Research Project: Sustainable Management of Arthropod Pests in Horticultural Crops

Location: Horticultural Crops Disease and Pest Management Research Unit

2024 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 report documents FY 2024 progress for project 2072-22000-044-000D, “Sustainable Management of Arthropod Pests in Horticultural Crops”, which began in October 2020. For Sub-objective 1A, research on the development of RNA interference (RNAi)-based spotted-wing drosophila (SWD) control is nearly complete. When RNAi materials, double-stranded RNA (dsRNA) molecules, are ingested by flies, the biological molecules must be protected from a degrading enzyme, dsRNA nuclease, in the midgut. Two dsRNA nucleases are in the midgut and actively degrading dsRNA molecules. Two approaches were conducted: 1) blocking two genes of these enzymes, 2) delivering encapsulated dsRNA into the fly using nano-lipid particles. Additional midgut-specific upregulated genes were identified, focusing on neuropeptides and their receptors, as potential targets for the development of SWD control methods. Under Sub-objective 1B, research was completed with the identification and characterization of two diuretic hormones (DHs, DH31 and DH44) and their corresponding receptors, seven G protein-coupled receptors (GPCRs), in spotted-wing drosophila. There are two GPCRs for DH31 and five GPCRs for DH44. Insect DH GPCRs use cyclic adenosine monophosphate (cAMP) as second messenger. Surprisingly, one of the DH31 GPCRs in the fly uses both calcium ions and cAMP for its second messenger. Urine production (diuresis) is one of the most important physiological processes for water regulation in insects for environmental adaptation. Injection of either DH31 or DH44 peptide increased urination, and injection of the mixture of the two DH peptides into the fly reduced survival. In support of Sub-objective 1C, ARS researchers conducted the pre-screening with 25 bioactive peptides injected into the grey garden slug and evaluated the impacts on the slug. Six of the peptides caused significant weight loss, a series of distinct, atypical behaviors, and secretion of copious milky mucus within 30 minutes after injection. One of these peptides was dissolved in the water or mixed with nano-particles, and applied topically to a lettuce leaf, which the slug fed on for 24 hours. The leaf consumption by the slug was significantly reduced on leaves treated with the bioactive peptide compared to the control leaves. This result indicates that bioactive peptides inhibit slug feeding, which can be developed into a slug repellent. For Sub-objective 1D, ARS researchers completed development of a molecular identification marker for the western flower thrips (WFT) using the internal transcribed spacer (ITS) gene. The identification of two genes producing multiple neuropeptides, four capacity (CAPA), and three pyrokinin (PK) peptides were completed. In addition, the distribution map of these peptides in the brain and other neural tissues of WFT were completed. Exploring their corresponding receptors at the molecular level provides information to identify specific biological functions that will be biological targets for the development of new management tools for WFT. Under Sub-objective 2A, research is nearly complete on the impacts of erythritol. Erythritol is a food-grade insecticide alternative for spotted-wing drosophila (SWD) control. Six years of field studies in blueberry and cherry show that erythritol sprays can reduce SWD infestation or oviposition. A two-year field study shows that erythritol has no discernable impact on mold development, nor fruit quality (flesh firmness, skin firmness, or sweetness), nor plant health (stomatal conductance, relative water content, osmolality and photosynthetic potential). Lastly, while erythritol is sweet and can be fed upon by beneficial insects, it had minimal impacts on honeybee adults and larvae, and a parasitic wasp of SWD particularly if other preferred sugar sources were present. In support of Sub-objective 2B, research was conducted to evaluate releases of parasitoid wasps, Pachycrepoideus vindemiae and Muscidifurax raptorellus, which attack spotted-wing drosophila (SWD) pupae. Modest release rates of P. vindemiae in caneberry hoop houses resulted in higher parasitism rates demonstrating 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, ARS researchers found that floral nectar benefitted Trissolcus japonicus, an egg parasitoid of the brown marmorated stink bug, and could be established using insectary plants. Methods were developed to study dispersal of T. japonicus. Research examined the use of fluorescent water on marking persistence and impacts on fitness. Marking did not affect longevity, parasitism rates, nor flight behavior of the parasitoid. This marking method was successful in tracking wasp dispersal for two weeks in the field. Under Sub-objective 2D, ARS researchers completed studies on protecting rhododendron plants from azalea lace bug via silicon supplementation. Silicon or calcium supplementation reduced herbivory and reproduction rates when applied via foliar or soil drench application, especially when lace bugs could move freely between plants. 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. Rather, supplementation used for promoting plant health would have the added benefit of conferring plant protection from herbivores.

Accomplishments
1. Silicon fertilization can reduce herbivore infestation on rhododendrons. The azalea lace bug is a serious pest of azaleas and rhododendrons, feeding damage on leaves renders plants unmarketable. ARS researchers in Corvallis, Oregon discovered that silicon fertilization led to reduced infestation by azalea lace bugs and rhododendron aphids when insects could freely move between treated and untreated plants. Silicon promotes plant health by strengthening plant tissue and disease resistance. This result shows an added benefit of pest protection with fertilization which is being recommended by landscape managers.

2. Bioactive peptides from spotted-wing drosophila (SWD) and western flower thrips (WFT). SWD and WFT are major global pests on small fruits and nursery crops, respectively. ARS researchers in Corvallis, Oregon, screened over 100 bioactive peptides for SWD and WFT using a new technology, receptor interference (Receptor-i). Target-specific bioactive peptides in the injection feeding tests reduced the survival of both insects. The structural modification of these peptides has potential for the development of new active compounds to control both SWD and WFT. A part of these results has been approved with a new invention disclosure and is being prepared for a U.S. patent application.

3. Flourophore-water marking successfully tracks dispersal of small parasitic wasps. Understanding insect dispersal is critical for monitoring and predicting their spread. Natural enemies are often released to control pests without knowing how far they spread. A fluorophore-water marking technique was found to persist on small parasitic wasps without detriment to their longevity, parasitism activity, movement, or flight. This mark is also more convenient and economical to detect compared to conventional insect marking methods. As a result, other researchers are using this technique to study dispersal of small parasitoids.

4. Immune-related genes from the grey garden slug identified. Slugs represent the most significant pest affecting nursery and field crops. Various nematodes are used as biological agents to control slugs in the field. When attacked by nematodes, the slug activates a specific immune response. If the slug's immune genes are either knocked out or disrupted, it will reduce their survival. ARS researchers in Corvallis, Oregon, identified ten immunity-related genes, including toll-like receptors, and confirmed them to be actively expressed in the nematode infected-slug. The identification of immune-related genes and their physiological mechanisms will integrate with using nematodes for slug management in the field.

Review Publications
Sriram, A., Voyvot, S., Johnson, B., Chowdhury, S., Fanning, P., Lee, J.C. 2023. Mesh covers on sentinel parasitoid traps can restrict Drosophila suzukii movement and allow parasitism by Ganaspis brasiliensis and Pachycrepoideus vindemiae. Biocontrol Science and Technology. 33(11):1030-1040. https://doi.org/10.1080/09583157.2023.2272227.
Yoon, H., Price, B.E., Parks, R., Ahn, S., Choi, M.Y. 2023. Diuretic hormone 31 activates two G protein-coupled receptors with differential second messengers for diuresis in Drosophila suzukii. Insect Biochemistry and Molecular Biology. 162. Article 104025. https://doi.org/10.1016/j.ibmb.2023.104025.
Lee, J.C. 2024. Flourishing with sugars - following the fate of parasitoids in the field. Current Opinion in Insect Science. 61. Article 101158. https://doi.org/10.1016/j.cois.2023.101158.
da Costa Dias, S., Brida, A.L., Jean-Baptiste, M.C., Leite, L.G., Ovruiski, S.M., Lee, J.C., Mello Garcia, F.R. 2024. Compatibility of entomopathogenic nematodes with chemical insecticides for the control of Drosophila suzukii (Diptera: Drosophilidae). Plants. 13(5). Article 632. https://doi.org/10.3390/plants13050632.
Hafeez, M., Mc Donnell, R., Colton, A., Howe, D., Denver, D., Martin, R., Choi, M.Y. 2024. Immune-related gene profiles and differential expression in the grey garden slug Deroceras reticulatum infected with the parasitic nematode Phasmarhabditis hermaphrodita. Insects. 15(5). Article 311. https://doi.org/10.3390/insects15050311.
Chinta, S., Vander Meer, R.K., O'Reilly, E.E., Choi, M.Y. 2023. Insecticidal effects of Receptor-i isolated bioactive peptides on fire ant colonies. International Journal of Molecular Sciences. 24(18). Article 13978. https://doi.org/10.3390/ijms241813978.
Lee, J.C., Price, B.E., Adams, C., Rutkowski, E.M., Choi, M.Y. 2023. Erythritol sprays reduce Drosophila suzukii infestation without impacting honey bee visitation nor fruit quality. Pest Management Science. 79(12):4990-5002. https://doi.org/10.1002/ps.7701.
Paul, R.L., Hagler, J.R., Janasov, E.G., McDonald, N.S., Voyvot, S., Lee, J.C. 2024. An effective fluorescent marker for tracking the dispersal of small insects with field evidence of mark-release-recapture of Trissolcus japonicus. Insects. 15(7). Article 487. https://doi.org/10.3390/insects15070487.
Graham, K.V., Janasov, E.G., Paul, R.L., Scagel, C.F., Lee, J.C. 2024. Silicon supplementation can reduce infestation by azalea lace bug-(Hemiptera: Tingidae). Journal of Economic Entomology. 117(5):1948-1958. https://doi.org/10.1093/jee/toae164.