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ARS Home Pacific West Area Maricopa, Arizona U.S. Arid Land Agricultural Research Center Pest Management and Biocontrol Research Research





- Brent, Colin
- Hull, Joe
- Fabrick, Jeffrey
- Hagler, James
- Hepler, James
- Zilnik, Gabriel
- 2023
- 2022
- 2021
- Publications
- Crop Protection & Quarantine (304)

Research Project: Sustainable Pest Management for Arid-Land Agroecosystems **Location:
                            Pest Management and Biocontrol Research** 2021 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**
                                

Objective 1: Biological control-informed thresholds, which determine pesticide treatment using the density of pests and their predators, will be developed for L. hesperus in cotton using experimental field research and data mining. Densities of L. hesperus and natural enemy communities will be manipulated and monitored to identify key predators of L. hesperus. Predictions of ratios that enable biological control will be tested and compared to conventional threshold models. Companion plantings of vernonia and marigold will be tested, with lab and field approaches, for their efficacy in protecting cotton by drawing pests away from the crop and towards areas with high predator density. Protein marking will be used to track movement and predator feeding patterns on all life stages, and to determine whether the impact of drought-tolerant cotton isolines on pest colonization and predator success.

Objective 2: Cotton engineered to express the Bacillus thuringiensis (Bt) toxin selective for L. hesperus will be tested for non-target effects on natural enemies. Field studies will compare Bt and non-Bt cottons with and without additional insecticides. Sweep net sampling and sticky cards will measure the abundance of common predators of L. hesperus and B. tabaci. Biological control function will be assessed using established thresholds for B. tabaci and direct measures of predation. The impact of insecticidal seed treatments on the natural enemies of B. tabaci and L. hesperus in cotton will be assessed using field-based inclusion cage studies with young cotton plants containing whitefly eggs exposed to adult and immature thrips. To assess early-season and season-long efficacy and non-target impacts of cotton seed-treatments, field studies will compare population densities of B. tabaci, thrips, and other arthropods exposed to cotton with and with seed treatment.

Objective 3: The efficacy of oral RNAi will be assessed in L. hesperus by feeding or injecting dsRNA for genes involved in ovary function. To determine if digestive tract nucleases destroy dsRNA before it can be effective, luminal contents and gut homogenates will be assessed for enzymatic activity. To identify genes involved in dsRNA uptake from the gut, homologs of endocytotic pathway genes will be identified then silenced by RNAi to determine function. The role of parental RNAi will be tested by injecting adult L. hesperus females with dsRNA targeting the eye pigmentation genes and examining embryo eye color. Sex determination gene homologs in L. hesperus will be identified, their expression measured, and function determined by RNAi. CRISPR/Cas gene driver methods will be optimized for L. hesperus, using injections and electroporation to modify embryos. Bt toxin resistance mechanisms in pink bollworm and corn earworm relying on mutations in the ABC transporter and midgut cadherin genes will be examined by toxicity screening and cellular localization. Determination of whether a fitness tradeoff occurs in the corn earworm with Bt toxin resistance will be made in susceptible and resistant strains fed toxic and non-toxic diets by comparing life history traits and flight performance. **Progress Report**
                                

Under Sub-objective 1Bi, preliminary tests were conducted to measure lygus bug and whitefly olfactory response to various types of plants. The methods used for testing included a Y-tube olfactometer and behavior monitoring software. The tests revealed that the computer automated system could effectively track the insect’s response to the various plant odors. Currently, a wide variety of plant species are being grown in the greenhouse. These plants will be tested to determine which might attract or repel major cotton pests. The goal is to find plants that can be planted adjacent to cotton (companion plants) that attract (lure) the pests away from cotton. Ultimately, companion planting practices could reduce pesticide use in cotton. Progress has been slowed by maximized telework and reduced occupancy limits so that the study is not expected to be completed until fiscal year (FY) 22.

Under Sub-objective 1Bii, a novel feeding choice test arena was conceptualized, constructed, and tested for efficacy at measuring lygus bug and whitefly host plant preference for feeding and egg laying activity. The novel testing arena, coined a “guillotine” arena, was very effective, and should enhance research productivity. Currently, a wide variety of plant species are being grown in the greenhouse. Using the new arena, these plants will be tested for their attractiveness to these major cotton pests. Progress has been slowed by maximized telework and reduced occupancy limits so that the study is not expected to be completed until FY22.

Under Sub-objective 1C, a newly developed feeding choice test arena is being used to measure lygus bug and whitefly host plant feeding preference for four different isolines of cotton. These various cotton isolines are being concurrently screened for desert adaptivity traits (e.g., heat and drought tolerance) by an ARS cotton geneticist in Maricopa, Arizona. Each variety also has traits (e.g., various trichome densities [hairiness], waxy leaf surfaces, rough leaf surfaces, etc.) that may make them more or less attractive to lygus bug and whitefly, the two most common cotton pests. Progress has been slowed by maximized telework and reduced occupancy limits so that the study is not expected to be completed until FY22.

Under Sub-objective 2A, replicated large field plot studies were conducted to assess the non-target impacts of a new transgenic cotton with efficacy against Lygus hesperus and thrips and to assess impacts on biological control services. Treatments included: transgenic Bacillus thuringiensis (Bt) cotton (MON88702), that produces an endogenous toxin, and its near isoline (DP393) without additional insecticides; both cultivars with the addition of a material that selectively controls thrips; and a positive control of DP393 sprayed with a broad-spectrum material that represents an alternative control agent for Lygus. Bt and non-Bt seeds did not have added insecticidal seed treatments as is common for the industry. These treatment combinations enabled comparison of the Bt trait alone on non-targets, a comparison of the Bt trait to a conventional control alternative, and comparison of the thrips trait and a conventional control alternative on the abundance of natural enemies and potential biological control function on key pests. Because thrips are also predators in the system, this will also allow us to evaluate if the planting of this new Bt cotton poses any risks to current levels of biological control on whitefly and mites. Extensive sampling quantified the abundance of pests and natural enemies in the system, and the data have been compiled and entered and are currently being analyzed. Preliminary results suggest that biological control function, measured on sentinel whitefly prey, is not altered by the Bt crop and that the technology is moderately effective in control of Lygus and thrips.  

Under Sub-objective 2B, the intended studies were not initiated due to a critical scientific vacancy in the unit. These studies will be completed when the necessary personnel are in place.

Under Sub-objective 3A, double stranded RNAs (dsRNA), which are used to reduce gene function, were designed to target genes known to influence Lygus hesperus ovarian development. These dsRNAs were either directly injected into or fed to young adult females. Several days after treatment, ovarian development in the injected group was significantly impaired relative to controls, whereas no changes in development were observed in Lygus fed the dsRNAs. Similarly, target gene transcripts were significantly reduced in Lygus injected with dsRNAs but were comparable to controls in Lygus fed dsRNAs. Transcriptome data mining identified two enzymes that could potentially degrade oral dsRNAs, one of which is expressed in the salivary gland and is activated during feeding. However, even after reducing the expression of these enzymes, orally administered dsRNAs targeting ovarian development in the nuclease impaired Lygus remained ineffectual, suggesting that biocontrol through transgenic plants expressing dsRNAs that target Lygus may not be possible.

Under Sub-objective 3C, experiments were able to demonstrate that Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can be used as a tool to assess gene function in the plant bug Lygus hesperus. Methods were optimized for embryonic delivery of a gene editing complex and guides to target the eye pigmentation genes, cinnabar and cardinal. Injected individuals that lived to adulthood and exhibited altered eye pigmentation relative to control insects were separated and allowed to reproduce. Molecular analysis of cinnabar and cardinal genes in the descendants revealed mutations specific to the target region, indicating successful gene editing and validating this tool for future studies of gene function.

Under Sub-objective 3Di, experiments using CRISPR/Cas9 gene editing were able to modify the adenosine triphosphate (ATP)-binding cassette gene ABCA2 in a susceptible strain of pink bollworm. The resultant insects were found to be resistant to Cry2Ab, a toxin found in transgenic Bt crops. Resistant individuals had mutations that disrupted the function of the ABCA2 gene, with 26 different disruptive mutations identified. These data not only confirm the genetic basis of resistance to Cry2Ab in pink bollworm, but also shows that CRISPR/Cas9 gene editing is useful for validating the function of genes involved in resistance to Bt transgenic crops.

Under Sub-objective 3Dii, plasmid DNA was created that could be inserted into cells to study expression and localization of cadherins, transport proteins implicated in Bt toxin resistance. These plasmids contained cadherin gene sequences from either susceptible or resistant strains and a gene sequence for Venus, a protein that fluoresces and serves as a location marker. Several stable insect cells lines have been produced and are ready for immunofluorescence experiments. Plasmids are currently being constructed to produce the PgCad1 proteins without the Venus marker protein. Progress has been slowed by maximized telework and reduced occupancy limits so that the study is not expected to be completed until FY22.

Under Sub-objective 3Diii, the studies to determine if toxin resistance alters the flight capability or propensity of the corn earworm have been delayed due to the maximized telework requirement and reduced occupancy limits. They are now scheduled for completion in FY22. **Accomplishments**
                                

1. Transgenic cotton and sterile insect releases synergize eradication of pink bollworm from the United States. The pink bollworm is one of the world’s most invasive insects and has been a major pest of cotton in the United States since 1917. However, decades of effort and implementation of the Binational Pink Bollworm Eradication Program culminated in the USDA Secretary of Agriculture officially declaring the pink bollworm eradicated from the cotton-growing regions of the continental United States in 2018. ARS researchers in Maricopa, Arizona, and collaborators from USDA, Animal and Plant Health Inspection Service, the Arizona Cotton Research and Protection Council, and the University of Arizona used models to demonstrate that eradication was made possible by the synergistic interaction of Bacillus thuringiensis cotton and sterile insect releases. They determined that eradication saved cotton growers in the United States $224 million from 2014 to 2020 and was associated with an 82% reduction in insecticide use for all cotton pests in Arizona during this same period. The economic and social benefits achieved have wide sweeping impacts on agriculture and society and demonstrate the benefits of using agricultural biotechnology in concert with classical pest control tactics. 2. Gene editing in pink bollworm provides direct validation of gene function and their involvement in resistance to transgenic cotton. Genetically engineered crops that produce insecticidal proteins from Bacillus thuringiensis (Bt) are important globally for managing insect pests. However, the evolution of pest resistance to Bt crops reduces their benefit. Understanding the genetic basis of such resistance is needed to better monitor, manage, and counter pest resistance to Bt crops. An ARS researcher in Maricopa, Arizona, and collaborators at the University of Arizona used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to introduce mutations in the adenosine triphosphate (ATP)-binding cassette protein ABCA2 gene in a susceptible strain of pink bollworm, which resulted in resistance to Cry2Ab, a Bt protein produced in transgenic cotton. Overall, 26 different disruptive mutations were found, which, together with previous results, provide the first case of practical resistance to Cry2Ab where evidence identifies a specific gene in which disruptive mutations can cause resistance and are associated with resistance in field-selected populations. This study not only confirms the genetic basis of resistance to Cry2Ab in pink bollworm, but also shows that CRISPR/Cas9 gene editing is useful for the direct functional validation of genes involved in pest resistance to Bt transgenic crops. 3. Valuation of biological control in the Asia-Pacific region. Biological control of insect pests has immense economic value in many agricultural systems throughout the world. Still, this value is under appreciated by many involved in spurring innovation in and adoption of biological control research and technology. An ARS researcher in Maricopa, Arizona, in collaboration with researchers from the Chinese Academy of Sciences, Fuijan Agricultural/Forestry University, and Zhejiang University, in China, University of Queensland, Australia, and the Centre for Agriculture and Bioscience International, in the United Kingdom, estimated the economic impact of classical (introductory) biological control against 43 insect pests in food, feed, and fiber crops in the Asia-Pacific region of the world at $17.1-22.7 billion U.S. dollars, annually. In addition, biological control was shown to promote rural growth and prosperity even in marginal, poorly endowed, non-rice environments. This research provides lessons for future efforts to mitigate invasive species, restore ecological resilience, and sustainably increase the output of global food systems. 4. A desert-adapted insect pest trap crop for the cotton agroecosystem. Cotton is vulnerable to a wide array of insect pests. However, pest damage to cotton can be reduced by planting a more attractive host plant adjacent to cotton (a companion trap crop). An ARS researcher in Maricopa, Arizona, showed that vernonia, a desert-adapted plant, is strongly attractive to cotton pests and harbors an abundance of natural enemies. Further, very few arthropods were captured beyond the vernonia trap crop after being tracked using a protein immunomarking technique. The arthropods’ strong attraction and fidelity to vernonia indicate that it could serve as a trap crop for the cotton pest complex and as a refuge for natural enemies. 5. Development of a new method to study predation on all the life stages of a major cotton pest. Biological control of insect pests has immense economic value in many agricultural systems, but knowledge is limited about which predator species are most important for a given pest. ARS researchers in Maricopa, Arizona, developed a novel method for studying predation on the various life stages of Lygus hesperus, a major pest of cotton and other crops. The method tagged Lygus eggs, immatures, and adults with unique proteins that were subsequently detectable in predator guts. They found that big-eyed bugs and spiders were not only the numerically dominant predator taxa in the cotton field but most frequently preyed on immature lygus. Results also showed that collops beetles and fire ants are adept at preying on the cryptic eggs, and that adult lygus, albeit at low frequencies, engaged in cannibalism on immatures. The methods described may have value in the study of predator-prey dynamics in many agroecosystems. 6. Use of artificial attractants to improve biological control in cotton. Biological control by naturally occurring predators and parasitoids is a key component of existing management programs in cotton. Plants can respond to herbivore damage by releasing volatile compounds that are attractive to a variety of natural enemies, but how these can be used to improve biological control is not well understood. ARS researchers in Maricopa, Arizona, showed that deployment of a commercially available compound (methyl salicylate) produced by damaged plants in large replicated plots failed to increase natural enemy populations, repel pest species, or improve predator to prey ratios indicative of enhance biological control in cotton. This finding was both consistent and inconsistent with similar studies in other cropping system and will be useful to pest management professionals making decisions on how to enhance management of natural enemies in cropping systems. 7. Diapause provides short and long-term benefits for a key insect pest. Lygus hesperus, a major cotton pest, survives winter conditions by entering diapause, a dormancy that limits reproductive development. Once environmental conditions are favorable, the bugs become reproductively active. An ARS researcher in Maricopa, Arizona, examined whether there are consequences for diverting resources towards diapause rather than normal development. Females that had gone through diapause were larger, had greater stores of fat, were able to mobilize the resources necessary for egg production faster than non-diapausers, and lived longer. Overall, the results indicate that short-term diapause does not have a negative impact. The resulting extra stored resources accrued may allow females to quickly take advantage of improved environmental conditions and may prolong life by shielding them against stressors. Disruption of diapause regulatory processes may render Lygus more susceptible to those stressors. **Review Publications**
                            *Tabashnik, B.E., Liesner, L.R., Ellsworth, P.C., Unnithan, G.C., Fabrick, J.A., Naranjo, S.E., Li, X., Dennehy, T.J., Antilla, L., Staten, R.T., Carriere, Y. 2021. Transgenic cotton and sterile insect releases synergize eradication of pink bollworm a century after it invaded the United States. Proceedings of the National Academy of Sciences(PNAS). 118(1). Article e2019115118. https://doi.org/10.1073/pnas.2019115118.*
                                *Brent, C.S. 2021. Diapause termination and post-diapause in lygus hesperus (heteroptera: miridae). Journal of Insect Science. 21(1). Article 4. https://doi.org/10.1093/jisesa/ieaa142.*
                                *Naranjo, S.E., Hagler, J.R., Byers, J. 2021. Methyl salicylate fails to enhance arthropod predator abundance or predator to pest ratios in cotton. Environmental Entomology. 50(2):293-305. https://doi.org/10.1093/ee/nvaa175.*
                                *Hagler, J.R., Casey, M.T., Hull, A.M., Machtley, S.A. 2020. A molecular approach for detecting stage-specific predation on Lygus hesperus. Journal of Insect Science. 20(6). Article 35. https://doi.org/10.1093/jisesa/ieaa136.*
                                *Hagler, J.R., Thompson, A.L., Machtley, S.A., Miles, C.T. 2021. Arthropod demography, distribution, and dispersion in a novel trap-cropped cotton agroecosystem. Journal of Insect Science. 21(1). Article 20. https://doi.org/10.1093/jisesa/ieab010.*
                                *Bordini, I., Naranjo, S.E., Fournier, A., Ellsworth, P. 2020. Novel insecticides and generalist predators support conservation biological control in cotton. Biological Control. 154. Article 104502. https://doi.org/10.1016/j.biocontrol.2020.104502.*
                                *Fabrick, J.A., Leroy, D.M., Mathew, L.G., Wu, Y., Unnithan, G.C., Yelich, A., Carriere, Y., Li, X., Tabashnik, B.E. 2021. CRISPR-mediated mutations in the ABC transporter gene ABCA2 confer pink bollworm resistance to Bt toxin Cry2Ab. Scientific Reports. 11. Article 10377. https://doi.org/10.1038/s41598-021-89771-7.*
                                *Guan, F., Hou, B., Dai, X., Liu, S., Liu, J., Gu, Y., Yang, Y., Fabrick, J.A., Wu, Y. 2021. Multiple origins of a single point mutation in the cotton bollworm tetraspanin gene confers dominant resistance to Bt cotton. Pest Management Science. 77(3):1169-1177. https://doi.org/10.1002/ps.6192.*
                                *Wyckhuys, K., Lu, Y., Zhou, W., Cock, M., Furlong, M., Naranjo, S.E. 2020. Ecological pest control fortifies agricultural growth in Asia-Pacific economies. Nature Ecology and Evolution. 4:1552-1530. https://doi.org/10.1038/s41559-020-01294-y.*
                                *Jin, H., Abouzaid, M., Lin, Y., Hull, J.J., Ma, W. 2021. Cloning and RNAi-mediated three lethal genes that can be potentially used for Chilo suppressalis (Lepidoptera: Crambidae) management. Pesticide Biochemistry and Physiology. 174. Article 104828. https://doi.org/10.1016/j.pestbp.2021.104828.*
                                *Naranjo S.E. 2014. Effects of GM crops on non-target organisms. In: Ricroch A., Chopra S., Fleischer S., editors. Plant Biotechnology. Springer, Cham. p. 129-142. https://doi.org/10.1007/978-3-319-06892-3_11.*
                                *Fleischer S.J., Hutchison W.D., Naranjo S.E. 2014. Sustainable management of insect-resistant crops. In: Ricroch A., Chopra S., Fleischer S., editors. Plant Biotechnology. Springer, Cham. p. 115-127. https://doi.org/10.1007/978-3-319-06892-3_10*
                                *Hull, J.J., Gross, R.J., Brent, C.S., Christie, A.E. 2021. Filling in the gaps: A reevaluation of the Lygus hesperus peptidome using an expanded de novo assembled transcriptome and molecular cloning. General and Comparative Endocrinology. 303. Article 113708. https://doi.org/10.1016/j.ygcen.2020.113708.*


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