Research Project: Sustainable Pest Management for Arid-Land Agroecosystems

Location: Pest Management and Biocontrol Research

2023 Annual Report

Objectives
Objective 1: Investigate the behavior, biology, demography and ecology of the major pests, and their natural enemies, of cotton and other western U.S. crops, with emphasis on pest movement, feeding, ecology, and conservation of natural enemies. Sub-objective 1A: Develop biological control-informed thresholds for L. hesperus in cotton (Naranjo, Vacant Entomologist) Sub-objective 1B: Characterize the demographics and dispersal patterns of B. tabaci and L. hesperus, natural enemies, and pollinators in a cotton field embedded with push and pull companion plants (Fabrick, Hagler, Vacant Entomologist) Sub-objective 1C: Identify arthropod demography and life stage-specific predation on L. hesperus inhabiting desert-adapted cotton breeding lines (Hagler, Vacant Entomologist) Sub-objective 1D: Test the efficacy on CSB of insecticides typically used in cotton pest management systems. (Brent, Vacant Entomologist) [NP304, C3, PS3A, 3B, and 3C] Objective 2: Examine non-target effects of new GE crops and determine efficacy and non-target effects of insecticidal seed treatments. Sub-objective 2A: Assess effects of Lygus-active Bt cotton on the pests L. hesperus and B. tabaci, and on the natural enemy community and its biological control function (Naranjo, Vacant Entomologist) Sub-objective 2B: Determine the contribution of F. occidentalis on B. tabaci control and the impact of insecticidal seed treatments on the natural enemy community associated with B. tabaci and L. hesperus in cotton (Naranjo, Vacant Entomologist) Objective 3: Investigate the physiology, biochemistry, and molecular biology of major pests of cotton and other arid land crops to develop new and improve existing management approaches such as those based on gene silencing or editing. Sub-objective 3A: Evaluate oral RNAi in L. hesperus (Brent, Fabrick, Hull) Sub-objective 3B: Identify and functionally characterize sex determination genes in L. hesperus (Brent, Fabrick, Hull) Sub-objective 3C: Develop and use CRISPR/Cas gene editing to create gene knockouts in L. hesperus (Brent, Fabrick, Hull) Sub-objective 3D: Identify Bt resistance mechanisms and fitness costs in the lepidopteran cotton pests, Pectinophora gossypiella and Helicoverpa zea (Fabrick, Hull, Naranjo) Sub-objective 3E: Develop tools for the genetic-based manipulation of CSB development for future use in precision-guided biorational pest management. [NP304, C3, PS3A, 3B, and 3C]

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
This report documents fiscal year (FY) 2023 progress for project 2020-22620-023-000D, “Sustainable Pest Management for Arid-Land Agroecosystems”, which began in July 2020. Under Sub-objective 1Bi, ARS researchers in Maricopa, Arizona, conducted Y-tube olfactometer bioassays to measure lygus bug responses to the odor of various plants to identify which species attract or repel major cotton pests. The goal is to find plants that can be placed adjacent to cotton (companion plants) that attract (lure) the pests away from cotton. Ultimately, companion planting practices could reduce pesticide use in cotton. An additional host plant test was initiated to examine lygus response to stinknet, an aggressive invasive weed. There is anecdotal evidence that this weed might serve as an overwintering host for lygus and its natural enemies. For Sub-objective 1Bii, ARS researchers developed a large-scale feeding choice arena to measure whitefly host plant preference and dispersal behavior in a greenhouse containing cotton (the target agronomic crop), vernonia (a preferred host plant), and basil (a non-preferred host plant). The three plant types were strategically placed within the greenhouse to simulate a real-world cropping system. The study showed that marked whiteflies released in the greenhouse's center preferentially dispersed to vernonia, suggesting that the plant has potential as a cotton companion crop. A similar study has been initiated testing the host plant preferences of lygus. If found to lure both species away from cotton, such a system could reduce pesticide usage. Under Sub-objective 1D, the invasive cotton seed bug (CSB) has been tested for its susceptibility to twelve contact insecticides typically used in a cotton cropping systems, many of which are effective against other hemipterans. The standard petri dish assay technique was adapted for the test, and three experimental replicates have been completed. For each insecticide and dosage (0, 1, 10, 100, and 1000 parts per million), five petri dishes, each containing five adult CSB, were tested for the effect on insect mortality. Several insecticides were identified that are very effective even at low dosages. The most promising five candidate insecticides will be tested now under lab conditions with open cotton bolls that have been sprayed. This will simulate more natural conditions prior to actual in situ testing. In support of Objective 2, the examination of potential effects of new genetically engineered crops and insecticidal seed treatments on non-target insect species, has now been fully completed, with all collected data published. Under Sub-objective 3A, the efficacy of oral RNA interference (RNAi) in Lygus hesperus was found to be limited by two main factors, extra-oral enzymatic degradation of dsRNAs and limited uptake of dsRNAs by the midgut. Although the degradative effects of salivary gland nucleases can be bypassed via administering dsRNAs in sugar water, those dsRNAs fail to undergo uptake by midgut cells. Midgut nuclease activity, however, had limited effects on dsRNA stability and searches of Lygus transcriptomic data has consistently failed to identify a quality candidate for SID-1, a channel protein critical in cellular uptake of dsRNAs. Delivery of dsRNAs in conjunction with various nanoparticles that enhanced dsRNA stability and uptake in other systems also failed to promote consistent uptake or target transcript knockdown. Topical application of dsRNA-linked nanoparticles mixed with various surfactants likewise was ineffective at producing an RNAi effect. Similarly, injection of dsRNAs into gravid females as a means of inducing RNAi in oocytes was ineffective. The only viable approach appears to be injection, limiting the utility of RNAi to studies of gene function rather than control applications. For Sub-objective 3B, Lygus hesperus transcriptomic datasets were searched for potential homologs of sex determination genes structurally and functionally conserved in model insects (e.g., fruitfly, red flour beetle, silkmoth). Searches have identified 20 of the 24 genes that comprised the initial search set and include early sex determination genes (sex lethal, transformer 2, doublesex, and fruitless) as well as important downstream genes such as intersex. These five key genes have been cloned, sequence validated, sex-specific expression assessed. Studies to determine the developmental effects of gene knockdown have been initiated. Under Sub-objective 3C, ARS researchers validated the viability of applying clustered regularly interspaced short palindromic repeats CRISPR/Cas9-mediated gene editing to study gene function in Lygus. Cardinal and cinnabar, two genes previously shown to play a role in Lygus eye pigmentation, were targeted by designing single guide RNA (sgRNA) for each. This was injected, along with one of two Cas9 enzymes, into newly laid Lygus eggs. Clear phenotypic effects were observed in developing embryos with experimental eggs exhibiting pink eyes, whereas control eggs were characterized by brown eyes. Atypical coloration in the experimental groups largely persisted throughout the nymphal stages and into early adult development, which is clear evidence of a persistent loss of gene function. Under Sub-objective 3Di, experiments using CRISPR/Cas9 gene editing to modify the ATP-binding cassette gene ATP-binding cassette sub-family A member 2 (ABCA2) in a susceptible strains of pink bollworm and corn earworm have been completed and the work has been published. The work demonstrated the power of using CRISPR/Cas9 gene editing for validating the in vivo function of genes involved in resistance to Bacillus thuringiensis (Bt) transgenic crops and showed that ABCA2 is a functional receptor of Cry2Ab in two important agricultural insect pests. For Sub-objective 3Dii, cellular localization of the wild-type pink bollworm cadherin (PgCad1_s) transmembrane protein and four mutant cadherins (PgCad1_r1, r2, r3, and r4) was determined and immunofluorescence methods were optimized and used to screen cell lines for cross-reactivity to PgCad1 antibodies. Plasmids corresponding to each of the five PgCad1-Venus fusion proteins and mCherry-labeled cellular organelle marker proteins were constructed and sequence-verified. Whereas wild-type PgCad1_s-Venus localized primarily within the plasma membrane of stably transformed Tni cells, the four mutant cadherins were retained intracellularly within the endoplasmic reticulum. These results indicate that the cadherin mutations alter the cellular trafficking of the known Cry1Ac midgut receptor, thereby contributing to functional resistance. To confirm this within actual pink bollworm midgut tissue, fixation and embedding methods that are compatible with immunostaining with custom anti-PgCad1 antibodies is required. Hence, cell lines expressing each of the five untagged cadherin proteins were provided to a University of Arizona collaborator, who tested numerous methods of cell fixation and embedding. Five different custom-made, anti-PgCad1 antibodies were tested against cultured cells expressing wild-type PgCad1_s under various fixation/embedding conditions. At least one PgCad1 antibody and a fixation/embedding method has been identified for future immunohistochemistry experiments with Cry1Ac-resistant pink bollworm strains harboring mutant PgCad1 alleles. In support of Sub-objective 3Diii, the studies to determine if toxin resistance alters the flight capability or propensity of the corn earworm were delayed primarily to allow for the rebuild of a new, improved flight mill apparatus. Specifically, research published by ARS scientists highlight the efficiency and ease of use for this newly built flight monitoring system. Diet bioassay experiments and flight mill tests to compare GA with GA-R and GZ with GZ-R corn earworm strains will be initiated in FY24.

Accomplishments
1. Increase in global resistance to genetically engineered Bacillus thuringiensis (Bt) crops. Transgenic plants producing insecticidal proteins from Bacillus thuringiensis (Bt) are grown widely to control pests, but evolution of insect resistance has reduced the efficacy of Bt crops. An ARS scientist at Maricopa, Arizona, and collaborators at the University of Arizona, analyzed global resistance monitoring data for the first 25 years of cultivation of Bt crops including corn, cotton, soybean, and sugarcane. A total of 73 cases were examined including 24 pest species from 12 countries with cases of practical resistance arising from three in 2005 to 26 in 2020. Practical resistance was documented in some populations of 11 pest species, collectively affecting nine widely used crystalline (Cry) Bt toxins in seven countries. Conversely, 30 cases reflect no decrease in susceptibility to Bt crops in populations of 15 pest species in nine countries. The remaining 17 cases provide early warnings of resistance, which entail genetically-based decreases in susceptibility without evidence of reduced efficacy. Numerous factors that can lead to sustained susceptibility to Bt crops were reviewed, providing improvements for the sustainability of current and future transgenic insecticidal crops.

2. CRISPR/Cas9 gene editing feasible in Lygus. CRISPR/Cas9-mediated gene editing is the foremost tool available for functional genomics approaches and targeted genomic engineering applications; however, the technique is species-specific and often requires significant optimization, especially in non-model organisms such as Lygus hesperus. The applicability of the CRISPR/Cas9 system for this species was successfully demonstrated by a team of ARS researchers in Maricopa, Arizona. Eggs injected with the Cas9 enzyme and sgRNAs targeting either cardinal or cinnabar, two eye pigmentation genes, developed red eyes instead of brown eyes typical of wildtypes. Although atypical coloration in both experimental groups largely persisted throughout the nymphal stages, adult manifestation of the phenotype was limited to the cinnabar group, and atypical eye coloration was associated with disruptions in the sequence of the respective target genes. The heritability and persistence of the red eye phenotype across multiple generations demonstrated that clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can be applied to L. hesperus and that eye pigmentation genes are useful for tracking genetic manipulation.

3. Gene editing identifies a key receptor gene of pest resistance to transgenic Bacillus thuringiensis (Bt) crops. The corn earworm, Helicoverpa zea, is a major New World pest of many crops that has evolved resistance to transgenic corn and cotton producing different Bt protein toxins. Understanding of 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, showed that mutations introduced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing in the corn earworm gene encoding an ATP-binding cassette protein (HzABCA2) causes resistance to the Cry2Ab Bt toxin. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains in which all resistant individuals tested had disruptive mutations in HzABCA2. The results demonstrate that HzABCA2 mutations can cause high levels of resistance to Cry2Ab, and that this gene is a leading candidate for monitoring corn earworm for Cry2Ab resistance in the field.

4. Complex molecular basis of lab- and field-selected pink bollworm resistance to transgenic Bacillus thuringiensis (Bt) cotton. Cotton has been genetically engineered to produce insect-killing proteins from the bacterium Bacillus thuringiensis (Bt) to control major pests that include the pink bollworm, one of the most damaging pests of cotton world-wide. Genetically engineered Bt crops have many advantages, including enhanced pest suppression, improved yields, increased farmer profits, and decreased use of conventional insecticides thereby benefiting the environment and human health; however, these benefits are reduced when pests evolve resistance to Bt crops. The pink bollworm rapidly evolved resistance to Bt cotton in India, but not in China or the United States where this invasive pest was eradicated using Bt cotton, sterile moth releases, and other tactics. An ARS researcher from Maricopa, Arizona, and collaborators from the University of Arizona, compared the genetic profiles of Bt-resistant pink bollworm taken from lab-selected strains from the United States and China and field-selected populations from India. Testing showed the genetic basis of resistance was similar across countries and between resistance selected in the lab versus the field suggesting that lab selection can be useful for identifying genes likely to be important in field-evolved resistance to Bt crops. Likely, differences in management practices among countries caused the different outcomes.

5. Development of a method to track insect dispersal. Tracking dispersal patterns in agroecosystems is essential for efficiently managing arthropod pests and conserving natural enemies and pollinators. An ARS researcher at Maricopa, Arizona, developed and refined a method to tag key cotton pests (lygus and whitefly), natural enemies, and pollinators with a fluorophore that glows brightly under ultraviolet light. The key features of this novel marking procedure are that it is inexpensive, persistent, easily detected, does not affect insect behavior, and works equally well on large and small insects (e.g., whiteflies and parasitoids). The novel fluorophore marking method will expedite future dispersal research on whiteflies and various other pest and beneficial arthropod species.

6. Monitoring lygus bug and natural enemy movement in trap-cropped strawberry fields. The lygus bug is the major strawberry pest, but prefer to feed on alfalfa and many types of native weeds. As such, intercropping strips of alfalfa (trap cropping) within strawberry production fields and allowing weeds to flower adjacent to the fields can serve as a sink for both lygus and its natural enemies. An ARS scientist at Maricopa, Arizona, teamed up with University of California scientists to study the population dynamics and dispersal characteristics of lygus and its associated predator complex in organically grown strawberry fields embedded with strips of alfalfa and surrounded by weedy vegetation. The research showed that the lygus population was up to 2.5 times higher in alfalfa and that most marked lygus and predators that emigrated from senescing weeds were recovered from alfalfa rather than strawberry. Moreover, the marked predator-to-lygus ratio found in alfalfa compared to strawberries was 5:1. Trap cropping effectively reduced the infestation of lygus in strawberries demonstrating that converting weedy areas to native perennial plantings could mitigate the risk of pest migration while conserving beneficial insects.

Review Publications
Hull, J.J., Brent, C.S., Fu, T., Wang, G., Christie, A.E. 2022. Mining Lygus hesperus (western tarnished plant bug) transcriptomic data for transient receptor potential channels: Expression profiling and functional characterization of a painless homolog. Comparative Biochemistry and Physiology. 44. Article 101027. https://doi.org/10.1016/j.cbd.2022.101027.
Guo, H., Liu, X., Long, G., Gong, L., Zhang, M., Ma, Y., Hull, J.J., Dewer, Y., He, M., He, P. 2022. Functional characterization of developmentally critical genes in the white-backed planthopper: Efficacy of nanoparticle-based dsRNA sprays for pest control. Pest Management Science. 79(3):1048-1061. https://doi.org/10.1002/ps.7271.
Fabrick, J.A., Heu, C.C., LeRoy, D.M., DeGain, B.A., Yelich, A.J., Unnithan, G.C., Wu, Y., Li, X., Carriere, Y., Tabashnik, B.E. 2022. Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea. Scientific Reports. 12. Article 16706. https://doi.org/10.1038/s41598-022-21061-2.
Tabashnik, B.E., Fabrick, J.A., Carriere, Y. 2023. Global patterns of insect resistance to transgenic Bt crops: The first 25 years. Journal of Economic Entomology. 116(2):297-309. https://doi.org/10.1093/jee/toac183.
Stahlke, A.R., Chang, J., Chudalayandi, S., Heu, C.C., Geib, S.M., Scheffler, B.E., Childers, A.K., Fabrick, J.A. 2023. Chromosome-scale genome assembly of the pink bollworm, Pectinophora gossypiella, a global pest of cotton. G3, Genes/Genomes/Genetics. 13(4). Article jkad040. https://doi.org/10.1093/g3journal/jkad040.
Fabrick, J.A., Li, X., Carriere, Y., Tabashnik, B. 2023. Molecular genetic basis of lab- and field-selected Bt resistance in pink bollworm. Insects. 14(2). Article 201. https://doi.org/10.3390/insects14020201.
Guo, H., Long, G., Liu, X., Ma, Y., Zhang, M., Gong, L., Dewer, Y., Hull, J.J., Wang, M., Wang, Q., He, M., He, P. 2023. Functional characterization of tyrosine melanin genes in the white-backed planthopper and utilization of a spray-based nanoparticle-wrapped dsRNA technique for pest control. International Journal of Biological Macromolecules. 230. Article 123123. https://doi.org/10.1016/j.ijbiomac.2022.123123.
Tabashnik, B.E., Carriere, Y., Wu, Y., Fabrick, J.A. 2023. Global perspectives on field-evolved resistance to transgenic Bt crops: A special collection. Journal of Economic Entomology. 116(2):269-274. https://doi.org/10.1093/jee/toad054.
Wang, M., Wang, J., Yasen, A., Fan, B., Hull, J.J., Shen, X. 2023. Determination of key components in the Bombyx mori p53 apoptosis regulation network using Y2H-Seq. Insects. 14(4). Article 362. https://doi.org/10.3390/insects14040362.
Weaver, M.A., Callicott, K.A., Mehl, H.L., Opoku, J., Park, L.C., Fields, K., Mandel, J.R. 2022. Characterization of the Aspergillus flavus population from highly aflatoxin-contaminated corn in the United States. Toxins. 14(11). Article 755. https://doi.org/10.3390/toxins14110755.
Ma, Y., Gong, L., Zhang, M., Liu, X., Guo, H., Hull, J.J., Long, G., Wang, H., Dewer, Y., Zhang, F., He, M., He, P. 2023. Two antennae-enriched carboxylesterases mediate olfactory responses and degradation of ester volatiles in the German cockroach Blattella germanica. Journal of Agricultural and Food Chemistry. 71(12):4789-4801. https://doi.org/10.1021/acs.jafc.2c08488.
Teng, H., Zuo, Y., Yuan, J., Fabrick, J.A., Wu, Y., Yang, Y. 2022. High frequency of ryanodine receptor and cytochrome P450 CYP9A186 mutations in insecticide-resistant field populations of Spodoptera exigua from China. Pesticide Biochemistry and Physiology. 186. Article 105153. https://doi.org/10.1016/j.pestbp.2022.105153.
Tabashnik, B.E., Unnithan, G.C., Yelich, A., Fabrick, J.A., Dennehy, T.J., Carriere, Y. 2022. Responses to Bt toxin Vip3Aa by pink bollworm larvae resistant or susceptible to Cry toxins. Pest Management Science. 78(10):3973-3979. https://doi.org/10.1002/ps.7016.
Bordini, I., Naranjo, S.E., Fournier, A., Ellsworth, P. 2023. Spatial scale of non-target effects of cotton insecticides. PLOS ONE. 18(5). Article e0272831. https://doi.org/10.1371/journal.pone.0272831.
Naranjo, S.E., Canas, L., Ellsworth, P.C. 2022. Mortality dynamics of a polyphagous invasive herbivore reveal clues in its agroecosystem success. Pest Management Science. 78(10):3988-4055. https://doi.org/10.1002/ps.7018.
Liu, X., Long, G., Guo, H., Ma, Y., Gong, L., Zhang, M., Hull, J.J., Dewer, Y., Liu, L., He, M., He, P. 2023. Functional characterization of five developmental signaling network genes in the white-backed planthopper: Potential application for pest management. Pest Management Science. 79(8):2869-2881. https://doi.org/10.1002/ps.7464.