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


2017 Annual Report

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.

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 control of the invasive spotted wing drosophila (SWD), we studied their flight behavior, physiological basis of attraction, host use of fruits, RNA interference (RNAi) target genes, identification with molecular tools, and olfactory genes. We found that SWD can fly up to 1.8 km on a flight mill in a single flight bout (Objective 1.1). Also, flight of SWD was enhanced with access to fruit juice and blossom nectar when compared to water starved flies (Objective 1.2). This shows the potential for SWD to disperse over landscapes with access to food. Regarding attraction, we found that hungry flies, virgin, or females that have laid few eggs were much more responsive to a fermentation-based commercial SWD lure than their counterparts that were well-fed, mated, or had laid many eggs. Thus selective baiting is useful for early season monitoring or developing attract-and-kill approaches that targets females that are likely hungry and not yet laying eggs. Regarding host use, we found that SWD often infests the same raspberry fields as brown marmorated stink bug, the latter which can taint fruit with a foul odor. If SWD avoids tainted fruit, there is concern that overall damage would increase synergistically. We found SWD to use tainted and untainted fruit at similar rates. For the RNAi approach (disrupting specific genes in a species), significant progress was made toward the selection of SWD RNAi target genes, and characterizing the biological functions in SWD. Over 20 RNAi candidates were identified from SWD genes related to housekeeping, neuropeptide hormones, neuropeptide receptors, and olfaction (Objective 3.1). We completed the identification and characterization of the following from SWD and compared it all to other Drosophila species: two G-protein coupled receptors (GPCR), pyrokinin and diapause receptors, their functional expression, and binding assay (Objective 4.1). We also designed specific primers, optimal fragment length, and polymerase chain reaction (PCR) amplicons for each target to synthesis double-stranded RNA (dsRNA) in vitro. Early detection and identification technology is critical to prevent the introduction of exotic species and establishment of exotic and native pests which would result in costly control measures. We investigated SWD unique gene(s) using SWD transcriptome data and developed a loop-mediated isothermal amplification (LAMP) method, to identify SWD. This technology provides high sensitivity (~femtogram, 10-12 g level) and rapid turnaround (10-30 minute incubation in a water bath) without sophisticated equipment required for standard PCR. This technology is now widely applied in many fields for on-site detection. We developed a novel molecular diagnostic method to detect SWD species and potential RNAi targets (Objective 3.1). To better understand how SWD identifies hosts, we characterized the transcriptome of female and male antennae using next generation sequencing (NGS). We dissected more than 2,000 antennae from male and female adults and extracted total RNA to prepare the transcriptome analysis in order to elucidate specific olfactory genes in SWD. This data can also be used for potential RNAi targets in the future. To better understand and control the new invasive brown marmorated stink bug (BMSB), we studied their nutrient requirements and the parasitic wasp that attacks their eggs. Regarding nutrition, we found that overwintering adults exit their shelter once their energetic reserves become depleted based on a comparison of adults that remained inside or exited their shelters. When female adults have a steady diet, the nutritional content that they allocate to their offspring remains steady as the mother ages. Regarding biological control, we are monitoring the longevity of Trissolcus japonicus, a parasitic wasp from the native range of Asia, in the presence of floral nectar (Objective 2.2). Sugar supplementation may be critical to conserve these wasps in the wild, or ensure that released wasps are effective. To improve the control of azalea lace bug, a pest of azaleas and rhododendrons, we compared the release of green lacewings at the egg or larval stage. We found that egg releases reduced pest numbers compared to larval releases (Objective 2.3).

1. Development of biologically-based control for spotted wing drosophila. Non-toxic insecticides especially those available for organic growers are being sought for spotted wing drosophila control. ARS scientists in Corvallis, Oregon, found that combinations of sucrose and erythritol have insecticidal effects against the fly. In the lab, two sucrose/erythritol formulations resulted in the highest mortality and the lowest fecundity on adults. These two formulations were further evaluated on blueberry bushes and fruits in the greenhouse; fly mortality was higher with a lower sucrose concentrations. For practical application, the sucrose/erythritol combination would be more effective than erythritol alone because the combination tastes sweeter and elicits more feeding. This erythritol formulation can be a potential insecticide used alone or as a delivery agent combined with conventional or biological insecticides to enhance their efficacy.

2. Impact of brown marmorated stink bug (BMSB) in nursery crops. While brown marmorated stink bug (BMSB) prefers to feed on (and damages) fruits, the impacts of BMSB feeding on the shoots of ornamental plants were not known. ARS scientists in Corvallis, Oregon, tested growth in apple, cherry, elm, gingko, hawthorn, lilac, linden, and maple seedlings and branches with and without the stink bug. The presence of stink bugs had little impact on plant growth. As a result, growers with non-fruiting ornamental plants have decided not to manage this pest when seen in their fields, reducing additional pesticide sprays.

3. Water spray for azalea lace bug. The azalea lace bug is a pest that damages leaves on rhododendron and azalea plants. Park managers have requested non-toxic control measures to use in public gardens next to waterways. ARS scientists in Corvallis, Oregon, tested pressurized water sprays on infested rhododendron plants. Biweekly water sprays resulted in lower pest density and fewer visibly damaged leaves than unsprayed control leaves. As a result, growers have adjusted their irrigation system to remove lace bugs from their plants as part of their management system.

4. Development of the early detection for spotted wing drosophila. Spotted wing drosophila is a serious pest of small fruits. Using spotted wing drosophila transcriptome data, ARS scientists in Corvallis, Oregon, first identified a gene (Ds10_00012111) that is present in this fly’s genome but has not been found in any other insect species. This gene can be used to develop a rapid and highly accurate detection using loop-mediated isothermal amplification (LAMP) assays. The minimum amount of genomic DNA required for the LAMP assay is 1.0 picogram, and time is 30 minutes with temperatures ranging from 58 to 62 degrees Celsius. The method can detect SWD genomic DNA from all geographical strains collected from eight different locations in Asia, Europe, Hawaii, and the United States. This LAMP assay could be a useful detection tool for identifying spotted wing drosophila rapidly in the field.

5. Identification and characterization of RNAi targets for spotted wing drosophila. RNA interference (RNAi) has great potential as a biologically-based control option for a target specific pest. ARS scientists in Corvallis, Oregon, searched and identified potential spotted wing drosophila RNAi target genes. RNAi specific to SWD targets caused 34-61% mortality in adult flies through the 1st screening with injection of double stranded RNA. Targets were narrowed down for further evaluation and feeding assays. Positive outcomes indicate that these RNAi targets are ready to be evaluated for impacts on mortality and fertility on adults. Spotted wing drosophila is a serious pest of small fruits.

6. Interaction between spotted wing drosophila and brown marmorated stink bug. Both spotted wing drosophila and brown marmorated stink bug are economic pests that feed on small fruits. Brown marmorated stink bug is known to taint fruit. If spotted wing drosophila avoids tainted fruit and selectively finds undamaged fruit, then growers are concerned that overall damage from both pests is synergistic. ARS scientists in Corvallis, Oregon, compared how the fly laid eggs on raspberry and blueberry fruit on fruit previously or simultaneously exposed to the stink bug. The fly used recently exposed and unexposed fruit similarly. This outcome suggests that if both pests are present in the field, typical (not synergistic) damage is expected, and both pests are to be managed as usual.

7. Identification of neuropeptides and their receptors. Neuropeptides are the largest group of insect hormones known to regulate a variety of physiological functions. Receptors of insect neuropeptides belong to the family of G protein-coupled receptors (GPCRs) which are involved in almost all biological functions through the insect life stages. Therefore, these receptors might be good targets to develop insect control method. ARS scientists in Corvallis, Oregon identified and characterized two neuropeptide genes and their receptors. These genes may offer a novel control method for this pest insect in the future.

8. RNAi-based method to control lepidopteran moth pests. Moth crop pests cause billions of dollars in crop damage and control costs each year. Chemical pesticides are the primary tools used to combat these insect pests; however, use of chemical pesticides has disadvantages, including non-target effects on neutral or beneficial animals. RNA interference (RNAi) is a new direction for insect pest control. ARS scientists in Corvallis, Oregon discovered RNAi of the PBAN/Pyrokinin gene to develop a novel biologically-based control method for corn earworm. Interference with this target gene and production of the neurohormones produced by this gene, or interference with their receptor(s), would result in incomplete development of immatures and negatively affect essential pheromonal communication in adults. The U.S. Patent and Trademark Office issued a patent for this novel method (Patent No. US 9,617,542, “Lepidopteran Moth Control Using Double-Stranded RNA Constructs”).

Review Publications
Kim, Y., Hur, J., Lee, G., Choi, M.Y., Koh, Y. 2016. Rapid and highly accurate detection of Drosophila suzukii, spotted wing Drosophila (Diptera: Drosophilidae) by loop-mediated isothermal amplification assays. Journal of Asia-Pacific Entomology. 19(4):1211-1216. doi: 10.1016/j.aspen.2016.10.015
Abram, P.K., Hoelmer, K.A., Acebes-Doria, A., Andrews, H., Beers, E., Bergh, C.J., Bessin, R., Biddinger, D., Botch, P., Buffington, M.L., Cornelius, M.L., Costi, E., Delfosse, E., Dieckhoff, C., Dobson, R., Donais, Z., Grieshop, M., Hamilton, G., Haye, T., Hedstrom, C., Herlihy, M.V., Hoddle, M., Hooks, C., Jentsch, P., Neelandra, J., Kuhar, T., Lara, J., Legrand, A., Lee, J.C., Leskey, T.C., Lowenstein, D., Milnes, J., Maistrello, L., Morrison III, W.R., Nielsen, A.L., Ogburn, E., Pickett, C., Poley, K., Pote, J., James, R., Shrewsbury, P., Talamas, E.J., Tavella, L., Walgenbach, J., Waterworth, R., Weber, D.C., Welty, C., Wiman, N.G. 2017. Integrative review of indigenous arthropod natural enemies of the invasive brown marmorated stink bug in North America and Europe. Journal of Pest Science. 90(4):1009-1020.
Ahn, S., Martin, R.C., Rao, S., Choi, M.Y. 2017. Neuropeptides predicted from the transcriptome analysis of the gray garden slug Deroceras reticulatum. Peptides. 93:51-63. doi: 10.1016/j.peptides.2017.05.005.
Ahn, S., Martin, R.C., Rao, S., Choi, M.Y. 2017. The complete mitochondrial genome of the gray garden slug Deroceras reticulatum (Gastropoda: Pulmonata: Stylommatophora). Mitochondrial DNA Part B. 2(1):255-256. doi: 10.1080/23802359.2017.1318677.
Choi, M.Y., Joon-Ahn, S., Kim, A., Koh, Y. 2017. Identification and characterization of pyrokinin and CAPA peptides, and corresponding GPCRs from spotted wing drosophila, Drosophila suzukii. General and Comparative Endocrinology. 246:354-362. doi: 10.1016/j.ygcen.2017.01.011.
Choi, M.Y., Tang, S.B., Ahn, S., Amarasekare, K.G., Shearer, P.W., Lee, J.C. 2017. Effect of non-nutritive sugars to decrease the survivorship of spotted wing drosophila, Drosophila suzukii. Journal of Insect Physiology. 99:86-94. doi: 10.1016/j.jinsphys.2017.04.001.
Klick, J., Yang, W., Lee, J.C., Bruck, D. 2016. Reduced spray programs for Drosophila suzukii management in berry crops. International Journal of Pest Management. 62(4):368-377: doi: 10.1080/09670874.2016.1222105.
Woltz, M.J., Lee, J.C. 2017. Pupation behavior and larval and pupal biocontrol of Drosophila suzukii in the field. Biological Control. 110:62-69. doi: 10.1016/j.biocontrol.2017.04.007.
Woltz, M., Wiman, N., Lee, J.C. 2017. Two pests overlap: Drosophila suzukii (Diptera: Drosophilidae) use of fruit exposed to Halyomorpha halys (Hemiptera: Pentatomidae). Journal of Economic Entomology. doi: 10.1093/jee/tox156.