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

Location: Horticultural Crops Research Unit

2020 Annual Report

Objectives
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. Obj. 1.3: Monitor and mitigate impacts of invasive and emergent pests. 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. Collaborative work on pests such as spotted wing drosophila, brown marmorated stink bug, and azalea lace bug includes improving our knowledge of insect behaviors, life cycle and host preference such that alternative tools can be developed to mitigate the their impacts in the landscape. Collaborative work on monitoring systems will assist decision support for pest management. 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
This is the final report for project 2072-22000-040-00D, "Biologically-based Management of Arthropod Pests in Small Fruit and Nursery Crops." Research will be continued in a new project, which is currently undergoing NP304 Office of Scientific Quality Review. For Objective 1, research was conducted on spotted-wing drosophila (SWD) using a flight mill. Flight capacity was tested between SWD of varying ages, sex, wild vs. colony source, rearing histories, and seasonal morphologies. Females flew more than males, and no differences were found between winter or summer morphs or between ages from one to seven days for short-term flight. Related to Sub-objective 1.1, researchers learned that SWD can fly up to 1.8 km on a flight mill in a single flight bout, and SWD expend lipid or sugar stores during flight on a tethered flight mill. Humidity had minimal impact on short duration flights, and flight of SWD was enhanced with access to fruit juice and blossom nectar compared to water starved flies (Sub-objective 1.2). This shows the potential for SWD to disperse over landscapes with access to food. For Sub-objective 2.1, research was conducted on the biology and biological control of SWD. SWD were found to predominantly leave the fruit as larva and pupate in the soil beneath the crop. This is important because it suggests that further research on biological control and cultural practices might be directed on the ground surface. For biological control, naturally occurring predators reduced infestations in fruits by 19-49% and removed 61-91% of pupae placed on the soil in fruit fields. Conservation of these predators will be important in unmanaged areas or before or after the main growing season. For Sub-objective 2.2, research was conducted on the biological control of brown marmorated stink bug (BMSB). Methyl salicylate (oil of wintergreen) could increase predation rates on BMSB eggs. This is a potentially quick non-toxic management tool. Adult female BMSB fed a steady diet were found to produce eggs of similar nutritional quality as they aged. This is important to know when rearing parasitoids on BMSB egg masses collected from a lab colony. Studies focused on Trissolcus japonicus, a parasitic wasp that attacks their eggs. 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. This knowledge enables more parasitoids to be reared with less effort. For Sub-objective 2.3, research continued on enhancing biological control of azalea lace bug, a pest of azaleas and rhododendrons. Green lacewings are common predators of azalea lace bugs. Green lacewing larvae readily consumed the immature stages of the pest, but had difficulty consuming the adult stage. In the field, we compared the release of green lacewings at the egg or larval stage. Egg releases reduced pest numbers compared to larval releases. To enhance control, we combined water sprays to dislodge pest nymphs and adults, followed by predator egg releases to control hatching pests. 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. Aside from augmentative releases of green lacewings, we tested plant volatiles as a more convenient tool to attract naturally occurring predators. Several combinations of plant volatiles effectively attracted naturally occurring green lacewings. For Objective 3, research was conducted to develop an RNAi technology to control insect pests, including SWD. Related to Sub-objective 3.1, more than 30 potential RNAi target genes, including the housekeeping, neuropeptides, and receptor genes, were identified from SWD adults. Double-stranded RNAs (dsRNAs) between 200 and 400 nucleotides were constructed for the target genes and then tested and evaluated on SWD adults, in support of Sub-objective 3.2. We developed a method for the large-scale dsRNA production using microbial-based dsRNA production using L4440 vector and HT115 (DE3) E. coli, and isolated a considerable amount of dsRNA (19.5 µg/mL) from bacterial culture. This work is related to Sub-objective 3.3. For Sub-objective 3.2, researchers evaluated the impact of RNA interference (RNAi) on the development of SWD. Direct injection of double-stranded RNA (dsRNA) into SWD adults negatively impacted flies causing a 60% mortality rate. In addition, the introduction of dsRNA molecules into Drosophila cell lines resulted in cell death and growth inhibition. However, feeding dsRNA to adult SWD had minimal impacts. Some activity of dsRNA degradation enzymes were found in the midgut. DsRNA molecules transferred in the midgut need protection from the attacking endonuclease, then pass through the hemolymph to target cells. Identification of dsRNA degradation enzymes and formulation of dsRNA with different lipid nanoparticles will continue to be evaluated on SWD. For Objective 4, research was conducted to develop receptor-based insecticides by finding and evaluating insect neuropeptides (NPs) and their G-protein coupled receptors (GPCRs) receptors to disrupt critical physiological signals in the pests. Related to Sub-objective 4.1, we identified a number of the target genes of the NPs and GPCRs from SWD and pest slugs, and expressed functionally in an insect cell-based expression system. The target genes were characterized to specific biological functions such that receptor interference technology can be applied to control these pests. For Sub-objective 4.2, research continued to identify molecular targets for SWD control. Advanced genomics/proteomics tools allowed us to screen functionally new receptor-based targets, such as neuropeptides and GPCRs, that regulate key biological functions for insect life stages. A novel receptor interference (Receptor-i) technology was developed using the biopanning method to screen small peptides using insect cell and phage peptides. Application of Receptor-i technology with the SWD GPCR target will continue to be used to identify small bioactive peptides as control agents for SWD.

Accomplishments
1. Receptor interference technology for biological insecticides. Neuropeptides (NPs) and their G-protein-coupled receptors (GPCRs) are involved in essential functions of insects, such as feeding, growth, and reproduction. Receptor-i is a key biological process where bioactive peptides are blocked in the target insect. ARS scientists in Corvallis, Oregon, led the identification of natural peptides from spotted-wing drosophila, brown marmorated stink bug, codling moths, and slugs, with over 100 NP and 20 GPCR targets. The results will provide select small peptides as bioactive peptides blocking the specific physiological function on targeting pests. This technology will provide a new avenue in integrated pest management.

2. Attracting predatory green lacewings. Green lacewings are an important predator, and can be released on azaleas and rhododendrons infested by azalea lace bug for biological control. However, augmentative releases can be costly and inconvenient. ARS scientists in Corvallis, Oregon, compared various plant volatile blends in attracting green lacewings. Two of the three-component plant volatile blends attracted adult green lacewings. The acetophenone blend was associated with a reduction of the pest. The results will assist landscape managers, who can purchase the lure components and combine them to increase biological control in fields where it is not convenient to make augmentative releases of predators.

Review Publications
Acebes-Doria, A.L., Agnello, A.M., Alston, D.G., Andrews, H., Beers, E.H., Bergh, J., Bessin, R., Blaauw, B.R., Buntin, G., Burkness, E.C., Chen, S., Cottrell, T.E., Daane, K.M., Fann, L.E., Fleischer, S.J., Guedot, C., Gut, L.J., Hamilton, G.C., Hilton, R., Hoelmer, K.A., Hutchison, W.D., Jentsch, P., Krawczyk, G., Kuhar, T.P., Lee, J.C., Milnes, J.M., Nielsen, A.L., Patel, D.K., Short, B.D., Sial, A.A., Spears, L.R., Tatman, K.M., Toews, M.D., Walgenbach, J.D., Welty, C., Wiman, N.G., Van Zoeren, J., Leskey, T.C. 2019. Season-long monitoring of the brown marmorated stink bug (Hemiptera: Pentatomidae) throughout the United States using commercially available traps and lures. Journal of Economic Entomology. 113(1):159-171. https://doi.org/10.1093/jee/toz240.
Cha, D.H., Skabeikis, D.D., Collignon, M.R., Siderhurst, M.S., Choi, M.Y., Vander Meer, R.K. 2019. Behavioral response of little fire ant, Wasmannia auropunctata (Hymenoptera: Formicidae), to trail chemicals laid on epiphytic moss. Journal of Insect Behavior. 32:145-152. https://doi.org/10.1007/s10905-019-09721-0.
Van Timmeren, S., Fanning, P.D., Schoneberg, T., Hamby, K., Lee, J.C., Isaacs, R. 2020. Exploring the efficacy and mechanisms of a crop sterilant for reducing infestation by spotted-wing drosophila (Diptera: Drosophilidae). Journal of Economic Entomology. 113(1):288-298. https://doi.org/10.1093/jee/toz245.
Dou, X., Liu, S., Ahn, S., Choi, M.Y., Jurenka, R. 2019. Transcriptional comparison between pheromone gland-ovipositor and tarsi in the corn earworm Helicoverpa zea. Comparative Biochemistry and Physiology, Part D: Genomics and Proteomics. 31:100604. https://doi.org/10.1016/j.cbd.2019.100604.
Ahn, S., Corcoran, J., Vander Meer, R.K., Choi, M.Y. 2020. Identification and characterization of GPCRs for Pyrokinin and CAPA peptides in the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae). Frontiers in Physiology. 11:559. https://doi.org/10.3389/fphys.2020.00559.
McIntosh, H., Skillman, V., Galindo, G., Lee, J.C. 2020. Floral resources for Trissolcus japonicus, a parasitoid of Halyomorpha halys. Insects. 11(7):413-422. https://doi.org/10.3390/insects11070413.
Ahn, S., Oh, H., Corcoran, J., Kim, J., Park, K., Park, C.G., Choi, M.Y. 2020. Sex-biased gene expression in antennae of Drosophila suzukii. Archives of Insect Biochemistry and Physiology. 104(1):e21660. https://doi.org/10.1002/arch.21660.