46:51-55. https://doi.org/10. 1016/j.phytol.2021.08.021.
Sen-Utsukarci, B., Kessler, S.M., Akbal-Dagistan, O., Estep Iii, A.S., Tabanca, N., Kurkcuoglu, M., Demirci-Kayarian, S., Eroglu-Ozkan, E., Gul, Z., Bardacki, H., Becnel, J.J., Kiemer, A.K., Mat, A., Husnu Can Baser, K. 2021. Chemical composition and biological activities of valeriana dioscoridis sm. roots. South African Journal of Botany. 141:306-312. https://doi.org/10.1016/j.sajb.2021.05.007.
Kilic, M., Orhan, I., Eren, G., Okudan, E., Estep Iii, A.S., Becnel, J.J., Tabanca, N. 2021. Insecticidal activity of forty-seven marine algae species from the Mediterranean, Aegean and Sea of Marmara in connection with their cholinesterase and tyrosinase inhibitory activity. Chemosphere. https://doi.org/10.1016/j.sajb.2021.06.038.
Roh, G., Kendra, P.E., Cha, D.H. 2021. Preferential attraction of oviposition-ready oriental fruit flies to host fruit odor over protein food odor. Insects. 12(10). Article 909. https://doi.org/10.3390/ insects12100909.
Wheeler, G.S., Kendra, P.E., David, A.S., Lake, E.C., Sigmon, J., Palacios, J.N. 2021. Community of bark and ambrosia beetles (Coleoptera: Curculionidae) infesting Brazilian peppertree treated with herbicide and the volatile tree response. Environmental Entomology. 50(6):1311⿿1321. https://doi.org/10.1093/ee/nvab096.
Park, M., Lee, B., Yang, J., Kim, B., Roh, G., Kendra, P.E., Cha, D.H. 2021. Ethyl formate as a methyl bromide alternative for fumigation of citrus: Efficacy, fruit quality, and workplace safety. Journal of Economic Entomology. 114(6):2290-2296. https://doi.org/10.1093/jee/toab175.
Demiray, H., Estep Iii, A.S., Tabanca, N., Becnel, J.J., Demirci, B. 2022. Chemical constituents from Rheum ribes shoots and its insecticidal activity against Aedes aegypti. Revista Brasileira de Farmacognosia. https://doi.org/10.1007/s43450-021-00224-8.
Tsikolia, M., Tabanca, N., Kline, D.L., Demirci, B., Yang, L., Linthicum, K., Bloomquist, J.R., Bernier, U.R. 2022. Studies on the volatiles composition of stored sheep wool, and attractancy toward Aedes aegypti mosquitoes. Insects. 13(2):1-9. https://doi.org/10.3390/insects13020208.

Progress 10/01/20 to 09/30/21

Outputs
Progress Report Objectives (from AD-416): 1. Identify semiochemicals that mediate the behavior and physiology of exotic insect pests and develop semiochemical-based tools that lead to products for pest detection, behavioral disruption, and surveillance. [NP 304, Component 4, Problem Statement 4A] 1.A. Document behavioral responses and olfactory chemoreception to semiochemicals that have potential application in pest monitoring and control systems. 1.B. Identify and quantify insect semiochemicals; develop improved systems to collect, analyze and identify semiochemicals. 1.C. Develop synthetic and natural product lures based on host location and/or feeding cues that are formulated for effective use in IPM programs. 2. Evaluate �next generation� technologies to advance methods for fruit fly detection and eradication, such as lasers, sonic methods, or nano- technologies. [NP 304, Component 4, Problem Statement 4A] 2.A. Compare available remote trapping systems that can automatically count and identify trapped insects; optimize automated detection systems for adult fruit fly pests. 2.B. Evaluate imaging techniques and non-destructive chemical analysis methods for detection of larval infestation within host fruit. 3. Evaluate essential oils for natural insect pest toxicants and elucidate the physiological mechanisms underlying resistance to conventional pesticides. [NP 304, Component 4, Problem Statement 4A] 3.A. Determine the molecular/physiological basis for development of pesticide resistance in adult fruit flies. 3.B. Identify plant essential oils and plant extracts that have insecticidal, repellent, or oviposition deterrent properties against adult fruit flies and other target pests. 3.C. Determine the specific chemical components responsible for the toxic/ repellent effects of essential oils and plant extracts. 4. Integrate tools, technologies and management strategies to reduce the threat of pest establishment and mitigate the impact of exotic pest incursions. [NP 304, Component 4, Problem Statement 4A] 4.A. Develop new approaches to assess efficacy of lures, repellents, and control practices based on spatial statistics and contour analysis of trap captures in the field. 4.B. Develop improved IPM protocols for tephritid fruit flies, ambrosia beetles, and other target pests by integrating semiochemical-based technologies with other management options, such as biological control, judicious alternating use of insecticides to manage pesticide resistance, sanitation and other cultural practices. Approach (from AD-416): Research will consist of field and laboratory experiments to investigate aspects of basic biology, physiology and chemical ecology that can be exploited to develop integrated pest management approaches for invasive insects that impact subtropical agriculture. Target species will include pests that threaten to invade or have recently established in south Florida, including fruit flies in the family Tephritidae (Oriental fruit fly, Medfly, and Anastrepha species) and ambrosia beetles that vector fungal pathogens (redbay ambrosia beetle and Euwallacea shot-hole borers). Strategies will include (1) identifying semiochemicals from natural product sources that can alter insect behavior, such as attractants, repellents, and oviposition deterrents; (2) developing new detection and delimitation tools that will include formulated lures, discrete attract- and-kill bait stations, next generation technologies like smart traps for automated pest surveillance, and non-destructive imaging techniques for detection of hidden infestation in agricultural commodities; (3) improving pesticide resistance management by identification of pesticide modes of action and physiological responses of fruit flies to toxicants; (4) evaluating plant essential oils as sources of natural toxicants (biopesticides) with less environmental impact; and (5) developing new approaches to assess efficacy of lures, repellents, and control practices based on spatial statistics and contour analysis of trap captures in the field. Progress was made on Sub-objective 1a: Document behavioral responses and olfactory chemoreception to semiochemicals that have potential application in pest monitoring and control systems and Sub-objective 1b: Identify and quantify insect semiochemicals; develop improved systems to collect, analyze and identify semiochemicals. The following research was conducted by ARS researchers in Miami, Florida: Redbay ambrosia beetle and Euwallacea shot hole borers are vectors of laurel wilt and Fusarium dieback, two fungal diseases of avocado. Incorporation of beetle repellents into pest management programs may reduce the incidence of these diseases. Previously, ARS researchers in Miami, Florida, identified piperitone as a new repellent for Euwallacea. Subsequent studies compared piperitone to verbenone and a-farnesene. Euwallacea captures in traps baited with lures were compared to captures in traps containing lures plus a repellent. Farnesene was ineffective; however, piperitone and verbenone were equally effective, reducing captures by 50-70% for 10-12 weeks. Field tests in progress indicate that piperitone is also effective against redbay ambrosia beetle. Since piperitone is less expensive than verbenone, the standard beetle repellent, these studies identify an economical alternative for management of both pests. Manuscripts are in preparation to report this work. The invasive lychee erinose mite (LEM, Aceria litchii), recently introduced to Florida, is a high priority pest with potential to devastate the state�s lychee production. LEM primarily attacks new leaf growth and flower buds, thereby preventing fruit development. At present, there are no attractants for monitoring tools. ARS scientists in Miami, Florida, in collaboration with the University of Florida, initiated research to identify potential host kairomones by comparing volatiles emitted from various lychee tissues (new flush leaves, older leaves, and flower buds). Chromatographic analysis indicated that the attractive tissue samples were rich in sesquiterpenoids. Behavioral assays are ongoing to identify specific kairomones. The hibiscus bud weevil (Anthonomus testaceosquamosus) is a new invasive pest that negatively impacts the hibiscus industry in south Florida, resulting in large economic losses. Females oviposit in flower buds and larvae develop inside the bud, causing bud drop prior to flowering. In collaboration with the University of Florida, volatile emissions from leaves, buds, and open flowers from Hibiscus were distilled, analyzed, and identified. Qualitative and quantitative differences were observed in all three samples and saturated and polyunsaturated long-chain fatty acids were the dominant compounds. Work is ongoing to identify the volatile components that attract females to hibiscus buds. In collaboration with Odessa National Polytechnic University (Ukraine), research is being conducted with a series of thymol and carvacrol esters (propyl-, butyl, octyl- and benzyl-) to assess relative attraction of medfly. Combining behavioral bioassays with electroantennography to quantify olfactory response, four new male attractants were identified, with benzyl and octyl esters of thymol and carvacrol analogs being the most potent. Chemical analyses identified key structural features (specific functional groups attached to the aromatic ring) associated with attraction. This work increases our understanding of structure- function relationships in insect kairomones, and helps direct future research on development of improved medfly lures. Medfly is a serious pest of fruits and vegetables worldwide. Trimedlure is a synthetic male attractant currently used to monitor for this pest. However, APHIS recently identified a need for new male attractants. In collaboration with national and international scientists, ARS researchers in Miami, Florida, evaluated a series of essential oils for potential new attractants, including Tetradenia riparia (native to southern Africa), Magnolia citrata (Vietnam), Tanacetum annuum (Morocco), and three species of Pulicaria (Saudi Arabia). Studies included behavioral bioassays to assess male attraction and gas chromatography-mass spectrometry (GC-MS) analyses to document chemical constituents. Results show that several of these oils are highly attractive to male medfly. This work has been reported in several publications in 2021; however, investigations are still ongoing to identify specific attractive chemicals. Since essential oils are natural plant products, safe to the environment and inexpensive to produce, this research will lead to promising alternatives to Trimedlure for medfly detection and management. Black pod disease (Phytophthora palmivora) and insect pests cause a severe reduction in cacao production. Beetle holes were observed on infected pods but not on healthy pods, indicating that pod-boring beetles are likely attracted to lesions. Therefore, research was initiated by ARS researchers in Miami, Florida, to identify potential beetle attractants emitted by Phytophthora. Headspace volatiles were collected using solid- phase microextraction-gas chromatography-mass spectrometry (HS-SPME/GC-MS). However, the spectral complexity of isomers and large number of unknown compounds posed a significant challenge. Several synthetic chemicals were purchased to facilitate identification, but this is still in progress. This study complements data on disease-resistant cacao cultivars and a survey of bark and ambrosia beetles in Ecuador. The giant African snail (GAS) is an invasive pest that causes feeding damage to a wide variety of agricultural, ornamental, and native plants. In recent years, GAS has established in Florida, prompting an extensive eradication program. Preliminary studies showed that canine dogs can locate GAS on properties where previous human surveys did not. In collaboration with APHIS-PPQ, research was initiated to determine if signature volatiles are emitted from the mucus trails of GAS. If so, these chemical signals could provide the basis for detection of GAS by trained dogs. Although conventional distillation procedures were not successful, modified procedures were able to detect a series of semivolatile aliphatic aldehydes and acids. This project recently was awarded USDA Farm Bill funding. Caribbean fruit fly is a pest in Florida, the Caribbean, and Central America to over 100 hosts, particularly citrus and guava. Insecticidal baits are the primary method to control this pest. However, intensive insecticide uses can lead to an increase in resistance in pest populations. A protocol was developed to identify the level of resistance in wild flies to insecticides (organophosphate, carbamates etc.). Lab bioassays were conducted on adults (insecticide free strain) to establish baseline susceptibility to insecticide (i.e., methomyl) and compare to susceptibility of F2 generation collected from the field. Tests are ongoing to determine the resistance level of wild flies to various classes of insecticide. Progress was made on Sub-objective 3a: Determine the molecular/ physiological basis for development of pesticide resistance in adult fruit flies, Sub-objective 3b: Identify and quantify insect semiochemicals; develop improved systems to collect, analyze and identify semiochemicals, and Sub-objective 4b, Develop improved IPM protocols for tephritid fruit flies, ambrosia beetles, and other target pests by integrating semiochemical-based technologies with other management options, such as biological control, judicious alternating use of insecticides to manage pesticide resistance, sanitation and other cultural practices. The Caribbean fruit fly is a pest of numerous tropical and subtropical fruits. Extensive use of insecticides has led to environmental concerns and an increase in pesticide resistance. ARS scientists in Miami, Florida, conducted lab research to evaluate anisole, a plant based volatile compound, for toxicity to Caribbean fruit fly. Results showed that anisole was effective against all insect stages: eggs, larvae, pupae, and adults via contact, fumigation, and residue tests. Anisole treatments caused 100% mortality in all tests within 24 hours using appropriate concentrations. Results indicate that anisole has good potential as an environmentally sound alternative to conventional insecticides and fumigants. Tephritid fruit flies are serious pests of fruits and vegetables worldwide, causing severe export restrictions. Concerns about the impact of conventional pesticides on the environment and an increase in pesticide resistance in fruit flies have prompted research to identify environmentally sound alternatives. In collaboration with scientists from Turkey, ARS scientist in Miami, Florida, initiated an investigation of Juniperus foetidissima fruit and leaf essential oils for potential toxicity to adult female Caribbean fruit fly (Anastrepha suspensa). Commercial insecticides (i.e., methomyl, fenthion) were also tested as baselines to compare the toxicity of the two oils. Tests are ongoing to determine lethal effect of the oils at different concentrations. In collaboration with Animal and Plant Health Inspection Service (APHIS), ARS scientists in Hilo, Hawaii and Miami, Florida, developed postharvest fumigation protocols using ethyl formate to control Oriental fruit fly (Hilo) and Caribbean fruit fly (Miami). The objective is to develop standardized fumigation protocols applicable for multiple tephritid species. Results demonstrated that ethyl formate is effective against immature stages of both species. An additional test combining ethyl formate with anisole (a volatile plant oil) showed a synergistic effect for Caribbean fruit fly. Record of Any Impact of Maximized Teleworking Requirement: The COVID-19 pandemic and maximized telework resulted in delays for most of our research projects, particularly in the chemistry lab. It also delayed laboratory and field research for our collaborators at ARS-Hilo, Hawaii and at the University of Florida. Cancellation of conferences and workshops prevented exchange of information within the scientific community. ACCOMPLISHMENTS 01 Evaluation of synthetic fruit fly lures. The Caribbean fruit fly (Anastrepha suspensa) is a quarantine pest of citrus and a production pest of guava and other specialty fruits in Florida. Early pest detection and management requires effective lures. In collaboration with APHIS, ARS scientist in Miami, Florida, conducted lab and field studies to compare synthetic 2- and 3-component (2C, 3C) cone lures, the current standards for fruit fly monitoring. In field tests, 2C lures captured significantly more flies than 3C lures. Field longevity was 8 weeks for 2C lures and 6 weeks for 3C lures. In addition, scientists used new ion chromatography methods to measure lure emissions and relate levels of ammonia, putrescine, and trimethyl amine with fly attraction observed in the field. This study provides data requested by APHIS and benefits the regulatory agency with design of field monitoring protocols for pest fruit flies.

Impacts
(N/A)

Publications

Stappen, I., Wanner J., Tabanca, N., Bernier, U.R., Kendra, P.E. 2021. Blue Tansy Essential Oil: Chemical Composition, Repellent Activity Against Aedes aegypti and Attractant Activity for Ceratitis capitata. Nat. Prod. Commun. 16(2): 1�8. https://doi.org/10.1177/1934578X21990194
Luu-Dam, N., Tabanca, N., Estep Iii, A.S., Nguyen, D., Kendra, P.E. 2021. Insecticidal and attractant activities of magnolia citrata leaf essential oil against two major pests from diptera: aedes aegypti (culicidae) and ceratitis capitata (tephritidae). Molecules. 26(8): 2311. https://doi.org/ 10.3390/molecules26082311.
Kendra, P. E., N. Tabanca, W. S. Montgomery, J. Niogret, D. Owens, and D. Carrillo. 2020. Chapter 18. Essential oils as lures for invasive ambrosia beetles. Pp. 495-513 In: K. H. C. Baser and G. Buchbauer (eds.), Handbook of Essential Oils: Science, Technology, and Applications, 3rd edition. CRC Press, Boca Raton, FL. (ISBN 9780815370963)
Blythe, E.K., Tabanca, N., Demirci, B., Kendra, P.E. 2020. Chemical composition of essential Oil from Tetradenia riparia and its attractant activity for Mediterranean fruit fly, Ceratitis capitata. Nat. Prod. Commun. 15(9): 1�6. https://doi.org/10.1177/1934578X20953955
Hoeferi, M., Wanner, J., Tabanca, N., Abbas, A., Gochev, V., Schmidt, E., Kaul, V.K., Singh, V., Jirovetz, L. 2020. Biological Activity of Matricaria chamomilla Essential Oils of Various Chemotypes. Planta Medica International Open. 7: e114�e121. https://doi.org/10.1055/a-1186-2400.
Kwon, T.H., Park, C.G., Lee, B., Zarders, D.R., Roh, G.H., Kendra, P.E., Cha, D.H. 2020. Ethyl formate fumigation and ethyl formate plus cold treatment combination as potential phytosanitary quarantine treatments of Drosophila suzukii in blueberries. Journal of Asia Pacific Entomology. 24(1):129-135. https://doi.org/10.1016/j.aspen.2020.11.008.
Cruz, L. F., O. Menocal, P. E. Kendra, and D. Carrillo. 2021. Phoretic and internal transport of Raffaelea lauricola by different species of ambrosia beetle associated with avocado trees. Symbiosis. https://doi.org/ 10.1007/s13199-021-00776-2.
Al-Massarani, S.M., El-Gamal, A.A., Al-Rehaily, A.J., Al-Sheddi, E.S., Al- Oqail, M.A., Farshoori, N.N., Estep Iii, A.S., Tabanca, N., Becnel, J.J. 2021. Insecticidal activity and free radical scavenging properties of isolated phytoconstituents from the saudi plant nuxia oppositifolia (hochst). Molecules. 26(4):1-8. https://doi.org/10.3390/molecules26040914.
Tsikolia, M., Opatz, T., Kauhl, U., Tabanca, N., Demirci, B., Tenbroeck, S. , Linthicum, K., Bernier, U.R. 2021. Trials for gathering information on an unknown peak in the GC-MS spectra of horse and pony hair extracts. Advances in Entomology. 9(2):100-111. https://doi.org/10.4236/ae.2021. 92009.
Yang, X., Liu, Y.-B. 2021. Anisole is an environmentally friendly fumigant for postharvest pest control. Journal of Stored Products Research. 93. Article 101842. https://doi.org/10.1016/j.jspr.2021.101842.