Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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. Artificial Intelligence (AI)/Machine Learning (ML) In collaboration with an Oregon State University researcher, deep learning was used to classify spotted-wing drosophila (SWD) from the closely resembling Drosophila melanogaster. Both species may visit the same traps, and distinction between the two species is important for pest management since growers actively monitor and control SWD. Drosophila melanogaster is a nuisance, and not a pest of harvestable fruit. Computing resources of the university collaborator were used. The image capture platform was re-designed and numerous training sets were run to distinguish between male and female SWD, which will benefit monitoring efforts and understanding behavioral patterns in the field. For in silico 3D modeling of SWD DH31 and DH44 GPCRs, ARS researchers in Corvallis, Oregon, have used two modeling databases, the Swiss model and AlphaFold. The Swiss model predicted the 3D structures of the two DH31 GPCRs well compared to the other DH receptors found in insects and mammals. The 3D structures of the two DH31-Rs are identical, except for their C-terminal structures, where different amino acid sequences are observed that are exactly matched with their 2D structures and the protein sequences. The 3D structures provide critical information between two receptors that are differentially activated by the DH31 peptide. Computational modeling facilitates the identification of the binding pocket structure of the specific receptor for bioactive peptides. ACCOMPLISHMENTS 01 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. 02 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. 03 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. 04 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.
Impacts
(N/A)
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. Article toae164. https://doi.org/10.1093/jee/toae164.
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Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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. Artificial Intelligence (AI)/Machine Learning (ML) In collaboration with an Oregon State University researcher, deep learning was used to classify spotted-wing drosophila (SWD) from the closely resembling Drosophila melanogaster. Both species may visit the same traps, and distinction between the two species is important for pest management since growers actively monitor and control SWD. D. melanogaster is a nuisance, and not a pest of harvestable fruit. Computing resources of the University collaborator were used. Redesign of the image capture platform and further deep learning are planned to distinguish between male and female SWD, which will benefit monitoring efforts and understanding behavioral patterns in the field. For in silico 3D modeling of insect GPCRs and bioactive peptides from SWD, thrips, and slug, ARS researchers in Corvallis, Oregon, have used two modeling databases, the Swiss model and AlphaFold for G Protein- Coupled Receptors (GPCRs), and the PEP-FOLD model for bioactive peptides. Along with our functional bioassays of GPCRs/bioactive peptides identified from SWD, thrips, and slug, the AI-based 3D modeling of GPCRs and bioactive peptides will further advance the goal of finding agonists and antagonists for the pests. ACCOMPLISHMENTS 01 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. 02 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. 03 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.
Impacts
(N/A)
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.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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. 02 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. 03 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. 04 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.
Impacts
(N/A)
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.
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Progress 10/01/20 to 09/30/21
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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. Record of Any Impact of Maximized Teleworking Requirement: The maximized telework requirement had a negative impact on the lab�s ability to conduct laboratory studies and maintain colonies/plants/cell lines to conduct studies. Sub-objective 1A required testing materials on target insects and Sub-objectives 1B and 1C screened bioactive peptides using insect cell lines have been temporarily lost because the experiments can only be done with appropriate equipment in the laboratory. We will try later with a fully recovered insect colony and cell lines. Sub-objective 2B required a robust parasitoid colony, and the colony was diminished when reared at home for telework due to mold development. Releases had to be done with a commercially available parasitoid wasp instead of the intended species. ACCOMPLISHMENTS 01 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. 02 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. 03 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. 04 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.
Impacts
(N/A)
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.
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