Research Project: Sustainable Management of Arthropod Pests in Horticultural Crops

Location: Horticultural Crops Disease and Pest Management Research Unit

2023 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
For Sub-objective 1B, researchers completed identifying and characterizing three diuretic hormones (DHs, DH31 and DH44) for water excretion, and seven receptors, G Protein-Coupled Receptors (GPCRs) from spotted-wing drosophila (SWD), Drosophila suzukii. Among the seven GPCRs, two GPCRs are for the DH31 hormone, and five GPCRs are for the DH44 hormone. We determined that both DH31 GPCRs are active, which means DH31 and the two GPCRs are binding well. Of the five GPCRs for DH44, however, only one GPCR is a functional receptor, and the other four GPCRs are not active. We are investigating more biological aspects of these GPCRs. Usually, insect DH GPCRs belong to the Class B GPCR family, which uses cAMP as a second messenger. But, one of the DH31 GPCRs uses both calcium ion (Class A type GPCR) and cAMP (Class B type GPCR) for the second messenger, which is unusual. We injected DH31 and DH44 hormones into SWD adults to increase water excretion in the fly. For Sub-objective 1C, researchers injected bioactive peptides into the slug (Deroceras reticulatum) and found significant weight loss, a series of distinct, atypical behaviors, and secretion of copious milky mucus. Researchers designed 23 different bioactive peptides, synthesized them, and measured any negative impacts on the slug. Six peptides with significant weight loss within 30 min after injection were selected. One of these peptides was dissolved in the water, applied topically to the lettuce leaf, and fed on the slug for 24 hours. The leaf consumption by the slug was significantly reduced on leaves treated with the bioactive peptide compared to the control, water-treated leaves. This result indicates that the bioactive peptides have an anti-feeding effect on the slug. For Sub-objective 1D, two neurohormones (produced from the brain or nerve tissues) named CAPA and PK peptides were identified and characterized in western flower thrips (WFT; Frankliniella occidentalis). The gene structure and differential gene expression were determined in various adult tissues and during the life stages. The study mapped the distribution of neurons releasing these peptides in the central nervous system of WFT. Exploring the biological processes at the molecular level provides information to identify biological targets for managing thrips in the field. For Sub-objective 2A, research is ongoing on the impacts of erythritol sprays on plants; erythritol is an food-grade insecticide alternative for SWD control. A two-year field study in multiple blueberry and cherry fields show no discernable impact of sprays on mold development, nor fruit quality in terms of flesh firmness, skin firmness, or sweetness. A first-year study in blueberry, wild blackberry, and cherry fields showed no discernible impact of sprays on stomatal conductance, relative water content, osmolality and photosynthetic potential. For Sub-objective 2C, methods were developed to study dispersal of Trissolcus japnoicus, an egg parasitoid of brown marmorated stink bug. Research examined the use of fluorescent water on marking persistence and impacts on fitness. To-date, marking does not affect longevity, parasitism rates, or flight behavior of the parasitoid. This result enables us to continue with mark-release-recapture studies in the field. For Sub-objective 2D, research was conducted on protecting rhododendron plants from azalea lace bug via silicon supplementation. A three-year trial was completed. Silicon or calcium supplementation reduced herbivory and reproduction rates when applied via foliar or soil drench application, especially in choice testing conditions. The process of supplementing plants did not elevate calcium nor silicon content in leaf tissue. While supplementation can confer protection from lace bugs, the protection was not consistent to warrant use specifically for lace bug management. Supplementation was observed twice to lower infestation by aphids on rhododendrons.

Accomplishments
1. Erythritol formulations have minimal impacts on beneficial insects. Spotted-wing drosophila (SWD) is an economic pest of small fruits and cherries. A food-grade insecticide such as erythritol is a sustainable way to manage SWD provided there are minimal non-target impacts. Honey bee adults were not impacted by direct feeding and would typically not forage on crops as erythritol is sprayed post-bloom, but the impact on immature bees was unknown if some adults carried this back to the hive. ARS researchers in Corvallis, Oregon, found that erythritol formulations introduced to honey bee brood had no discernible impact on survival. Also, feeding on erythritol by a parasitoid of SWD had minimal impacts on survival especially since wasps prefer to feed on other sugar sources such as floral nectar. With minimal impacts on honey bees, companies can consider registering erythritol in new alternative formulations. This technology will improve SWD control and reduce the use of chemical insecticides by fruit growers.

2. Bioactive peptides from SWD and thrips. Spotted-wing drosophila (SWD) and western flower thrips (WFT) are major global pests on small fruits and nursery crops, respectively. Insect neuropeptides (small protein molecules produced in the brain or nerve tissues) are potential targets for new insecticides because they are involved in most essential biological processes during life stages. ARS researchers in Corvallis, Oregon, identified seven short bioactive peptides from SWD and WFT using an insect cell-based in vitro assay. The binding activities of these peptides to their receptors were similar to their natural ligands. Although injection or feeding of the bioactive peptides did not significantly affect the survival of the two insects. These results will be used to develop new insecticidal active ingredients to be used by nursery and small fruit growers to control both thrips and SWD.

3. Development of nano-injection method using thrips. Micro-injection techniques are invaluable in entomological research as they allow for direct delivery of biological compounds into the hemocoel of specimens at controlled volumes. Nearly all micro-injection methods utilize forcibly immobilized insects that could affect specimen fitness and physiology. Also, most current micro-injections for live insects are restricted to insects over 4 mm in size. ARS researchers in Corvallis, Oregon, improved the nano-volume injection technique for micro-insects (under 3 mm) using the western flower thrips as a model. The method, without sedation and physical injury, will be used by researchers working with the injection of nano-volumes into live micro-insects, such as mosquitoes, bedbugs or ticks.

Review Publications
Price, B.E., Breece, C., Galindo, G., Greenhalgh, A., Sagili, R., Choi, M.Y., Lee, J.C. 2022. Nonnutritive sugars for spotted-wing drosophila (Diptera: Drosophilidae) control have minimal nontarget effects on honey bee larvae, a pupal parasitoid, and yellow jackets. Environmental Entomology. 52(1):47-55. https://doi.org/10.1093/ee/nvac095.
Yoon, J., Ahn, S., Choi, M.Y. 2023. Selection and comparative gene expression of midgut-specific targets for Drosophila suzukii. Insects. 14(1). Article 76. https://doi.org/10.3390/insects14010076.
Lee, J.C., Rodriguez-Saona, C., Zalom, F. 2022. Introductory remarks: Spotlight on spotted-wing drosophila. Journal of Economic Entomology. 115(4):919-921. https://doi.org/10.1093/jee/toac041.
Paul, R.L., Abram, P.K., Lee, J.C. 2022. Host patch quality increases parasitoid locomotor activity despite risk of egg limitation. Ecological Entomology. 47(5):810-821. https://doi.org/10.1111/een.13171.
Raffin, C., Price, B.E., Yun, S., Choi, M.Y. 2022. Nano-injection method for micro-insects without sedation using the western flower thrips, Frankliniella occidentalis. Journal of Applied Entomology. 146(9):1200-1206. https://doi.org/10.1111/jen.13063.
Khan, F., Kim, K., Sung, J., Lim, H., Kim, S., Choi, M.Y., Kim, Y. 2023. A novel physiological function of pheromone biosynthesis-activating neuropeptide in production of aggregation pheromone. Scientific Reports. 13. Article 5551. https://doi.org/10.1038/s41598-023-32833-9.
Yun, S., Jang, H., Ahn, S., Price, B.E., Hasegawa, D.K., Choi, M.Y. 2023. Identification and characterisation of PRXamide peptides in the western flower thrips, Frankliniella occidentalis. Insect Molecular Biology. https://doi.org/10.1111/imb.12859.
Cha, D.H., Skabeikis, D.D., Kim, B., Lee, J.C., Choi, M.Y. 2023. Insecticidal properties of erythritol on four tropical tephritid fruit flies, Zeugodacus cucurbitae, Ceratitis capitata, Bactrocera dorsalis, and B. latifrons (Diptera: Tephritidae). Insects. 14(5). Article 472. https://doi.org/10.3390/insects14050472.
Maestas, L.P., Lee, J.C., Choi, M.Y. 2023. Effect of erythritol and sucralose formulation on the survivorship of the mosquito Aedes aegypti. Florida Entomologist. 106(2):129-132. https://doi.org/10.1653/024.106.0210.