Research Project: Mitigation of Invasive Pest Threats to U.S. Subtropical Agriculture

Location: Subtropical Horticulture Research

2024 Annual Report

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 ‘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
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 Report
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.

Accomplishments
1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

Review 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.