Research Project: Biologically-based Management of Arthropod Pests in Small Fruit and Nursery Crops

Location: Horticultural Crops Research Unit

2018 Annual Report

Objectives
Objective 1: Determine risk across landscapes to improve management of pests such as spotted wing drosophila by understanding their behaviors (i.e., host range and preference, dispersal). Obj. 1.1: Describe flight parameters and energy used in flight of spotted wing drosophila. Obj. 1.2: Compare effects of temperature and sugar feeding on spotted wing drosophila. Objective 2: Develop biological control strategies for pests such as spotted wing drosophila, brown marmorated stink bug, and azalea lace bug, using commercially available and endemic natural enemies and habitat conservation. Obj. 2.1: Study endemic natural enemies of spotted wing drosophila, and pupation habits of spotted wing drosophila. Obj. 2.2: Study endemic natural enemies and using the volatile methyl salicylate for brown marmorated stink bug. Obj. 2.3: Study endemic natural enemies, augmentative release and methyl salicylate for azalea lace bug. Objective 3: Develop RNAi technology to control pests such as spotted wing drosophila by finding and evaluating target genes, and developing large-scale production methods. Obj. 3.1: Identify potential RNAi target genes from spotted wing drosophila. Obj. 3.2: Evaluate RNAi impact(s) on development of spotted wing drosophila. Obj. 3.3: Develop large-scale dsRNA production methods. Objective 4: Develop receptor-based drug discovery for pests such as spotted wing drosophila by finding and evaluating insect neuropeptides and receptors to disrupt critical physiological signals in the pests. Obj. 4.1: Cloning and functional expression of the specific G-protein coupled receptors from spotted wing drosophila and other pests. Obj. 4.2: Develop receptor-based screening method.

Approach
Objective 1 includes studying spotted wing drosophila (SWD) on a flight mill or other suitable arenas and measuring their nutrient reserves, such that basic flight parameters and energetics can be obtained. For SWD, its movement across landscapes may be influenced by its ability to find resources in the surrounding habitat and temperature and nutrient stores. Objective 2 includes surveying the endemic natural enemies present that attack important pests such as SWD, brown marmorated stink bug (BMSB), and azalea lace bug (AzLB). Trials will determine if the release of predators or use of attractive plant volatiles can improve control of these pests in nursery and fruit crop fields. Objective 3 will identify genes in SWD for RNAi targets, and measure the impact on treatment on SWD longevity and activity. Also, a large-scale method to produce dsRNA will be tested using E. coli with inserted vectors. Objective 4 will screen G-protein coupled receptors that are important in the development or adult stages of SWD, and will clone and test expression of them.

Progress Report
To improve management of the invasive spotted wing drosophila (SWD), ARS scientists in Corvallis, Oregon, studied their flight under various humidity ranges, interaction with Botrytis, which causes grey mold in fruit, and an erythritol formulation to induce fly mortality and reduce fecundity in the lab and greenhouse. SWD was sometimes found to expend lipid or sugar stores during flight on a tethered flight mill (Sub-objective 1.1), and humidity had minimal impact on short duration flights (Sub-objective 1.2). The erythritol formulation was found to be a potential insecticide used alone and/or as a delivery agent combined with biological insecticides to enhance their efficacy. ARS scientists synthesized and purified dsRNAs (RNAi material) for 30 genes of SWD using an in vitro system (Sub-objective 3.1), injected into the adult flies with a nano-injection under a microscope, and monitored their potential impacts (Sub-objective 3.2). SWD was found to be a potential vector of Botrytis. SWD exposed to blueberries infected with a fluorescent Botrytis strain were observed to transfer the spores to a new substrate. ARS scientists identified and characterized two neuropeptide (NP) genes and their receptors that were produced in the central nervous system of the fly. These genes can be potentially utilized to search for biological targets for SWD management (Sub-objective 4.1). To control the invasive brown marmorated stink bug (BMSB), ARS scientists studied Trissolcus japonicus, a parasitic wasp that attacks their eggs (Sub-objective 2.2). Because mass rearing the parasitoid requires a constant stock of BMSB eggs, the suitability of variously collected and stored egg masses were compared for rearing T. japonicus. BMSB eggs frozen within three days of oviposition were suitable for parasitism with no apparent negative impact on sex ratio or wasp size. Providing T. japonicus with buckwheat flowers extends their longevity, and may be an ideal companion plant to improve biological control. ARS scientists identified 10 BMSB genes for RNAi targets and synthesized their dsRNAs to inject into BMSB. ARS scientists also identified two neuropeptides, and characterized their biological functions in BMSB, which may be developed as biological targets. To improve the control of azalea lace bug, a pest of azaleas and rhododendrons, studies examined the efficacy of pairing water sprays to dislodge pest nymphs and adults, followed by predator egg releases to control hatching pests (Sub-objective 2.3). A substantial reduction in pest counts and leaf damage occurred on treated rhododendrons for five weeks after the treatment application. This provides growers and homeowners a non-toxic alternative to control lace bugs without frequent treatment applications.

Accomplishments
1. Flight of spotted wing drosophila (SWD). Spotted wing drosophila (SWD), a serious pest of small fruits and cherries, is known to move from wild fruits into crop fields, but their dispersal capacity is unknown. Therefore, ARS scientists in Corvallis, Oregon, and Maricopa, Arizona, tested SWD on a flight mill. Flies could fly 1.8 kilometers in a short-term bout. Flies with access to blossom nectar or fruit exudates flew longer and farther than starved flies, since flight expends sugar reserves. This research helps elucidate the spatial dynamics of infestation by indicating that most flies will remain within a limited area, such as a field, and a few may disperse long distances to other fields.

2. Development of a non-toxic sugar formulation to control spotted wing drosophila (SWD). Spotted wing drosophila (SWD) is a serious pest of fruits, which is commonly controlled by insecticides. ARS scientists in Corvallis, Oregon, found that an erythritol-sucrose formulation induces mortality and decreases fecundity in SWD, yet the mechanisms were unknown. Physiological studies were conducted which suggested that erythritol (a sugar alcohol) might be directly transported from the midgut without being metabolized and stored, but is accumulated in the hemolymph, which in turn elevates the osmotic pressure in the fly hemolymph (a fluid similar to blood in most invertebrates). This non-toxic sugar formulation is a potential insecticide that can be used alone or as a delivery agent combined with conventional or biological insecticides to enhance efficacy. The formulation is currently being evaluated in the field, and for impacts on honeybees.

3. Early season monitoring of spotted wing drosophila (SWD). An abundance of research has focused on trapping the serious fruit pest, spotted wing drosophila (SWD), throughout the season, but most growers rely on monitoring SWD for initiating treatment programs. ARS scientists in Corvallis, Oregon, in collaboration with scientists from Wisconsin, Michigan, and Georgia, compared various lures for early season efficacy. A fermentation-based commercial lure was most effective. ARS scientists further found that flies typically present in spring that were hungry or with limited reproductive experience would be more responsive to this lure. This lure is now being recommended for early season monitoring for SWD at the eOrganic website hosted by Purdue University Extension.

4. Enhancing aphid parasitoids with marigold. Aphids are ubiquitous pests that can be attacked by naturally-occurring parasitoid wasps. Floral nectar has often been suggested to enhance parasitism rates, but very few studies have examined the use of marigolds combined with the neotropical parasitoid, Aphidius platensis. ARS scientists in Corvallis, Oregon, in collaboration with scientists from Brazil found that marigold extended the longevity of this wasp, and improved their search and parasitism rate on aphids. Growers in South America are encouraged to incorporate marigolds in their field for improving biological control.

5. Identification of RNAi targets to develop spotted wing drosophila (SWD) control. There is a need for non-toxic control measures for spotted wing drosophila (SWD), since insecticide residue can affect human health and also affect exports markets for harvested fruit. To develop a non-toxic and specific control for SWD, ARS scientists in Corvallis, Oregon, identified more RNAi target genes, and evaluated potential RNAi impacts on SWD. Once RNAi targets were identified, dsRNA was injected into adult flies causing up to 60% mortality. Selected RNAi targets were then mixed into the diet of adult flies and evaluated for mortality and fecundity. RNAi directly injected into hemolymph caused fly mortality, leading to evaluations on delivering RNAi via feeding systems.

6. Nutrient physiology of brown marmorated stink bug (BMSB). The brown marmorated stink bug (BMSB) is a major pest worldwide capable of overwintering and reproducing in its introduced range, but few studies have examined the energetic expenditures of these essential activities. ARS scientists in Corvallis, Oregon, in collaboration with scientists from Oregon State University, examined the energetic requirements of overwintering and reproduction of wild stink bug populations. Nutritional depletion along with temperature and photoperiod cues cause BMSB to emerge from diapause. Providing aging females with a steady diet ensured the production of eggs with consistent nutritional quality. This information is important to help predict emergence periods and produce consistent egg masses needed for mass-rearing biological control agents.

7. Identification and characterization of neuropeptide hormones of brown marmorated stink bug (BMSB). The brown marmorated stink bug (BMSB) is a common pest which can attack over 200 kinds of plants. Current control methods depend on non-specific chemical insecticides that negatively impact the environment and human health. Thus, there is a need for biologically-based control alternatives. One such alternative targets insect neuropeptides. The capa and pyrokinin (PK) genes produce CAPA and PK neuropeptides, which regulate a variety of physiological functions in insects. ARS scientists in Corvallis, Oregon, identified and characterized neuropeptides related to endocrine regulation and feeding behaviors, with the goal of targeting these specific neutopeptides for BMSB control.

8. Identification of the neuropeptide profile of the gray garden slug. The gray garden slug is a destructive and omnivorous pest on a variety of crops, including nursery crops. The economic cost of management and loss by the slugs is estimated at over $50 million for the seed industry. Studies on the slug have mainly focused on its ecology, biology, pest status, and control, but virtually nothing is known about peptide hormones and their biological signaling. ARS scientists in Corvallis, Oregon, identified and characterized the entire profile of neuropeptide genes by RNA-sequencing technique. This study provides comprehensive transcriptome data of the gray garden slug. This data is essential for a better understanding of slug physiology and to develop novel biologically-based control methods in the future.

Review Publications
Bushakra, J., Dossett, M., Carter, K., Vining, K., Lee, J.C., Bryant, D., Vanburen, R., Lee, J., Mockler, T., Finn, C.E., Bassil, N.V. 2018. Characterization of aphid resistance loci in black raspberry (Rubus occidentalis L.). Molecular Breeding. 38:83. https://doi.org/10.1007/s11032-018-0839-5.
Gundersen, D.E., Adrianos, S.L., Allen, M.L., Becnel, J.J., Chen, Y., Choi, M.Y., Estep, A., Evans, J.D., Garczynski, S.F., Geib, S.M., Ghosh, S.B., Handler, A.M., Hasegawa, D.K., Heerman, M.C., Hull, J.J., Hunter, W.B., Kaur, N., Li, J., Li, W., Ling, K., Nayduch, D., Oppert, B.S., Perera, O.P., Perkin, L.C., Sanscrainte, N.D., Sim, S.B., Sparks, M., Temeyer, K.B., Vander Meer, R.K., Wintermantel, W.M., James, R.R., Hackett, K.J., Coates, B.S. 2017. Arthropod genomics research in the United States Department of Agriculture-Agricultural Research Service: Applications of RNA interference and CRISPR gene editing technologies in pest control. Trends in Entomology. 13:109-137.
Jaffe, B.D., Avanesyan, A., Bal, H.K., Grant, J., Grieshop, M.J., Lee, J.C., Liburd, O.E., Rhodes, E., Rodriguez-Saona, C., Sial, A.A., Feng, Y., Zhang, A., Guedot, C. 2018. Multistate comparison of attractants and the impact of fruit development stage on trapping Drosophila suzukii (Diptera: Drosophilidae) in raspberry and blueberry. Environmental Entomology. https://doi.org/1093/ee/nvy052.
Nielson, A., Fleischer, S., Hamilton, G., Hancock, T., Krawczyk, G., Lee, J.C., Ogburn, E., Pote, J., Raudenbush, A., Rucker, A., Saunders, M., Skillman, V., Sullivan, J., Timer, J., Walgenbach, J., Wiman, N., Leskey, T.C. 2017. Phenology of brown marmorated stink bug described using female reproductive development. Ecology and Evolution. 7(17):6680-6690. https://doi.org/10.1002/ece3.3125.
Skillman, V., Lee, J.C. 2017. Evaluating impacts of brown marmorated stink bug on non-fruiting nursery crops. Journal of Environmental Horticulture. 35(3):93-98.
Skillman, V., Lee, J.C. 2017. Nutrient content of brown marmorated stink bug eggs and comparisons between experimental uses. Journal of Insect Science. 17(6):120. https://doi.org/10.1093/jisesa/iex098.
Souza, I.L., Marucci, R.C., Paterno Silveira, L.C., Paixão De Paulo, N.C., Lee, J.C. 2018. Effects of marigold on the behavior, survival, and nutrient reserves of Aphidius platensis. Biocontrol. 63(4):543-553. https://doi.org/10.1007/s10526-018-9882-8.
Tang, S., Lee, J.C., Jung, J., Choi, M.Y. 2017. Effect of erythritol formulation on the mortality, fecundity and physiological excretion in Drosophila suzukii. Journal of Insect Physiology. 101:178-184. https://doi.org/10.1016/j.jinsphys.2017.07.015.
Wong, J., Cave, A., Lightle, D., Mahaffee, W.F., Naranjo, S.E., Wiman, N., Woltz, M., Lee, J.C. 2018. Drosophila suzukii flight performance reduced by starvation but not affected by humidity. Journal of Pest Science. 91(4):1269-1278. https://doi.org/10.1007/s10340-018-1013-x.
Wong, J., Wallingford, A., Loeb, G., Lee, J.C. 2018. Physiological status of Drosophila suzukii (Diptera: Drosophilidae) affects their response to attractive odours. Journal of Applied Entomology. 142(5):473-482. https://doi.org/10.1111/jen.12497.