Research Project: Biologically-Based Products for Insect Pest Control and Emerging Needs in Agriculture

Location: Biological Control of Insects Research

2023 Annual Report

Objectives
Objective 1: Identify new molecular components of insect immune signaling. Objective 2: Determine the biology of insect cell line establishment and associated cryopreservation technologies to establish next-generation insect cell lines to meet specific needs of academic and indus¬trial partners, such as the need for honey bee cell lines. Objective 3: Identify genomic structural variants and metabolites contributing to corn rootworm toxin resistance phenotypes and develop genetic markers to assess rootworm resistance. Objective 4: Determine the influence of microbiomes on the performance of selected agricultural pests, including the spotted wing Drosophila.

Approach
1A: Existing A. tristis genetic databases will be used to identify candidate PGF2a synthase genes based on sequence similarities to known PGF2a synthase genes from other organisms. 1B: Utilizing standard molecular biology techniques, candidate PGF2a synthase genes will be cloned and their biological function will be verified experimentally. 1C: Advanced molecular biology techniques and chemical agents will be used to impair PGF2a synthase function and any effects on immune system function and reproductive health will be measured experimentally. 2A: Variations in DNA, RNA and protein expression patterns will be compared between primary cultures and replicating cell lines using next-generation bioinformatic tools and analyses. 2B: Cell lines will be genetically engineered through the introduction of plasmid DNA coding for proteins of interest, and the expression and proper function of the introduced proteins will be verified in cell-based fluorescent reporter assays. 2C: Various techniques and proprietary technologies will be tested for their ability to improve the survival rate of cells as they are frozen and stored for long time periods. 3A: Structural variants of ABC transporters will be identified from genetic data produced from Bt toxin-resistant and toxin-sensitive lab colonies of corn rootworm. Changes in ABC transporter expression levels will be evaluated following exposure of corn rootworms to Bt toxins. The functional role of these variants in Bt toxin resistance will be evaluated by gene knockdown experiments. 3B: Genetic markers for variants of ABC transporters, as well as other genes, associated with Bt toxin resistance will be identified from genetic databases, and their presence in field populations and other corn rootworm strains will be evaluated using standard molecular biology techniques. 3C: Metabolites that are up- or down-regulated in Bt-resistant and Bt-sensitive lab colonies of corn rootworm will be measured using standard analytical chemistry techniques. Compounds with significantly different expression patterns will be linked to metabolic pathways and their functional role in Bt toxin resistance or sensitivity will be evaluated experimentally. 4A: The gut microbiome of wild Drosophila suzukii will be compared to a microbial database to characterize its community structure. Statistical modeling will be used to further determine whether certain microbes tend to co-occur. Further models will test whether individual and co-associated groups of microbes seem particularly suited to colonizing the wild fly gut. 4B: Lipid content of the fly guts will be measured. Statistical analyses will then be used to determine whether specific microbes are associated with increased or diminished fat content in the fly host. Germ-free flies will be generated and a portion of them will be exposed to select microbes to verify their influence on the host. 4C: The genomes of microbes of interest will be sequenced and genes and genetic pathways will be associated with host lipid content. Finally, germ-free flies will be exposed to bacteria modified to lose or gain these genes to verify their influence on the host.

Progress Report
In support of Objective 1, Goal 1.1., we established nervous tissue cell lines from the international pest, the fall armyworm, Spodoptera frugiperda. Fall armyworms are serious pests in the United States, Mexico, South America, and, since 2016, African countries. Nervous tissue cell lines from the international pest, the fall armyworm, were used to continue research into prostaglandin signaling in pest insect biology. Combined treatments with a prostaglandin biosynthesis inhibitor, indomethacin (INDO), sharply reduced cellular prostaglandin concentrations, plus the effect of the molting hormone, 20-hydroxyecdysone (20E) on protein expression in the nervous system cell lines. At 24 hours post-treatment, significant increased expression of 222 proteins that act in a wide range of biological functions was observed. This work considerably broadens understanding of prostaglandin signaling in insects. Through their influence on expression of many genes and proteins in nervous cell lines, prostaglandins and 20E operate in a very wide range of insect biology. Also, in support of Objective 1, insect immune responses to Tomato Spotted Wilt Virus (TSW) infection were studied. TSW is a plant virus that causes massive economic damage to high-value crops, including tomatoes and peppers. This virus is transmitted by a small insect, the western flower thrip. The virus is acquired by young larvae while feeding on infected host plants. The virus infects cells of the thrip alimentary canal via two proteins. We identified these proteins as Fo-Gn and Fo-Cyp1. They are necessary and sufficient for viral infection of the alimentary canal. The virus multiplies within the thrip and is transmitted to healthy plants when the thrips feed on the plants. Silencing the gene encoding Fo-Gn and, separately, Fo-Cyp1, blocks viral infection and subsequent transmission of the virus. We then investigated the thrip immune reactions to Tomato Spotted Wilt Virus infections. We first recorded viral infections in midgut and viral multiplication in salivary glands. The virus is transmitted from the salivary glands to uninfected plants while the thrips feed on host plants. In larval thrips, the viral infections lead to release of a specific protein from the alimentary canal into circulation. This protein is called dorsal switch protein 1. It is a damage signal that activates biosynthesis of prostaglandins and other eicosanoids which signal thrip immune responses to viral infections. While these immune responses are not sufficient to block viral transmission, they are now targets that may be developed to enhance thrip immune reactions to viral infections, which may also be applied to other insect defense systems against viral infections. In support of Objective 2, Goal 2.1, research efforts continued to sequence the genome of the grape mealybug; a draft genome was completed by a commercial service provider and is currently facilitating identification of various olfactory genes. Further efforts are underway, both internally and externally, to produce a chromosome-level genomic assembly for this agricultural pest. In support of Goal 2.2, researchers generated transcriptomic libraries from three mealybug species (grape, vine and citrus mealybugs) which are currently being used to identify and evaluate the expression levels of olfactory genes in males and females of each species. For Goal 2.3, researchers have begun cloning mealybug odorant receptors of interest so they can be heterologously expressed in cell lines for functional characterization. In support of Objective 3, Goal 2, research efforts directed at understanding the resistance of western corn rootworm (WCR) to commercial entomotoxic proteins was continued. Histological studies demonstrated that susceptible WCR larval midgut responses to feeding on moderate concentrations of a single entomotoxic protein causes extensive damage to the intestinal lining and cells, resulting in high mortality. Resistant WCR larvae do not exhibit the tissue damage seen in susceptible larvae. We previously discovered that knockdowns of one ABC transporter gene restores susceptibility to resistant larvae, increasing mortality. The WCR genome encodes at least 65 unique ABC transporters in addition to the one that we have previously identified as important to the toxicity of an entomotoxic protein. In order to determine whether other ABC transporters, and additional entomotoxins, were similarly involved in this unique response, the coordinate expression of all 65 were studied following larval consumption of two separate entomotoxic proteins. We observed that 32 of these partitioned into three expression groups: upregulated, downregulated, and unchanged by feeding on toxins. Double stranded RNAi knockdowns were designed for eight of the best upregulated candidates, and the growth and mortality response of knocked down larvae to entomotoxins were measured. Knockdown of the newly selected ABC transporters exhibited no significant effect on resistant mortality or larval growth. Further, the chosen candidates also did not demonstrate a reversal of resistance to toxin observed with the previous best candidate. The results confirm that in WCR larvae, a single ABC transporter has a unique involvement in the response to a single entomotoxin, and that other similar ABC transporters have no contribution to resistance. In support of Objective 4, Goal 4.1, working relationships were established with local farms and vineyards to monitor spotted wing Drosophila (SWD). Collaborations with other ARS units (USDA ARS Corvallis, OR; USDA ARS Wapato, WA) and universities (University of Oregon, Eugene, OR; University of Florida, Gainesville, FL; Lincoln University, Jefferson City, MO; University of Missouri, Columbia, MO; Cornell University, Ithaca, NY; Baylor University, Waco, TX; Brigham Young University, Provo, UT) were initiated to assist with specimen collection and data analyses. Additional funds were obtained from the Missouri Grape and Wine Institute to facilitate and enhance objectives and a postdoctoral fellow was hired via the SCINet/AI Center of Excellence funds. ARS scientists and collaborators have collected, dissected, and extracted DNA from flies, and have extracted and cultured the fly-associated microbes. Flies were captured from grapes, blueberries, raspberries, blackberries, cherries, strawberries, and elderberries. Samples were submitted for full shotgun metagenomic sequencing and pan-genome assembly. In support of Goal 4.2, four laboratory, and two field-derived colonies of SWD were established, and optimized axenic and gnotobiotic techniques were developed. Axenic and gnotobiotic techniques are undergoing optimization. Preliminary computational correlative models were constructed.

Accomplishments
1. Modeled the community assembly of the Spotted wing Drosophila microbiome. Early work in SWD has demonstrated acetic acid microbiome-related effects on metabolism and show that microbes are necessary for proper fly development. These same acetic acid bacteria cause sour rot in grapes, and result in massive economic damage to the agricultural industry. These bacteria convert alcohols into acetic acid which can ruin entire batches of wine. However, there remains poor understanding of which factors lead to successful colonization and persistence of microbes within flies. The knowledge gained from these models will help determine whether there are microbes that can be used to reduce the pest fitness of SWD as well as inhibit sour rot microbes in grape vineyards. ARS researchers in Columbia, Missouri, sequenced the microbiomes for spotted wing Drosophila collected from grapes, blackberries, blueberries, and elderberries to yield communities of microbes that may compete against sour rot bacteria. These microbes may potentially be used in precision microbial sprays to enhance existing integrative pest management efforts and reduce pesticide resistance in flies.

Review Publications
Xiao, K., Wu, C., Yang, L., Wang, J., Song, Q., Stanley, D.W., Wei, S., Zhu, J. 2022. Comparative genomic analysis of ABC transporter genes in Tenebrio molitor and four other tenebrionid beetles (Coleoptera: Tenebrionidae). Archives of Insect Biochemistry and Physiology. 111 (3). Article 21916. https://doi.org/10.1002/arch.21916.
Wang, Y., Li, G., Li, L., Song, Q., Stanley, D.W., Wei, S., Zhu, J. 2022. Genome-wide and expression-profiling analyses of the cytochrome P450 genes in Tenebrionidea. Archives of Insect Biochemistry and Physiology. 111(3). Article 21954. https://doi.org/10.1002/arch.21954.
Corcoran, J., Han, X. 2022. Improved cryopreservation media formulation reduces costs of maintenance while preserving function of genetically modified insect cells. In Vitro Cellular and Developmental Biology - Animal. 58:867-876. https://doi.org/10.1007/s11626-022-00741-3.
Ji, J., Liu, Y., Zhang, L., Cheng, Y., Stanley, D.W., Jiang, X. 2022. The clock gene, period, influences migratory flight and reproduction of the oriental armyworm, Mythimna separata (Walker). Insect Science. 30(3):650-660. https://doi.org/10.1111/1744-7917.13132.
Khan, F., Stanley, D.W., Kim, Y. 2023. Two alimentary canal proteins, Fo-GN and Fo-Cyp1, act in Western Flower Thrips, Frankliniella occidentalis TSWV infection. Insects. 14(2). Article 154. https://doi.org/10.3390/insects14020154.
Stanley, D.W., Haas, E.J., Kim, Y. 2023. Beyond cellular immunity: On the biological significance of insect hemocytes. Cells. 12(4). Article 599. https://doi.org/10.3390/cells12040599.
Corcoran, J., Hamiaux, C., Faraone, N., Löfstedt, C., Carraher, C. 2023. Structure of an antennally-expressed carboxylesterase suggests lepidopteran odorant degrading enzymes are broadly tuned. Current Research in Insect Science. 3. Article 100062. https://doi.org/10.1016/j.cris.2023.100062.
Zou, D., Coudron, T.A., Zhang, L., Gu, X., Xu, W., Wu, H. 2018. Performance of Arma chinensis reared on an artificial diet formulated using transcriptomic methods. Bulletin of Entomological Research. 109:24-33. https://doi.org/10.1017/S0007485318000111.
Morales Ramos, J.A., Rojas, M.G., Coudron, T.A., Huynh, M.P., Zou, D., Shelby, K. 2022. Artificial diet development for entomophagous arthropods. In: Morales-Ramos, J.A., Rojas, M.G., Shapiro-Ilan, D.I., editors. Mass Production of Beneficial Organisms: Invertebrates and Entomopathogens. 2nd edition. San Diego, CA: Academic Press. p.233-260.
Chen, L., Lang, K., Zhang, B., Shi, J., Ye, X., Stanley, D.W., Fang, Q., Ye, G. 2022. iVenomDB: a manually curated database for insect venom proteins. Insect Science. 30(1):264-266. https://doi.org/10.1111/1744-7917.13054.
Li, L., Wang, Y., Li, G., Song, Q., Stanley, D.W., Wei, S., Zhu, J. 2022. Genomic and transcriptomic analyses of chitin metabolism enzymes in Tenebrio molitor. Archives of Insect Biochemistry and Physiology. 111(3). Article 21950. https://doi.org/10.1002/arch.21950.
Paddock, K.J., Dellamano, K., Hibbard, B.E., Shelby, K. 2023. eCry3.1Ab-resistant western corn rootworm larval midgut epithelia respond minimally to Bt intoxication. Journal of Economic Entomology. 116(1):263-267. https://doi.org/10.1093/jee/toac191.
Yang, L., Li, G., Li, X., Wu, C., Wang, J., Song, Q., Stanley, D.W., Wei, S., Zhu, J. 2022. The Wnt gene family in Tenebrio molitor and other coleopterans. Archives of Insect Biochemistry and Physiology. 111(3). Article 21915. https://doi.org/10.1002/arch.21915.
Yang, Y., Li, X., Wang, J., Song, Q., Stanley, D.W., Wei, S., Zhu, J. 2022. Comparative genomic analysis of carboxylesterase genes in Tenebrio molitor and four other tenebrioinids. Archives of Insect Biochemistry and Physiology. 111(3). Article 21967. https://doi.org/10.1002/arch.21967.
Kang, D.S., Kim, Y., Stanley, D.W. 2022. What is in a model. Archives of Insect Biochemistry and Physiology. 112(1). Article 21972. https://doi.org/10.1002/arch.21972.
Pereira, A.E., Huynh, M.P., Paddock, K.J., Ramirez, J.L., Caragata, E.P., Dimopoulos, G., Krishnan, H.B., Schneider, S.K., Shelby, K., Hibbard, B.E. 2022. Chromobacterium Csp_P biopesticide is toxic to larvae of three Diabrotica species including strains resistant to Bacillus thuringiensis. Scientific Reports. 12. Article 17858. https://doi.org/10.1038/s41598-022-22229-6.
Wu, C., Xiao, K., Wang, L., Wang, J., Song, Q., Stanley, D.W., Wei, S., Zhu, J. 2022. Identification and expression profiling of serine protease-related genes in Tenebrio molitor. Archives of Insect Biochemistry and Physiology. 111(3). Article 21963. https://doi.org/10.1002/arch.21963.
Zou, D., Coudron, T.A., Zhang, L., Xu, W., Xu, W., Wang, M., Xiao, X., Wu, H. 2021. Effect of prey species and prey densities on the performance of adult Coenosia attenuata. Insects. 12(8). Article 669. https://doi.org/10.3390/insects12080669.
Zou, D., Coudron, T.A., Wu, H., Zhang, L., Wang, M., Xu, W., Xu, J., Song, L., Xiao, X. 2022. Differential proteomics analysis unraveled mechanisms of Arma chinensis responding to improved artificial diet. Insects. 13(7). Article 605. https://doi.org/10.3390/insects13070605.
Li, X., Song, L., Coudron, T.A., Zuo, T., Chen, Y., Zhang, Y., Wu, S. 2019. Effects of two natural diets on the response of the predator Arma chinensis (Hemiptera: Pentatomidae: Asopinae) to cold storage. Applied Ecology and Environmental Research. 17(6):15329-15347. https://doi.org/10.15666/aeer%2F1706_1532915347.
Zou, D., Coudron, T.A., Xu, W., Xu, J., Wu, H. 2020. Performance of the tiger-fly Coenosia attenuata Stein reared on the alternative prey, Chironomus plumosus (L.) larvae in coir substrate. Phytoparasitica. 49:83-92. https://doi.org/10.1007/s12600-020-00866-9.
Chul-Young, K., Abmed, S., Stanley, D.W., Kim, Y. 2023. HMG-like DSP1 is a damage signal to mediate the western flower thrips, Frankliniella occidentalis, immune responses to tomato spotted wilt virus infection. Developmental and Comparative Immunology. 144. Article 104706. https://doi.org/10.1016/j.dci.2023.104706.
Li, J., Yin, L., Bi, J., Stanley, D.W., Feng, Q., Song, Q. 2022. The TGF-beta receptor gene Saxophone influences larval-pupal-adult development in Tribolium castaneum. Molecules. 27(18). Article 6017. https://doi.org/10.3390/molecules27186017.
De Clercq, P., Courdron, T.A., Riddick, E.W. 2023. Production of heteropteran predators. In: Morales-Ramos, J.A., Rogas, M.G., Shapiro-Ilan, D.I., editors. Mass Production of Beneficial Organisms. 2nd edition. San Diego, CA: Academic Press. p. 57-100.
Li, J., Bo, L., Bi, J., Shan, R., Stanley, D.W., Feng, Q., Song, Q. 2023. Partner of neuropeptide bursicon homodimer pburs mediates a novel antimicrobial peptide Ten3LP via Dif/Dorsal2 in Tribolium castaneum. International Journal of Biological Macromolecules. 247. Article 125840. https://doi.org/10.1016/j.ijbiomac.2023.125840.
Li, G., Yang, L., Xiao, K., Song, Q., Stanley, D.W., Wei, S., Zhu, J. 2022. Characterization and expression profiling of serine protease inhibitors in the yellow mealworm Tenebrio molitor. Archives of Insect Biochemistry and Physiology. 111(3). Article e2194. https://doi.org/10.1002/arch.21948.
Arya, S.K., Goodman, C.L., Stanley, D.W., Palli, S.R. 2022. A database of crop pest cell lines. In Vitro Cellular and Developmental Biology. 58(8):719-757. https://doi.org/10.1007/s11626-022-00710-w.
Flick, A.J., Coudron, T.A., Elderd, B.D. 2020. Intraguild predation decreases predator fitness with potentially varying effects on pathogen transmission in a herbivore host. Oecologia. 193:789-799. https://doi.org/10.1007/s00442-020-04665-1.
Lund, M., Brainard, D., Coudron, T.A., Szendrei, Z. 2020. Predation threat modifies Pieris rapae performance and response to host plant quality. Oecologia. 193:389-401. https://doi.org/10.1007/s00442-020-04686-w.
Wu, H., Coudron, T.A., Zhang, L., Aldrich, J.R., Xu, W., Xu, J., Wang, H., Zou, D. 2019. Identification and field verification of aggregation-sex pheromone from the predaceous bug, Arma chinensis. Chemoecology. 29:235-245. https://doi.org/10.1007/s00049-019-00292-2.