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ARS Home » Southeast Area » Gainesville, Florida » Center for Medical, Agricultural and Veterinary Entomology » Chemistry Research » Research » Research Project #430032

Research Project: Insect, Nematode, and Plant Semiochemical Communication Systems

Location: Chemistry Research

2020 Annual Report

1. Develop new improved attractants for weevils (Anthonomus pepper and cranberry weevils and Sitophilus maize and rice weevils) based on combinations of host plant kairomones and/or aggregation pheromones. 2. Develop pheromones and kairomones to improve the efficacy of mass-reared entomophagous nematodes used in biocontrol. 3. Develop new technologies to detect and control invasive arthropod pests. 3A. Develop kairomone-based attractants and repellants to control arthropod pests of honey bees, including the Varroa mite and the small hive beetle. 3B. Identify microbe-generated semiochemicals that influence insects or microbes, for example nectar microbes that increase pollinator visits to flowering crops. 3C. Identify volatile biomarkers for insect-infested crop products, such as fruit fly infested tomatoes, bananas, and mangoes.

Develop new and improved attractants for pest weevils based on combinations of host plant kairomones and/or aggregation pheromones. Develop pheromones and kairomones to improve the efficacy of mass-reared entomophagous nematodes used in biocontrol. Develop and test host plant volatile- and/or pheromone-based attractants and/or repellants to control arthropod pests of honey bees, including varroa mite and small hive beetle. Elucidate kairomone-based communication systems of tephritid fruit flies and the impact of kairmones on accelerated development of sexual signaling and reproductive maturity. This research will utilize numerous interactive laboratory- and field-based bioassays with insects, mites, nematodes, and plants, as well as purified biochemicals and other organisms. Isolation and identification of new bioactive chemicals that mediate arthropod and nematode behaviors and plant-arthropod/nematode interactions will be achieved using a combination of approaches including preparative GC, HPLC, preparative flash chromatography, GC-MS, FT0IR, NMR, micro-degradation, and synthesis where applicable. Major target insects for this research will include pest Coleoptera and Diptera that attack fruit and vegetable, Coleoptera and Acarina that impact honey bees, and Nematoda that control root insects. Other target insects may be selected as needed during progression of the project.

Progress Report
This is the final report for this 5-year research project. No host plant volatiles were identified as improving the attraction of the cranberry weevil aggregation pheromone. After evaluating trap location and best trap design, the attractant pheromone was optimized by field testing of various component blends. For pepper weevil, female-produced oviposition deterring pheromones were identified and the blend optimized in laboratory assays, with candidate blends being forwarded for evaluation in field trapping studies. A previously established host plant-based attractant gave mixed results in field trials. The attractant blend was re-developed by combining damage induced, fresh damaged related, and fruit associated volatiles. Laboratory olfactometer-guided testing resulted in a more complex blend with significantly attraction. The blend will be further optimized and field tested in south Florida. Both pheromone blends will be evaluated for patentability prior to publication. ARS researchers at Gainesville, Florida, have previously established that host insect-related attractants have significant importance in successful entomopathogenic nematode host infestation, and that only a subset of nematodes needed to experience those signals to promote a mass infestation. ARS researchers at Gainesville, Florida, have discovered that two distinctive types of nematode-produced pheromones are involved in this “follow the leader” process: a more generalized trail pheromone guiding the host searching behavior; and, a host specific recruitment or deterrent pheromone allowing a large enough number of conspecific nematodes to invade and overcome a host insect’s defensive system, while simultaneously avoiding competition by other entomopathogenic nematodes. ARS researchers at Gainesville, Florida, also discovered that previously identified dispersal pheromones promote a general host searching behavior and improve infectivity of host insects. The last discovery has been published while methods have been established to isolate the two new types of pheromones. Their identities have yet to be elucidated. ARS researchers at Gainesville, Florida, have further discovered that entomopathogenic nematodes can be reared to innately respond to specific volatile cues. In collaboration with researchers at the University of Florida, Gainesville, Florida, ARS scientists have exposed honey bees infested with Varroa mites to varying levels of carbon dioxide. Preliminary results suggest that carbon dioxide sensitivity of the Varroa mite is much lower than that of the honey bee. Mite infested honey bees that were exposed to low-doses of carbon dioxide caused the mite to become anesthetize and drop from the honey bee. The sedated honey bees are able to recover unharmed, however there is high mite mortality. It is essential to develop pesticide-free mite control due to mite pesticide resistance. The Varroa mite threatens the survival of honey bees throughout the world. This method will offer beekeepers an inexpensive application for Varroa mite control within the hive. Future research will focus on carbon dioxide thresholds levels that will be tolerated within an entire hive. Our research objective was to gain knowledge and disseminate this information regarding current honey bee control measures for safe, cost effective, and efficacious management of Varroa mites. Pollen and microbes are the most frequently observed contaminants found in floral nectar and both can impact nectar chemistry and bee preferences. Pollen can also support the growth of microbes within nectar by providing a source of amino acids. ARS scientists, at Gainesville, Florida, examined the effects of sunflower (Helianthus annuus) pollen and nectar yeast in a model nectar system. Nectar chemistry, including volatile emission and sugar content were examined. Both pollen and yeast scented nectar, whereas surprisingly, only pollen impacted sugar content. Honey bee foraging assays revealed bees avoided foraging from nectar containing yeast but did not discriminate based on pollen content. This research provides better understanding of the interactive effects of pollen and yeast in nectar, and represents an improved model system for studying floral microbes, as pollen can be used as a reproducible and relevant source of plant metabolite which may impact microbe growth and metabolism. ARS scientists, at Gainesville, Florida, completed honey bee foraging bioassays for an ongoing project examining the effects of floral microbes on sunflower volatile emission and pollinator preferences. Volatile analysis methods have been developed and optimized for entire flowers, pollen, and nectar. Separate analysis of floral rewards along with total floral aroma will provide richer understanding of the specific volatile cues bees use to locate resources and the effects of floral microbes on these cues. ARS scientists, at Gainesville, Florida, have designed and validated a new volatile collection sorbent mixture to simultaneously trap microbial and plant volatiles. Plant volatile collection methods do not always capture microbial volatiles due to their small size. However, the new collection sorbent was found to perform comparably to other widely available and currently employed methods for microbial volatile capture when used to analyze nectar containing yeast and pollen. The major benefits of this new approach are its improved sample stability after collection, thus allowing field sampling of plant and microbe volatile emission, and its efficient capture of both plant and microbial volatiles. These results introduce a new and valuable tool for researchers interested in plant-microbe interactions, within and beyond the floral context. In the spring of this year, ARS scientists, at Gainesville, Florida, in collaboration with researchers at the University of Florida began sampling blueberry flowers to discover the microbial communities which spontaneously occur in flowers. Flowers from two plant cultivars were sampled from four Florida farms, grown under either organic or conventional management techniques. Native, wild blueberries were also sampled from two natural areas. In total, approx. 500 nectar microbes were isolated and preserved in cryoculture from 200 blueberry flowers. These microorganisms will be identified and analyzed for their effects on floral reward chemistry, pollinator response, and their capacity to prophylactically prevent bee and plant pathogen infection. Isolated microorganisms will be contributed to national microbe collections for use by researchers throughout the world. ARS scientists, at Gainesville, Florida, investigated optimal volatile collection and desorption methods capable of extreme conditions while shipped locally and globally. Using data from a previous years’ infestation of local, seasonal fruit with the insect pest Caribbean fruit fly, Anastrepha suspensa, ARS researchers analyzed and compared data from non-infested peaches. Statistical analyses provided volatile biomarkers signaling infestation. Detected biomarkers will provide researchers with specific volatile profiles that can be used by APHIS scientists to detect infested fruit that is in transit or being imported.

1. Pepper weevil oviposition deterring pheromone. Pepper weevils, as well as many other weevil species, are major insect pests of several important agricultural commodities. Efforts for efficacious control have been limited, and as such interest in weevil pheromones or host plant odors are an important source for natural biological control management tools. ARS researchers in Gainesville, Florida, have progressed studies of combining the weevil pheromone with an ARS discovered pepper plant-based attractant blend. This combination is expected to significantly aid in the control of this devastating agricultural pest.

2. Explore attractants for manipulation of entomopathogenic nematodes (EPNs). Root feeding insects are difficult to control, and infestations are often not detected before plants are irreversibly damaged. EPNs can be very effective biological control agents for root feeding insects, but the level of control can be unpredictable despite significant progress in understanding plant-insect nematode interactions. An understanding of nematodes innate and learned responses to host related cues, as well as their pheromone regulated group behaviors are crucial for successful biological control of plant root pests such as the generalist pest Diaprepes abbreviates, yet the objective is to optimize EPN attraction and infectivity to any pest insect. Therefore, new EPN discoveries, such as their group behavior and the existence of trail and recruitment pheromones, can separately be considered as steppingstones in a growing field of research; but, more importantly, the pheromones ARS Gainesville, Florida, scientists have discovered can be used to attract and maintain nematode populations in a field for extended times, thus control anticipated insect infestations. Furthermore, ARS scientists, at Gainesville, Florida, anticipate their techniques of rearing nematodes to innately respond to novel volatile cues can be used to produce specialized strains of EPNs that can be used in control of new and still evolving pest insect-plant interactions, such as the recently discovered D. abbreviates devastation of blueberry plant roots.

3. Varroa mite sensitivity to carbon dioxide. Varroa mites feed and live on adult honey bees and feed and reproduce on larvae and pupae in the developing brood, causing deformity and weakening of honey bees by transmitting numerous viruses, thus contributing to a loss of 43.7% of U.S. managed honey bee colonies from April 2019 to April 2020. The American Beekeepers Federation estimates that honey bees contribute nearly $20 billion annually to the value of U.S. crop production. ARS researchers in Gainesville, Florida, in collaboration with researchers at the University of Florida have discovered a vulnerability in the Varro mite, Varroa destructor, that can be exploited to remove them from the honey bee. Research performed on honey bees and Varroa mites has determined that the carbon dioxide sensitivity of the Varroa mite is much lower than that of the honey bee. The result of low-dose carbon dioxide exposure to infested honey bees caused the mite to become anesthetized and drop from the honey bee. This technique will be evaluated for its application to the entire hive. Alternative control measures are needed to replace current pesticide use due to resistance and honey bee exposure.

4. Yeast-bacteria interactions in floral nectar. In nature, nectar may play host to large microbe populations which are commonly comprised by a small number (1-2) of species, and with dominant species competing fiercely with one another and would-be invaders. Microbe-microbe competitive interactions may be harnessed for the control of bee and plant pathogens; however, most studies of nectar microbes have focused on only one species. ARS scientists in Gainesville, Florida, in collaboration with colleagues at the University of California, Davis evaluated single and binary mixtures of nectar yeast and bacteria. When microbes were co-inoculated to a synthetic nectar, yeast contributed a greater number and abundance of volatiles to nectar aroma than bacteria. Yeast populations were lower in cocultures than in monocultures, while bacteria growth was improved when yeast was present, suggesting the occurrence of competitive interactions. Honey bees exhibited subtle preferences for microbe-free nectar compared to the yeast- or bacteria-inoculated nectars. These results are the first to simultaneously describe volatile emission, microbial population dynamics, sugar metabolism, and pollinator response to nectar microbe mixtures, and represent a critical next step in improving our understanding of microbe-microbe and microbe-pollinator interactions occurring in nectar.

Review Publications
Ray, H., Stuhl, C.J., Gillett-Kaufman, J. 2019. Rapid collection of floral fragrance volatiles using a headspace volatile collection technique for GC-MS thermal desorption sample analysis. Journal of Visualized Experiments. (154).
Stuhl, C.J. 2020. Attract-and-Kill bait for controlling the Small Hive Beetle (Coleoptera: Nitidulidae). Apidologie. 51,(3)428-435.
Rering, C.C.; Vannette, R.L.; Schaeffer, R.N.; Beck, J.J. Microbial co-occurrence in floral nectar affects metabolites and attractiveness to a generalist pollinator. J. Chem. Ecol. 2020, 46, 659-667.
Molyneux, R.J.; Beck, J.J.; Colegate, S.C.; Edgar, J.; Gaffield B.; Gilbert, J.; Hofmann, T.; McConnell, L.; Schieberle, P. Guidelines for unequivocal structural identification of compounds with biological activity of significance in food chemistry. Pure Appl. Chem. 2019, 91, 1417-1437.
Hunter III, C.T., Block, A.K., Christensen, S.A., Li, Q., Rering, C.C., Alborn, H.T. 2020. Setaria viridis as a model for translational genetic studies of jasmonic acid-responsive insect defenses in Zea mays. Plant Science. 291, February 2020, 110329.
Burge, D.O., Beck, J.J. 2019. Dispersal of spicebush (Calycanthus occidentalis, Calycanthaceae) by yellow jackets (genus Vespula; Hymenoptera: Vespidae). Madrono. 66(2):41-46.
Rodriguez-Saona, C., Alborn, H.T., Oehlschlager, C., Calvo, C., Kyryczenko-Roth, V., Sunil Tewari, S., Sylvia, M.M., Averill, A.L. 2020. Fine-tuning the composition of the cranberry weevil (Coleoptera Curculionidae) aggregation pheromone. Journal of Applied Entomology. 2020;144:417–421.
Burks, C.S., Higbee, B.S., Beck, J.J. 2020. Traps and attractants for monitoring navel orangeworm (Lepidoptera: Pyralidae) in the presence of mating disruption. Journal of Economic Entomology. 113(3):1270-1278.
Shapiro Ilan, D.I., Kaplan, F., Hofman, C.O., Schliekelman, P., Alborn, H.T., Lewis, E. 2019. Conspecific pheromone extracts enhance entomopathogenic infectivity. Journal of Nematology. (51)1-5.
Rering, C.C., Williams, K.L., Redman, Z.C., Hengel, M.J., Wong, J.W. 2019. Analysis of pesticides in plant foods by QuEChERS and gas chromatography-mass spectrometry: An undergraduate laboratory experiment. Journal of Chemical Education. 2020, 97, 1, 226–233.
Kihika, R.; Tchouassi, D.P.; Ng’ang’a, M.M.; Hall, D.R.; Beck, J.J.; Torto, B. Compounds associated with infection by root-knot nematodes influence the ability of infective juveniles to recognize host plants. J. Agric. Food Chem. 2020, 68, 9100-9109.