Location: Horticultural Crops Disease and Pest Management Research Unit
2022 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 1A, ARS researchers in Corvallis, Oregon, found that RNA interference (RNAi) through oral delivery of double-stranded RNA (dsRNA) was low, which could be a result of dsRNA degradation in the fly gut. The researchers identified two dsRNA degradation enzymes from spotted-wing drosophila (SWD), and published the results. To increase RNAi efficacy, dsRNA molecules ingested by SWD must be protected from enzyme attack for delivery to the target cells to take place. Therefore, stopping enzyme activity is critical for SWD RNAi. Three dsRNAs were designed, synthesized, and evaluated. The survival rates of SWD adults injected with various dsRNAs and combinations were not significant compared to the controls. We continued to test a lipid-based dsRNA formulation on insects and cell line-based RNAi effect.
For Sub-objective 1B, researchers continued to identify diuretic hormones (DHs, DH31 and DH44) and seven corresponding receptors, G Protein-Coupled-Receptors (GPCRs) from SWD. After SWD adults fed on the non-caloric sugars, erythritol or sucralose, the expression of the DH receptors was very different among these flies, indicating that the DHs are involved in the regulation of the water balance in the body. We found that the DH GPCRs belong to the Class B GPCR family which uses cyclic adenosine monophosphate (cAMP) as a second messenger. This was unexpected, and it is an unusual GPCR family that needs a different measurement method: a luminescent-based binding assay to confirm the pairing of the DHs and GPCRs expressed in insect cell lines from the armyworm, Spodoptera frugiperda (Sf9). The development of a new method for the binding assay is ongoing. A florescent-based measurement is a common method to determine binding affinities of neuropeptides and their GPCRs using calcium ions (Ca2+) as a second messenger in insects. The two DHs and GPCRs were investigated with their gene expressions from various tissues, including the brain, midgut, hindgut, Malpighian tubules, and fatty body, and during the fly life stages.
For Sub-objective 1C, researchers designed amino acid sequences of the slug myomodulin family of peptides consisting of 7 amino acids. These have strong GPCR binding properties and pronounced physiological effects on the slugs, including body paralysis, mucus production, weight loss, and dehydration. The amino acids in the peptide sequence were modified by reducing amino acids at the N- or C- termini, or by replacing target amino acids with alanine (A). The modified peptides were tested for binding affinity to the slug GPCRs expressed in Sf9 cells to determine the essential functional core of the peptide.
For Sub-objective 1D, researchers used internal transcribed spacer 2 (ITS2) gene as a DNA marker for molecular identification of western flower thrips (WFT). ITS gene is the spacer DNA situated between the small-subunit ribosomal RNA (rRNA) and large-subunit rRNA genes. To amplify the ITS region (475 nucleotides) by the polymerase chain reaction (PCR) method, researchers: 1) isolated total RNAs from thrips colonies in the greenhouse and field; and 2) designed primer sets to amplify the WFT ITS gene. The PCR products amplified with DNAs were gel electrophoresed and confirmed the DNA sizes, then digested by a restriction enzyme (RsaI) to see the digested pattern. The result showed that the Oregon strain (OR) is exactly the same as the WFTs identified in other areas previously; thus, the thrips colony we collected in Oregon is the WFT (Frankliniella occidentalis). The ITS sequences were cloned and sequenced to confirm the nucleotides.
For Sub-objective 2A, researchers continued to test formulations with erythritol as an alternative insecticide for SWD. Erythritol is detrimental to SWD and safe for humans. Adding a phagostimulant such as sucrose or sucralose increases the propensity of the fly to feed on the solution, die quicker and lay fewer eggs. Field tests are ongoing to test formulations with sucrose or sucralose on blueberry and blackberry. Previously, some discoloration on leaves sprayed with erythritol was observed, and this summer, researchers are examining the impact of sprays on the osmotic potential, pore stomatal conductance, and light reflectance on leaves. Researchers also tested the impact of erythritol solutions when fed to honeybee brood larvae and found no impact of the larvae surviving to adulthood.
For Sub-objective 2B, research was completed on releases of Pachycrepoideus vindemiae in caneberry hoop houses for control of SWD pupae. New trials have started with releasing the imported parasitoid, Ganaspis brasiliensis, onto wild Himalaya blackberry habitat near grower fields. These releases are to establish the parasitoid in the landscape in an area that is not sprayed and will reliably have SWD hosts to reproduce on.
For Sub-objective 2C, research was conducted to test the host specificity of a parasitoid wasp, Trissolcus japonicus, which attacks brown marmorated stink bug eggs. Wasps appear to attack the first hosts encountered, regardless of species. Other stink bug species lay smaller eggs, and researchers are examining whether there is a fitness cost of developing in smaller hosts. Lastly, releases of T. japonicus will rely on dispersal and retention of the parasitoid and can be examined by mark-release-recapture studies. Researchers are comparing various protein marks and fluorescent water marks on marking persistence and whether it affects the longevity, reproduction, and flight behavior of wasps. Results will guide planned mark-release-recapture studies.
For Sub-objective 2D, researchers are continuing to supplement rhododendrons with silicon fertilization, and determine whether it protects leaves from herbivory by azalea lace bug. This summer, calcium silicate is being evaluated, as well as calcium carbonate, as both a foliar spray or soil drench. The second treatment was evaluated in case calcium in the product, and not silicon, is influencing herbivory. Results can be used by growers and homeowners to fertilize their plants while enhancing pest control.
Accomplishments
1. Sucralose added to erythritol controls SWD. Growers have requested safer alternative products to control spotted-wing drosophila (SWD), a fly which attacks small fruits and cherries. Erythritol is a human-safe non-caloric sugar that is toxic to SWD and is easily sprayed. ARS scientists in Corvallis, Oregon, found that adding sucralose to erythritol enhances its sweetness, causing flies to feed more on it, die quicker and lay fewer eggs. Also, sucralose is not metabolized into useable carbohydrates by SWD, and causes the flies to lose moisture. Sucralose erythritol formulations are being examined by other scientists in field trials and by companies as a potential product to control SWD.
2. Releases of Pachycrepoideus vindemiae in protected environments. Organic growers have one primary pesticide to control spotted-wing drosophila (SWD), a major pest of fruit crops, and are interested in biological control options. Releases of parasitic wasps that attack SWD may be helpful in protected environments where movement is limited. ARS scientists in Corvallis, Oregon, and Albany, California, as well as University of Minnesota, tested releases of the parasitic wasp (Pachycrepoideus vindemiae) in protected caneberries. Modest release rates of the wasp combined with an augmentorium resulted in higher parasitism rates. Also, releases of the commercially available parasitic wasp, Muscidifurax raptorellus, resulted in successful parasitism, which is the first documentation of parasitism in the field. Research results are guiding growers on wasp releases in their fields.
3. Molecular identification method for western flower thrips (WFT). Thrips damages hundreds of host plants, including many ornamental and nursery crops and vegetables in the greenhouse and field. This insect not only causes direct damage from feeding and laying eggs on leaves, flowers and fruits, but also transmits tomato spotted wilt virus. Due to their small size, rapid life cycle, and broad host range, detecting and preventing the spread of WFT is difficult. ARS scientists in Corvallis, Oregon, developed a sustainable mass rearing system, and established a molecular identification tool using DNA. This simple and cost-effective rearing method and accurate identification of thrips species is critical and is being used by other groups to rear thrips and quickly confirm thrips species to plan appropriate control measures for growers.
4. Activity of stink bug parasitoid. The brown marmorated stink bug damages many plant species and requires frequent insecticide applications to control. Biological control with Trissolcus japonicus, a parasitic wasp from the pest’s native range, is a sustainable way to lower pest populations. ARS scientists in Corvallis, Oregon, in collaboration with scientists in AgCanada, explored the activity of newly emerged and aged female T. japonicus. Aged females had matured eggs and were much more active than new females. Research results allow practitioners to produce wasps of the appropriate age leading to improved efficacy in the field.
Review Publications
Lee, J.C., Flores, S.M., Graham, K.V., Skillman, V.P. 2022. Methyl salicylate can benefit ornamental pest control, and does not alter per capita predator consumption at close-range. Frontiers in Ecology and Evolution. 9. Article 788187. https://doi.org/10.3389/fevo.2021.788187.
Price, B.E., Yoon, J., Choi, M.Y., Lee, J.C. 2021. Effects of non-nutritional sugars on lipid and carbohydrate content, physiological uptake, and excretion in Drosophila suzukii. Archives of Insect Biochemistry and Physiology. 109(2). Article e21860. https://doi.org/10.1002/arch.21860.
Choi, M.Y. 2022. Pheromone biosynthesis activating neuropeptide (PBAN) in insects. Korean Journal of Applied Entomology. 61(1):15-28. https://doi.org/10.5656/KSAE.2022.02.0.013.
Abram, P.K., Wang, X., Hueppelsheuser, T., Franklin, M.R., Daane, K.M., Lee, J.C., Lue, C., Girod, P., Carrillo, J., Wong, W.H., Kula, R.R., Gates, M.W., Hogg, B.N., Moffat, C.E., Hoelmer, K.A., Sial, A., Buffington, M.L. 2022. A coordinated sampling and identification methodology for larval parasitoids of spotted-wing drosophila. Journal of Economic Entomology. https://doi.org/10.1093/jee/toab237.
Hogg, B.N., Lee, J.C., Rogers, M., Worth, L., Nieto, D., Stahl, J.M., Daane, K.M. 2022. Releases of the parasitoid Pachycrepoideus vindemiae for augmentative biological control of spotted wing drosophila, Drosophila suzukii. Biological Control. 168. Article 104865. https://doi.org/10.1016/j.biocontrol.2022.104865.
Hull, J.J., Brent, C.S., Choi, M.Y., Miko, Z., Fodor, J., Fonagy, A. 2021. Molecular and functional characterization of pyrokinin-like peptides in the western tarnished plant bug lygus hesperus (Hemiptera: Miridae). Insects. 12(10). Article 914. https://doi.org/10.3390/insects12100914.
Price, B.E., Lee, J.C., Choi, M.Y. 2021. Erythritol combined with non-nutritive sucralose increases feeding by Drosophila suzukii, quickens mortality and reduces oviposition. Crop Protection. 150. Article 105812. https://doi.org/10.1016/j.cropro.2021.105812.
Tait, G., Mermer, S., Stockton, D.G., Lee, J.C., Avosani, S., Abrieux, A., Anfora, G., Beers, E., Biondi, A., Burrack, H.J., Cha, D.H., Chiu, J., Choi, M.Y., Cloonen, K., Crava, C.M., Daane, K., Dalton, D.T., Diepenbrock, L., Fanning, P., Ganjisaffar, F., Gomez, M., Gut, L., Grassi, A., Hamby, K., Hoelmer, K.A., Ioriatti, C., Isaacs, R., Klick, J., Kraft, L., Loeb, G.M., Rossi-Stacconi, M.V., Nieri, R., Pfab, F., Puppato, S., Rendon, D., Renkema, J., Rodriguez-Saona, C., Rogers, M., Sassu, F., Schoneberg, T., Scott, M., Seagraves, M., Sial, A., Van Timmeren, S., Wallingford, A., Wang, X., Yeh, D., Zalom, F., Walton, V.M. 2021. Drosophila suzukii (Diptera: Drosophilidae): A decade of research towards a sustainable integrated pest management program. Journal of Economic Entomology. 114(5):1950-1974. https://doi.org/10.1093/jee/toab158.
Price, B.E., Raffin, C., Yun, S., Velasco-Graham, K., Choi, M.Y. 2022. A sustainable mass rearing method for western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae). Florida Entomologist. 105(2):170-173. https://doi.org/10.1653/024.105.0211.
