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

Location: Invasive Insect Biocontrol & Behavior Laboratory

2022 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
Sub Objective 1A: ARS scientists in Beltsville, Maryland, led field experiments with the previously synthesized striped cucumber beetle aggregation pheromone, vittatalactone, in several states. Vittatalactone proved to be the male-produced aggregation pheromone of the western striped cucumber beetle, Acalymma trivittatum, as was previously shown for the (eastern) striped cucumber beetle, Acalymma vittatum. A synthetic vittatalactone is attractive to both sexes of both species in the field, as demonstrated by trapping using baited and unbaited sticky panels in California and in Maryland. Females of both species do not produce detectable vittatalactone. We have discovered that vittatalactone is attractive to other cucurbit pests as well: the squash bug Anasa tristis, and the horned squash bug Anasa armigera, and the spotted cucumber beetle, Diabrotica undecimpunctata. ARS chemists in Beltsville, Maryland, achieved a new synthesis of the flea beetle pheromone (+)-(6R,7S)-himachala-9,11-diene based on the (-)-a-himachalene obtained by fractional distillation of an essential oil. This is also the starting material for the related pheromone of the striped crucifer flea beetle, (6R,7S)-10-hydroxyhimachalan-9-one; thus, the diene is valuable for attraction of both key pest species of flea beetles (Phyllotreta) that attack cole crops, canola, and other mustard-family crops. Sub Objective 1B: ARS scientists in Beltsville, Maryland, with university collaborators, are examining the pest management implications of the non-target attraction of vittatalactone, the aggregation pheromone of striped cucumber beetle, and its combination with plant-produced floral volatiles as well. Further research is examining the strength of different timing, placement, and combination with cucurbitacin-containing baits for an attract-and-kill approach that is protective of essential pollinators. Sub Objective 1C: ARS scientists in Beltsville, Maryland, completed the development of the controlled-release dispenser for optimization of spotted wing drosophila (SWD) attraction and application. Specifically, ARS scientists in Beltsville, Maryland, collaborated with scientists at Towson University and the University of Maryland Eastern Shore and developed a push-and-pull strategy utilizing an optimized controlled-release dispenser for spotted wing drosophila (SWD) attraction and a powerful SWD repellent, methyl benzoate (MB), to reduce the SWD population and infestation in blueberry plantations. Because the natural semiochemicals were used in push-and-pull strategy, growers were provided with an efficient, convenient, safe, and environmentally friendly SWD-control alternative. ARS scientists in Beltsville, Maryland, also continued to evaluate methyl benzoate and its analogs as insecticides, with efficacy against bed bugs, thrips, white flies, nematodes, and some stored product insect pests under laboratory conditions. ARS scientists in Beltsville, Maryland, screened naturally occurring products for toxicology against SWD in respirometry assays. Transcriptome analysis of exposed SWD to the compound of interest were initiated to determine potential genetic targets of the natural product. Sub Objective 2A: ARS scientists in Beltsville, Maryland, completed a two-year project to evaluate the effects of host plant species on the host finding behavior of H. pennsylvanicus, the key egg parasitoid of squash bug species. Results suggest that parasitoids were able to specifically orient to the combination of host plant and host cues to locate the pest egg masses. Hadronotus pennsylvanicus parasitizes polyphagous pest species in the genus Leptoglossus as well as the cucurbit specialist squash bugs (genus Anasa). Preliminary results indicate that H. pennsylvanicus may prefer Leptoglossus oppositus to the two squash bug species in paired choice tests. 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 L. oppositus and related Leptoglossus species which are pests of vegetable, fruit, and nut (pomegranate, pistachio) and conifer seed crops. The first year of a two-year study was completed to evaluate parasitism by the tachinid fly Trichopoda pennipes on adult squash bugs in different cucurbit crops. Differences in parasitism rates will be compared by squash bug species, sex, and habitat. Sub Objective 3A: Four genome contig assemblies of Pentatomidae were generated under 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. Sub Objective 3B: Analysis of the bagrada bug transcriptome was delayed during the reporting period, as its scope was expanded to include analysis of transcripts derived from eggs. Their integration into the project required a full reworking of the global transcriptome assembly, quantitative analysis (at both gene and transcript levels) and qualitative analysis of virus-related content and key resistome gene families: glutathione S-transferases, carboxylesterases and cytochrome P450s. These gene families have now been phylogenetically analyzed in the context of four select hemipteran and five coleopteran comparator species. In collaboration with Virginia Tech, a family of isoprenyl diphosphate synthase-like genes from the brown marmorated stink bug were further characterized in terms of their evolutionary histories. Sub Objective 4A: ARS scientists in Beltsville, Maryland, have generated high quality genomic sequence data for 16 isolates of Bacillus thuringiensis used in a multi-lab NIFA project to identify and develop Bt toxins effective against the Asian citrus psyllid. The isolates included examples of Bt varieties kurstaki, pakistani, toumanoffi, israelensis, thuringiensis, finitimus and entomocidus. In most cases, the plasmids of the isolates were fully assembled. Although the isolates were geographically diverse, the plasmid complement within a given varieties appeared to be generally conserved, including smaller plasmids not encoding Cry toxins. A number of apparent genes for novel toxins were found. In collaboration with the European Biological Control Laboratory, ARS scientists in Beltsville, Maryland, have sequenced the genome of a bacterium belonging to the genus Serratia associated with several braconid parasitoids of the olive fruit fly. The bacterium was determined to represent a new species by comparing its genome sequence with those of other Serratia species. The new species joins several other species of Serratia that are either pathogens, symbionts or otherwise associated with insects or other invertebrates. Sub Objective 4B: As part adult moth transcriptomic analysis, the Lymantria dispar iflavirus genome was detected in adults of all four populations (2 Chinese, 2 USA) at a high level, and iflavirus genome sequences indicate that Lymantria dispar iflavirus in Chinese and U.S. moths have diverged from each other. Sub Objective 4C: Genome sequences were determined for isolates of seven alphabaculoviruses and one betabaculovirus. Three of the alphabaculovirus isolates and the betabaculovirus isolate represent previously undescribed species of baculoviruses. Detailed analysis of isolate 435 from larvae of the brown tussock moth revealed that it belonged to a novel species in a group of viruses that infect the spongy moth and related moths. Twelve alphabaculovirus isolates and two betabaculovirus isolates were tested for insecticidal activity against larvae of spongy moth, diamondback moth, and cabbage looper. Inoculation with three alphabaculovirus isolates from eastern tent caterpillar killed spongy moth larvae, though it appears that contaminating Bacillus thuringiensis bacteria in these isolates may be responsible for their insecticidal activity. Sub Objective 4D: ARS scientists in Beltsville, Maryland, selected immune transcripts from Bagrada hilaris transcriptome as gene targets for the development of dsRNAs. 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. Adult moth transcriptomes from four different populations of the spongy moth, Lymantria dispar L., two from China and two from the U.S., were developed and analyzed. Differences in gene expression between the Chinese and North American populations were characterized. An international collaboration with Beijing Forestry University on comparative transcriptomics of European and Asian spongy/ gypsy moth geographical isolates has resulted in preparation of a manuscript recently submitted to a peer-reviewed research journal: ARS-115: #394096. Wang, Y.-M., Sparks, M.E., Harrison, R.L. and Shi, J. (2022) Analyses of adult transcriptomes from four different populations of the spongy moth, Lymantria dispar L., from China and the USA.

Accomplishments
1. Development of new trapping system for spotted wing drosophila pest management. ARS scientists in Beltsville, Maryland, collaborated with scientists at Towson University in Maryland, and others in France, Germany, Spain, and Italy, to develop a dry trap baited with an optimized controlled-release dispenser for spotted wing drosophila (SWD). The new trapping system achieved earlier SWD detection compared to the conventional liquid traps that are currently used in orchards and farms. Due to the simplicity of the dry trap design with controlled release dispenser, growers are provided with an efficient, convenient, and easy to process tool for SWD infestation detection and control.

2. Discovery of a new class of safer insecticides against yellow fever mosquito. ARS scientists in Beltsville, Maryland, have discovered novel composition of chemicals and application for repelling, knocking down, and killing adult female yellow fever mosquito, Aedes aegypti. Methyl benzoate (MB) and several analogs have been shown to have spatial repellent properties, knockdown effect, and lethal activity against Ae. aegypti and compared with the “gold standard” mosquito repellent, DEET, under laboratory conditions. Several compounds not only can repel but also knock down and kill Ae. aegypti efficiently. These compounds are the most promising DEET alternatives and have high potential to be used in development of new products, providing improved real-world protection against Ae. aegypti as well as other mosquito pests and reducing their damage to human health.

Review Publications
Haber, A.I., Weber, D.C. 2021. Both male and female-produced pheromones influence Colorado potato movement in the field. Pest Management Science. https://doi.org/10.1002/ps.6739.
Malek, R., Kaser, J.M., Anfora, G., Ciolli, M., Khrimian, A., Weber, D.C., Hoelmer, K.A. 2021. Trissolcus japonicus foraging behavior: Implications for host preference and classical biological control. Biological Control. https://doi.org/10.1016/j.biocontrol.2021.104700 .
Cornelius, M.L., Haber, A.I., Weber, D.C. 2022. Host finding behavior of the parasitoid Hadronotus pennsylvanicus (Hymenoptera: scelionidae) for egg masses of the squash bugs Anasa tristis and Anasa armigera (Hemiptera: coreidae) in squash and cucumber fields. Environmental Entomology. https://doi.org/10.1093/ee/nvac018.
Weber, D.C., Konstantinov, A.S., Khrimian, A., Bier, A.D., Lubenow, L., Knodel, J., Haber, A.I., Wallingford, A.K., Mason, J., Kuhar, T. 2022. Trapping of crucifer flea beetles (Phyllotreta spp.)(Coleoptera: chrysomelidae) with pheromones and plant kairomones. Journal of Economic Entomology. 115:1-9. https://doi.org/10.1093/jee/toac042.
Boyle, S.M., Alford, A.M., Mcintyre, K.C., Weber, D.C., Kuhar, T.P. 2022. Effect of plastic mulch colors on Anasa tristis (Hemiptera: coreidae) population dynamics in summer squash (Cucurbita pepo). Journal of Economic Entomology. https://doi.org/10.1093/jee/toac036.
Popham, H.R., Rowley, D.L., Harrison, R.L. 2021. Insecticidal properties of isolates of Spodoptera frugiperda multiple nucleopolyhedrovirus against corn-strain and rice-strain fall armyworm, and complete genome sequences of three isolates from the USA and Colombia. Journal of Invertebrate Pathology. https://doi.org/10.1016/j.jip.2021.107561.
Tanaka, S., Harrison, R.L., Hiroshi, A., Katayama, Y., Mizutani, T., Inoue, M.N., Miles, J.E., Marshall, S.D., Kitalong, C., Nakai, M. 2021. Confirmation of Oryctes rhinoceros nudivirus infections in G-haplotype coconut rhinoceros beetles (Oryctes rhinoceros) from Palauan PCR-positive populations. Scientific Reports. https://doi.org/10.1038/s41598-021-97426-w.
Harrison, R.L., Rowley, D.L. 2022. The complete genome sequence of an alphabaculovirus from the brown tussock moth, Olene mendosa Hübner, expands our knowledge of lymantriine baculovirus diversity and evolution. Virus Genes. https://doi.org/10.1007/s11262-022-01899-4.
Pinero, J.C., Shapiro Ilan, D.I., Cooley, D.R., Tuttle, A., Eaton, A., Drohan, P., Leahy, K., Zhang, A., Hancock, T., Wallingford, A.K., Leskey, T.C. 2020. Toward the integration of an attract-and-kill approach with entomopathogenic nematodes to control multiple life stages of plum curculio (Coleoptera: Curculionidae). Insects. https://doi.org/10.3390/insects11060375.
Larson, N.R., Strickland, J.A., Zhang, A., Feldlaufer, M.F. 2020. Behavioral activity of methyl benzoate against bed bug adults. Journal of Entomological Science. 55(3):344-349.
Larson, N.R., Nega, M., Zhang, A., Feldlaufer, M.F. 2021. Toxicity of methyl benzoate and analogs to adult Aedes aegypti. Journal of the American Mosquito Control Association. 37(2):83-86.
Andreazza, F., Valbon, W.R., Wang, Q., Liu, F., Xu, P., Bandason, E., Chen, M., Wu, S., Smith, L.B., Scott, J.G., Jiang, Y., Jiang, D., Zhang, A., Oliveira, E.E., Dong, K. 2021. Sodium channel activation underlies transfluthrin repellency in Aedes aegypti. Proceedings of the National Academy of Sciences (PNAS). https://doi.org/10.1371/journal.pntd.0009546.
Lawrence, S.D., Farrar, R.R., Blackburn, M.B., Shao, J.Y., Novak, N.G. 2022. The response to cabbage looper infestation in Arabidopsis is altered by lowering levels of Zat18 a Q-type C2H2 zinc finger protein. Journal of Plant Interactions. https://doi.org/10.1080/17429145.2021.2024285.
Wu, S., Blackburn, M.B., Mizell, R.F., Duncan, L.W., Toews, M.D., Sparks, M., El-Borai, F., Bock, C.H., Shapiro Ilan, D.I. 2021. Pupal cell antibiosis suppresses plant and insect pathogenic fungi and is associated with a bacterium related to Serratia nematodiphila i. Journal of Invertebrate Pathology. 184/107655. https://doi.org/10.1016/j.jip.2021.107655.
Grodowitz, M.J., Gundersen, D.E., Elliot, B., Evans, R.M., Sparks, M., Reed, D.A., Miles, G.P., Allen, M.L., Perring, T.M. 2022. Trypanosomatids associated in the alimentary canal of Bagrada hilaris (Hemiptera: Pentatomidae). Journal of Insect Science. 22(1):1-6. https://doi.org/10.1093/jisesa/ieab110.
Wang, X., Ramualde, N., Desurmont, G., Smith, L., Gundersen, D.E., Grodowitz, M.J. 2021. Reproductive traits of the egg parasitoid Aprostocetus fukutai a promising biological control agent for invasive citrus longhorned beetle Anoplophora chinensis. Biocontrol. https://doi.org/10.1007/s10526-021-10118-2.