Location: Invasive Insect Biocontrol & Behavior Laboratory
2017 Annual Report
Objectives
Objective 1: Identify, synthesize, and develop semiochemicals (pheromones, kairomones, plant volatiles, and other attractants and repellants) of significant insect crop pests (such as harlequin bug, bagrada bug, spotted wing drosophila, striped cucumber beetle, kudzu bug) and their natural enemies for use in integrated pest management.
Subobjective 1a: Develop attractant volatiles and trap designs to monitor and manage the harlequin bug.
Subobjective 1b: Assess known stink bug attractants for value in monitoring and/or managing bagrada bug.
Subobjective 1c: Identify plant volatiles that attract squash bugs.
Subobjective 1d: Identify plant and/or insect volatiles for detection and attraction of kudzu bug.
Subobjective 1e: Improve lures for spotted wing drosophila by identification of fruit-based attractants.
Subobjective 1f: Synthesize and test aggregation pheromone in combination with baits and traps for striped cucumber beetle management.
Objective 2: Develop arthropod biological controls for managing key vegetable pests such as stink bugs, squash bug, and cucumber beetles, including integration of natural enemies with other tactics such as microbial control, semiochemicals, and cultural pest controls.
Subobjective 2a: Evaluate the efficacy of the squash bug natural enemy complex for use in pest management and identify the most effective biological control agents.
Subobjective 2b: Assess the value of native egg parasitoids to suppress BMSB.
Objective 3: Advance effective microbial controls for key crop pests such as stink bugs, noctuid moths, and other seedling pests, including discovery of molecular and ecological mechanisms for sustained field reproduction and persistence, using diverse Bacillus thuringiensis (Bt) strains, Chromobacterium spp., baculoviruses, and other entomopathogens.
Subobjective 3a. Discover additional environmental isolates of C. subtsugae and related species, and characterize their insecticidal properties.
Subobjective 3b. Determine ability of Bt strains to persist in common vegetable mulches.
Subobjective 3c. Develop effective baculoviruses for key pest diamondback moth.
Objective 4: Discover naturally occurring biopesticides (such as botanical compounds and/or RNAis) targeting key vegetable pests such as stink bugs and cucumber beetles.
Objective 5: Determine and strengthen the genetic basis of plant defense mechanisms (e.g., pest aversion and resistance) for protection of high-value crops such as cole crops on small farms or gardens against destructive insects, and determine the influence of the microflora of pest insects and/or plants on the plant defense response.
Subobjective 5a: Determine the effect of knocking out or over-expressing the infestation responsive TF StZFP2 in its native species, and in Arabidopsis.
Subobjective 5b: Determine how infestation inducible Q-type C2H2 TFs affect resistance to pest insects in Arabidopsis and B. oleracea.
Subobjective 5c: Determine the influence of plant and insect microflora on the infestation response of Arabidopsis to T. ni.
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 and urban gardens, both organic and non-organic. Bio-based 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 including use of their semiochemicals; (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. 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 crop pests in urban small farms and gardens.
Progress Report
Objective 1: Identify, synthesize, and develop semiochemicals (pheromones, kairomones, plant volatiles, and other attractants and repellants) of significant insect crop pests and their natural enemies for use in integrated pest management. The aggregation pheromone of the harlequin bug (Murgantia histrionica) is a powerful tool for monitoring and managing this important vegetable pest. The pheromone identified earlier consists of two principal components, both of which are important for the attractiveness. To allow an efficient scale-up of pheromone production, ARS scientists in Beltsville, Maryland, developed a new synthetic procedure that provides both components in improved yields for bioassays. Due to the overlap in chemical composition with brown marmorated stink bug pheromone, this new synthesis is valuable to management of both species. Field attraction of bagrada bug (Bagrada hilaris) to 10,11-epoxy-1-bisabolen-3-ols which contain pheromones of the brown marmorated stink bug and harlequin bug, was not reproduced by California cooperators, and therefore an APHIS-permitted colony of this western vegetable pest was obtained for further ecological, behavioral, and semiochemical study starting in July 2017. A study of the ovipositional preferences of two squash bug species, Anasa tristis and Anasa armigera, for different cultivars and species of plants in the family Cucurbitaceae was completed. This study provided evidence that A. tristis had an induced preference for the cultivar it was reared on compared with several other cultivars of the same species. Also, A. tristis had a significant preference in paired choice tests for Cucurbita pepo over cucumber and watermelon, but not C. maxima or C. moschata. This study is the first experimental evidence of the ovipositional preferences of the horned squash bug, A. armigera. Horned squash bugs did not show any preference for cucumber over C. pepo, regardless of which species they were reared on. However, they did show a significant preference for cucumber over watermelon and C. moschata, and they preferred C. maxima to cucumber. Ovipositional preference tests provided the groundwork for further research on the responses of squash bugs to host plant volatiles. Methyl benzoate isolated from apple juice, and naturally-occurring in many other plants, was found to possess toxicity against various stages of a variety of insect pests, including the invasive brown marmorated stinkbug (BMSB) and spotted wing drosophila (SWD). Methyl benzoate was at least 5 to 20 times more toxic than a conventional pyrethroid (ß-cyfluthrin), sulfur & pyrethrin mixture, and some commercial organic products available on the market against BMSB eggs and SWD larvae and adults. ARS obtained a U.S. Patent (US 9629362 B1) covering this technology. In addition, an attractant blend was developed for spotted wing drosophila (Patent applied for). SWD research is supported by the USDA NIFA CPPM grant, “Managing an Invasive Drosophilid Species in Agriculture using Innovative Behavioral Manipulation.” A pheromone was identified for the peanut burrower bug, the first for this family of true bugs (Cydnidae). Research is supported by the USDA NIFA CPPM grant, “Development of Risk Assessment and Monitoring Tools for Peanut Burrower Bug in Southeastern US Peanut Production Systems.”
Objective 2: Develop arthropod biological controls for managing key vegetable pests such as stink bugs, squash bug, and cucumber beetles, including integration of natural enemies with other tactics such as microbial control, semiochemicals, and cultural pest controls. A two year study of parasitism and predation rates on wild and sentinel eggs of the squash bug, Anasa tristis, in squash fields at BARC was completed and the results were published in 2016. Gryon pennsylvanicum accounted for 99% of the egg parasitism. The egg parasitism rate peaked in late July at 72.8%. In addition, laboratory studies evaluating the efficacy of Gryon pennsylvanicum as a biological control agent have been completed. In the laboratory, the average parasitism rate of a single female without prior exposure to eggs was 59.6%, resulting in a squash bug nymphal survival rate of 22.9%. There were significant differences in the parasitism rate based on egg size of squash bug egg masses, with parasitism rates ranging from < 20% for the largest masses and > 90% for the smallest. A two-year field study of sentinel egg masses of the pest brown marmorated stink bug, and the beneficial stink bug, spined soldier bug (SSB, Podisus maculiventris), deployed in 2015-16 to field sites in Maryland, Virginia, DC, Delaware, and West Virginia, has yielded lesser numbers of the adventive Asian parasitoid, Trissolcus japonicus, in the DC region, but new state records for Delaware and West Virginia. This makes the redistribution of the parasitoid within these states allowable for BMSB suppression. However, T. japonicus only attacks BMSB in certain habitats and is not yet abundant. It successfully attacks the beneficial sentinel egg masses of SSB, however, from laboratory rearings these eggs are too small for optimal parasitoid success. Successful emergence of some native egg parasitoids and higher rates of egg mass consumption by native predators was also notable. Further research is supported by USDA NIFA SCRI CAP grant, “Management of BMSB in US Specialty Crops,” through North Carolina State University in cooperation with several other universities and ARS labs.
Objective 3: Advance effective microbial controls for key crop pests such as stink bugs, noctuid moths, and other seedling pests, including discovery of molecular and ecological mechanisms for sustained field reproduction and persistence, using diverse Bacillus thuringiensis (Bt) strains, Chromobacterium spp., baculoviruses, and other entomopathogens. Chromobacterium species were isolated from diverse aquatic habitats and characterized by genomic sequencing, biochemical properties, and their insecticidal properties against diamondback moth, seedcorn maggot and red flour beetle. Two previously unknown species toxic to seedcorn maggot were identified in FY17 by comparing their genomic sequences with known Chromobacterium species. These newly described species can be developed into organic insecticides. To date, four new species of insecticidal Chromobacterium have been identified with lepidopteran and/or dipteran toxicity. The phylogenetic relationship of these species with other species Chromobacterium has been analyzed by analysis of their genomic sequences. A manuscript describing the first of these, C. sphagni, has been accepted for publication. The target pests for Bacillus thuringiensis toxins or modified toxins has been broadened by ARS work with University of Florida with support from a USDA NIFA SCRI grant, “Bt toxin-based strategies for management of Diaphorina citri and citrus greening.” Toxins with promise for control of psyllids, including for control of Asia citrus psyllid, the vector of the devastating citrus greening disease, huanglongbing, will be used to develop resistance in citrus. Determination and analysis of the genomes of two baculoviruses from the true armyworm were completed. A novel virus gene that may be involved in protection of viral DNA during infection was identified. Evidence emerged of extensive exchange of genes among the genomes of different true armyworm and Oriental armyworm baculoviruses. Old World bollworm betabaculovirus isolates from the IIBBL collection were evaluated for pathogenicity in bioassays against bollworm larvae. A group of isolates deposited in 1973, 1978, and 1993 killed larvae with symptoms previously reported for this baculovirus.
Objective 4: Discover natural biopesticides (such as botanical compounds and/or RNAi’s) targeting key vegetable pests such as stink bugs and cucumber beetles. ARS scientists in Beltsville, Maryland, made progress in developing RNA interference (RNAi) tools for use with harlequin bug. RNAi deactivates or silences specific genes to disable a target organism. The transcriptome, or the total complement of active genes in the harlequin bug, was determined for different life stages and both sexes of the adult stage. Intraspecific comparison of transcripts was made to identifying a wide variety of differentially expressed transcripts and a reliable source to determine genes involved in key physiological processes. Distinct transcripts associated with particular life stages or sexes, or associated with highly specific biological systems, for example semiochemistry-related genes expressed in adult male during pheromone biosynthesis, were identified. These represent candidate harlequin bug-specific gene targets that may be involved in key synthesis of semiochemicals or may serve as candidates for disruption by molecular biopesticide technologies useful in managing the negative impacts of this insect pest. Specific RNAi- inducing dsRNAs for harlequin bug were developed and used to evaluate RNAi efficacy in vivo.
Objective 5: Determine and strengthen the genetic basis of plant defense mechanisms (e.g., pest aversion and resistance) for protection of high-value crops such as cole crops on small farms or gardens against destructive insects. In order to determine the role of the potato gene StZFP2 in resistance to insects or pathogens, transgenic potato lines were produced knocking down or over-expressing StZFP2. The StZFP2 gene in potato is turned on during numerous plant stresses such as insect feeding and late blight infection. StZFP2 encodes a transcription factor that regulates the expression of numerous genes. These transgenic lines have been tested for their level of resistance to potato late blight (Phytophthora infestans) and infestation by Manduca sexta. Several of the over-expressing lines show increased resistance to late blight and several of the knock-down lines are significantly less resistant compared to non-transformed.
Accomplishments
1. Key genes important for biochemical pathways and pest management identified in harlequin bug by comparative transcriptome analysis. Murgantia histrionica (Hahn), the harlequin bug, is an invasive insect that is a major vegetable insect pest in the United States. Harlequin bug is a piercing/sucking feeder that poses a considerable ecological and economic threat to mustard-family crops (Brassicaceae) especially in the southern U.S. ARS scientists in Beltsville, Maryland, determined the total complement of active genes the transcriptome of different harlequin bug life stages and both sexes of the adult stage. Intraspecific comparison of these transcriptomes identified a wide variety of genetic components involved in key physiological processes. Some genetic components were associated with particular life stages or sexes, or were associated with highly specific biological systems, for example semiochemistry in adult male during pheromone biosynthesis. These represent candidate harlequin bug-specific gene targets that may be candidates for disruption by biopesticide technologies to help manage the negative impacts of this insect pest. This information will be useful to understand genetic components involved in pheromone biosynthesis and pesticide detoxification.
2. Methyl benzoate found toxic to invasive insect pests. ARS scientists in Beltsville, Maryland, discovered that methyl benzoate, isolated from apple juice and naturally-occurring in many other plants, was toxic to various stages of a variety of insect pests, including the invasive brown marmorated stinkbug (BMSB) and spotted wing drosophila (SWD). Against BMSB eggs and SWD larvae and adults, methyl benzoate was found to be 5 to 20 times more toxic than a conventional treatments. Because methyl benzoate is considered environmentally friendly, it has great potential to be used as a safer alternative to synthetic pesticides for sustainable agriculture.
Review Publications
Harrison, R.L., Rowley, D.L., Funk, C. 2016. The complete genome sequence of Plodia interpunctella granulovirus: Discovery of an unusual inhibitor-of-apoptosis gene. PLoS One. 11(7):e0160389.
Feng, Y., Zhang, A. 2017. A floral fragrance, methyl benzoate, is an efficient green pesticide. Scientific Reports. 7:42168.
Harrison, R.L., Rowley, D.L., Mowery, J.D., Bauchan, G.R., Theilman, D.A., Rohrmann, G.F. 2017. The complete genome sequence of a second distinct betabaculovirus from the true armyworm, Mythimna unipuncta. PLoS One. 12(1):e0170510.
Weber, D.C., Morrison III, W.R., Khrimian, A., Rice, K.B., Leskey, T.C., Rodriguez-Saona, C., Nielsen, A.L., Blaauw, B.R. 2017. Chemical ecology of Halyomorpha halys: Discoveries and applications. Journal of Pest Science. 90:989-1008.
Abram, P.K., Hoelmer, K.A., Acebes-Doria, A., Andrews, H., Beers, E., Bergh, C.J., Bessin, R., Biddinger, D., Botch, P., Buffington, M.L., Cornelius, M.L., Costi, E., Delfosse, E., Dieckhoff, C., Dobson, R., Donais, Z., Grieshop, M., Hamilton, G., Haye, T., Hedstrom, C., Herlihy, M.V., Hoddle, M., Hooks, C., Jentsch, P., Neelandra, J., Kuhar, T., Lara, J., Legrand, A., Lee, J.C., Leskey, T.C., Lowenstein, D., Milnes, J., Maistrello, L., Morrison III, W.R., Nielsen, A.L., Ogburn, E., Pickett, C., Poley, K., Pote, J., James, R., Shrewsbury, P., Talamas, E.J., Tavella, L., Walgenbach, J., Waterworth, R., Weber, D.C., Welty, C., Wiman, N.G. 2017. Integrative review of indigenous arthropod natural enemies of the invasive brown marmorated stink bug in North America and Europe. Journal of Pest Science. 90(4):1009-1020.
Blackburn, M.B., Sparks, M., Gundersen, D.E. 2016. The genome of the insecticidal chromobacterium subtsugae PRAA4-1 and its comparison with that of chromobacterium violaceum ATC12472. Genomics. doi: 10.1016/j.gdata.2016.08.013.
Cornelius, M.L., Dieckhoff, C., Vinyard, B.T., Hoelmer, K.A. 2016. Parasitism and predation on sentinel egg masses of the brown marmorated stink bug (Hemiptera: Pentatomidae) in three vegetable crops: Importance of dissections for evaluating the impact of native parasitoids on an exotic pest. Environmental Entomology. 45(6):1536-1542.
Dimeglio, A.S., Wallingford, A.K., Weber, D.C., Kuhar, T., Mullins, D. 2016. Supercooling points of Murgantia histrionica (Heteroptera: Pentatomidae) and field mortality in the Mid-Atlantic United States following lethal low temperatures. Environmental Entomology. 45(5): 1294-1299.
Meyer, S.L., Chauhan, K.R., Macdonald, M.H. 2016. Evaluation of roselle (Hibiscus sabdariffa) leaf and pomegranate (Punica granatum) fruit rind for activity against Meloidogyne incognita. Nematropica. 46(1):85-96.
Shirali, S., Guzman, F., Weber, D.C., Khrimian, A. 2017. Expedient synthesis of bisabolenol stink bug pheromones via stereodefined cyclohex-2-enones. Tetrahedron Letters. 58:2066-2068.
Short, B.D., Khrimian, A., Leskey, T.C. 2016. Pheromone-based decision support tools for management of Halyomorpha halys in apple orchards: development of a trap-based treatment threshold. Journal of Pest Science. doi: 10.1007/s10340-016-0812-1.
Sparks, M., Rhoades, J.H., Nelson, D.R., Kuhar, D.J., Lancaster, J., Lehner, B., Tholl, D., Weber, D.C., Gundersen, D.E. 2017. A transcriptome survey spanning life stages and sexes of the Harlequin bug, Murgantia histrionica. Insects. doi: 10.3390/insects8020055.
Ze, S., Zhuang, L., Wen, Z., Huanan, J., Hao, L., Aiming, Z., Zhang, A., Man-Qun, W. 2016. Temporal interactions of plant - insect - predator after infection of bacterial pathogen on rice plant. Scientific Reports. 6:26043.
Sun, X., Zeng, F., Yan, M., Zhang, A., Wang, M. 2016. Interactions of two odorant-binding proteins from Cnaphalocrocis medinalis Güenée (Lepidoptera: Pyralidae). Insect Molecular Biology. 25(6):712-723.
Sun, X., Zhao, Z., Zeng, F., Zhang, A., Lv, Z., Wang, M. 2016. Functional characterization of a pheromone binding protein from rice leaf-folder cnaphalocrocis medinalis in detecting pheromones and host plant volatiles. Bulletin of Entomological Research. 106(6):781-189.
Thangaiah, S., Webb, M.Z., Ganga, B., Chauhan, K.R. 2016. Synthesis of southern corn root worm pheromone from S-Citronellol and its field evaluation. Journal of Agricultural Chemistry and Environment. 5:223-230.
