Research Project: Sustainable Management of Arthropod Pests in Horticultural Crops

Location: Horticultural Crops Disease and Pest Management Research Unit

2021 Annual Report

Objectives
This Project Plan focuses on invertebrate pests important to small fruit and nursery growers and are also highly visible to the public. While an Integrated Pest Management (IPM) program is desired, growers still follow calendar-based insecticide sprays. To promote sustainable management, our objectives include biological control agents currently present in the field, convenient strategies for growers to apply such as erythritol and silicon, and longer-term molecular approaches for developing species-specific biologically-based insecticides. Objective 1: Develop and improve molecular-based management tools for control of arthropod pests in the Pacific Northwest that affect horticultural crops, especially spotted-wing drosophila (SWD), slugs, and thrips, with an emphasis on new technologies. • Sub-objective 1A: Develop delivery methods for biological agents such as RNAi to SWD: 1) Identify and characterize RNase in SWD; 2) Formulate dsRNA with lipid nanoparticle materials. • Sub-objective 1B: Identify bioactive peptides using GPCR-based screening in SWD. • Sub-objective 1C: Identify bioactive peptides to control slugs. • Sub-objective 1D: Identify molecular markers and neuropeptides in western flower thrips (WFT). Objective 2: Develop and integrate management strategies for arthropod pests in the Pacific Northwest that affect horticultural crops such as blueberries, raspberries, and wine grapes, especially spotted-wing drosophila (SWD) and brown marmorated stink bug (BMSB), with an emphasis on biological control. • Sub-objective 2A: Explore erythritol for managing SWD. • Sub-objective 2B: Develop augmentative biological control of SWD in protected environments. • Sub-objective 2C: Improve conservation biological control of BMSB. • Sub-objective 2D: Test silicon supplementation for azalea lace bug (AzLB) control.

Approach
Obj. 1A hypothesizes that protecting dsRNA in the midgut will enhance RNAi impact on SWD. We will first identify RNAse in SWD using a BLAST search and DNA sequencing. Then we will characterize dsRNA enzymatic acitivty in the midgut, and formulate dsRNA with nanoparticle materials. Formulated dsRNAs will be injected into or fed to SWD flies, and the phenotypic impacts will be monitored. If the target RNAi does not work, we will continue to search for other RNAi targets expressed in the midgut membrane. Obj. 1B hypothesizes that receptor interference using small peptides will negatively affect SWD, and Obj. 1C tests slugs. First, G-protein coupled receptors (GPCR) will be identified and expressed, and then screened using a biopanning technique where where a peptide or protein is fused with the coat protein of a bacteriophage. After screening, a small amount of peptides (< 5 mg) will be synthesized and injected into SWD adults or slugs and monitored for survival. If efficacy is low, the small peptides will be modified with hydrophobic side chains such as cysteine bonds or formulated with lipid nanoparticles. Obj. 1D hypothesizes that internal transcribed spacer genes can be used as a molecular marker for western flower thrips identification. Digested patterns will be compared to other thrips species. If these genes are not suitable, we will try cytochrome oxidase genes as an identifying marker. Obj. 2A hypothesizes that the non-nutritive sweetener sucralose is phagostimulative, and field applications of erythritol will lower SWD infestation in the field and have minimal non-target effects. Flies will be fed various solutions to determine phagostimulation. Blueberry plants will be sprayed with erythritol formulations, and resulting pest infestation and visits by non-target insects will be monitored. Natural infestation rates may vary, and cage studies may be done to monitor impact. Obj. 2B hypothesizes that releases of the parasitoid Pachycrepoideus vindemiae will lower SWD infestation. Augmentative releases will be made in small fruits grown in hoop houses, and resulting parasitism in sentinel traps, and infestation among fruit monitored. Natural infestation may get too high causing the grower to spray, in which case, studies may be repeated in smaller scale experimental plots. Obj. 2C hypothesizes that the imported parasitoid of BMSB, Trissolcus japonicus, will benefit from floral supplementation. Wasps will be fed various floral species, with their longevity and nutrient storage measured. The most beneficial flowers will be seeded in the field to measure impact on parasitism. Obj. 2D hypothesizes that supplementing rhododendron plants with silicon will result in uptake in plant tissues, and make plants less susceptible to azalea lace bug herbivory. Plants will be supplemented as recommended, and lace bug feeding and reproduction will be monitored on plants. Plants may not take up silicon in the tissues, but supplementation may still deter herbivory. If this occurs, we will examine the impact of silicon on settling preference of lace bugs on treated versus untreated surfaces.

Progress Report
This is a new project in Year 1 for 2072-22000-044-00D, and continues studies from the expired project 2072-22000-040-00D, “Biologically-based management of arthropod pests in small fruit and nursery crops.” For Objective 1, research was conducted to develop and improve molecular-based management tools for control of arthropod pests in the Pacific Northwest that affect horticultural crops, especially spotted-wing drosophila (SWD), slugs, and thrips, with an emphasis on new technologies. For Sub-objective 1A, two dsRNase genes were identified from SWD adults that have enzymatic activity in the midgut and their gene expression profiles were determined. The dsRNA degradation activity was evaluated using the extracts of the SWD midgut tissue or Sf9 cells expressing dsRNA genes for the enzyme, and both extracts showed dsRNA degradation activity. The expression levels of two genes in the tissue- and development-specific stages of SWD were investigated. The dsRNA enzymes were produced in the midgut only, and not in other tissues. They were expressed in the feeding stages, such as the larva and adult, but not expressed in the non-feeding egg stage. This makes sense that SWD can protect against foreign RNA molecules received from various diets. Research results have been published in a peer-reviewed journal. For Sub-objectives 1B and 1C, diuresis is important to regulate water and ion balance between the fly midgut and Malpighian tubules (similar to the kidney) in SWD. After SWD adults feed on the non-caloric sugars erythritol or sucralose, the flies need excessive water to excrete the non-metabolized carbohydrates. This water imbalance in the flies causes mortality. We expect diuretic hormones (Dhs) and corresponding receptors will control SWD water balance in the body. We identified two Dhs (SWD31Dh and SWDDh44) as natural ligands, and seven neuropeptide receptors, G-protein-coupled receptors (GPCRs), from SWD adults. Studies to characterize these hormones and receptors and gene expressions in flies is ongoing. The work is also related to Sub-objective 2A. For Sub-objective 1C, we identified specific GPCRs for the gray garden slug that will be used for receptors and small peptide binding tests to screen bioactive peptides. For Sub-objective 1D, work was done to establish western flower thrips (WFT) colonies in the lab and isolate total RNA from various life stages and tissues. To initiate a WFT colony, thrips were collected from soybean plants in the greenhouse and the species was identified. The study includes developing a DNA molecular identification (ID) tool for micro-insects which are difficult to identify to species by morphological traits. The study of the molecular ID includes extraction of genomic DNA, synthesis of cDNA from the total RNA, primer design for the DNA marker gene, polymerase chain reaction (PCR) amplification, cloning of DNA gene, sequencing, blast search against National Center for Biotechnology Information (NCBI) database, and analysis. For Sub-objective 2A, researchers continued to develop formulations with erythritol as an alternative insecticide for SWD that is non-toxic to humans. Sucralose, a non-caloric sugar, has phagostimulative properties and could improve the effectiveness of erythritol sprays by adding sweetness. SWD that feed on this also die quicker than the previous erythritol formulation with sucrose. Newer erythritol formulations reduced oviposition activity of SWD. This occurred when erythritol was ingested and flies were given untreated berries, and when flies were fed sugars and given treated berries. Erythritol formulations affect the physiology of flies, and erythritol-coated fruit also deters oviposition. For Sub-objective 2B, research was conducted to evaluate releases of parasitoid wasps, Pachycrepoideus vindemiae and Muscidifurax raptorellus, which attack SWD pupae. Modest release rates of P. vindemiae in caneberry hoop houses resulted in higher parasitism rates. This demonstrated that augmentative releases can be contained in protected environments. However, infestation by and prevalence of adult SWD did not differ between hoop houses with and without wasp releases, so the benefits of such releases has not been demonstrated. Releases of M. raptorellus only increased parasitism rates on one date despite much higher release rates. For Sub-objective 2C, research was conducted to enhance the parasitoid wasp, Trissolcus japonicus, which attacks brown marmorated stink bug eggs in the field. Alyssum was previously found not to improve wasp longevity, while buckwheat did. In planted fields, wasps were released and their retention on various floral species was monitored. Overall, retention of wasps was low, and they were found on all flower species. For Sub-objective 2D, research started on supplementing rhododendrons with silicon fertilization, and determining whether it protects leaves from herbivory by azalea lace bug. Thus far, silicon supplementation appears to reduce egg laying and feeding by adult lace bugs. Additional work is examining whether supplementation should occur with foliar sprays or via soil drench.

Accomplishments
1. Identification and characterization of SWD dsRNase. The current control for spotted-wing drosophila (SWD), a serious pest that can infest and destroy fruits of many plants, primarily relies on chemical insecticides, despite having potential negative effects. RNA interference (RNAi) is a novel alternative control that is specific to SWD and safe. Double stranded (ds) RNA may be applied in a field for SWD to ingest as a spray/bait, but its efficacy is limited due to dsRNA degradation in the fly’s gut. ARS scientists in Corvallis, Oregon, identified two dsRNase genes from SWD’s gut and investigated gene expression profiles during SWD life stages. dsRNases were produced in larval and adult stages during feeding periods. Research results are being used by scientists to improve RNAi application strategies for SWD control.

2. Identification of floral species that benefit a parasitic wasp of brown marmorated stink bug. Brown marmorated stink bug is an invasive pest of tree fruits and vegetables and can cause significant crop loses. The bug eats many plant species; therefore insecticides applied to the plants are frequently used to control the pest. Biological control with Trissolcus japonicus, a parasitoid from the pest’s native range, is a sustainable way to lower pest populations in the surrounding landscape. ARS scientists in Corvallis, Oregon, screened various flowering plant species that could help conserve this parasitoid. Alyssum, a common insectary plant, was not beneficial, while buckwheat, dill and cilantro enhanced longevity and nutrient reserves of the parasitoid. Growers and other researchers are using these results to implement beneficial floral borders to provide a refuge for parasitoids during insecticide sprays, and to monitor parasitoid dispersal into the field for pest control.

3. Bioactive peptides control slugs. Slugs are a worldwide problem and estimated to cost the seed industry over $50 million annually in Oregon. Currently, common slug control methods rely on chemical pesticides that are mixed in pellet bait-based products. This control method is limited in terms of delivery and efficacy, and is not sustainable for environmental and human health. ARS scientists in Corvallis, Oregon, identified a group of small molecules from the gray garden slug that are important for normal body functioning for the slug. This is the first report on this specific group of molecules in the garden slug. Results will be used by scientist to develop novel pesticides to control slugs.

4. A novel method to identify western flower thrips. Thrips, small flying insects, attack hundreds of host plants, including ornamental and nursery crops, and vegetables in the greenhouse and field. Western flower thrips (WFT) is one of the most economically important thrips, not only because the insect directly damages the plant, but also because it transmits damaging viruses to plants. Thrips are a few millimeters in length, adults are difficult to identify to species, and immature stages cannot be identified with current methods. ARS scientists in Corvallis, Oregon, identified a gene from WFT that can be used as a molecular identification tool to identify thrips species collected in the field. Accurate identification of thrips species is critical for novel approaches for pest management.

Review Publications
Ahn, S., McDonnell, R., Corcoran, J., Martin, R.C., Choi, M.Y. 2020. Identification and functional characterization of the first molluscan neuromedin U receptor in the slug, Deroceras reticulatum. Scientific Reports. 10. Article 22308. https://doi.org/10.1038/s41598-020-79047-x.
Burrack, H., Lee, J.C., Loeb, G., Rodriguez-Saona, C. 2021. Progress and challenges in building monitoring systems for Drosophila suzukii. In: Garcia F.R.M., editor. Drosophila suzukii Management. Cham, Switzerland: Springer. p. 111-132. https://doi.org/10.1007/978-3-030-62692-1_6.
Choi, M.Y., Vander Meer, R.K. 2021. GPCR-based bioactive peptide screening using phage-displayed peptides and an insect cell system for insecticide discovery. Biomolecules EISSN 2218-273X. 11(4). Article 583. https://doi.org/10.3390/biom11040583.
Graham, K.V., Choi, M.Y., Lee, J.C. 2020. Attracting Chrysopidae with plant volatiles for lace bug (Hemiptera: Tingidae) control in rhododendrons and azaleas. Journal of Insect Science. 20(5). https://doi.org/10.1093/jisesa/ieaa078.
Reich, I., Jessie, C., Ahn, S., Choi, M.Y., Williams, C., Gormally, M., McDonnell, R. 2020. Assessment of the biological control potential of common carabid beetle species for autumn and winter active pests (Gastropoda, Lepidoptera, Diptera: Tipulidae) in annual ryegrass in Western Oregon. Insects. 11(11). Article 722. https://doi.org/10.3390/insects11110722.
Wang, X., Daane, K.M., Hoelmer, K.A., Lee, J.C. 2021. Biological control of spotted-wing drosophila - an update on promising agents. In: Garcia, F.R.M., editor. Drosophila suzukii Management. Cham, Switzerland: Springer. p. 143-167. https://doi.org/10.1007/978-3-030-62692-1_8.
Wang, X., Lee, J.C., Daane, K.M., Buffington, M.L., Hoelmer, K.A. 2020. Biological control of Drosophila suzukii. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources. 15. Article 054. https://doi.org/10.1079/PAVSNNR202015054.
Wiman, N.G., Andrews, H., Rudolph, E., Lee, J.C., Choi, M.Y. 2020. Fatty acid profile as an indicator of larval host for adult drosophila suzukii. Insects. 11(11). Article 752. https://doi.org/10.3390/insects11110752.
Yoon, J., Ahn, S., Flinn, C.M., Martin, R.R., Choi, M.Y. 2021. Identification and functional analysis of dsRNase genes in drosophila suzukii. Archives of Insect Biochemistry and Physiology. 107(4). Article e21822. https://doi.org/10.1002/arch.21822.