Research Project: Ecologically Based Pest Management in Western Crops Such as Cotton

Location: Pest Management and Biocontrol Research

2018 Annual Report

Objectives
1: Improve biological control of key pests by quantifying interactions between prey & generalist predators, including predators occupying different trophic levels, using molecular marking & gut content assays in the field & defining impacts of transgenic crops on non-target species through meta-analyses. Sub-objective 1A though 1B: See uploaded project plan. Sub-objective 1C: Examine temporal and spatial dynamics of whitefly, Lygus, predator and pollinator movements between cotton and Vernonia, a new industrial crop.(New, May, 2018) Sub-objective 1D: Assess the risk of disruption of biological control of whiteflies by the introduction of a new Bt cotton with activity against Lygus bugs and thrips.(New, May, 2018) 2: Refine resistance management strategies based on improved knowledge of host (species & phenology) & environmental (temperature) influences on inducible mechanisms of stress response in whitefly & lygus & of Cry-toxin binding & mechanisms of Bt (Bacillus thuringiensis) toxin resistance in pink bollworm. Sub-objective 2A through 2C: See uploaded project plan. 3: Refine knowledge of factors regulating mate-finding & the dynamics of reproduction in lygus & whitefly by optimizing lygus sex pheromone doses & component ratios, defining insect phenology-dependent roles of short-range cues of lygus mating receptivity, & quantifying impacts of host, environmental, & population density-based factors on whitefly sex ratios. Sub-objective 3A through 3D: See uploaded project plan. 4: Define key life history parameters including the development & survival strategies of lygus & key species of beneficial insects in relation to the environment by quantifying consequences of extreme thermal environments & defining insect stage- dependent & environment-dependent diapause responses & associated transcriptional- based & endocrine-based patterns in lygus. Sub-objective 4A through 4C: See uploaded project plan. 5: Describe molecular genetic responses, facilitating survival & adaptation in pest insects by identifying lygus & whitefly transcripts responsive to xenobiotics & environmental (thermal, water, oxidative) stressors, identify molecular targets for disruption by chemical or genetic agonists or antagonists, & develop methods to deliver dsRNA for functional disruption of aquaporins or other targets essential to maintain homeostasis. Sub-objective 5A through 5B: See uploaded project plan. Sub-objective 5C: Examine the potential of an ornamental plant to disrupt the osmotic water permeability of B. tabaci aquaporin water channel proteins using cage studies and in vitro heterologous insect cell expression functional assays.(New, May, 2018)

Approach
Interactions among key prey and predator species will be quantified using molecular marking and gut content assays in laboratory, greenhouse and field experiments. Meta-analyses of updated databases will examine the impacts of transgenic Bt crops on non-target arthropod abundance, community diversity, and biological control services. Insecticide susceptibility of whitefly in relation to host and environmental conditions will be determined using laboratory assays of field-collected insects. Results of field studies will guide controlled experiments to determine mechanisms by which host condition, population density, and temperature influence susceptibility to insecticides, including expression of detoxification enzymes. Inheritance, dominance, and allelism of Cry2Ab resistance in the pink bollworm will be determined using crosses among laboratory strains of the insect. Roles of pink bollworm cadherin and ABC transporter protein as functional receptors of Cry-toxins will be examined by fluorescent imaging of cell cultures transfected with tagged clones of the target cDNA. Cytotoxicity of Cry-proteins will be determined for each putative receptor. Seasonal patterns in whitefly sex ratios will be documented in the field and association of symbionts with sex ratio shifts will be examined using PCR. Respective roles of male availability and copulation interference in determining sex ratios will be evaluated in greenhouse studies. Potential insect- and plant-derived semiochemicals for manipulating or monitoring whitefly will be identified by GC and screened using olfactometry. Attractiveness of the recently identified sex pheromone of Lygus hesperus will be optimized using electro-antennographic detection followed by field trap studies and experiments to determine the diel pattern of pheromone emission. Influences of male lygus reproductive phenology, time since mating, and concentration of a chemical inhibitor of mating on mating frequency will be determined in laboratory assays. Also, potential of the mating inhibitor as a mating disruptant will be evaluated based on responses of insects to treated substrates. Influences of environmental extremes on development and survival of lygus and selected predators, and on mating, reproduction, and longevity of lygus adults, will be examined in controlled studies incorporating constant and variable temperature regimes. Stage-specific sensitivity of the lygus diapause response will be examined in photoperiod-switching and controlled environment experiments. Companion studies will examine hemolymph protein and transcript profiles to identify potential molecular markers indicative of diapause. Molecular responses of whitefly and lygus to xenobiotic and environmental stressors, especially temperature, will be assessed based on transcriptomic responses to experimentally induced stress, and links between stress responses and susceptibility to insecticides will be examined in bioassays. dsRNA will be used against selective targets to silence genes important to biological fitness in lygus and whitefly.

Progress Report
Substantial progress was made on all five objectives, which fall under National Program 304, Component 3, Insects and Mites. This project focuses on Problem A2, the need for an improved systems approach to environmentally sound pest management. Objective 1: Progress was made to better understand insects that promote healthy crop ecosystems, including pollinators and predators. A universal food immunomarking technique was developed to detect immuno-labelled food items in arthropods, which is being used to investigate: (1) feeding activity on eggs of Lygus hesperus, a major pest of cotton, (2) cannibalism, and (3) scavenging by the cotton predator community. Additional studies are evaluating insect dispersal patterns in various cropping systems using a protein immunomarking technique (PIT). One crop is Vernonia, a desert-adapted plant with potential as a trap crop for cotton. Temporal and spatial dynamics of whitefly, Lygus, predator and pollinator movements between cotton and Vernonia are being examined and early results indicate all are attracted to the crop. The PIT is also being used to study dispersal of spotted wing Drosophila in blueberry fields, in collaboration with scientists at Michigan State University, East Lansing Michigan and, dispersal of blue orchard bees, bumble bees, and leafcutter bees in cooperation with ARS researchers at Logan, Utah. Another monitoring technique was developed to non-destructively monitor bee movement across the landscape. ARS researchers at Maricopa and Tucson, Arizona, developed innovative methods to study varroa mite dispersal. Several publications have resulted from these advances and others are in preparation. A database of non-target arthropod field studies in Bt crops (cotton, eggplant, rice, potato) was compiled and is nearing completion which complements an existing database for Bt maize. The latter database has been fully analyzed and manuscripts are under development. Replicated field plot studies were conducted, and are being repeated, to examine the non-target impacts of several new insecticides on arthropod predators in cotton and to examine the effect of plot size on non-target assessment. Other field studies are being conducted to examine the non-target impacts of a new Bt cotton formulated to target Lygus bugs and thrips, particularly about natural enemy species that help suppress whitefly populations. This research will be enhanced this summer and in the next 3 years with additional funding from Monsanto and a recently awarded USDA National Institute of Food and Agriculture Biotechnology Risk Assessment grant. Objective 2: Individual insect cell lines have been generated that each produce one of seven forms of the pink bollworm midgut cadherin, including the normal protein from individuals susceptible to Bt toxins, and six mutant forms (four from Arizona and two from China) isolated from populations resistant to Bt toxins. Of the five cadherin proteins from the U.S., only the non-mutant form was found to be expressed on the surface of the insect cells, which effectively prevents using this experimental system to test toxicity. However, this observation led to a new research hypothesis that cadherin gene mutations that prevent the proper localization of the protein on the surface of gut cells are responsible for generating resistance. Collaborative work with scientists from China and the University of Arizona, Maricopa, Arizona, showed that mutations in pink bollworm cadherin can disrupt movement of the protein, resulting in Bt toxin resistance. Additional work is underway to verify that resistance occurs when cadherins and another class of transporter proteins are not expressed appropriately, including use of gene silencing and a newly obtained anti-cadherin antibody. Objective 3: Examination of how aging influences mating activity in L. Hesperus has faced impediments. Experiments were undertaken to assess how males of different ages would respond to an anti-aphrodisiac. This chemical message is normally produced by other males and transferred to females during mating, rendering those females unattractive. Unexpectedly, a synthetic version of the anti-aphrodisiac failed to inhibit mating attempts and further tests revealed that males of the stock colony being used had lost their capacity to respond to the odor. Tests using males from field populations indicate that they and their offspring are still responsive to the odor, so a new laboratory population has been established and is currently being used to collect data on age-related mating motivation, persistence of topically applied anti-aphrodisiac, and minimum effective concentration. Tests should be completed by the end of the year. Objective 4: Substantial progress has been made in determining the factors influencing life history traits of L. hesperus. An examination was completed of the temperature-dependence of Lygus nymph development. Patterns of development were similar to those previously observed for eggs; at a low temperature, 59 degrees Fahrenheit (F), nymphs developed more slowly when the temperature was constant compared with when the temperature varied daily from 45 to 75 degrees. The opposite trend was observed for nymphs held at a high average temperature (84 degrees); nymphs held at a constant temperature developed more rapidly compared with nymphs held under temperatures that varied daily from 70 to 99 degrees. Development of nymphs held at a moderate temperature (72 degrees) developed at approximately the same rate whether temperatures were constant or variable. Survival was similar under all temperature conditions except the low, constant conditions, where nymph survival was lower compared with the other temperatures. These results demonstrate only modest negative influences on nymph development when crop canopy temperatures are high because of drought conditions but suggest that daily temperature fluctuations during the cooler winter months allow higher survival of overwintering bugs than are indicated by constant temperature studies. Companion studies of adult Lygus reproductive development were initiated using the same temperature conditions that were used for nymphs. These results have important application to interpreting population patterns of Lygus in irrigated crops, and to the ecology of Lygus overwintering. Studies to quantify instar- or stage-specific diapause responses of Lygus were analyzed and published. Diapause is a spontaneous period of suspended animation that seems to happen in response to adverse environmental conditions. Experiments to examine the influence of temperature on the incidence of diapause in Lygus were completed and indicated that Lygus diapause is relatively insensitive to temperature. Because the ability to accurately distinguish the diapause status of individual insects would be a valuable tool for examining the molecular biology of diapause, efforts were made to determine distinct markers in Lygus. Protein markers of diapause found circulating in the blood have been identified for several insect species. However, an assessment of protein diversity in the blood of diapausing and non-diapausing female and male Lygus indicated only one difference, which was a large molecular weight protein found only in reproductive females. The size of the protein and its association with egg production suggest the substance is an egg yolk component. The absence of this protein in diapausing and non-diapausing males precludes its use as a useful marker of Lygus diapause status. Unlike other species, a diapause-linked/sex-independent hemolymph protein is either absent in L. hesperus or is sufficiently below a detectability threshold to render it impractical for use as an indicator of diapause status. Nevertheless, a novel method of nondestructively identifying Lygus adults in diapause using body dimensions was developed and validated. Accuracy of the new method is nearly 100 percent for females and about 85 percent for male bugs. Host-free survival patterns of adult Lygus classed as reproductive or diapausing using the new method indicated clearly different survival potential for the two classes of bugs. Diapausing males and females held without food at a relatively high temperature (80 degrees F) lived for an average of 32 and 38 days, respectively, whereas their reproductive counterparts survived for only 5 to 8 days. Objective 5: Continued progress has been made toward locating genes that respond to xenobiotics and environmental stressors, in our quest to identify targets for chemical or genetic disruption in controlling cotton pests. L. hesperus were exposed to a variety of thermal conditions, and high-throughput ribonucleic acid (RNA) sequencing indicated many genes that differ in their expression levels depending on treatment. However, subsequent attempts to confirm that environmental stressors directly promote the expression of these identified genes has only been successful for two of the candidates. The discrepancy in the two types of expression data suggest that the initial identification process may have been flawed or may reflect significant genetic variability in the Lygus population assessed. Gene silencing is being used to validate the role of the two confirmed genes in mitigating thermal stress.

Accomplishments
1. New mechanism of resistance to Bt crops involves mis-localization of cadherin protein receptor. Bt crops are named for Bacillus thuringiensis (Bt), a bacterium that protects crops by naturally producing a crystal protein that is toxic to many pest insects. Bt crops are genetically engineered to produce the same toxin as Bt in every cell of the plant, with the goal of protecting the crop from pests. Pink bollworm, a common pest, have become resistant to the toxins of Bt cotton via mutations to the cadherin proteins that are thought to prevent toxin binding to the insect midgut. ARS scientists in Maricopa, Arizona, and researchers from China and the University of Arizona demonstrated that cadherin mutations affect protein trafficking and therefore reduced binding of the toxin due to the loss of an available receptor. Mutations that affect various regions of the cadherin protein can alter expression on the cell surface, which may be a common underlying mechanism promoting the development of resistance to Bt crops, in addition to mutations that directly affect toxin binding. These results are valuable for scientists concerned with understanding the mechanisms of resistance, for biotechnology companies developing new strategies to target pests, and for government authorities responsible for regulating transgenic crops.

2. Development of a novel non-destructive marking technique for tracking bees. Studies of bee movement and activities across a landscape are important for developing an understanding of their behavior and their ability to withstand environmental stress. Recent research has shown that proteins, are effective for mass-marking bees; however, current techniques require sacrificing individual bees during data collection. ARS scientists in Logan, Utah, and Maricopa, Arizona, developed a nonlethal sampling method for protein mark-capture research on bumble, blue orchard, and leafcutter bees. The technique consists of catching marked bees in the wild, immersing them momentarily in a buffer to extract any potential egg albumin mark, and then releasing them. Results showed that an egg albumin-specific assay was 100 percent effective at detecting the protein on bees and did not have an impact on bee survivorship. These methods are currently being used throughout the U.S. to study the dispersal patterns of these important pollinators.

3. Retrospective analysis of a classical biological control program. Classical biological control has been a key technology in the management of invasive arthropod pests globally for over 120 years, yet rigorous quantitative evaluations of program success or failure are rare. An ARS scientist in Maricopa, Arizona, used long term (15 years) life table data and matrix model analyses to quantitatively assess a classical biological control program for the sweetpotato whitefly, a key invasive insect pest in the western U.S. Analysis showed that the introduction of two parasitoid species failed to increase mortality or reduce population growth of the pest in cotton. Instead, native arthropod predators were found to inflict heavy mortality on the pest and are the key to managing this insect in western cotton production systems. The research demonstrated a robust approach to assessing biological control programs and will be of interest to researchers and policy makers involved in developing and implementing classical biological control programs globally.

4. Diapause and overwinter survival of the boll weevil. Understanding overwintering strategies of the boll weevil is important to completion of eradication efforts in the U.S. and expansion of eradication into the subtropics and tropics. Recent reports have characterized the winter-dormancy of the boll weevil as a quiescence, whereupon overwintering weevils immediately begin reproduction upon encountering fruiting cotton. Further reports have concluded the dormancy is absent in the subtropics; instead, the weevil relies on non-cotton hosts for winter survival. ARS researchers in College Station, Texas, and Maricopa, Arizona, unambiguously demonstrated the boll weevil dormancy is a diapause, or a period of suspended development, that is induced by impending maturity of the cotton crop. The diapause is relatively insensitive to temperatures typical of fall and early-winter months and provides the weevil the capacity to survive the subtropical or tropical non-cotton season in the absence of cotton or other putative alternative food sources. These discoveries warn against the practice of extending spray intervals during the cooler fall months and emphasize the importance of eradication and management programs for reducing populations of diapausing weevils before they abandon maturing cotton fields.

5. Important facets of western tarnished plant bug (Lygus) diapause and overwintering. Management efforts focused on reducing overwintering populations of Lygus bugs have been attempted with little success. One factor limiting these efforts may be an insufficient understanding of the diapause, a period of suspended animation that facilitates overwinter survival of the bugs. ARS researchers at Maricopa, Arizona elucidated key aspects of the Lygus diapause, and demonstrated that only a portion of the field population enters diapause in the fall. The diapause is induced by exposing large (4th stage) nymphs to short day lengths (12 hours or less), is maintained by continued exposure to short days, and enables the diapausing adults to survive without food for more than 40 days even at relatively high temperatures. Lack of a winter-time pattern in the incidence of diapause combined with observations of diapausing bugs in early spring indicate that southern populations of Lygus likely have more than one diapausing generation each year. These findings provide new insights into the survival strategies of Lygus bugs and are important in the development of improved management tactics targeting the overwintering population.

Review Publications
Kang, P., Chang, K., Liu, Y., Bouska, M., Karashchuk, G., Thakore, R., Wang, W., Post, S., Brent, C.S., Li, S., Tatar, M., Bai, H. 2017. Drosophila Kruppel homolog 1 represses lipolysis through interactions with dFOXO. Scientific Reports. 7:16369.
Prabhaker, N., Naranjo, S.E., Perring, T., Castle, S.J. 2017. Comparative toxicities of newer and conventional insecticides against four generalist predator species. Journal of Economic Entomology. 110(6):2630-2636.
Naranjo, S.E., Ellsworth, P. 2017. Methodology for developing life tables for sessile insects in the field using the Whitefly, Bemisia tabaci, in cotton as a model system. Journal of Visualized Experiments. 129:e56150. doi:10.3791/56150.
Spurgeon, D.W., Suh, C.P. 2018. Starvation-induced morphological responses of the boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae). Journal of Cotton Science. 21:275-283.
Tian, J.C., Wang, X.P., Chen, Y., Romeis, J., Naranjo, S.E., Hellmich, R.L., Wnag, P., Shelton, A.M. 2018. Bt cotton producing Cry1Ac and Cry2Ab does not harm two parasitoids, Cotesia marginiventris and Copidosoma floridanum. Scientific Reports. 8:307.
Wang, L., Ma, Y., Wan, P., Liu, K., Xiao, Y., Wang, J., Cong, S., Xu, D., Wu, K., Fabrick, J.A., Li, X., Tabashnik, B.E. 2018. Resistance to Bacillus thuringiensis linked with a cadherin transmembrane mutation affecting cellular trafficking in pink bollworm from China. Insect Biochemistry and Molecular Biology. 94:28-35. https://doi.org/10.1016/j.ibmb.2018.01.004.
Spurgeon, D.W., Suh, C.P. 2017. Temperature influences on diapause induction and survival in the boll weevil (Coleoptera: Curculionidae). Journal of Insect Science. 17(6):124.
Spurgeon, D.W. 2017. Instar- and stage-specific photoperiodic diapause response of Lygus hesperus (Hemiptera: Miridae). Journal of Insect Science. 17(6):125.
Rendon, D., Hagler, J.R., Taylor, P., Whitehouse, M. 2018. Integrating immunomarking with ecological and behavioural approaches to assess predation of Helicoverpa spp. larvae by wolf spiders in cotton. Biological Control. 122:51-59. https://doi.org/10.1016/j.biocontrol.2018.03.019.
Wong, J., Cave, A., Lightle, D., Mahaffee, W.F., Naranjo, S.E., Wiman, N., Woltz, M., Lee, J.C. 2018. Drosophila suzukii flight performance reduced by starvation but not affected by humidity. Journal of Pest Science. 91(4):1269-1278. https://doi.org/10.1007/s10340-018-1013-x.
Boyle, N.K., Machtley, S.A., Hagler, J.R., Pitts-Singer, T. 2018. Evaluating the persistence of fluorescent and protein powders on adult blue orchard bees, Osmia lignaria (Hymenoptera: Megachilidae), for mark-capture studies. Apidologie. 49(3):378-385. https://doi.org/10.1007/s13592-018-0564-4.
Boyle, N.K., Tripodi, A.D., Machtley, S.A., Pitts-Singer, T., Strange, J.P., Hagler, J.R. 2018. A nonlethal method to examine non-Apis bees for mark-capture research. Journal of Insect Science. 18(3):10. https://doi.org/10.1093/jisesa/iey043.
Naranjo, S.E. 2018. Retrospective analysis of a classical biological control programme. Journal of Applied Ecology. 55:2439-2450.
Vandervoet, T., Ellsworth, P., Carriere, Y., Naranjo, S.E. 2018. Quantifying conservation biological control for management of Bemisia tabaci (Hemiptera: Aleyrodidae) in cotton. Journal of Economic Entomology. 111:1056-1068.
Hagler, J.R., Thompson, A.L., Stefanek, M.A., Machtley, S.A. 2018. Use of body-mounted cameras to enhance data collection: an evaluation of two arthropod sampling techniques. Journal of Insect Science. 18(2):1-8.
Spurgeon, D.W., Suh, C.P. 2018. Morphology, diet, and temperature dependent host-free survival of the boll weevil, Anthonomus grandis (Coleoptera: Curculionidae). Journal of Insect Science. 18(3):8. https://doi.org/10.1093/jisesa/iey047.
Luo, J., Li, Z., Ma, C., Zhang, Z., Hull, J.J., Lei, C., Jin, S., Chen, L. 2017. Knockdown of a metathoracic scent gland desaturase enhances the production of (E)-4-oxo-2-hexenal and suppresses female sexual attraction in the plant bug, Adelphocoris suturalis. Insect Molecular Biology. 26(5):642-653.
Christie, A.E., Yu, A., Pascual, M.G., Roncalli, V., Cieslak, M.C., Warner, A.N., Lameyer, T.J., Stanhope, M.E., Dickinson, P.S., Hull, J.J. 2018. Circadian signaling in Homarus americanus: Region-specific de novo assembled transcriptomes show that both the brain and eyestalk ganglia possess the molecular components of a putative clock system.. Marine Genomics. 40:25-44. https://doi.org/10.1016/j.margen.2018.03.002.
Fodor, J., Hull, J.J., Koblos, G., Jacquin-Joly, E., Szlanka, T., Fonagy, A. 2018. Identification and functional characterization of the pheromone biosynthesis activating neuropeptide receptor isoforms from Mamestra brassicae. General and Comparative Endocrinology. 258:60-69. https://doi.org/10.1016/j.ygcen.2017.05.024.
Brent, C.S. 2018. Mating and social contact change egg production and longevity in adult females of the mirid Lygus hesperus. Entomologia Experimentalis et Applicata. 166:545-554. https://doi.org/10.1111/eea.12683.
Hogg, B.N., Nelson, E.H., Hagler, J.R., Daane, K.M. 2018. Foraging distance of the Argentine ant in California vineyards. Journal of Economic Entomology. 111:672-679.
Irvin, N.A., Hagler, J.R., Hoddle, M.S. 2018. Measuring natural enemy dispersal from cover crops in a California vineyard. Biological Control. 126:15-25.