Research Project: Sustainable Insect Pest Management for Urban Agriculture and Landscapes

Location: Invasive Insect Biocontrol & Behavior Laboratory

2023 Annual Report

Objectives
Objective 1: Discover, characterize, develop and/or promote field adoption of pheromones and other behavior-modifying semiochemicals of key vegetable and fruit insect pests such as cucumber beetles, crucifer flea beetles, stink bugs, squash bugs, and spotted-wing drosophila. Objective 2: Characterize and evaluate native and non-native biological control agents for management of key vegetable pests such as stink bugs, squash bugs, & leaf-footed bugs. Objective 3: Decipher genomes, biochemical and molecular processes of invasive insect pests in order to mitigate damage by crop and landscape pests, such as stink bugs, gypsy moth, and other invasive species. Objective 4: Discover and develop microbial and molecular-based biopesticide agents for control of invasive forest and crop insect pests such as gypsy moth and true bugs, and including model species for evaluation. Objective 5: Utilize molecular approaches to enhance plant defense against destructive insect pests by manipulating the plant defense pathways and insect microflora with a focus on cole crop pests such as those of cabbage and broccoli.

Approach
The project brings together a research team with diverse expertise for multiple approaches to insect management. The proposed project will focus on control of key insect pests in small farms, urban gardens and landscapes, both organic and non-organic. Biobased integrated pest management approaches to be developed will include: (i) discovery and deployment of natural insect attractants and repellents; (ii) conservation and augmentation of beneficial insects; (iii) pest-specific microbial controls [bacteria (including Bacillus thuringiensis strains and Chromobacterium spp.) and baculoviruses]; and, (iv) crop genetic resistance using molecular-based gene discovery. Research will target insect pests that cause major damage to key crops such as cucurbits and cole crops, although other important crops such as small fruit, beans, and potatoes may receive attention for specific problems. Research will also include insect pests that cause major damage to woody plants in the urban landscape. The combination of semiochemical approaches, biological controls, molecular techniques, and crop resistance, will offer a range of non-chemical tactics useful to integrated pest management strategies for major pests in small farms, urban gardens and landscapes.

Progress Report
In support of Subobjective 1A: ARS scientists in Beltsville, Maryland, led field experiments with the previously synthesized striped cucumber beetle aggregation pheromone, vittatalactone, in several states. We have discovered that vittatalactone is attractive to other cucurbit pests as well the squash bug Anasa tristis, the horned squash bug Anasa armigera, and the spotted cucumber beetle, Diabrotica undecimpunctata. We have demonstrated a dose-response and compared effectiveness of several traps for capture of stiped and spotted cucumber beetles. We have also devised attract-and-kill bait stations combining pheromone with the feeding stimulant cucurbitacins with organic-approved toxins for selective control. Furthermore, we have worked with collaborators to show that the pheromone can be combined with inexpensive plant-derived volatiles (both floral and foliar) to increase cost-efficient attraction. ARS scientists have commissioned synthesis of decemdiol, the male-produced aggregation pheromone of the notorious Colorado potato beetle, in gram quantities, to allow testing by ARS and collaborators for trapping, trap cropping, and other potential control tactics. Studies are also underway for combination with repellants and/or antifeedants to constitute a push-pull strategy for this key potato, tomato, and eggplant pest. ARS chemists in Beltsville, Maryland, achieved a new synthesis of the crucifer flea beetle pheromones (+)-(6R,7S)-himachala-9,11-diene and (6R,7S)-10-hydroxyhimachalan-9-one; field trials are planned in 2024. This is valuable for attraction of both key pest species of flea beetles (Phyllotreta) that attack cole crops, canola, and other mustard-family crops. In support of Subobjective 1B: ARS scientists in Beltsville, Maryland, are working to develop formulation for the harlequin and brown marmorated stink bug pheromones, to commercialize attract-and-kill strategies, especially for harlequin bug. We are also collaborating in studies to attempt identification of the aggregation pheromones of the leaf-footed bugs (Leptoglossus) that are pests of forestry, fruit and vegetable crops in eastern and western United States as well as invaded regions of Europe, Asia, and South America. In support of Subobjective 1C: ARS scientists in Beltsville, Maryland, completed the development of attractant for Spotted wing drosophila (SWD) management. ARS scientists continued to develop the application of push-and-pull strategy (attractant to pull Spotted wing drosophila away from the orchard and repellent, methyl benzoate and methyl 2-methoxy benzoate, to push Spotted wing drosophilaout of the orchard) to manage Spotted wing drosophila population and protect the crop damages. ARS scientists also continued to evaluate sprayable controlled release formulations (microencapsulation and emulsion of methyl benzoate and its analogs as repellents/insecticides), with efficacy against bed bugs, mosquitoes, thrips, white flies, nematodes, and some stored product insect pests under laboratory conditions. In support of Subobjective 2A: ARS scientists in Beltsville, Maryland, are continuing studies with the biological control agent Hadronotus pennsylvanicus, the key egg parasitoid of squash bug and other leaf-footed bug (coreid) species. Hadronotus pennsylvanicus parasitizes polyphagous pest species in the genus Leptoglossus as well as the cucurbit specialist squash bugs (genus Anasa). This information is valuable in providing the groundwork for evaluating how parasitoids find host eggs in multiple habitats and if H. pennsylvanicus can play a role in suppressing populations of Leptoglossus species which are pests of vegetable, fruit and nut (pomegranate, pistachio) and conifer seed crops. Studies during summer 2023 will clarify the value of early-season augmentation of H. pennsylvanicus to suppress squash bug population in cucurbits, and also, whether the parasitoid is attracted to male or female squash bugs or leaf-footed bugs (Leptoglossus) to crop areas where it then parasitizes the bugs’ eggs. In support of Subobjective 3A: Four genome contig assemblies of Pentatomidae were generated by the Ag100Pest Initiative by ARS scientists in Beltsville, Maryland, Hilo, Hawaii, and Montpellier, France, including Halyomorpha halys, Murgantia histrionica, and two geographical isolates of Bagrada hilaris. All assemblies were generated using PacBio HiFi data generated from DNA from a single specimen. Data were filtered for adapters prior to assembly with the HiFiASM package. Hi-C libraries were generated and are in queue for sequencing in order to scaffold the assemblies after which they will be deposited into NCBI for automated gene predictions. Subsequently they will be incorporated into the i5k Workspace where they will be functionally annotated and incorporated into genome browsers for data visualization and manual curation. In support of Subobjective 3B: Analysis of the bagrada bug transcriptome was successfully performed in the reporting period, and a manuscript describing both quantitative analysis (at both gene and transcript levels) and qualitative analysis (including RNAi- and virus-related content, terpene-based sex pheromone biosynthetic genes and the three principal resistome gene families: glutathione S-transferases, carboxylesterases and cytochrome P450s) is to be submitted in FY23. The spongy moth subspecies present in the United States is characterized by flightless female moths. However, two Asian subspecies, collectively known as the flighted spongy moth, pose a significant invasive threat and are expected to have a much wider distribution in the United States due to its flightworthy females. Identifying the genetic differences between spongy moth and flighted spongy moth will assist in surveillance and identification of invading flighted spongy moth populations. Towards this end, ARS researchers in Beltsville, Maryland, together with Beijing Forestry University, Beijing, China collaborators, assembled and compared gene expression profiles of two United States populations of spongy moth and two Chinese populations of flighted spongy moth from high-throughput sequencing of female moth RNA. Distinct differences in gene expression between the United States and Chinese populations were identified. In addition,a follow-up study revealed that all four populations contained surprisingly high loads of a persistent RNA virus, with variant viral sequences that were associated with specific host populations. The population-specific virus and moth sequence data generated during this research are available at the National Center for Biotechnology Information( NCBI) Genbank and Sequence Read Archive databases and can be mined for genetic markers to be used in flighted spongy moth surveillance. In support of Subobjective 4A: An article describing full genome sequences, comprising sequences of plasmids encoding pesticidal proteins, of 14 strains of Bacillus thuringiensis was published in FY23. A manuscript describing a novel species, proposed as Chromobacterium ocalense, is currently under peer review. Lack of laboratory access due to COVID-19 building occupancy limitations and subsequent flooding of IIBBL laboratory space severely limited progress on Objective 4A. In lieu of genomic analysis of the IIBBL Bacillus thuringiensis collection, analysis of publicly available Bt genome sequences was initiated and is ongoing. In support of Sub-objective 4B: The impact of the antiviral compound ribavirin on the growth and viability of two iflavirus-infected spongy moth cell lines was assessed. Cell growth and viability was reduced at concentrations between twenty and 200 micrograms/milliliter of Ribavirin, with higher concentrations causing more severe reductions in growth and viability. In support of Sub-objective 4C: DNA samples from 62 baculovirus isolates of the IIBBL virus collection were subjected to high-throughput sequence determination. Draft genome sequence assemblies have been completed for 17 of the isolates. Seven of the isolates did not yield usable sequence data. A full analysis was completed on the genome of an alphabaculovirus from the tufted apple bud moth, and results indicate that it represents a novel alphabaculovirus lineage. The tufted apple bud moth alphabaculovirus did not exhibit insecticidal activity against larvae of diamondback moth, cabbage looper, or spongy moth in bioassays. In support of Sub-objective 4D: ARS scientists in Beltsville, Maryland, selected immune transcripts from Bagrada hilaris transcriptome as gene targets for the development of dsRNAs to induce detrimental effects. In vitro transcribed double-stranded RNA targets were tested in vivo in both injection and feeding studies and assayed for mortality and expression levels of gene targets, with those eliciting the greatest response having potential and being developed further for use in biocontrol.

Accomplishments
1. Discovery of striped cucumber beetle pheromone non-target attraction of four other vegetable pests. Behavioral control of pests using their own pheromones is a powerful tool for pest management. However, many pheromones are not known and even those that are known, have not been tested for their potential application pest management. ARS scientists in Beltsville, Maryland, in collaboration with scientists at University of California-Davis, California, Virginia Tech, Blacksburg, Virginia and Cornell University, Ithaca, New York, and University of New Hampshire Durham, New Hampshire, have shown that the male-produced aggregation pheromone of the striped cucumber beetle (synthesized by ARS) is also attractive to four other cucurbit (vegetable) pests: spotted cucumber beetle, squash bug, horned squash bug, and western striped cucumber beetle. This attraction allows development of traps, baits, or other attract-and-kill tactics useful in integrated pest management for cucurbit pests, for future commercialization.

2. Development of push-and-pull strategy for spotted wing drosophila pest management. Push-pull strategies use attractants and repellents to manage pests. Effective push-pull systems can divert pests away from crops and control them without broad-spectrum pesticides. ARS scientists in Beltsville, Maryland, continued collaboration with scientists in Towson University Baltimore, Maryland, Rutgers University New Brunswick, New Jersey, and University of Maryland Eastern Shore, Princess Anne, Maryland, to develop a push-and-pull strategy utilizing an optimized controlled-release dispenser for spotted wing drosophila (SWD) attraction and powerful SWD repellents, methyl benzoate (MB) and analog microcapsules, to reduce the SWD population and infestation in blueberry orchards. Because the natural semiochemicals were used in push-and-pull strategy, it has provided growers/farmers an efficient, convenient, safe, and environmentally friendly Spotted wing drosophila-control alternative.

3. Resources for surveillance of flighted spongy moth. Trees and forests in the northeastern United States suffer tremendous damage from outbreaks of the spongy moth, a defoliating pest. The spongy moth subspecies present in the United States is characterized by flightless female moths. However, two Asian subspecies, collectively known as the flighted spongy moth, pose a significant invasive threat and are expected to have a much wider distribution in the United States due to its flightworthy females. Identifying the genetic differences between spongy moth and flighted spongy moth will assist in surveillance and identification of invading flighted spongy moth populations. Towards this end, ARS researchers in Beltsville, Maryland, together with Beijing Forestry University collaborators, assembled and compared gene expression profiles of two U.S. populations of spongy moth and two Chinese populations of flighted spongy moth from high-throughput sequencing of female moth RNA. Distinct differences in gene expression between the United States and Chinese populations were identified. All four populations contained surprisingly high loads of a persistent RNA virus, with variant viral sequences that were associated with specific host populations. The population-specific virus and moth sequence data generated during this research are available at the NCBI National Center for Biotechnology Information Genbank and Sequence Read Archive databases and can be mined for genetic markers to be used in flighted spongy moth surveillance.

Review Publications
Rebholz, Z., Lancaster, J., Larose, H., Khrimian, A., Luck, K., Sparks, M., Gendreau, K., Shewade, L., Köllner, T., Weber, D.C., Gundersen, D.E., O'Maille, P., Morozov, A., Tholl, D. 2022. Ancient origin and conserved gene function in terpene pheromone evolution of stink bugs and hemipteran insects. Insect Biochemistry and Molecular Biology. https://doi.org/10.1016/j.ibmb.2022.103879.
Cornelius, M.L., Vinyard, B.T., Weber, D.C. 2022. Annual and seasonal changes in parasitism rates by Hadronotus pennsylvanicus (Hymenoptera: Scelionidae) on the squash bug Anasa tristis (Hemiptera: Coreidae) in squash fields: Implications for augmentative releases. Insects. https://doi.org/10.3390/insects13110984.
Haber, A.I., Pasteur, K., Guzman, F., Boyle, S.M., Kuhar, T.P., Weber, D.C. 2023. Spotted cucumber beetle (Diabrotica undecimpunctata howardi) is attracted to vittatalactone, the pheromone of striped cucumber beetle (Acalymma vittatum). Journal of Pest Science. https://doi.org/10.1007/s10340-022-01586-1.
Weber, D.C., Haber, A.I., Pasteur, K., Boyle, S.M., Kuhar, T.P., Cornelius, M.L. 2022. Both the squash bug Anasa tristis and horned squash bug Anasa armigera are attracted to vittatalactone, the aggregation pheromone of striped cucumber beetle. Environmental Entomology. https://doi.org/10.1093/ee/nvac079.
Blackburn, M.B., Sparks, M., Mishra, R., Bonning, B.C. 2023. Genomic sequencing of fourteen Bacillus thuringiensis isolates: Insights into geographic variation and phylogenetic implications. BMC Research Notes. https://doi.org/10.1186/s13104-023-06411-1.
Oliveira, H.P., Harrison, R.L., Ribeiro, B.M., Ardisson-Araujo, D.P. 2022. Identification and analysis of putative tRNA genes in baculovirus genomes. Virus Research. https://doi.org/10.1016/j.virusres. 322; Article 198949
Harrison, R.L., Rowley, D.L. 2022. The Parapoynx stagnalis nucleopolyhedrovirus (PastNPV), a divergent member of the alphabaculovirus group I clade, encodes a homolog of Ran GTPase. Viruses. https://doi.org/10.3390/v14102289.
Sparks, M., Wang, Y., Shi, J., Harrison, R.L. 2023. Lymantria dispar iflavirus 1 RNA comprises a large proportion of RNA in adult L. dispar moths. Insects. https://doi.org/10.3390/insects14050466.
Wang, Y., Sparks, M., Harrison, R.L., Shi, J. 2022. Analyses of adult transcriptomes from four different populations of the spongy moth, Lymantria dispar L., from China and the USA. Scientific Reports. https://doi.org/10.1038/s41598-022-18377-4.
Niogret, J., Ekayanti, A., Zhang, A. 2022. Sex pheromone of cocoa pod borer, Conopomorpha cramerella: field activity evaluation of pheromone formulations in Indonesia plantation. Insects. 13(8):663. https://doi.org/10.3390/insects13080663.
Villena, O.C., Sullivan, J.H., Landa, E.R., Yarwood, S.A., Torrens, A., Zhang, A., Leisnham, P.T. 2022. The role of tire leachate in condition-specific competition and the persistence of a resident mosquito from a competitively superior invader. Insects. https://doi.org/10.3390/insects13080663.
Yan, J., Zhou, Y., Jiang, D., Lu, Y., Liu, Y., Yu, M., Zhang, A., Yan, S. 2022. Evaluation of trap efficiency for the Asian longhorned beetle, Anoplophora glabripennis. Journal of Forestry Research. https://doi.org/10.1007/s11676-022-01550-w.
Amalin, D.M., Arcelo, M., Almarinez, B.J., Castillo, R.C., Legaspi, J.C., Tavera, M.A., Janairo, J.B., Zhang, A. 2023. Field evaluation of the sex pheromone of the cacao pod borer, Conopomorpha cramerella (Snellen) in the Philippines. Journal of Asia-Pacific Entomology. https://doi.org/10.3389/fagro.2023.1165299.
Ivanauskas, A., Zhang, A., Zhao, Y., Wei, W. 2023. Exploring changes in Volatile Organic Compound (VOC) profiles of tomato plants infected with phytoplasma. Phytopathogenic Mollicutes. 13(1):5-6. https://doi.org/10.5958/2249-4677.2023.00003.8.
Weber, D.C., Khrimian, A., Ramirez Bonilla, J.P., Grettenberger, I.M., Guzman, F., Haber, A.I. 2023. Vittatalactone is the male-produced aggregation pheromone of the western striped cucumber beetle, Acalymma trivittatum. Journal of Chemical Ecology. https://doi.org/10.1007/s10886-023-01437-y.
Strickland, J., Larson, N.R., Feldlaufer, M.F., Zhang, A. 2022. Characterizing the repellencies of methyl benzoate and its analogs against the common bed bug Cimex lectularius. Journal of Economic Entomology. 13(11):1060. https://doi.org/10.3390/insects13111060.
Niogret, J., Kendra, P.E., Ekaynati, A., Zhang, A., Marelli, J., Tabanca, N., Epsky, N.D. 2022. Development of a kairomone-based attractant as a monitoring tool for the cocoa pod borer, Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillariidae). Insects 13(9): 813.