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

Location: Pest Management and Biocontrol Research

2016 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. 1A: Quantify interactions among prey & predators occupying different trophic levels using molecular & gut content assays. 1B: Define impacts of Bt crops on non-target arthropod abundance, community diversity & biological control function. 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. 2A: Evaluate insecticide susceptibility of Bemisia tabaci in relation to temperature & host plant condition. 2B: Genetically characterize in-house Cry2Ab-resistant Pectinophora gossypiella; determine inheritance & whether in-house strain shares a genetic locus with the Univ. of Az Cry2Ab-resistant strain. 2C: Establish whether P. gossypiella cadherins & ABCC transporters function as Bt Cry-1Ac & Cry2Ab toxin receptors. 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. 3A: Evaluate sex ratio variability in the haplodiploid whitefly B. tabaci. 3C: Optimize sex pheromone doses & components to attract Lygus hesperus. 3D: Investigate responses of male L. hesperus to myristyl acetate & anti-mating activity of surface-applied myristyl acetate. 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. 4A: Quantify influences of variable temperatures on development & survival of L. hesperus stages, & on eggs & nymphs of key predators. 4B: Determine effects of mating & temperature on reproduction & longevity of L. hesperus. 4C: Define key aspects of L. hesperus diapause. 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. 5A: Describe transcriptional & biological responses of L. hesperus to stressors. 5B: Develop methods to knockdown L. hesperus & B. tabaci gene targets using dsRNA.

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
Progress was made on all five objectives, which fall under National Program 304, Component 3, Insects and Mites. Progress on this project focuses on Problem A2, the need for an improved systems approach to environmentally sound pest management. Under Objective 1A, progress was made to better understand insects that promote healthy crop ecosystems, including pollinators and predators. Protein markers were used to study the foraging behavior of insect pollinators, essential knowledge for conserving pollinators and increasing their ecosystem services. In cooperation with ARS scientists in Logan, Utah, and scientists at Michigan State University and Arizona State University, pollinator movement was studied in blue orchard bees, bumble bees, and honey bees. Field studies using protein markers also showed that lygus bugs and western flower thrips readily colonize cotton from surrounding cotton and alfalfa sources and provide preliminary evidence that a natural refuge strategy may function to manage resistance to Bacillus thuringiensis (Bt) cottons with activity against lygus and thrips. Initial studies of vernonia, a desert-adapted industrial crop, indicate it may function as a trap crop for cotton pests. A novel gut content assay procedure was developed to detect foodstuffs (e.g., insect prey, weed seeds, blood meals) in insect carnivores, omnivores, and parasites (bed bugs). The assay proved to be superior to the conventional gut assay procedure (DNA-specific assays), and was used to examine complex food web interactions (e.g., hyperpredation, scavenging, cannibalism, and hematophagy). Under Objective 1B, literature searches for non-target arthropod field studies in Bt crops (cotton, eggplant, rice, potato) were conducted and are being screened for relevance and entry into an existing database that already includes Bt maize. Meta-analyses have been conducted on Bt maize studies to predict risks to non-target arthropods. Progress has been made toward refining resistance management strategies. Under Objective 2A, a total of 71 whitefly populations have been sampled in cotton from across Arizona to evaluate susceptibilities to seven insecticides, using seasonal conditions and pesticide use patterns as evaluation criteria. Similarly, glassy-winged sharpshooter populations have been evaluated in Kern County, California, for susceptibility to six insecticides out of concern that recent population expansion is due in part to insecticide resistance. Under Objective 2B, we worked in cooperation with scientists from the University of Arizona to examine strains of pink bollworm that are resistant to the Cry toxins produced in Bt cotton. We found that the genes providing resistance can be either recessive or partially dominant depending on the strain, and that some families share at least one resistance loci for toxin resistance. We also sequenced genes expressed in the guts of six pink bollworm strains, some of which are resistant to the Cry toxins. This was used to produce the first large-scale molecular resource for this species, and will help us to better understand the underlying functions of pink bollworm digestion and interactions with Cry toxins. Under Objective 2C, we are developing new tools for determining the interactions between potential receptor proteins and Cry toxins and how these may govern susceptibility. Despite successfully obtaining five genes encoding variant cadherin receptor proteins, not all were appropriately expressed in cells, prohibiting the assessment of toxicity. New insect cell lines and expression vectors are currently being evaluated for these assays. We have obtained complementary DNA for two ATP-Binding Cassette (ABC) transporter genes, which will be used to produce these putative receptors for Cry toxins in cell lines. We have been optimizing conditions and protocols to measure cytotoxicity in such cells. We have advanced our understanding of the factors that impact whitefly mate finding in sex ratios under Objective 3A. Whitefly populations were sampled from 16 cotton fields in the summer of 2015, various hosts in October, 2015, and from melons in spring of 2016 to evaluate the incidence and diversity of secondary endosymbionts in whitefly populations. Secondary endosymbionts can alter sex ratio dynamics in some insect species and may have a similar effect on whitefly populations. Identifying candidate species is a crucial step towards developing a better understanding of the potential influence that secondary endosymbionts have on whitefly sex ratios. Ultimately we are interested in drivers of whitefly population dynamics, of which sex ratio variability can have a profound influence on rates of population increase. We have also enhanced our knowledge of factors influencing life history traits of a key pest species. Under Objective 4B, we have determined that females of the western tarnished plant bug, Lygus hesperus, produce eggs at a higher rate after mating and the more frequently a female mates, the higher the overall rate she sustains. Even the presence of a male without mating promoted egg production. However, mated females had shorter lifespans than virgins, possibly due to the rapid depletion of essential resources leading to increased susceptibility to environmental stressors. In the course of such studies we also determined that evaluations of the accuracy of L. hesperus egg counts accentuated the role of sampler experience, and indicated counts of 3-day-old eggs were more accurate than counts of newly laid eggs. These results can be used to minimize sampling error in studies of Lygus fecundity. Under Objective 4C, we have worked toward determining the critical life stage at which a Lygus bug needs to receive the photoperiodic cue to induce diapause. Results indicated a graded response instead of a single receptive life stage, with the incidence of diapause increasing with earlier and longer exposure to a short photoperiod. A separate study was initiated to examine morphological consequences of switching from short to long day photoperiod on adult reproductive development. These results will provide insights into diapause termination and should provide phenotypes to monitor in efforts to disrupt clock genes. Preliminary studies indicated L. hesperus head capsule widths along with other characters can be used to unambiguously identify instar, but wing pad coloration was a reliable indicator of impending adult eclosion in only 40% of the population. These results will be used to improve the efficiency of diapause and thermal studies that rely on accurate identification of Lygus physiological age. We have also tentatively identified proteins circulating in the blood of insects that might be used as indicators to distinguish reproductive from diapausing insects, in at least one gender. Identification of key biochemical markers of diapause may provide targets for molecular disruption or phenotypes to monitor during efforts to manipulate gene expression. We have also progressed toward locating genes that respond to xenobiotics and environmental stressors, in our quest to identify targets for chemical or genetic disruption. Under Objective 5A, we have produced gene expression data from Lygus bugs exposed to thermal or oxidative stress. After assembling and annotating the data, we have identified 14 genes that were responsive to temperature challenges, and five that responded to oxidative agents in the diet. The identities of these genes and their responses to stressors are currently being validated. Five candidate stress response genes have been cloned into various expression vectors for subsequent functional characterization using either bacteria-based or insect cell culture-based expression. Working under Objective 5B, we have developed and optimized methods for injecting double-stranded RNA to knock down several genes important for L. hesperus development and survival. These include the “Halloween” genes, of which “spookiest” is critical for Lygus development and reproduction. We have further shown knockdown of another gene transcript that is involved in molting and reproductive maturation. Because knockdown of these genes produced highly tractable phenotypes, we also determined the longevity, specificity, and physiological and behavioral effects of the knockdowns to better understand how this approach might be used to develop new control approaches. Separately, we have also examined the impact of herbivore-induced plant defenses on pest insect survival and development. This was done with a diet bioassay and by measuring changes to the expression of detoxification genes. Results have been inconsistent but overall showed negative effects on Lygus survival and development when plant defensive responses were first induced by caterpillar feeding.

Accomplishments
1. New mode of action for Bacillus thuringiensis (Bt) toxins discovered. Resistance to Bt transgenic crops is increasing globally, threatening the benefits of these effective and environmentally-friendly crops. An ARS scientist at Maricopa, Arizona, and a team of university collaborators discovered a new model of Bt intoxication (the “dual” model) in which Bt protoxins and activated toxins kill insects via different pathways. The research demonstrated that protoxins killed some Bt resistant insects better than the corresponding activated toxins. This suggests that a previously unknown mode of action for Bt toxins exists and that there may be utility in using protoxins in transgenic Bt crops to enhance and/or sustain their efficacy.

2. Novel method for identifying predators in agroecosystems. Insect predators and spiders provide valuable ecosystem services in agriculture settings, however, identifying the main predators of pests by direct observation is difficult because arthropods are small and elusive. The most popular indirect method to assess predation is by examination of a predator’s stomach contents for the presence of prey-specific DNA, but these assays are complex, expensive, and labor intensive. An ARS scientist in Maricopa, Arizona, has developed an alternative approach that uses generic immunological assays designed to detect unique protein marks applied to the prey and subsequently transferred to the predator during feeding. Research showed that the generic assay was more sensitive and reproducible than the DNA assay for detecting predation while being less expensive and time consuming than the DNA assay. The generic gut assay procedure has been adopted by researchers throughout the world to study various aspects of predation. Using this method to pinpoint the main predators in an agroecosystem setting will lead to better conservation biological control practices.

3. Long-term dynamics of insect parasitoids attacking sweetpotato whitefly. Sweetpotato whiteflies are a pest of global significance and negatively impact many field and horticultural crops. Whiteflies are attacked by a broad range of natural enemies, including insect parasitoids. An ARS scientist in Maricopa, Arizona, conducted a long-term 15-year study to examine interactions between sweetpotato whitefly and several parasitoid species that generally specialize on whiteflies. This time period coincided with a USDA led, large-scale program in conjunction with the Animal and Plant Health Inspection Service (APHIS) to introduce exotic parasitoid species for biological control of sweetpotato whitefly. These introductions dramatically changed the parasitoid species composition in the cotton system by replacing three native species with two exotic species, however, the introductions did not change the levels of parasitism and have thus not contributed to increased biological control of the pest in Arizona cotton. Results are of interest to scientists studying introductory biological control and pest management and should help to inform future large-scale biological control programs.

4. Field validation of insecticide efficacies against Cucurbit Yellow Stunting Disorder Virus (CYSDV) in cantaloupes. Cantaloupe production in the southwestern U.S. has been severely impacted by epidemics of CYSDV since 2006 when it was first detected in Arizona. Infection by CYSDV causes yield reduction and poor fruit quality, especially when plants are infected early in their development. A series of greenhouse tests demonstrated the capacity of certain insecticides to prevent transmission of CYSDV to cantaloupe plants by viruliferous whiteflies. To evaluate their performance in an agricultural setting, ARS scientists in Maricopa, Arizona, conducted field studies on cantaloupes treated with five systemic insecticides. Under heavy whitefly pressure, two of the five insecticides significantly reduced the number of infections and the rate at which they became infected. These findings will provide critical guidance to growers and pest consultants seeking to both reduce and delay the incidence of CYSDV in cantaloupe fields.

5. Transcriptomic analysis of four species of xylem-sap feeding insects. Specialized feeding on nutrient-deficient xylem sap is relatively rare among insect herbivores, and only limited genomic and transcriptomic information has been generated for xylem-sap feeders. Various species of spittle bugs and sharpshooters are well-known xylem-sap feeders and vectors of the bacterium (Xylella fastidiosa) that causes Pierce’s disease in grapevines and other crop diseases. ARS scientists in Maricopa, Arizona, generated transcriptome assemblies for three sharpshooter species and one spittle-bug species for a more comprehensive understanding of biochemical adaptations and symbiotic relationships that support their unique living habits. Information contained in the transcriptomes of the four xylem-sap feeding insects will provide additional knowledge and insight into the survival of ecological specialists on a nutritionally poor dietary source.

None.

Review Publications
Fabrick, J.A., Unnithan, G.C., Yelich, A.J., Degain, B., Masson, L., Zhang, J., Carriere, Y., Tabashnik, B.E. 2015. Multi-toxin resistance enables pink bollworm survival on pyramided Bt cotton. Scientific Reports. 5:16554. doi: 10.1038/srep 16554.
Naranjo, S.E., Li, S. 2016. Long term dynamics of aphelinid parasitoids attacking Bemisia tabaci. Biological Control. 93:56-64.
Tassone, E.E., Geib, S.M., Hall, B., Fabrick, J.A., Brent, C.S., Hull, J.J. 2016. De novo construction of an expanded transcriptome assembly for the western tarnished plant bug, Lygus hesperus. Gigascience. 5:6.
Spurgeon, D.W. 2016. Captures of boll weevils (Coleoptera: Curculionidae) in relation to trap orientation and distance from brush lines. Journal of Economic Entomology. 109(2):676-683. doi: 10.1093/jee/tov392.
Wang, Y., Kaftanoglu, O., Brent, C.S., Page Jr., R.E., Amdam, G.V. 2016. Starvation stress during larval development reveals predictive adaptive response in adult worker honey bees (Apis mellifera). Journal of Experimental Biology. 219:949-959.
Guo, Y., Tian, J., Shi, W., Dong, X., Romeis, J., Naranjo, S.E., Hellmich Ii, R.L., Shelton, A. 2015. The interaction of two-spotted spider mites, Tetranychus urticae Koch, with Cry protein production and predation by Amblyseius andersoni (Chant) in Cry1Ac/Cry2Ab cotton and Cry1F maize. Transgenic Research. 25(1):33-44.
Katayama, Y., Suzuki, T., Ebisawa, T., Ohtsuka, J., Wang, S., Natsume, R., Lo, Y., Senda, T., Nagamine, T., Hull, J.J., Matsumoto, S., Nagasawa, H., Nagata, K., Tanokura, M. 2016. A class-A GPCR solubilized under high hydrostatic pressure retains its ligand binding ability. Biochimica et Biophysica Acta. 1858(9):2145–2151.
Spurgeon, D.W., Brent, C.S. 2015. Diapause response to photoperiod in an Arizona population of Lygus hesperus (Hemiptera: Miridae). Journal of Entomological Science. 50:238-247.
Tassone, E.E., Zastro-Hayes, G., Mathis, J., Nelson, M., Wu, G., Flexner, L.J., Carriere, Y., Tabashnik, B.E., Fabrick, J.A. 2016. Sequencing, de novo assembly, and annotation of a pink bollworm larval midgut transcriptome. Gigascience. 5:28. doi: 10.1186/s13742-016-0130-9.
Hagler, J.R., Tassone, E.E., Coffelt, T., Lipka, A.E. 2016. Host plant preference of Lygus hesperus exposed to three desert-adapted industrial crops. Industrial Crops and Products. 89:363-367. doi: 10.1016/j.indcrop.2016.05.041.
Brent, C.S., Katelyn, M., Connor, V., Brittany, M., Bronwen, S., Brent, K.G., Rachna, N. 2016. Regulatory roles of biogenic amines and juvenile hormone in the reproductive behavior of the western tarnished plant bug (Lygus hesperus). Journal of Comparative Physiology. 186(2):169-179.
Carriere, Y., Fabrick, J.A., Tabashnik, B. 2016. Can pyramids and seed mixtures delay resistance to Bt crops? Trends in Biotechnology. 34(4):291-302. doi: 10.1016/j.tibtech.2015.12.011.
Hagler, J.R., Blackmer, F., Spurgeon, D.W. 2015. Accuracy of a prey-specific DNA assay and a generic prey-immunomarking assay for detecting predation. Methods in Ecology and Evolution. 6(12):1426-1434. doi: 10.1111/2041-210X.12436.
Hagler, J.R., Machtley, S.A. 2016. Administering and detecting protein marks on arthropods for dispersal research. Journal of Visualized Experiments. 107:e53693. doi: 10.3791/53693(2016).
Tabashnik, B.E., Zhang, M., Fabrick, J.A., Wu, Y., Gao, M., Huang, F., Wei, J., Zhang, J., Yelich, A., Unnithan, C., Bravo, A., Soberon, M., Carriere, Y., Li, X. 2015. Dual mode of action of Bt proteins: Protoxin efficacy against resistant insects. Scientific Reports. 5:15107. doi: 10.1038/srep15107.
Blubaugh, C.K., Hagler, J.R., Machtley, S.A., Kaplan, I. 2016. Cover crops increase foraging activity of omnivorous predators in seed patches and facilitate weed biological control. Agriculture, Ecosystems and Environment. 231:264-270.
Brent, C.S., Wang, M., Miao, Y., Hull, J.J. 2016. Ecdysteroid and chitinase fluctuations in the western tarnished plant bug (Lygus hesperus) prior to molt indicate roles in development. Archives of Insect Biochemistry and Physiology. 91(5):108-126.
Hagler, J.R., Blackmer, F. 2015. Evidence of intraguild predation on a key member of the cotton predator complex. FOOD WEBS. 4:8-13.
Zinna, R., Gotoh, H., Brent, C.S., Dolezal, A., Kraus, A., Niimi, T., Emlen, D., Lavine, L. 2016. Endocrine control of exaggerated traits in rhinoceros beetles. Integrative & Comparative Biology. 56(2):247-259.
Fu, T., Hull, J.J., Yang, T., Wang, G.R. 2016. Identification and functional characterization of four TRPA1 variants in Apolygus lucorum (Meyer-Dür). Insect Molecular Biology. 25(4):370–384.
Klick, J., Yang, W., Walton, V., Dalton, D., Hagler, J.R., Dreves, A., Lee, J.C., Bruck, D. 2016. Distribution and activity of Drosophila suzukii in cultivated raspberry and surrounding vegetation. Journal of Applied Entomology. 140:37-46. doi: 10.1111/jen.12234.
Aslimwe, P., Ellsworth, P.C., Naranjo, S.E. 2016. Natural enemy impacts on bemisia tabaci dominate plant quality effects in the cotton system. Ecological Entomology. 41:642-652.