Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
May 1, 2019
Project End Date
Apr 30, 2024
Grant Year
Project Director
Tooker, J. F.
Recipient Organization
408 Old Main
Performing Department
Non Technical Summary
Recent research has revealed that goldenrod (Solidago altissima) can perceive the pheromones of its co-evolved herbivore and respond by priming its defense response, presumably because the pheromone serves as a reliable cue associated with impending herbivory. Our preliminary data indicate that American cranberry (Vaccinium macrocarpon) can similarly detect and respond to a blend of three sex pheromones being developed for a mating disruption program against three key, co-evolved lepidopteran pest species. Caterpillars that fed upon pheromone-exposed cranberry plants ate less tissue, gained less weight, and suffered greater mortality than caterpillars on control plants. Notably, cranberry plants exposed to the pheromone blend also grew more than unexposed plants, a result also found with S. altissima that suggests some plant species may not exhibit expected ecological tradeoffs between growth and defense. Building on these initial observations, we propose to (i) more fully characterize the influence of pheromone exposure to cranberry on caterpillar herbivory and survival and plant growth; (ii) elucidate key defense mechanisms underlying cranberry's responses to the three caterpillar species following exposure to the pheromones; and (iii) document field-scale effects of pheromone exposure on commercial cranberries. Our proposal aligns well with the priorities of the "Pest and Beneficial Species" program because we will study novel, recently revealed ecological interactions, and their mechanistic underpinnings, in a high-value crop species. Moreover, our research has strong potential to reveal key, basic details of pheromone-induced defense priming and evolution of pheromonal communication systems, while contributing significantly to development of an innovative pest management strategy.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
Our specific objectives are to:In the lab, fully document the influence of pheromone exposure to cranberry on herbivore performance and growth responses of cranberries (V. macrocarpon). In this objective, we will use a mating disruption-based, three-species lepidopteran pheromone blend and focus on organismal-level outcomes, including insect growth and survival, plant damage and growth, tradeoffs (e.g., growth/defense), and reproductive (fitness) consequences of pheromone-induced priming for cranberry. We will also test blueberry (Vaccinium angustifolium) for similar responses because it is a congener of cranberries, is native to similar habitats in North America, and shares multiple pest species with cranberries.Elucidate key biochemical and molecular mechanisms underlying cranberry's responses to the lepidopteran pheromones. Part of this work will focus on the species-specific effects of pheromone/caterpillar identity on induction of phytohormone-mediated signaling pathways and expression of key defense-related genes.Document field-scale effects of pheromone exposure on cranberry growth as well as performance and survival of sentinel larvae in commercial fields. This work will dovetail with ongoing mating disruption programs, and assess the impacts of chronic pheromone exposure on pest populations, damage from pest species, and plant productivity.
Project Methods
To more fully document the details of this interaction, we will conduct a large-scale factorial experiment in environmental chambers in WI to evaluate (i) success of Sparganothis caterpillars on pheromone-exposed and unexposed V. macrocarpon plants and (ii) growth and productivity of exposed and unexposed V. macrocarpon with and without Sparganothis caterpillar feeding. Methods: As described for our preliminary data, we will plant three cranberry plugs (same genetic background as Exp. #1) in each pot and let them establish for 6 wk. In glass jars (Fig. 3), we will then expose 60 pots to the lepidopteran pheromone blend for 5 d, using slow-release pellets (a newer release method that is easier to use than SPLAT®), while an additional 60 control plants are exposed to pheromone-free, control pellets. This experiment will progress in uniform conditions in balanced batches of plants based on the number of glass jars we have available. As described in our preliminary experiments, we will standardize plant size, then confine 10 Sparganothis neonates (from a WI lab colony) to half of the plants in each exposure treatment for 21 d, resulting in four treatments (N = 30 for each): (i) unexposed/no caterpillars; (ii) unexposed/caterpillars; (iii) exposed/no caterpillars; (iv) exposed/caterpillars. Afterward we will track herbivory and caterpillar development, plant growth and flower and seed production. As V. macrocarpon is an obligate outcrosser, we will introduce two bumble bee hives (Koppert Biological Systems, Inc) to facilitate fruit and seed production (we have used bumble bees with success in previous greenhouse experiments; they perform better than honeybees in such confined spaces).We hypothesize that caterpillar feeding on pheromone-exposed plants induces stronger concentrations of the defense phytohormone jasmonic acid. To test this hypothesis in cranberry, we will analyze biochemical and molecular mechanisms underlying enhanced plant defenses by assessing phytohormone levels and defense gene expression in pheromone-exposed and unexposed plants subject to herbivory. In preparation for these biochemical and molecular analyses, we will collect and freeze plant tissue from our other experiments. In light of our previous work with S. altissima and previous studies with JA in cranberries, we will focus on a suite of defense-related phytohormones, genes in the octadecanoid pathway and others know to be involved in JA-mediated induced defenses, including terpene synthases, which are important anti-herbivore defenses for V. macrocarpon. We will harvest and freeze damaged and undamaged leaves from each treatment at five time points (0, 6, 12, and 24, 48 h) for phytohormone and gene-expression assays. For each timepoint, we will destructively sample in individual pots from randomly selected uprights and harvest eight similar sized leaves (four with equivalent damaged and four undamaged from the same positions along the upright; undamaged leaves on plants with caterpillars will be used to characterize the systemic defensive response of plants); these leaves will be divided equally for phytohormone analysis and gene expression. Levels of phytohormones and gene expression will be compared across timepoints using ANOVA or Student's t-test as appropriate for the design of each experiment.At four large commercial cranberry marshes (= farm) in central WI (where we have ongoing mating disruption trials with treated/untreated blocks), we will embed plots for the pheromone-induced defense/growth experiments. Within each of the four marshes, there will be a single pair of very large blocks: one will be the mating disrupted block and the other, a 'standard grower practice' (no mating disruption) control block. We will apply pheromone using described protocols (Steffan et al. 2017) to provide uniform rates and spatial distributions of the pheromone carriers (a slow-release wax matrix). We will deploy pheromone-baited traps to assess degree of disruption for all three moth species. Embedded within each mating disrupted block and control block at each marsh will be 12 plots (24 total plots per marsh; across the four marshes, 48 plots). In each plot will be a 1×1-m mesh exclusion-cage. Half the cages in any given block (i.e., 6 cages) will be populated with 10 sentinel 2nd instar sparganothis larvae; the other cages in the block (6 cages) will have no larvae added. Thus, within the mating-disruption treatment factor (pheromones present/absent), there will be a nested herbivore treatment, resulting in four treatments (n = 6 each): pheromones/herbivores, pheromones/no herbivores, no pheromone/herbivores, no pheromones/no herbivores. Prior to adding larvae, we will "Dvac" the enclosed cranberry canopy to minimize background arthropod abundance. We will initiate the trial mid- to late-June of yr 2 and 3. Mid- to late June represents the likely period in which sparganothis adults would be flying and ovipositing. The duration of this trial will be weather-dependent since larval growth is temperature-mediated. We will conclude the experiment when larvae in control plots (no pheromones present) begin to reach the 5th instar, which would likely require 10-14 days. Response variables and statistical analyses: We will measure larval survival per cage, mean dry-weight, and developmental status (instar distribution among the surviving larvae). We will count cranberry upright density per square meter, and take subsamples of canopy biomass (dry-weight per 0.25 sq meters). We will analyze plant response metrics via 2-way ANOVA, looking for main and interactive effects between the pheromone and herbivore factors. The insect responses will be analyzed via 1-way ANOVA to assess the effects of the pheromone treatment on the larval metrics.

Progress 05/01/22 to 04/30/23

Target Audience: The primary audience is the cranberry industry (growers, packers, processors) and associated stakeholders (consultants, chemical companies, commodity groups). Changes/Problems:Delay on our progress are due to tight restrictions on research during COVID. We are working to compensate for time lost during lock down. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?

What was accomplished under these goals? Objective 1: In a greenhouse experiment, we examined the capacity of MeJA to induce defenses responses against a major insect pest species of cranberries. Specifically, we measured the effects of MeJA-induced defenses on growth of larvae of Sparganothis fruitworm (Sparganothis sulfureana). Using 4-L glass vessels, we place three cranberry plants in each of 12 glass vessels. We exposed half (N = 6) to MeJA for 48 h and the other half were left as untreated controls. The experiment included two cranberry cultivars: 'Stevens' and 'Ben Lear.' After MeJA exposure, we allowed plants to off-gas for 24 h, then placed Sparganothis larvae (5 per vessel) on cranberry foliage to establish and feed. For the following two weeks, we counted larvae every 2-3 days. In control vessels, approximately 60% of the larvae survived, while in the treated vessels, no larvae survived. Our results clearly indicate that MeJA-induced defenses of the cranberry plant can be significant. Our next step will be to establish if MeJA-induced defenses are more significant following exposure to moth pheromones.


    Progress 05/01/21 to 04/30/22

    Target Audience:The primary audience is the cranberry industry (growers, packers, processors) and associated stakeholders (consultants, chemical companies, commodity groups). Changes/Problems:COVID restrictions have stymied our progress. We hope our one-year extension will allows us to make significant progress. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Due to COVID restrictions, our target audience has not learned much about our project. Co-PI Steffan continues to interact with cranberry growers in Wisconsin, but his meetings were restricted to phone calls and online forums. Nevertheless, he continues to promote the potential benefits of our research project to cranberry growers, and these stakeholders are enthusiastic about the potential of pheromone-mediated defenses. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we hope to be able to promote our research results and share a message that this alternative form of insect control has potential to reduce reliance on insecticides and help improve environmental and human health in and around cranberry production areas.

    What was accomplished under these goals? The goal of this project is to explore and understand the capacity of cranberry plants (Vaccinium macrocarpon) to detect the sex pheromones of their native lepidopteran pest species and alter their defensive status to better protect themselves against caterpillar feeding. Our progress in the last year was severely limited by institutional COVID restrictions which made research challenging. In particular, we struggled to establish and maintain a robust colony of Sparganothis fruitworm, the main pest species that we have been studying. Our approved one-year no-cost extension should make up for this limitation and allow us to make important progress on our project. Nevertheless, by using spongy moth caterpillars (Lymantria dispar), a generalist caterpillar species that is an occasional pest of cranberry, we have made some progress. Via experimental work in the lab, we discovered that spongy moth caterpillars feeding on cranberry plants exposed to pheromones of Sparganothis fruitworm did not grow well and lost weight compared to caterpillars that fed upon plants that were not exposed to pheromone. Moreover, cranberry plants exposed to pheromones and fed upon by caterpillars showed a trend for having altered phytohormone profiles, which are key for better resisting caterpillar feeding. In the field, large-scale deployment of pheromones has been shown to reduce the survival of Sparganothis fruitworm larvae feeding within the cranberry canopy. Our work in the next year will seek to clarify and expand on these results.


      Progress 05/01/20 to 04/30/21

      Target Audience:The primary audience is the cranberry industry (growers, packers, processors) and associated stakeholders (extension professionals, consultants, chemical companies, commodity groups), as well as scientists that work with pest management in food crops, particularly entomologists and plant scientists. Changes/Problems:COVID restriction greater limited our progress on our project.We were not able to complete field work and only conducted minimal greenhouse-based research. We hope research will be easier in the coming year. What opportunities for training and professional development has the project provided?In Pennsylvania, postdoc Eric Yip encountered significant challenges to our research progress. He showed creativity and flexibility by looking into alternative herbivore species and collaborating with colleagues on campus to secure a steady supply of gypsy moth caterpillars. Eric trained an Penn State undergraduate student (John Sauter, who earned a minor in entomology) to conduct phytohormone analyses and help with behavioral bioassays. In Wisconsin, two undergraduate students (Molly Bidwell, Ellen Koch) were trained on how to sample cranberry insects within pheromone-disrupted blocks at commercial cranberry marshes. This work was part of an ongoing mating disruption project that has been documenting efficacy within large-scale pheromone deployment systems. These students worked part-time, for approximately 6 weeks during May and June, 2020. How have the results been disseminated to communities of interest?Results have been presented at the 2020 Wisconsin State Cranberry Growers Association (WSCGA) winter meeting, the spring mini-clinic, and the fall WSCGA Research Roundtable. What do you plan to do during the next reporting period to accomplish the goals?With COVID restrictions easing at our universities and the USDA, we are optimistic that we can resume our planned investigations. In Pennsylvania, we will re-establish cranberry plants in our greenhouse, and re-start a colony of sparganothis fruitworm. We will begin again plant assays with caterpillars feeding on pheromone-exposed and unexposed plants, which we evaluate for feeding damage and phytohormone production. In Wisconsin, we are planning another year of field evaluation of the influence of mating disruption on caterpillar growth and mortality.

      What was accomplished under these goals? Due to COVID-imposed constraints, we struggled to remain productive. We performed limited greenhouse work because we did not have a consistent source of insects to study.Keeping their colonies going was challenging with COVID, and we were not allowed to travel to replenish our colony.No field work involving pheromone-induced plant defenses was attempted during the spring or summer of 2020. These are the items we were able to accomplish despite COVID restrictions: Created new means of loading pheromones into fertilizer granules, then did small-scall tests of pheromone release longevity. Established in Wisconsin new cultures ofSparganothis sulfureana(sparganothis fruitworm) andSystena frontalis(flea beetles) for subsequent screening of plant defense lethality. Because COVID restrictions limited our ability to maintain populations of cranberry pests, we used gypsy moth (Lepidoptera: Erebidae:Lymantria dispar dispar) caterpillars as our focal herbivore species. Previous research has shown that gypsy moth caterpillars can be susceptible to inducible defenses of cranberry plants. With this new focal species, our experiments revealed that cranberry plants exposed to pheromones of sparganothis fruitworm had significantly elevate defenses because gypsy moth caterpillars that feed upon pheromone-exposed plants gained significantly less mass than caterpillars feeding on plants that were only exposed to the solvent control. Our phytohormone analyses have not revealed which compounds may be involved in this response.


        Progress 05/01/19 to 04/30/20

        Target Audience:Audiences include scientists, the cranberry industry (growers, packers, processors) and associated stakeholders (consultants, chemical companies, commodity groups). Changes/Problems:We have encountered some difficulties with cranberry culturing in the greenhouse and rearing S. sulfureana in the laboratory, but feel we have developed a system that will be successful in the coming year. More importantly, the COVID-19 outbreak has limited our ability to conduct experiments this spring, but we hope to begin in earnest once restrictions are lifted. What opportunities for training and professional development has the project provided?At USDA/Wisconsin, undergraduate researchers assisted with field experiments gaining valuable research experience. At Penn State, a MS student and postdoc are active on the project and these two individuals have been able to experience some of the challenges of establishing protocols for raising new plants species and rearing new research species. The MS student gave her first talk at a scientific conference and excelled. The postdoc is gaining experience helping to supervise and advise the MS student. How have the results been disseminated to communities of interest?Results have been presented at the North American Cranberry Research and Extension Workers meeting, as well as at three grower meetings hosted by the Wisconsin State Cranberry Growers Association. Further, a new manuscript has been drafted for publication in a peer-reviewed journal. Theoretical justification, experimental plan, and preliminary results were shared with the Annual Meeting of the Entomological Society of America in St. Louis in November 2019. What do you plan to do during the next reporting period to accomplish the goals?We plan to pursue lab and greenhouse experiments to gain insight on the plant defense response, its specificity, and its effectiveness in protecting cranberries against herbivory.

        What was accomplished under these goals? 1) Documented evidence that a key pest of cranberries (caterpillars of the moth, Sparganothis sulfureana) suffered increased mortality when caged on plants that had been exposed to S. sulfureana sex pheromones. This represented the first evidence that an agricultural crop could eavesdrop on courtship pheromones of its herbivores, then deploy defensive responses that significantly reduced pest survivorship. 2) Field-scale mating disruption system in which lepidopteran larvae (Sparganothis sulfureana) were impacted by the induced defenses of the cranberry plant. The defenses were induced by the broad deployment of insect sex pheromones at commercial cranberry marshes in Wisconsin. Larvae had been caged on cranberry uprights (4 larvae/ cage), and the mean larval density in cages in pheromone-treated beds was 0.85 larvae, compared to 2.3 larvae/cage in the control beds. This finding mirrored prior work, though smaller scale, within a greenhouse setting. In the greenhouse, significant mortality was found in S. sulfureana larvae that had been reared on cranberry plants that had been previously dosed for 48 hrs with insect sex pheromones. 3) Altogether, this represented evidence of increased efficacy of pheromone-based mating disruption. Mating disruption programs interfere with mate finding, but ultimately mating often does occur, even under ideal circumstances. Importantly, the present study has provided evidence that even the "escapees" (the progeny of those moths that did find mates) can be suppressed by induced defensive responses in the plant. 4) In our efforts to understand mechasnisms involved in the plant response, the Tooker lab has been working to understand the logistics of growing cranberries and rearing Sparganothis sulfureana. After some trial and error, we have established effective protocols for raising plants and rearing caterpillars. We have also determined the best length for greenhouse feeding trials (48 h) so that plants experience enough damage and plant defense pathways are triggered. We have also determined an effective way to confine caterpillar to plants and not letting them escape without limiting their access to food.


        • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Golinski, J. E., E. Yip, and J. F. Tooker. Plant defense by eavesdropping on the communication of herbivores: Is it common or rare? Annual Meeting of the Entomological Society of America, St. Louis, MO, November 2019.