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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

2019 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
Replicate field tests were conducted in avocado groves to compare efficacy of four trap designs for detection of ambrosia beetles. In traps baited with the 2-component lure (a-copaene plus quercivorol), there were significant differences in captures of Euwallacea near fornicatus, the vector of Fusarium dieback disease. With this beetle, two trap types were more effective than Lindgren funnel traps, the current standard for pest monitoring. In traps baited with ethanol lures (a general attractant for ambrosia beetles), a community of 20 beetles was detected, including species known to be secondary vectors of laurel wilt. Efficacy of trap design varied by species, but in general sticky panel traps were more effective than Lindgren funnel traps. Tests are ongoing to determine the effect of ethanol dose on the number of beetles captured and the species detected in groves with laurel wilt. Incorporation of a repellent in management programs for E. nr. fornicatus may reduce the incidence of Fusarium dieback in avocado groves. In FY18, we identified piperitone as a new repellent for E. nr fornicatus. Ongoing studies are comparing efficacy of piperitone to two other repellents, verbenone and a-farnesene, all formulated in plastic bubble dispensers. Field tests compared beetle captures in traps baited with 2-component lure versus captures in baited traps that also contained a repellent dispenser. In addition, chemical analyses are underway to quantify repellent emissions. Initial results indicate that farnesene is ineffective; however, efficacy of piperitone and verbenone is comparable, reducing captures by 50-70%. Since piperitone is less expensive than verbenone, this study identifies an economical alternative repellent for pest ambrosia beetles. During gallery excavation, female ambrosia beetles inoculate host trees with spores of symbiotic fungi, and subsequently ‘farm’ fungal gardens that provide their food. With E. nr. fornicatus, only two attractants are known, one of which is quercivorol (an isomer of p-menthenol), a food-based attractant emitted by fungal symbionts. In collaboration with the University of Florida, volatile emissions from six symbiont species have been collected, analyzed, and identified by ARS scientists in Miami, FL. Each species has different patterns of emissions and ratios of cis- and trans-p-menthenol. Work is ongoing to document temporal patterns in emissions and to determine if there are additional attractants emitted by Euwallacea symbionts. An evaluation of plant essential oils demonstrated that tea tree oil is highly attractive to male Mediterranean fruit flies. To identify the attractive component(s) in this oil, a bioassay-directed investigative approach was developed using thin-layer chromatography (TLC). Tea tree oil was fractionated by preparative TLC, yielding five fractions for further evaluation. In bioassays and electroantennographic analyses, one fraction was found to elicit strong attraction and strong olfactory responses. A second fraction was found to be moderately attractive. Work is in progress to isolate and identify the specific attractive chemicals in those fractions. In collaboration with APHIS-PPQ (Miami, Florida), a new ion chromatography (IC) method was developed to quantify 3-component (3C) cones containing biogenic amine salts (ammonium acetate, putrescine, and trimethylamine; the lure for Mediterranean fruit fly). The previous method required the use of two complex and expensive instruments [IC and Liquid Chromatography-Mass Spectrometry (LC-MS)], as well as double sample preparation. With the new method, all three components are quickly quantified using only IC, thus saving time and waste generation. The method was also validated so that low amounts of each analyte (i.e. volatile emissions) can be quantified. This improved method provides an easier, faster and more cost-effective means for quality control of the total content and emission rate of these lures. A similar method, now undergoing validation, was developed for 2C cones (ammonium acetate and putrescine; the lure for Anastrepha fruit flies), which will also reduce analysis time, cost and waste. As a first step in developing new attract-and-kill technologies for Oriental fruit fly, a collaboration with the University of Florida was initiated to compare efficacy of insecticides on pest populations 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 2018 field trial in a mango orchard, these insecticides were compared by tree canopy spraying, followed by population monitoring with baited traps for adults and fruit collection for larval detection. Of the four, Fenitrothion + Fenvalerate showed the most promise, achieving the lowest captures and lowest level of infestation. Replicate field trials are planned for 2019 to confirm these results. Cocoa pod borer is the most serious insect pest impacting cacao production in Southeast Asia. Pest detection currently uses a synthetic pheromone lure attractive to male moths, but the lures are expensive and products from different sources vary in efficacy. Under a previous Cooperative Research and Development Agreement, laboratory and field evaluations of plant essential oils and extracts were conducted in Indonesia, and a male-specific kairomone-based attractant was discovered. Research is ongoing to identify the attractive chemicals and to optimize dose, formulation, and other modifications to develop a kairomone lure equal to or better than the pheromone lure. An invention disclosure is in preparation to determine patent potential for this discovery. Fungal diseases and insect pests greatly compromise cacao production worldwide. Pod-boring beetles are attracted to lesions on cacao pods resulting from black pod disease (caused by Phytophthora palmivora). Following emergence from infected pods, beetles may act as vectors of black pod disease by transporting viable pathogen propagules. To identify potential beetle attractants emitted by Phytophthora, volatiles were collected from healthy and infected pods, and identification of compounds is in progress. The study complements data on disease resistant cultivars and a survey of pod-boring beetles in Ecuador. Recently established in Florida, the lychee erinose mite is a high priority pest with potential to devastate the state’s lychee production. The mite preferentially attacks new growth, including developing flower buds, thereby preventing fruit development. At present, there are no known attractants or monitoring tools. In collaboration with the University of Florida, research was initiated to identify potential host kairomones. Steam-distillation and solvent-free sample extraction techniques were developed to analyze various tissues from lychee (e.g. new flush leaves, older leaves, flower buds). Samples were found to be rich in sesquiterpenoids and analyses are in progress to identify chemical constituents in attractive tissues. Additionally, bioassays will be designed to determine specific compound(s) responsible for attraction. Giant African snails pose an imminent threat to U.S. agriculture and natural resources, directly through their feeding damage and indirectly through the costs incurred for control. In collaboration with APHIS-PPQ, research was initiated to determine if this pest emits unique volatile chemicals. If so, that chemical signature will be explored for potential use by trained dogs for improved pest detection.

1. Determined the sampling range of lures for pest Euwallacea ambrosia beetles. Invasive beetles similar to tea shot-hole borer (Euwallacea fornicatus, native to Asia) are now established in California and Florida. Members of this cryptic species complex (collectively E. near fornicatus) vector fungal pathogens that cause Fusarium dieback in host trees, including avocado. ARS researchers in Miami, Florida, identified a-copaene as a new attractant for Euwallacea and developed a 2-component lure that combines a-copaene with quercivorol (the only other known attractant). The combination lure is now used in Florida and Mexico. New research utilized (1) release-recapture experiments with marked beetles to calculate the effective sampling range (attractive distance) of the two attractants deployed alone and in tandem, (2) spatial analysis to determine the effects of wind speed and direction on female dispersal patterns and lure sampling range, and (3) laboratory flight mill studies to measure the flight capacity of host-seeking females. Results demonstrated that the 2-component lure provides more sensitive pest detection, capturing significantly more released beetles than either lure alone. In addition, results indicate that spacing should be 30-40 m between monitoring traps baited with the 2-component lure. This research provides data needed by action agencies for optimization of surveillance programs for pest Euwallacea.

2. Identified structure-activity relationships as predictors of kairomone activity. In collaboration with chemists from the Università di Napoli Federico II (Naples, Italy), ARS researchers in Miami, Florida, investigated a series of 29 structurally-related natural and synthetic aromatic compounds, which differed in functional groups, for potential attraction of the Mediterranean fruit fly. Using a combination of short-range laboratory bioassays and electroantennography (to quantify behavioral and olfactory responses, respectively), four new male attractants were identified, with o-eugenol being the most potent. Results demonstrated that the four compounds contain key structural features (specific functional groups attached to the aromatic ring) associated with attraction of this pest. This study increases our understanding of chemical structure-function relationships in insect kairomones (host-based attractants), and will help direct future research on development of improved lures for Mediterranean fruit fly.

Review Publications
Epsky, N.D., Niogret, J. 2017. Short range attraction of Ceratitis capitata (Diptera: Tephritidae) sterile males to six commercially available plant essential oils. Natural Volatiles & Essential Oils. 4(1):1-7.
Niogret, J., Epsky, N.D. 2018. Attraction of Ceratitis capitata (Diptera: Tephritidae) sterile males to essential oils: the importance of linalool. Environmental Entomology. 47(5):1287-1292.
Tsikolia, M., Bernier, U.R., Agramonte, N.M., Estep, A., Becnel, J.J., Tabanca, N., Linthicum, K., Gross, A.D., Guerin, P.M., Krober, T., Bloomquist, J.R. 2018. Insecticidal and repellent properties of novel trifluoromethylphenyl amides II. Pesticide Biochemistry and Physiology. 151:40-46.
Al-Massarani, S., Tabanca, N., Farshori, N. 2018. Headspace-SPME/GC-MS Analysis of the Anethum graveolens volatiles from Saudi Arabia with different fiber coatings. Natural Volatiles & Essential Oils. 5(4):29-34.
Tabanca, N., Demirci, B., Aytac, Z., Kemal Husnu, C. 2018. The chemical composition of Salvia verticillata L. subsp. verticillata from Turkey. Natural Volatiles & Essential Oils. 4(1):18-28.
Yusufoglu, H.S., Tabanca, N., Bernier, U.R., Li, A.Y., Salkini, M.A., Alqasoumi, S.I., Demirci, B. 2018. Mosquito and tick repellency of two Anthemis essential oils from Saudi Arabia. Saudi Pharmaceutical Journal. 26(6):860-864.
Stappen, I., Tabanca, N., Ali, A., Wanner, J., Lal, B., Jaitak, V., Wedge, D.E., Kaul, V.K., Schmidt, E., Jirovetz, L. 2018. Antifungal and repellent activities of the essential oils from three aromatic herbs from western Himalaya. Open Chemistry. 16:306-316.
Nesterkina, M., Bernier, U.R., Tabanca, N., Kravchenko, I. 2018. Repellent activity of monoterpenoid esters with neurotransmitter amino acids against yellow fever mosquito, Aedes aegypti. Open Chemistry. 16:95-98.
Altintop, M.D., Tabanca, N., Becnel, J.J., Bloomquist, J.J., Kaplancikli, Z.A., Özdemir, A. 2018. Synthesis and mosquitocidal activity of a series of hydrazone derivatives against Aedes aegypti. Letters in Drug Design & Discovery.
Tsikolia, M., Bernier, U.R., Wedge, D.E., Tabanca, N., Abboud, K.A., Linthicum, K. 2019. Fungicidal properties of some novel trifluoromethylphenyl amides. Chemistry and Biodiversity.
Owens, D., Seo, M., Montgomery, W.S., Rivera-Mamounis, M.J., Stelinski, L.L., Kendra, P.E. 2019. Dispersal behavior of Euwallacea nr. fornicatus (Coleoptera: Curculionidae: Scolytinae) in avocado groves and estimation of lure sampling range. Agricultural and Forest Entomology. 21(2):199-208.
Niogret, J., Ekayanti, A., Ingram, K., Lambert, S., Kendra, P.E., Alborn, H.T., Epsky, N.D. 2019. Development and behavioral ecology of Conopomorpha cramerella (Lepidoptera: Gracillariidae). Florida Entomologist. 102(2):382-387.