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Research Project: Methyl Bromide Replacement: Mitigation of the Invasive Pest Threat from the American Tropics and Subtropics

Location: Subtropical Horticulture Research

2020 Annual Report

1. Identify semiochemicals that mediate the behavior and physiology of exotic insects such as tephritid (Ceratitis and Anastrepha) and drosophilid (Zaprionus indianus) fruit flies, the redbay ambrosia beetle, the cocoa pod borer, and other new invasive pests from the Caribbean and Central and South America. 1.A. Determine and document behavioral and olfactory responses to semiochemicals that have potential use in monitoring and control systems. 1.B. Identify and quantify insect semiochemicals; develop improved systems to collect and identify semiochemicals. 2. Develop semiochemical- and/or chemical-based technologies that lead to products for detection, behavioral disruption, or surveillance of fruit flies. 2.A. Develop synthetic and natural product lures based on host location and/or feeding cues that are formulated for effective use in integrated pest management. 2.B. Develop optimized trapping and control systems based on chemical lures in combination with traps and bait stations. 2.C. Develop novel, high-tech solutions for OFF detection and eradication, such as lasers, sonic methods, or nano-technologies. For example, traps could be developed that can automatically identify trapped insects based on wing beat frequency, size, weight, or protein content. 3. : Develop practical systems for integrated pest management using semiochemical and other detection-based technologies to reduce the threat of importation and establishment of exotic pests from foreign tropical and subtropical environments by suppressing and/or controlling the population at the source. An insect toxicologist is needed to identify the modes of actions for different pesticide classes on the OFF, and the physiological mechanisms for pesticide resistance development. This research will lead to improved chemical control strategies and reduce pesticide resistance development. 3.A. Develop IPM tools for tephritid fruit flies using semiochemical-based technologies for use by regulatory agencies and growers; develop new approaches including improved protocols and spatial analysis techniques to assess monitoring approaches and control systems such as attract-and-kill technologies. 3.B. Stereochemistry of selected compounds and structural characteristics may play a highly significant role in efficacy of kairomones needed for pest detection and control. The correlations of active components such as configuration, functional groups and identification of cis-, trans- and chiral isomers can influence on the bioactivity. 3.C. Develop pesticide resistance management for fruit fly control by 1) identifying the modes of actions for different pesticide classes on OFF and 2) determining the physiological mechanisms for pesticide resistance development in Oriental fruit fly and other fruit flies.

Research will consist of field and laboratory experiments to determine the behavior, physiology and chemical ecology of insect pests that can be used in the development of monitoring and control techniques. Strategies will include developing new detection and delimitation tools that will include baits, discrete attract-and-kill devices for insect pests, next generation technologies such as detectors and robots, and network theory and optimization that will improve detection of the Oriental fruit fly and relatives; identifying pesticide modes of action on and physiological response of Oriental fruit fly and relatives for improved pesticide resistance management; identifying new semiochemicals through investigations of plant essential oils and stereochemistry of plant compounds, and discovering components in the insect's biology that can be exploited for control measures for exotic insect pests that affect plant production.

Progress Report
Objective 1: FY2020 concludes this 5-year research project. Identified a-copaene as a new attractant for Euwallacea nr. fornicatus. ARS scientists in Miami, Florida, discovered that a-copaene is attractive to E. nr. fornicatus, vector of Fusarium dieback of avocado in California and Florida. Euwallacea was first detected in Florida in 2012 at low levels, but since 2016 beetle numbers and tree damage continue to increase in commercial groves. Impact: Discovery of this new host-based attractant (kairomone) led to development of an improved two-component lure for E. nr. fornicatus, achieving more effective pest detection. Identified new reproductive hosts for Euwallacea. Avocado is a preferred host of E. nr. fornicatus. However, in a 12-month survey in south Florida, ARS scientists in Miami, Florida, identified new hosts, including the fruit crop soursop, Annona muricata, and native Lysiloma latisiliquum and Albizia lebbeck. Chemical analyses indicated that a-copaene (a known attractant) is high in avocado, but not in the other hosts, suggesting that additional kairomones are used by beetles for host location. Impact: The host range of E. nr. fornicatus is expanding in Florida and other cultivated and native trees are at risk for attack. Infested native trees may function as reservoirs for pest populations capable of invading adjacent avocado groves. Evaluated Mexican Lauraceae for susceptibility to redbay ambrosia beetle (RAB). All U.S. hosts of RAB are trees in the family Lauraceae. Since first detected in Georgia in 2002, RAB has invaded 10 additional states in the southeast. As its range expands, RAB will likely enter Mexico. In collaboration with the Instituto de Ecología (Veracruz, Mexico), 8 native Mexican laurels and Hass avocado (the major cultivar in Mexico and California) were evaluated by ARS scientists in Miami, Florida, for risk of RAB attack. Beetle preferences were then correlated with chemical content of the wood. Impact: This risk assessment identified new species highly attractive to RAB, including Hass avocado. Chemical analyses identified potential new kairomone attractants to incorporate into lures for improved RAB detection. Developed improved method to quantify attractants from fruit fly lures. With APHIS-PPQ collaborators, a new ion chromatography (IC) method was developed by ARS scientists in Miami, Florida, to quantify 3-component (3C) cones containing biogenic amine salts (ammonium acetate, putrescine, and trimethylamine; the lure for female Medfly). The previous method required two instruments [IC and Liquid Chromatography-Mass Spectrometry (LC-MS)], and double sample preparation. With the new method, all components are quantified using only IC, thus saving time and reducing waste. Impact: This improved method provides a faster and more cost-effective means for quality control of total content and emission rates of these lures. Objective 2: Developed an improved lure for redbay ambrosia beetle (RAB). Previous research by ARS scientists in Miami, Florida, identified cubeb oil as a new lure for RAB; however, the specific attractants were unknown. Fractional distillation was used to separate whole cubeb oil into 17 component fractions (based on molecular weight), which were then used for bioassays and electroantennography. Fractions high in a-copaene and a-cubebene were the most attractive. Based on these results, a prototype lure was developed containing an oil enriched to 50% a-copaene content. In field trials, the copaene lure captured significantly more beetles than cubeb lures and lasted for 3 months. Impact: The enriched a-copaene lure is highly attractive, long lasting, and has become the standard lure for RAB surveillance in the U.S. and Mexico. Developed a two-component lure for better detection of Euwallacea. After discovering that a-copaene is an attractant for E. nr. fornicatus, ARS scientists in Miami, Florida, evaluated the kairomone alone and in combination with quercivorol (the initial lure developed for this pest). Results indicated that the combination was synergistic, significantly increasing beetle captures compared to quercivorol alone. Impact: The two-component lure has become the standard for detection of Euwallacea. It has been used in Florida surveys to determine pest prevalence in the avocado production area; it also has been adopted by SAGARPA (Mexico's Secretary of Agriculture) for dual detection of E. nr. fornicatus and redbay ambrosia beetle. Identified piperitone as a new repellent for Euwallacea. Incorporation of a repellent into pest management programs may reduce the incidence of Fusarium dieback in avocado groves. In 2018, ARS scientists in Miami, Florida, identified piperitone as a new repellent for E. nr. fornicatus. Subsequent studies compared efficacy of piperitone to two other repellents, verbenone and a-farnesene. Beetle captures in traps baited with the 2-component lure were compared to captures in baited traps that also contained 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. Impact: Since piperitone is less expensive than verbenone, the standard repellent, this study identifies an economical alternative for management of E. nr fornicatus in avocado groves. Objective 3: Patented a wax-based attract-and-kill device for Medfly. Medfly is the most important pest of fruits and vegetables worldwide. ARS scientists in Miami, Florida, in collaboration with APHIS scientists in Guatemala, developed a Medfly bait station. It consists of a box-like device coated with a wax matrix containing a feeding stimulant (sugar), visual cue (green color), and pesticide. A three-component lure (invented by ARS scientists in Miami, Florida, and patented by ARS) is placed inside the device to attract flies. Once flies land, they detect the sugar and feed, obtaining a lethal dose of pesticide. Field tests indicate that these devices provide control equivalent to that of broadcast sprays. Impact: This device provides an effective, targeted, environmentally-sound method for Medfly suppression. U.S. patent issued 9 January 2018 (serial no. 9861090). Determined the sampling range of lures for Euwallacea. In 2017, ARS scientists in Miami, Florida, developed an improved lure for Euwallacea by combining a-copaene with quercivorol. The two-component lure is now used in Florida and Mexico. Subsequent research utilized a series of release-recapture experiments with marked beetles to calculate the effective sampling range (attractive distance) of the two attractants deployed alone and in tandem. Results demonstrated that the combination lure provides more sensitive pest detection, capturing significantly more released beetles than either lure alone. The estimated sampling range indicates 30-40 m spacing between monitoring traps baited with the two-component lure. Impact: Research provides data needed by action agencies for optimization of pest surveillance programs in avocado groves. Determined the stereochemistry of emissions from ambrosia beetle lures. A new lure for E. nr. fornicatus was developed by ARS scientists in Miami, Florida, by combining a fungal volatile (quercivorol) with an essential oil enriched in the host kairomone a-copaene. Both the oil and quercivorol contain multiple components. Laboratory analyses indicated that the attractive chemical in the oil is the negative enantiomer of a-copaene. The quercivorol lure contains 4 isomers, but the attractant is most likely a trans- isomer of p-menth-2-en-1-ol. Impact: Identification of the specific attractive components and their stereochemistry increases our understanding of ambrosia beetle semiochemicals and will facilitate future lure improvement. Identified structure-activity relationships as predictors of fruit fly attraction. In collaboration with the Università di Napoli Federico II (Naples, Italy), a series of 29 structurally-related aromatic compounds were investigated by ARS scientists in Miami, Florida, for potential attraction of Medfly. Using lab bioassays and electroantennography, four new male attractants were identified, with o-eugenol being the most potent. Chemical analyses identified key structural features (specific functional groups attached to the aromatic ring) associated with attraction of this pest. Impact: This study increases our understanding of chemical structure-function relationships in insect kairomones, and will help direct future research on development of improved lures for Medfly. Evaluated pesticides for Oriental fruit fly (OFF). A collaboration between scientists at ARS in Miami, Florida, and the University of Florida was initiated to compare efficacy of insecticides on OFF in Ghana. In lab evaluations of eight products, four insecticides were identified to achieve high mortality in both sexes (Fenitrothion + Fenvalerate, Emamectin benzoate, Lambda cyhalothrin, and Imidacloprid + Betacyfluthrin). In a field trial, Fenitrothion + Fenvalerate showed the most promise, achieving the lowest adult captures and lowest level of larval infestation. Impact: An effective pesticide was identified for OFF; future research will evaluate its utility in bait stations or as an option for alternate use in a resistance management program.

1. Developed a kairomone-based lure for cocoa pod borer. Cocoa pod borer is the most serious insect pest impacting cacao production in Southeast Asia. Pest detection currently relies on a synthetic pheromone lure attractive to male moths, but the lures are expensive and products from different sources are highly variable in efficacy. Under a Cooperative Research and Development Agreement, researchers at ARS in Miami, Florida, conducted laboratory and field evaluations of various plant essential oils and fruit extracts, and a male-specific kairomone-based attractant was discovered. Subsequent research optimized the dose, formulation, and other modifications to develop a kairomone lure that performs as well or better than the pheromone lure. Moreover, the kairomone lure has a significantly longer field life and costs much less to produce than the pheromone lure. A patent application has been submitted for this invention.

2. Compared trap designs to optimize detection of Euwallacea in avocado groves. Optimal pest detection requires not only a good lure, but also an appropriate trap design. ARS scientists in Miami, Florida, conducted field tests in avocado groves to compare efficacy of four trap types for detection of Euwallacea nr. fornicatus, vector of Fusarium dieback. All traps were baited equally with a two-component lure (a-copaene plus quercivorol). Two trap types were found to be more effective than the 8-unit Lindgren funnel trap (the current standard for monitoring ambrosia beetles), but highest captures were consistently obtained with sticky panel traps. In addition to better trapping efficacy, sticky panels are much less expensive than funnel traps. This research identified a trap-lure combination that is more effective and more economical than the standard Lindgren funnel trap. This will benefit growers and regulatory agencies with improved pest detection and reduced costs in surveillance programs for E. nr. fornicatus in commercial groves.

3. Developed a new method to isolate kairomones for Mediterranean fruit fly. The Mediterranean fruit fly (Medfly), Ceratitis capitata is one of the most destructive agricultural pests in the world. New attractants could improve early detection and suppression of this pest. In 2017, ARS scientists in Miami, Florida, discovered that tea tree oil is highly attractive to male C. capitata. To isolate the active components, a new method was developed using a combination of thin-layer chromatography (TLC) and laboratory bioassays to directly locate attractants on the TLC plate. When medflies were exposed to the TLC chromatogram, biologically active zones were clearly distinguished. In addition, electroantennography indicated that olfactory responses correlated well with bioassay results. This novel method provides not only a rapid screening tool for deciding which essential oils should be selected for further study but also provides identification of bioactive compounds for potential improvement of Medfly lures. This method will have broad application for semiochemical-based studies on a variety of plant extracts and insect pests.

Review Publications
Niogret, J., Ekayanti, A., Kendra, P.E., Ingram, K., Lambert, S., Epsky, N., Marelli, J. 2020. Host preferences of the cocoa pod borer, Conopomorpha cramerella (Lepidoptera:Gracillariidae), the main threat to cocoa production in Southeast Asia. Entomologia Experimentalis et Applicata. 168(3): 221-227.
Kendra, P.E., Montgomery, W.S., Narvaez, T.I., Carrillo, D. 2020. Comparison of trap designs for detection of Euwallacea nr. fornicatus and other Scolytinae (Coleoptera: Curculionidae) that vector fungal pathogens of avocado trees in Florida. Journal of Economic Entomology. 113(2): 980-987.
Ali, A., Tabanca, N., Demirci, B., Raman, V., Budel, J., Baser, K., Khan, I. 2020. Insecticidal and biting deterrent activities of Magnolia grandiflora essential oils and selected pure compounds against Aedes aegypti. Molecules. 25(6), 1359.
Carrillo, D., Cruz, L.F., Revynthi, A.M., Duncan, R.E., Bauchan, G.R., Ochoa, R., Kendra, P.E., Bolton, S.J. 2020. Detection of the lychee erinose mite, Aceria litchii (Keifer) (Acari: Eriophyidae) in Florida, USA: A comparison with alien populations. Insects. 11(4):235.
Tabanca, N., Niogret, J., Kendra, P.E., Epsky, N.D. 2020. TLC-based bioassay to isolate kairomones from tea tree essential oil that attract male Mediterranean fruit flies, Ceratitis capitata (Wiedemann). Biomolecules EISSN 2218-273X. 10(5): 683.
Tabanca, N., Nalbanstoy, A., Kendra, P.E., Demirci, F., Demirci, B. 2020. Chemical characterization and biological activity of mastic gum essential oils from Pistacia lentiscus var. chia from Turkey. Molecules. 25(9): 2136.
Tabanca, N., Masi, M., Epsky, N.D., Nocera, P., Cimmino, A., Kendra, P.E., Niogret, J., Evidente, A. 2019. Laboratory evaluation of natural and synthetic aromatic compounds as potential attractants for male mediterranean fruit fly, ceratitis capitata. Molecules. 24(13):2409.
Sen-Utsukarci, B., Tabanca, N., Estep, A.S., Akbal-Dagistan, O., Kessler, S.M., Ozturk, Z., Becnel, J.J., Kiemer, A.K., Mat, A. 2020. The cytotoxicity and insecticidal activity of extracts from Delphinium formosum Boiss. & Huet. Istanbul Journal of Pharmacy. 49(3):148-153.
Sen-Utsukarci, B., Taskin, T., Goger, F., Tabanca, N., Estep, A.S., Kessler, S.M., Akbal-Dagistan, O., Bardakci, H., Kurkcuoglu, M., Becnel, J.J., Kiemer, A.K., Mat, A. 2019. Chemical composition and antioxidant, cytotoxic, and insecticidal potential of Valeriana alliariifolia in Turkey. Archives of Industrial Hygiene and Toxicology. 70(3):207-2018.
Ali, A., Li, A.Y., Tabanca, N., Ali, Z., Khan, I.A. 2019. Insecticidal and repellent activities of cinnamates against mosquitoes and ticks. International Journal of Current Research. 11(09):6814-6818.
Al-Massarani, S., El-Shaibany, A., Tabanca, N., Ali, A., Estep, A.S., Becnel, J.J., Goger, F., Demirci, B., El-Gamala, A., Baser, K. 2019. Assessment of selected Saudi and Yemeni plants for mosquitocidal activities against the yellow fever mosquito Aedes aegypti. Saudi Pharmaceutical Journal. 27(7):930-938.
Revynthi, A. M., R. E. Duncan, C. Mannion, P. E. Kendra, and D. Carrillo. 2020. Post-harvest paraffinic oil dips to disinfest lychee fruit from lychee erinose mite. Florida Entomol. 103(2): 299-301.