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

Location: Pest Management and Biocontrol Research

2017 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
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 1A: Studies were conducted to better understand insects that promote healthy crop ecosystems, including pollinators and predators. In cooperation with ARS scientists in Logan, Utah, and scientists at Michigan State University, pollinator movement was studied in blue orchard bees, bumble bees, and honey bees. Field studies also showed that two key pest insects, the western tarnished plant bugs and western flower thrips, readily colonize cotton from surrounding source crops. This provides preliminary evidence that a natural refuge strategy may function to manage resistance to cotton crops expressing Bacillus thuringiensis (Bt) toxins. 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, and was used to examine complex food web interactions (e.g., hyperpredation, scavenging, cannibalism, and hematophagy). Objective 1B: Literature searches for non-target arthropod field studies in Bt crops (cotton, eggplant, rice, potato) were completed and about 50 percent have been screened and entered into an existing database that already includes Bt maize. Objective 2A: A total of 91 whitefly populations have been sampled in cotton across Arizona to evaluate susceptibilities to seven insecticides, using seasonal conditions and pesticide use patterns as evaluation criteria. Geospatial and statistical analysis of the data is ongoing. Related to this, greenhouse and field studies examined whether systemic insecticide treatment of cantaloupe plants results in higher incidence of Cucurbit Yellow Stunting Disorder Virus (CYSDV). Assessments of virus incidence indicate that infection rates were consistently lowest in plots treated with flupyradifurone, while dinotefuran was second in efficacy. Two other insecticides, imidacloprid and cyantraniliprole, exacerbated virus incidence in multiple trials. Flupyradifurone also held whitefly densities to their lowest numbers relative to the other treatments. Similarly, glassy-winged sharpshooter populations have been evaluated in California for susceptibility to six insecticides out of concern that recent population expansion is due in part to insecticide resistance. Advances were made toward refining resistance management strategies. Objective 2B: Cooperative research was conducted with scientists from the University of Arizona to examine strains of pink bollworm resistant to the Cry toxins produced in Bt cotton. Complementation tests to examine if resistance loci are shared in two Cry2Ab-resistant strains of pink bollworm were completed. Genetic crosses between a susceptible strain and the two Cry2A-resistant strains confirmed that resistance to Cry2Ab toxin is inherited as a recessive trait in one strain, but partially dominant in another. Single-pair crosses indicate that some families share at least one resistance loci for Cry2 resistance. Scientists have identified one of the shared genetic loci as the adenosine triphosphate (ATP) binding cassette (ABC transporters) A2 protein. Completed experiments showed a direct genetic link between a mutation in the ABCA2 gene and resistance to Cry2Ab. Under Objective 2C, new tools are being developed for determining the interactions between potential receptor proteins and Cry toxins and how these may govern susceptibility. Five transcripts encoding variant cadherin receptor proteins were cloned and verified for correct insertion; however, all mutant forms of the protein were retained within cells of expression system. This prohibited the direct assessment of toxicity, but has allowed for new research hypotheses. Namely, the incorrect expression of mutant receptor proteins (e.g. retention of proteins within cells) may prevent the proper localization of protein on the surface of gut cells and may be responsible for the decreased binding that results in resistance to Bt toxin. New cadherin antibodies are currently being produced that will permit testing for similar changes in cellular localization of cadherin receptor protein within midguts in homozygous resistant strains of pink bollworm. Furthermore, clonal cell lines specifically selected for enhanced cadherin expression on the cell surface are being generated. In addition, gene-editing experiments are being conducted to directly knockout the cadherin protein in Bt-susceptible pink bollworm to demonstrate function of these putative receptors in vivo. Objective 3D: An investigation of the chemical ecology of Lygus has been initiated. Male responses to surface-applied myristyl acetate, an anti-mating compound, has been examined. We found that under enclosed test conditions, the antiaphrodisiac loses its efficacy, likely due to sensory habituation. Trials using more field-like conditions are underway. Additionally, a nondestructive technique to age-grade the Lygus spermatophore by external observation of the female abdomen has been developed. Three criteria were evaluated that changed predictably, but differently, with increased time since mating. Validation of these results would provide a rapid, nondestructive method for distinguishing recently mated females, which may be necessary in applied evaluations of myristyl acetate as a mating disruptor. Objective 4B: Factors influencing life history traits of Lygus have been elucidated. The effect of temperature on reproductive development and life span have been examined. Relative to females reared under moderate temperature conditions, those under warmer conditions begin laying eggs sooner and at a faster rate, but die sooner and produce fewer eggs overall. Females raised under cooler conditions, produce eggs later in life and at a slower rate, but live much longer and have greater lifetime fecundities. Differences in females reared under constant versus variable temperature regimes have been noted. Objective 4C: Studies to quantify instar- or stage-specific diapause responses of Lygus have been completed. Lygus remained responsive to a short day length, so long as exposure occurred earlier than 5th instar. The diapause response was rarely detected in insects switched from short to long days irrespective of the age when the switch occurred. Adults reared under short day conditions and switched to long days rapidly initiated reproductive development compared with adults reared under long day conditions. Nymphs switched from short to long days at 2nd instar responded similarly to nymphs that remained under short days. When the photoperiod was switched during the 4th instar, incidence of diapause was diminished but not eliminated. Understanding the age-dependent responsiveness of Lygus to photoperiodic cues will be necessary to elucidate the underlying physiological and molecular biological mechanisms controlling the diapause phenomenon. Further, studies to nondestructively distinguish diapausing from reproductive Lygus adults suggested the ratio of head capsule width to abdomen length was a superior criterion for males, compared with the criterion of abdomen color. Additional data will be collected to ensure adequate sample sizes of non-diapausing bugs, after which results will be validated with independent data. The ability to nondestructively distinguish diapause status of individual insects will be of critical importance to interpretation of planned host-free survival studies. Preliminary data to distinguish physiological age of Lygus nymphs based on abdomen dimensions has also been collected. If the ratio of head capsule width to abdomen width can be modeled relative to chronological age, these measurements would nondestructively predict physiological age of individual insects. This ability will benefit ongoing evaluations of gene knock-down that rely on treatment application to a specific physiological age class. Objective 5: New methods have been developed and optimized to use a microinjection system for introducing nanoliter volumes of double-stranded ribonucleic acid (RNA) into both adult and nymph Lygus for RNA interference (RNAi) mediated knock-down of diverse target genes. Genes targeted include those involved in noxious compound detection, diapause development, reproductive tissue development, molting, and eye pigmentation. The viability of oral-based introduction of double stranded RNA (dsRNAs) to trigger the RNAi response is also being assessed. Although initial efforts have proven to be ineffective, possible reasons for the poor efficacy are currently being explored at the molecular level. Objective 5A: New genes that respond to xenobiotics and environmental stressors are being indentified. Gene expression data has been produced from Lygus bugs exposed to thermal or oxidative stress. The initial sequence reads have been made publically available via the short read archive. The identities of these genes and their responses to stressors are currently being validated.

Accomplishments
1. Patterns of endosymbiont infection in populations of whitefly and their parasitoids. Populations of whitefly, a major pest insect and a vector of viral plant diseases, are difficult to control, but their numbers and impact may be influenced by the symbiotic microorganisms living within each individual. An ARS scientist from Maricopa, Arizona, and researchers at Çukorova University in Adana, Turkey, showed that identification of the symbiotic species living within the whitefly and its parasitoids could be determined by DNA analysis. Examination of multiple populations from both Arizona and Turkey showed differences in the composition of the symbiotic species. Similarly, the symbiont composition for the parasitoids of those populations also differed. These differences may explain regional variation in whitefly sex ratios, which can impact the growth and spread of these populations. The results provide a novel avenue for manipulating the population dynamics of whitefly to mitigate their spread and damage.

2. Transgenic crops do not affect important insect parasitoids. Transgenic crops expressing Bacillus thuringiensis (Bt) toxins are cultivated on millions of hectares globally, but concerns remain about the risks these crops may impose on non-target organisms like insect parasitoids that supply important ecological services. An ARS scientist from Maricopa, Arizona, researchers at Cornell University, an ARS scientist from Ames, Iowa, and scientists at Agroscope in Zurich, Switzerland, showed that the development and reproduction of two species of parasitic wasps were not affected by ingestion of hosts that had fed on diets incorporating Bt cotton material. The host used was resistant to Bt proteins in the plant, eliminating host quality issues and allowing the direct effects of Bt proteins to be assessed under realistic exposure scenarios. Results are valuable to governmental authorities responsible for regulating transgenic crops, scientists concerned with developing integrated pest management strategies for pest control, and a general public concerned about the environmental effects of biotechnology.

3. Induced cotton plant defenses negatively affect growth and survival of Lygus bugs. With the widespread cultivation of cotton expressing Bacillus thuringiensis (Bt) toxins, several pests unaffected by Bt proteins have become more problematic. One possible reason is that the induction of inherent cotton plant defenses have been reduced because chewing caterpillars have been largely eliminated in Bt cotton production systems. An ARS scientist from Maricopa, Arizona, collaborated with researchers from Agroscope in Zurich, Switzerland, to show that cotton plants can be systemically induced to produce several terpinoid compounds by the feeding of caterpillar. This induction resulted in reduced growth and survival of Lygus bugs. However, follow-up diet studies showed that these effects were not due to two common terpinoids but by other induced defensive chemicals. Results support the hypothesis that lack of defense induction might contribute to non-target pest issues in Bt cotton, and are of interest to researchers examining insect plant interactions and to pest management of non-caterpillar pests in cotton.

4. Improved management of sweetpotato whitefly in cotton. While it is known that biological control by native arthropod predators is critical to effective control of sweetpotato whiteflies in cotton, current Integrated Pest Management (IPM) is based only on the scouting of pest abundance. An ARS scientist from Maricopa, Arizona, collaborated with researchers at the University of Arizona, to develop, test and implement economic thresholds that use predator-pest ratios for making pest control decisions in IPM. Results were presented to pest control advisors and growers through extension programs and were validated in grower fields in Arizona and Mexico. The new biological control based thresholds greatly improved decision-making, advancing spray decisions about 10 percent of the time (too little biological control) and deferring sprays nearly 60 percent of the time (adequate biological control). The findings promote the development of improved economic threshold models and reduce the cost and risk for growers managing whitefly.

Review Publications
Brent, C.S., Byers, J.A., Levi-Zada, A. 2017. An insect anti-antiaphrodisiac. eLife. doi:10.7554/eLife.24063.
Carrière, Y., Fabrick, J.A., Tabashnik, B.E. 2016. Advances in managing pest resistance to Bt crops: Pyramids and seed mixtures. In: Horowitz, A.R. and Ishaaya, I., editors. Advances in Insect Control and Resistance Management. New York/Switzerland: Springer International Publishing. p. 263-286.
Christie, A.E., Hull, J.J., Richer, J.A., Tassone, E.E., Geib, S.M. 2016. Prediction of a peptidome for the western tarnished plant bug Lygus hesperus. General and Comparative Endocrinology. 243:22-38. doi: 10.1016/j.ygcen.2016.10.008.
Cook, C.N., Brent, C.S., Breed, M.D. 2017. Octopamine and tyramine modulate the thermoregulatory fanning response in honey bees (Apis mellifera L.). Journal of Experimental Biology. 220:1925-1930.
Cui, J., Li, S., Spurgeon, D.W., Jia, W., Lu, Y., Gouge, D. 2016. Flight capacity of Sitophilus zeamais Motschulsky in relation to gender and temperature. Southwestern Entomologist. 41:667-674.
Eisenring, M.A., Meissle, M., Hagenbucher, S., Naranjo, S.E., Wettstein, F., Romeis, J. 2017. Cotton defense induction patterns under spatially, temporally and quantitatively varying herbivory levels. Frontiers in Plant Science. 8:234. doi: 10.3389/fpls.2017.00234.
Hagler, J.R. 2016. A false positive food chain error associated with a generic predator gut content ELISA. Entomologia Experimentalis et Applicata. 161(3):187-192.
Leboeuf, A.C., Waridel, P., Brent, C.S., Goncalves, A.N., Menin, L., Ortiz, D., Koto, A., Privman, E., Soares, Z.G., Miska, E.A., Benton, R., Keller, L. 2016. Oral transfer of chemical cues, growth proteins and hormones in social insects. eLife. 5:e20375. doi:10.7554/eLife.20375.
Mansfield, S., Hagler, J.R. 2016. Wanted dead or alive: scavenging versus predation by three insect predators. FOOD WEBS. 9:12-17.
Oliveira, R.C., Vollet-Neto, A., Oi, C.A., Van Zaweden, J.S., Nascimento, F., Brent, C.S., Wenseleers, T. 2017. Hormonal pleiotropy helps maintain queen signal honesty in a highly eusocial wasp. Scientific Reports. 7:1654.
Spurgeon, D.W., Brent, C.S. 2016. Development, survival and hatching periodicity of Lygus hesperus (Hemiptera: Miridae) eggs under constant and variable temperatures. Journal of Entomological Science. 51(4):292-304.
Spurgeon, D.W. 2016. Captures of boll weevils (Coleoptera: Curculionidae) in relation to trap distance from cotton fields. Journal of Economic Entomology. 109(6):2405-2410.
Suh, C.P., Spurgeon, D.W. 2016. Boll weevils (Coleoptera: Curculionidae) continue to release pheromone following host removal. Journal of Entomological Science. 51(4):332-335.
Traynor, K.S., Wang, Y., Brent, C.S., Amdam, G., Page, R.E. 2017. Young and old honey bee (Apis mellifera) larvae differentially prime the developmental maturation of their caregivers. Animal Behaviour. 124(1):193-202.
Van Ekert, E., Wang, M., Miao, Y., Brent, C.S., Hull, J.J. 2016. RNAi-mediated knockdown of the Halloween gene spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus. Insect Molecular Biology. 25(5):550-565.
Van Ekert, E., Chauvigne, F., Finn, R.N., Mathew, L.G., Hull, J.J., Cerda, J., Fabrick, J.A. 2016. Molecular and functional characterization of Bemisia tabaci aquaporins reveals the water channel diversity of hemipteran insects. Insect Biochemistry and Molecular Biology. 77:39-51.
Brent, C.S., Penick, C.A., Trobaugh, B., Liebig, J. 2016. Induction of a reproductive-specific cuticular hydrocarbon profile by a juvenile hormone analog in the termite zootermopsis nevadensis. Chemoecology. 26(5):195-203.
Eisenring, M., Romeis, J., Naranjo, S.E., Meissle, M. 2017. Multitrophic Cry-protein flow in Bt-cotton. Agriculture, Ecosystems and Environment. 247:283-289.
Fabrick, J.A., Hull, J.J. 2017. Transient expression and cellular localization of recombinant proteins in cultured insect cells. Journal of Visualized Experiments. 122:e55756. doi: 10.3791/55756.
Sivakoff, F.S., Jones, S.C., Machtley, S.A., Hagler, J.R. 2016. Protein self-marking by ectoparasites: a case study using bed bugs. Journal of Medical Entomology. 53(6):1370-1377.
Spurgeon, D.W., Van Ekert, E., Elhoff, L.K. 2017. Accuracy of Lygus hesperus Knight (Hemiptera: Miridae) egg counts improves with egg development. Journal of Cotton Science. 21:1-7.
Zhang, H., Yu, S., Shi, Y., Yang, Y., Fabrick, J.A., Wu, Y. 2017. Intra- and extracellular domains of the Helicoverpa armigera cadherin mediate Cry1Ac cytotoxicity. Journal of Insect Biochemistry and Molecular Biology. 86:41-49.