Location: Pest Management and Biocontrol Research2019 Annual Report
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)
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.
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. Under Objective 1, the compilation of a database of non-target arthropod field studies in Bacillus thuringiensis (Bt) crops (cotton, eggplant, rice, potato) is nearly complete. The database for Bt maize has been updated and analyses and publication of results are underway. Replicated field plot studies were repeated for a second year 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. Preliminary results show that several new insecticides are highly selective and will enable additional options for growers to selectively control whitefly while preserving natural enemy species. Preliminary results also suggest that medium size plots (18 x 18 meters) may be sufficient to accurately measure non-target effects for most species. The effects of insecticides and plot size on biological control function (predation and parasitism on sentinel whitefly) are currently being analyzed. Additional field studies were initiated to examine the non-target impacts of a new Bt cotton formulated to target Lygus bugs and thrips, particularly with regard to natural enemy species that help suppress whitefly populations. Studies examined impacts on individual species such as minute pirate bugs, which have been shown to be sensitive to the new Bt plants in laboratory studies. In addition, some thrips species act as predators of whitefly and other important pests in the cotton system in Arizona and California and there is concern that the new Bt crop may affect biological control of whitefly and other pests. Field research will address impacts on predator abundance and on their biological control function using sentinel whitefly prey. This research is being supported in part from a USDA-NIFA, Biotechnology Risk Assessment grant. Additionally, under this objective a “universal food immunomarking technique” (UFIT) has been developed to detect immuno-labelled food items in arthropods. Studies are in progress to investigate: (1) predator feeding activity specifically on the lygus egg stage, (2) predator feeding activity on each specific lygus life stage (egg, nymph and adult), (3) intraguild predation and cannibalism exhibited on lacewing by the cotton predator assemblage, and (4) predation on the bagrada bug egg stage. A field study was completed that evaluated pest, natural enemy, and pollinator dispersal patterns in various cropping systems using a protein immunomarking technique (PIT). The PIT is being used to study Vernonia, a desert-adapted plant, as a trap crop for cotton. Preliminary data shows that the Vernonia preferentially attracts lygus along with its predaceous natural enemies, as well as a wide variety of pollinators. Field collections and mark analyses (> 20,000 specimens) are complete, and data are being analyzed for publication. The PIT method was also being used in cooperation with an ARS scientist in Tucson, Arizona, to study varroa mite dispersal. Under Objective 2, studies were conducted to examine the effect of sublethal doses of two commonly used insecticides on the biology and flight behavior of Heliothis virescens. Such data will be important in understanding the effect of insecticide use in non-Bt refuge fields that are designed to supply Bt susceptible insects to mate with and dampen the potential development of resistance insects in the Bt crop. Data are currently being analyzed. Additionally, under Objective 2, we found that insect cell lines producing several mutant forms of the pink bollworm midgut cadherin (including all four known alleles from the U.S. and two from China) fail to translocate to the cell surface. This implicates a new mechanism of Bt resistance in which the underlying cause is due to lack of receptor availability and not direct loss of toxin binding. Although this effectively prevents using the insect cell experimental system to test toxicity, the data provide a previously unknown mechanism of Bt resistance. Additional work is underway to use gene-editing to target putative Bt receptors in vivo, which will provide an alternative strategy to directly determine receptor function. While performing these experiments, we selected a new lab strain of pink bollworm highly resistant to Cry1Ac Bt toxin that lacks mutations in the cadherin receptor gene, and identified a new mechanism that involves down-regulation of the cadherin receptor (both RNA and protein). The results reveal the remarkable adaptability of pink bollworm and other major pests to evolve resistance to Bt toxins via both qualitative and quantitative changes in receptor proteins and demonstrates the challenges for monitoring and managing resistance to Bt crops. Studies of host condition on susceptibility to insecticides and of thermal effects on insecticide susceptibility for Bemisia tabaci were not completed due to the retirement of the responsible ARS scientist. Under Objective 3, studies of influence of an exogenously applied anti-aphrodisiac, myristyl acetate (MA), on Lygus hesperus mating are nearing complete. It was found that although direct application of MA to a female limited the interest of males in mating, just having the MA in proximity to the female had no effect on male behavior, regardless of the dose being used. Preliminary evidence has also been found that MA expression by males may enhance the likelihood that a female will mate with them, possibly by signaling his readiness to provide sperm. Collectively the results suggest that MA would not be appropriate for use in population control via field sprays. Under Objective 4, studies of the temperature-dependence of Lygus adult reproductive development were completed. Development of male and female reproductive organs tended to be more rapid at a constant temperature of 84 degrees fahrenheit (F) compared with a variable temperature regime (average 84 degrees; daily range 70 to 99 degrees F), but this difference was not statistically significant. Development under the low variable temperatures (average 59 degrees F; daily range from 45 to 75 degrees F) was consistently more rapid compared with a constant temperature of 59 degrees F. These patterns of development were similar to those previously observed for eggs and nymphs, although differences between constant and variable temperature regimes were subtler for adults compared with the other stages. The collective results for all developmental stages (eggs, nymphs, and adults) suggest that previously reported population responses of Lygus bugs to irrigation levels in cotton are not caused by elevated canopy temperatures associated with moderate drought stress. Instead, differences corresponding to irrigation levels are likely related to host preferences of the immigrating adult bugs that are exhibited in small-plot field studies. Also, under this objective, studies to examine the modifying influence of temperature on the photoperiodic diapause response of Lygus hesperus were completed. Under the temperature conditions studied, rearing temperature had no demonstrable effect on the incidence of diapause. These results suggest that annual variations in late-summer and fall temperature conditions do not appreciably influence the timing of diapause induction in the field. Study of the hormonal mechanisms regulating diapause was hindered by the loss of a radioisotope-based assay. Effort was instead focused on creating a new measurement technique. A new liquid chromatography mass spectrometry (LC-MS) based hormonal assay has been successfully developed and validated and data collection for understanding diapause control will begin shortly. Study of temperature effects on the development of two predators of Lygus, Geocoris punctipes and Nabis alternatus, were prevented due to the retirement of the principal scientist examining the subject. Under Objective 5, the continued analysis of a transcriptomic database generated from L. hesperus exposed to differing thermal conditions facilitated the identification of a stress-regulated gene family that consists of seven related genes. Polymerase chain reaction (PCR) based expression profiling revealed up-regulation of one gene in response to heat stress, whereas a second gene was up-regulated in response to both heat and cold stress. Further examination of transcriptional responses to stress conditions is ongoing. Additionally, research has continued into the optimization of gene silencing methods via dsRNA-mediated RNA interference (RNAi) mechanisms. Oral-delivery of dsRNA in L. hesperus has proven to be ineffective. Unlike other species, extraoral and gut degradation of the dsRNA do not appear to be the principal factors limiting the efficacy of oral RNAi. The primary barrier may be due to poor persistence and uptake of the ingested dsRNAs. The effectiveness of modified RNAs, which have previously been reported in other species to exhibit greater stability/potency in triggering the RNAi response, failed to generate expected phenotypes in L. hesperus. Further research to elucidate the mechanism(s) underlying the poor response of oral RNAi is ongoing. Injected dsRNAs that target genes of interest remains highly effective for examining putative gene function as a suite of genes implicated in L. hesperus eye pigmentation. The visual phenotype that results from silencing these eye pigmentation genes can be used to assess the viability of gene-editing methods for L. hesperus.
1. Novel mechanism of pink bollworm resistance to Bacillus thuringiensis (Bt) crops. Transgenic crops engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) provide many benefits including pest suppression, increased yields and farmer profits, reduced conventional insecticide use, decreased harm to non-target species, and enhanced biological control. However, pest resistance to Bt crops decreases such benefits. Pink bollworm is a global pest of cotton and resistance to the toxins of Bt cotton involve mutations and/or cellular trafficking of a cadherin receptor protein thought to prevent toxin binding to the insect midgut. Findings by an ARS scientist from Maricopa, Arizona, and researchers from the University of Arizona, show that the down-regulation of cadherin transcript and protein is responsible for loss of the available receptor. These findings reveal the remarkable adaptability of pink bollworm and other major pests to evolve resistance to Bt toxins via both qualitative and quantitative changes in receptor proteins and demonstrates the challenges for monitoring and managing resistance to Bt crops. The results are valuable for scientists concerned with understanding the mechanisms of resistance, for private industry for developing new commercial strategies to target pests, and for government authorities responsible for regulating transgenic crops.
2. Flight mill technology for the study of insect flight behavior. Dispersal is a key component in the population ecology and dynamics of insects, yet it remains one of the most difficult and intractable ecological processes to study in the field. Thus, many researchers have looked to laboratory methods for investigating the many factors that influence an insect’s ability to move within its environment. A scientist in Maricopa, Arizona, reviewed and synthesized the global literature on the development, use, and data interpretation of insect flight mills for the study of flight behavior. The study provided details on the construction and operation of a flight mill developed by the ARS scientist, including access to websites and videos. The technology has been used by numerous scientists studying a wide range of insect pests and is of substantial value to those interested in used flight mills for the study of insect dispersal.
3. Efficacy and safety of Bacillus thuringiensis (Bt) eggplant in Bangladesh. Bangladesh approved the commercial cultivation of Bt eggplant in 2014 and is the first developing nation in the world to introduce a Bt vegetable crop. Bt eggplants produce an insecticidal protein that is toxic to certain caterpillar pests. A researcher in Maricopa, Arizona, in collaboration with scientists from Cornell University and the Bangladesh Agricultural Research Institute, conducted replicated plot studies at two sites in Bangladesh to assess the efficacy and environmental safety of four Bt eggplant varieties. All varieties provided high levels of control of the eggplant fruit and shoot borer, a key caterpillar pest, and resulted in substantial economic returns relative to conventional practice. In addition, there was no evidence that Bt eggplant negatively affected other arthropods, including non-target pests and many species of natural enemies. Results demonstrated that this highly selective and effective crop will benefit growers, consumers and the environment in Bangladesh.
4. Thermal ecology of Lygus hesperus. Irrigation management strategies in western crops are changing because of the increasingly limited supply and higher cost of water. Alternative strategies such as deficit irrigation result in periodic, moderate drought stress in cotton, and corresponding increases in canopy temperatures. Previous reports indicate an association between limited irrigation and lower Lygus population levels in cotton, but the controlling mechanism is not known. Knowledge of arthropod responses to changing irrigation practices can enhance practices supporting a successful integrated pest management program. ARS researchers at Maricopa, Arizona, showed that development of Lygus eggs, nymphs, and adults were similar between constant and variable temperature regimes when temperatures were moderate, but that low and high-variable temperature regimes typical of field conditions supported development at rates different from those observed under constant temperatures. These findings suggest that survival and development of overwintering Lygus are enhanced by daily variations in temperatures, and both are likely higher in the field than constant temperature studies have indicated.
5. Arthropod predation monitoring. Knowledge of arthropod predation is an important component of a successful integrated pest management program. The most popular indirect method to assess predation is by examination of a predator’s stomach contents for the presence of pest-specific DNA; however, this method is time consuming, costly, tedious, and has many limitations. An ARS researcher at Maricopa, Arizona, developed an immunological assay for detecting a unique protein biomarker applied to any targeted pest species that can be readily transferred to a predator during feeding. Studies revealed that the technique is effective at detecting protein-marked prey remains in the guts of all types of predators and that the protein marks should be applied internally to the prey (when possible) to maximize detection while minimizing the risk of false positive assay errors. The assay is less expensive, time consuming, and tedious than the conventional DNA assay method. Refinement of this gut assay procedure provides researchers with a reliable alternative technique for evaluating prey choice of predators in agroecosystems.
6. Tracking the dispersal of arthropod predators and parasitoids. Knowledge of arthropod predator and insect parasitoid dispersal patterns is critical for effective biological control of grape pests. An ARS researcher at Maricopa, Arizona, and scientists at the University of California, Riverside, developed a novel “triple mark” method to track arthropod movement in a vineyard containing a buckwheat cover crop. Arthropods were marked directly in the buckwheat plots using a “triple mark" solution containing yellow dye, casein protein, and albumin protein. The abundance of marked and unmarked natural enemies was recorded at a gradient of distances from the treated buckwheat plots into the vineyard. Results revealed that buckwheat refuges planted every sixth or tenth row within the vineyard could increase the biological control services rendered by the natural enemy complex on key grape pests.
7. Host preference and dispersal of spotted wing Drosophila. The spotted wing drosophila (SWD) fly is one of the worst invasive pest species in the U.S., in part because it feeds on the fruit of many different types of economically important crops (e.g., blueberry, strawberry, cherry, etc.). Knowledge of its host preference and dispersal ability are important for effective management. ARS researchers at Maricopa, Arizona, and Michigan State University, used protein marking to examine the movement of SWD from wild host plants in the landscape surrounding blueberry farms. Results identified five commonly infested wild fruits (honeysuckle, wild blackberry, wild raspberry, autumn olive, and American pokeweed) and showed that SWD disperses at an even rate throughout the blueberry farms. Moreover, early season flies were less likely to remain in the marked host compared to late season flies. Findings highlight the importance of wild hosts on local pest pressure from SWD and suggest that wild host management should be considered as part of integrated strategies for reducing the economic impacts of this pest.
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Cook, C.N., Mosquiero, T., Brent, C.S., Ozturk, C., Gadau, J., Pinter-Wollman, N., Smith, B.H. 2019. Individual differences in learning and biogenic amine levels influence the behavioural division between foraging honey bee scouts and recruits. Journal of Animal Ecology. 88:236-246.
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Dickinson, P.S., Hull, J.J., Miller, A., Oleisky, E.R., Christie, A.E. 2019. To what extent may peptide receptor gene diversity/complement contribute to functional flexibility in a simple pattern-generating neural network? Comparative Biochemistry and Physiology, Part D: Genomics and Proteomics. 30:262-282.
Dickinson, P.S., Dickinson, E.S., Bergstrom, M., Oleisky, E.R., Rivera, C.D., Stanhope, M.E., Stemmler, E.A., Hull, J.J., Christie, A.E. 2019. AMGSELFamide, a member of a broadly conserved peptide family, modulates multiple neural networks in Homarus americanus. Journal of Experimental Biology. 222:jeb194092-15. https://doi.org/10.1242/jeb.194092.
Luo, J., Ma, C., Li, Z., Zhu, B., Lei, C., Jin, S., Hull, J.J., Chen, L. 2018. Assessment of suitable reference genes for qRT-PCR analysis in Adelphocoris suturalis. Journal of Integrative Agriculture. 17(12):2745-2757.
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Mathew, L.G., Ponnuraj, J., Mallappa, B., Chowdary, L.R., Zhang, J., Tay, W., Walsh, T.K., Gordon, K.J., Heckel, D.G., Downes, S., Carriere, Y., Li, X., Tabashnik, B.E., Fabrick, J.A. 2018. ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm. Scientific Reports. 8:13531.