Location: Pest Management and Biocontrol Research2020 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 through 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.
This is the final report for project 2020-22620-022-00D "Ecologically Based Pest Management in Western Crops Such as Cotton", which has been replaced by new project 2020-22620-023-00D "Sustainable Pest Managment Arid-Land Agroecosystems". Under Sub-objective 1A, field studies using trapping grids, in-field sampling and protein markers showed that Lygus and western flower thrips colonize cotton from surrounding cotton and alfalfa and provide evidence that a natural refuge strategy may function to manage resistance to Bacillus thuringiensis (Bt) cottons with activity against Lygus and thrips. Data was used to support an EPA registration package and further data analyses and manuscript preparation are underway. For Sub-objective 1B, literature searches for non-target arthropod field studies in Bt crops (cotton, eggplant, rice, potato) were conducted and entered into a database that already includes Bt maize. Meta-analyses have been conducted on Bt maize studies to predict risks to non-target arthropods and a manuscript is in preparation. Under Sub-objective 1C, a molecular gut analysis procedure was developed. This universal food immunomarking technique (UFIT) facilitates study of arthropod carnivory, herbivory, and scavenging. Prey tagged with a foreign protein marker are exposed to predators in field cages. Predators are subsequently analyzed for marked prey remnants using an enzyme linked immunosorbent assay. UFIT use in several studies pinpointed life stage-specific predation events on members of the cotton arthropod complex, identified collops beetles and ants as effective predator of Lygus eggs, and detected intraguild predation and cannibalism among lacewings. Immunomarking was also used to investigate the efficacy of trap crops in controlling strawberry and cotton pests. Alfalfa was found to be a highly effective at retaining Lygus, while vernonia was found to be an effective trap when paired with cotton. Importantly, the pest and predator bug complex was retained in vernonia throughout the growing season, and pesticide application wasn’t needed. Manusripts have been published. For Sub-objective 1D, large plot field studies are being used to identify non-target impacts on natural enemy species of a new Bt cotton formulated to target Lygus bugs and thrips. Sampling includes sweep nets for a large community of arthropod predators, and plant washes and sticky traps for omnivorous thrips. Whiteflies are being used as sentinel prey in life table studies to measure natural enemy induced mortality and how it might be affected by the new Bt cotton. Two years of replicated field plot studies were also conducted to examine the non-target impacts of new insecticides on arthropod predators in cotton and to examine the effect of plot size on non-target assessment. Results indicate several new insecticides are highly selective and provide options for growers to control whitefly while preserving natural enemy species. Results also suggest that medium size plots may accurately measure non-target effects for most species. The effects of insecticides and plot size on biological control function are being analyzed and a manuscript is underway. Under Sub-objective 2A, studies were conducted to examine the effect of sublethal doses of two commonly used insecticides on the biology and flight behavior of Chloridea virescens. Results demonstrated effects on larval and pupal development and adult reproduction that could potentially disrupt synchronous mating between susceptible moths from non-Bt refuge fields and putatively resistant moths from Bt fields. Flight behavior assays using automated mills suggests that sublethal doses may affect male moth flight but not to such a degree that they would be unable to disperse from non-Bt fields and mate with potentially Bt resistant moths over a maximum distance of 800m. These studies have been published. For Sub-Objective 2B, complementation tests examined if resistance loci are shared in two Cry2Ab-resistant strains of pink bollworm. Genetic crosses between susceptible and resistant strains confirm that resistance to Cry2Ab toxin is 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. Molecular analysis confirmed that two resistant US laboratory strains and one from India all harbor mutations that disrupt the ATP-binding cassette transporter A2 (PgABCA2) gene. Results from inter-strain crosses also indicate that one US lab strain has at least one additional mechanism of resistance to Cry2Ab that does not involve PgABCA2 and is not completely recessive or autosomal. These studies have been published. Under Sub-objective 2C, functionality assays were conducted to determine if pink bollworm cadherin and ATP-binding cassette transporter C (ABCC) proteins were Bt Cry-toxin receptors based on binding and cytotoxicity of Cry1Ac and Cry2Ab. Insect cell lines producing several mutant forms of the pink bollworm midgut cadherin fail to translocate to the cell surface, suggesting a new mechanism of Bt resistance that is due to lack of receptor availability and not to direct loss of toxin binding. Evidence was also found supporting the hypothesis that cadherin is a functional receptor of Cry1Ac Bt toxin, with down-regulation of cadherin increasing resistance. No evidence was found that the ABCC transporter is involved with Bt mode of action. Studies have been published. Sub-objectives 3A-C were eliminated. Under Sub-objective 3D, assays found that when Lygus males transfer the antiaphrodisiac myristyl acetate (MA) into females while mating, they also deliver geranyl-geranyl acetate, which female convert to geranyl geraniol. This alcohol counteracts MA, serving as an anti-antiaphrodisiac. Tests to determine if MA could be used to control Lygus populations found that while direct application of MA to a female limits male sexual interest, just having MA in proximity to a female does not affect male behavior regardless of dosage or whether tests are conducted in enclosed or more field-like conditions. MA also degrades within hours under field conditions. Collectively the results suggest that MA is not suitable for population control via field sprays. Manuscripts have been published. Under Sub-objectives 4A & 4B, the development of Lygus embryos, nymphs and adult was examined under low, moderate or high temperatures with constant or variable regimes. Development rates increased with temperature. At high temperatures, development in all stages was more rapid under constant conditions, while at low temperatures development was faster under variable conditions. Relative to females under moderate temperatures, those under warmer conditions lay eggs sooner and faster but die earlier and produce fewer eggs overall. Females raised under cooler conditions produce eggs later and more slowly and live longer and have greater lifetime fecundities. Studies have been published. Under Sub-objective 4C, the effects of temperature, photoperiod and age on the diapause response of Lygus were studied. While temperature had little effect on diapause induction, a graded response was evident in nymphs of different instars. Exposure to short days had to occur prior to the 5th instar to induce diapause. Diapause rarely occurred when nymphs were switched from short to long days regardless of their age at the switch. Adults reared under short day conditions and switched to long days initiated reproductive development faster than adults reared under long day conditions. A search for molecular markers of diapause in Lygus identified only one protein, likely an egg yolk component. Absence of this protein in males precludes its use as a useful marker of Lygus diapause status. An alternative method for nondestructively identifying Lygus adults in diapause using body dimensions was developed and validated. Host-free survival patterns of adult Lygus classed using the new method indicated diapausing Lygus held without food at a relatively high temperature survived up to 7 times longer than non-diapausers. A manuscript is being published. Under Sub-objective 5A, Lygus hesperus transcriptomic data sets were generated under various thermal conditions and used to identify multiple stress-related genes. While numerous genes exhibit tissue-specific expression differences, the number of thermal-responsive genes remains ambiguous as results vary between transcriptomic datasets and PCR analyses. As validation of individual gene responses progresses, the transcriptomes have been used for comprehensive data mining and expression profiling, with two manuscripts currently in preparation and additional manuscripts expected depending on mining results. Under Sub-objective 5B, exploration of gene silencing methods in Lygus hesperus via dsRNA-mediated RNA interference mechanisms found that orally delivered dsRNAs is ineffective. Extraoral degradation of the dsRNA does not appear to be the main impediment, rather the barrier appears related to the poor persistence and uptake of the ingested dsRNAs in the gut. In contrast, injection of dsRNAs that target genes of interest was readily optimized and used to assess the in vivo functionality of genes implicated in noxious odor perception, egg laying, and desiccation tolerance. Studies have been published. For Sub-objective 5C, the potential of the marigold, Tagetes patula, to disrupt the osmotic water permeability of whitefly aquaporin water channel proteins was examined. On-plant cage tests and diet incorporation assays revealed that foliar extracts have insecticidal activity against both whitefly and Lygus which could be developed as novel alternatives to conventional chemical treatments. This study has been published. Marigold extract activity against Tni cell lines producing whitefly or Lygus aquaporins was not tested because Tni cell transformation requires further optimization.
1. Sublethal insecticide effects on Bt soybean refuge strategy. High dose and deployment of a non-Bt (Bacillus thuringiensis) crop refuge are the key strategies for delaying resistance to the Bt proteins in target pests. However, insecticide sprays are often needed in the non-Bt refuge to manage the target and other pests. A collaborative study between ARS scientists from Maricopa, and from the University of Sao Paulo, Brazil showed that sublethal doses of two common insecticides used in Brazilian soybean systems increased larval and pupal development times and reduced fecundity of a key soybean pest but only subtly affected the flight behavior of male moths. These biological effects could lead to asynchronous generations and the production of an insufficient number of susceptible moths coming out of refuge fields but should not alter the ability of moths to disperse between Bt and non-Bt fields. Results reinforce the value of using high dosage treatment and are of value to growers and regulators interested in sustaining the durability of this pest control technology.
2. Selective control of cotton pests facilitates biological control. A fundamental component of integrated pest management (IPM) is maximizing biological control from natural enemies such as arthropod predators. This conservation is predicated on the use of insecticides that selectively kill the pests while leaving the natural enemies unharmed. Collaborative field studies between ARS scientists in Maricopa, Arizona, and the University of Arizona showed that four new insecticides registered for use in cotton selectively killed whiteflies and Lygus bugs but did not harm predatory arthropod communities. The favorable ratio of the abundance of these conserved predators relative to whitefly pests (predator to prey ratios) in cotton field enabled further biological control of the pest. Results allow cotton growers to continue managing pest populations in the most efficient way possible while having additional selective insecticide options that will facilitate biological pest control.
3. Biological control of arthropod pests has large economic value. Biological control is an integral tactic of modern integrated pest management (IPM) but we have only a rudimentary knowledge of its economic value. Collaborative research between ARS scientists in Maricopa, Arizona, and the University of Arizona synthesized available economic data on projects targeting arthropod pests and found that introductory biological control of exotic pests is valued at $37.3M per project with an average benefit to cost ratio of 61:1. The augmentation biological control industry is valued at $1.7 billion and is growing at 15% annually, while the value of conservation biological control is around $74/ha. Interaction among diverse scientists and stakeholders will be required to ultimately measure the inclusive benefits and costs of biological control. However, focus on gaining greater accuracy in measurement should be balanced with additional effort to educate both end-users and public institutions about the immense value of biological control in order to spur greater adoption, and investment in research and implementation.
4. Diapause induction in the western tarnished plant bug (Lygus) is primarily light-induced and comes at little cost. Management efforts focused on reducing overwintering populations of Lygus bugs have been attempted with little success, previously limited by an insufficient understanding of the diapause that facilitates overwinter survival. ARS researchers at Maricopa, Arizona, found that temperature had a limited impact on diapause induction compared to daylength, and the sensitive period for exposure to short days that induces diapause starts during an early stage of development, the third nymphal instar. Further, despite redirecting resources to enhance overwintering survival, diapausing Lygus exhibit no negative impact in their development, post-diapause lifespan or lifetime egg production. In fact, the extra internal resources stored during adult diapause allows Lygus females to begin producing eggs more rapidly than if they were just newly emerged adults, which provides an advantage over non-diapausing females when environmental conditions become more hospitable. These findings provide new insights into the survival strategies of Lygus bugs, enhance the target value of diapausing individuals, and will be crucial for researchers developing improved management tactics targeting the overwintering population.
5. Pink bollworm resistance to Bt cotton involves multiple genetic and biochemical mechanisms. Transgenic Bacillus thuringiensis (Bt) crops are critical for the control of important insect pests, but evolving resistance threatens their continued use. Pink bollworm is a global pest of cotton and multiple strains have developed resistance to Bt toxins. Findings by an ARS scientist from Maricopa, Arizona, and researchers from the University of Arizona, show that in pink bollworm the primary mechanism is evolved mutations in the receptors that enable Bt toxin binding to the insect midgut. These mutations involve changes to the genetic sequence as well as to how the genes are translated into proteins. Reduced receptor expression and improper localization of those proteins on cell surfaces also can impair Bt toxin efficacy. These findings show the notable adaptability of pink bollworm to evolve resistance to Bt cotton 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.
6. Lygus transcriptomic data yield numerous potential control targets. The generation of transcriptomic datasets from Lygus exposed for set periods of time to different thermal environments has provided ARS researchers at Maricopa, Arizona, a better understanding of the molecular and cellular processes activated by Lygus in response to stress conditions. The datasets have provided crucial insights into the molecular basis of Lygus biology in general as well as that of other non-model agriculture insect pests. Dataset mining in conjunction with concurrent efforts to develop gene knockdown methods in Lygus to assess in vivo gene functionality has allowed researchers to identify numerous candidate genes for targeted disruption. More than 60,000 sequences have been made available via publicly accessible data repositories, which will facilitate the work of researchers looking for molecular targets for species-specific disruption that would support Lygus population control efforts in cotton and other commodity crops for which they are pests.
7. RNA interference (RNAi) is effective in Lygus by injection but not ingestion of dsRNA. The ingestion of double-stranded RNAs to trigger RNAi-mediated knockdown of target genes has been promoted as a novel control strategy for agricultural pests such as Lygus. However, studies exploring RNAi methods in Lygus by ARS researchers at Maricopa, Arizona, have established that current methods for oral delivery of dsRNAs are ineffectual. Unlike other species, extraoral degradation of dsRNAs does account for the poor efficacy, indicating that the barrier is related to the persistence and uptake of the ingested dsRNAs. In contrast, injected dsRNAs have proven to be effective and have been used to assess the function of several candidate genes identified for targeted disruption. Negative physiological impacts were observed with the successful knockdown of genes involved in reproductive tissue development, desiccation tolerance, egg laying, and noxious odor detection. The results will facilitate developing highly targeted control approaches for Lygus using gene manipulation, which would make it possible to reduce pesticide application for cotton and other commodity crops for which they are pests.
8. Development of a novel method to study arthropod feeding activity. While insect predators can provide biological control of pest species, they can undermine their efficacy by also engaging in intraguild predation (IGP) on other predator species and cannibalism. An ARS researcher in Maricopa, Arizona, developed a method for examining both IGP and cannibalism on a well-known predator, the green lacewing. The method entailed tagging young (third instar) and old (fifth instar) lacewing larvae with rabbit IgG and chicken IgY proteins, respectively. The uniquely marked lacewings were then introduced into caged arenas containing a cotton plant and an assemblage of predators. The predators were recaptured, and their gut contents examined for the presence of rabbit IgG- and chicken IgY-marked lacewing remnants by protein-specific immunological assays. The predator gut analyses detected incidences of both IGP and cannibalism, showing this technique to be useful for a variety of life stage-specific predation events. This method can be broadly applied to regions across the country, allowing researchers to identify which predators make substantial contributions to biological control so that their populations can be promoted to enhance pest population suppression.
9. Alfalfa is an effective trap crop for strawberry fields. The Lygus bug is a major strawberry pest but prefer to feed on alfalfa. As such, embedding strips of alfalfa within large strawberry production fields can serve as a sink for both Lygus and its predaceous natural enemies. An ARS researcher in Maricopa, Arizona, examined the population dynamics and dispersal characteristics of the Lygus predator complex in strawberry fields embedded with one row of alfalfa for every 49 rows of strawberry. The data revealed that the minute pirate bug was the numerically dominant predator taxon, comprising 84% of the predator population. Predator movement from a centralized alfalfa trap crop row, using a protein mark-capture procedure, showed that most predators dispersed less than 2-m, indicating that the prey reservoir found in trap crops often produces a predator sink. This study suggests that alfalfa is a useful cultural (trap cropping) and biological (refuge for natural enemies) control tactic for managing Lygus in strawberries, leading to less insecticide use and greater production of organically grown strawberries.
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