Progress 09/01/21 to 01/31/24

Outputs
Target Audience:During the award period, the PD mentored seven undergraduate students in biology, including professional development in insect rearing, plant propagation, and data collection techniques; reading scientific literature; and data analysis. The PD also reached basic and applied ecologists, plant biologists, and evolutionary biologists by presenting project results at three international conferences (American Society of Naturalists 2023, Ecological Society of America 2023, and New Phytologists Next Generation Scientists 2024) and three invited seminars (University of Tennessee, Knoxville Dept. of Ecology & Evolutionary Biology, Towson University Dept. of Biology, Illinois State University Dept. of Biology). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PD has mentored seven undergraduate students, including professional development in insect rearing, plant propagation, data collection techniques, and data analysis. The PD engaged in professional development through training in evidence-based STEM teaching (NSF CIRTL online courses), active participation in a theoretical ecology research group, a demographic modeling workshop at the Ecological Society of America annual meeting, and six professional presentations. How have the results been disseminated to communities of interest?We have shared the results of the project with audiences of plant biologists, ecologists, and evolutionary biologists at three professional meetings (American Society of Naturalists 2023, Ecological Society of America 2023, and New Phytologists Next Generation Scientists 2024) and three invited seminars (University of Tennessee, Knoxville Dept. of Ecology & Evolutionary Biology, Towson University Dept. of Biology, Illinois State University Dept. of Biology). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Measuring the effect of herbivore density on the plastic induction of defenses in plant populations through time. During the award period, we (1) conducted a field experiment to characterize the effect of herbivore density on the plastic induction of plant defenses through time using upland cotton, Gossypium hirsutum, (2) collected field data and processed leaf samples from experimental plants to measure physical and chemical defenses, (3) began data analysis, and (4) presented results at three international conferences and three invited seminars. Field experiment: During summer 2022 we conducted a field experiment at the UTIA's East Tennessee Research and Education Center to characterize the effect of herbivore density on the plastic induction of plant defenses through time in upland cotton, Gossypium hirsutum. Plants used for the experiment were propagated from seed acquired from the USDA Cotton Germplasm and originally collected from wild populations in Mexico, representing 45 different genotypes in total. We used 70 experimental populations of 10 plants each, split among four herbivore density treatments. We introduced second-instar beet armyworm (Spodoptera exigua) larvae into caged experimental populations, then allowed continuous feeding by the caterpillars within the cages. We collected leaf samples from all plants in experimental populations 2, 4, 7, 12, or 21 days after introducing caterpillars. Data collection and sample processing: In the field, we measured plant biomass and percent leaf damage across the growing season. Using leaf samples brought back to the lab, we measured plant resistance to herbivory at each timepoint as naïve beet armyworm performance (i.e., relative growth rates) and leaf area consumed in no-choice bioassays. With separate leaf samples, we photographed the leaf surface under magnification, then analyzed the images to estimate trichome and terpenoid gland densities. Leaf samples were flash-frozen in the field and held for chemical analysis at the Biological and Small Molecule Mass Spectrometry Core at UTK. Mass spectrometry was used to quantify the relative abundance of specializedterpenoid aldehydes (two isomers of hemigossypolone). At the end of the experiment, we harvested the plants and measured dry weight. Data analysis and preliminary results: We observed rapid induction of resistance in plant populations exposed to caterpillar feeding. Within 2-4 days, plants in herbivore addition treatments depressed growth and feeding of naïve caterpillars (by up to 156 % and up to 75 %, respectively) relative to plants in the no-herbivore control. Mean resistance was sustained at this level until about 12 days of caterpillar feeding, then declined back to baseline levels by day 21 (coincident with caterpillar pupation). During the period of induced resistance (days 2-12), mean resistance increased with herbivore density, suggesting that plant induced response depends on herbivore density or the extent of herbivore feeding, and that this relationship is detectable at the level of genetically variable plant populations. We found differences in the timing and duration of induction among the multiple defensive traits measured. Leaf physical defenses against herbivory, trichomes and terpenoid glands, were each induced for a short duration but differed in their timing: increased trichome density occurred only on day 2, while increased terpenoid gland density occurred only on day 7. Specialized chemical defenses, on the other hand, were induced consistently (for one hemigossypolone isomer) or intermittently (for the other hemigossypolone isomer) acrossat least one week, between days 4 and 12. For terpenoid glands and both hemigossypolone isomers, induced defenses increased with herbivore density (full analysis of trichome density is still pending). Together, this combination of short-duration, sequentially induced defenses and defenses induced over longer periods may help to maintain high resistance to herbivory through time (e.g., across herbivore development) while minimizing the energetic or ecological costs associated with particular inducible defense traits. Objective 2. Modeling the effect of defense plasticity on consumer-resource dynamics in annual and perennial plant populations. During the award period, we (1) developed a continuous time plant-herbivore population model where plants induce defenses in response to herbivore feeding, and where the plant population is demographically structured into seedling and reproductive life stages, (2) used numerical simulation to characterize equilibrium dynamics for plant populations across a fast-slow pace-of-life continuum, and (3) submitted a manuscript to a major journal for publication. Model development and analysis: We developed a continuous-time model to address how the timing and costs of inducible defenses interact with plant demographic rates to influence plant-herbivore dynamics. Our model describes change through time of five state variables: seedling biomass, reproductive plant biomass, mean induction levels of seedling and reproductive plant biomass, and herbivore abundance. In our model: (1) herbivore feeding removes plant biomass and increases defenses in both seedling and reproductive plants, (2) plant defenses reduce herbivore feeding (thus protecting existing plant biomass) and are costly to plants via reduced seed production, and (3) seedling germination and establishment are limited by intraspecific competition. Importantly, we use separate rates to describe plant fecundity, seedling maturation, and reproductive plant mortality, allowing us to model different plant life histories by manipulating these parameters in combination. For each plant life history (i.e., combination of demographic parameters), we used numerical simulation to characterize the effects of induction responsiveness (how strongly plant defenses are induced upon herbivore feeding), induction cost (how strongly inducible defenses reduce seed production), and induction effectiveness (how strongly inducible defenses reduce herbivore feeding) on equilibrium dynamics. Results: We show that plant life history alters the effects of inducible defenses on population persistence, stability, and equilibrium densities. In particular, several population-level outcomes of inducible defenses differed between plant populations with a fast pace-of-life (e.g., high mortality/high fecundity) and populations with low adult mortality, slow maturation, or both. Persistence in fast pace-of-life populations was highly sensitive to inducible defense efficacy and costs. When inducible defenses did enable persistence in these populations, greater inducible defenses consistently maximized total plant biomass, with equilibrium densities strongly dependent on seedling (but not mature plant) induction. In slow pace-of-life populations, on the other hand, inducible defenses strongly promoted persistence, but plant biomass was not always maximized at high induction strength. We observed two distinct types of cyclic dynamics in these populations - those driven by high herbivore population growth and those driven by high inducible defense costs. With these findings, we demonstrate that the combination of demographic transition rates that characterize stage-structured populations can strongly modify the effects of inducible defenses by changing the relative effect of new induction on mean inducible defense levels, and by determining which factors are most limiting to seedling densities. Manuscript: A manuscript reporting the findings of this study is currently in revision at a major peer-reviewed journal.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Mutz, J. and S. Kalisz. American Society of Naturalists, Asilomar, CA. Population-level consequences of defense plasticity: effects of herbivore density on induced resistance through time.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Mutz, J. and S. Kalisz. Ecological Society of America, Portland, OR. Sequential expression of multiple herbivore-induced defensive traits confers sustained resistance.
• Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Mutz, J. and K.C. Abbott. Life history modulates effects of inducible defenses on consumer-resource dynamics.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Mutz, J. and S. Kalisz. New Phytologist Next Generation Scientist Symposium, Duke University, NC. Sequential expression of multiple herbivore-induced defensive traits confers sustained resistance.

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:The PD reached basic and applied ecologists and evolutionary biologists by presenting results at the American Society of Naturalists annual meeting (January 2023) and Ecological Society of America annual meeting (August 2023). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the project, the PD has mentored three undergraduate students, including professional development on insect rearing, plant propagation, and data collection techniques. The PD engaged in professional development through training in evidence-based STEM teaching (NSF CIRTL online courses), experience in methods for chemical analyses using mass spectrometry, and gaining skills in theoretical ecology. How have the results been disseminated to communities of interest?During the reporting period, weshared preliminary results from Objective 1 at two scientific meetings: American Society of Naturalists annual meeting (January 2023) and Ecological Society of America annual meeting (August 2023). A manuscript with the results from Objective 2 is in the final stages of preparation for submission for publication in a peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Measuring the effect of herbivore density on the plastic induction of defenses in plant populatuions through time. We will finish image analyses and chemical analyses of leaf samples collected during the field experiment. PD will analyze data from the field experiment and prepare manuscript for publication, including mentoring an undergraduate student on data analysis. Objective 2. Modeling the effect of defense plasticity on consumer-resource dynamics in annual and perennial plant populations. We will submit the prepared manuscript to a peer-reviewed journal, and will revise as needed for publication.

Impacts
What was accomplished under these goals? Objective 1. Measuring the effect of herbivore density on the plastic induction of defenses in plant populatuions through time. During the current reporting period we have focused on completing data collectionandentry, and beginning data analyses, associated with the summer 2022 field experiment. To complete data colection, we photographed all leaf samples and have analyzed 780 of 1400 images to quantify trichome and terpenoid gland densities. Chemical analyses of 700leaf samples(via high-performance liquid chromatography) were delayed due to equipment maintenance and repair, but theseanalyses are currently underway. All available data have been entered, cleaned, and annotated. We have written and tested statistical models using a subset of the data. Preliminary results: The mean resistance level of plant population increased quickly upon herbivory, reaching its highest level after 4days. Mean resistance was sustained at this level until about 12 days after the introduction of caterpillars, then declined back to baseline levels by day 21. During the period of induced resistance (days 4-12), mean resistance increased with herbivore density, suggesting that the plants' induced response depends on herbivore density or the extent of herbivore feeding, and that this relationship is detectable at the level of genetically variable plant populations. Using only data from the high herbivore density treatment, we found that leaf trichome density peaks early (at day 4), then begins to decline, while terpenoid gland density peaks later (at day 7). These offsets in the timing of peak induction may hep to maintain high resistance to herbivory through time (e.g., across herbivore development) while minimizing the energetic or ecological costs associated with particular inducible defense traits. Objective 2. Modeling the effect of defense plasticity on consumer-resource dynamics in annual and perennial plant populations. Using the model developed during the previous reporting period, we simulated equilibrium dyanmicsfor plant populations occurring along a fast-slow pace-of-life continuum (by manipulating rates of plant fecundity, maturation, and mortality in tandem). We compared the results of these simulations to an analytically tractable model without inducible defenses to isolate the effect of inducible defenses per se. Results: We show that plant demography alters the effects of inducible defenses on the persistence, stability, and equilibrium densities of plant and herbivore populations. In particular, several population-level outcomes of inducible defenses we unique tofast pace-of-life (e.g., high mortality/high fecundity) or slow pace-of-life (e.g., low mortality/low fecundity) populations. In fast pace-of-life populations, we found that greater inducible defenses consistently maximized total plant biomass, with equilibrium densities strongly dependent on seedling (but not mature plant) induction. Persistent cycles were possible at high inducible defense costs, and these cycles could not be stabilized by increasing maturation rate. In slow pace-of-life populations, on the other hand, inducible defenses strongly promoted persistence. We observed two distinct types of cyclic dynamics in these populations - those driven by high herbivore population growth and those driven by high inducible defense costs. With these findings, we demonstrate that the demographic transitions inherent to stage-structured populations can strongly modify the effects of inducible defenses by changing the relative effect of new induction on mean inducible defense levels, and by determining which factors are most limiting to seedling dynamics. We have written a full draft of a manuscript presenting these results, which in the final stages of preparation for submission for publication in a peer-reviewed journal.

Publications

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:During the reporting period, the PD mentored five undergraduate students in biology (ecology & evolutionary biology concentration), including professional development in insect rearing, plant propagation, and data collection techniques; reading scientific literature; and data analysis. The PD also reached basic and applied ecologists and evolutionary biologists by presenting preliminary research results at an invited seminar at University of Tennessee, Knoxville (Dept of Ecology & Evolutionary Biology) and an informal presentation at Case Western Reserve University. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the project, the PD has mentored three undergraduate students, including professional developmenton insect rearing, plant propagation, and data collection techniques. The PD engaged in professional development throughtraining in evidence-based STEM teaching (NSF CIRTL online courses), experience in methods for chemical analyses using mass spectrometry, and gainingskillsin theoretical ecology. How have the results been disseminated to communities of interest?At this point we have only preliminary results, which were shared with an audience of ecology and evolutionary biology researchers at a University of Tennessee, Knoxville departmental seminar given by the PD and at an informal presentation at Case Western Reserve University. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Measuring the effect of herbivore density on the plastic induction of defenses in plant populatuions through time. We will finish image analyses and chemical analyses of leaf samples collected during the field experiment. PD will analyze data from the field experiment and begin manuscript preparation. PD will present results of field experiment at the January 2023 meeting of the American Society of Naturalists. Objective 2. Modeling the effect of defense plasticity on consumer-resource dynamics in annual and perennial plant populations. We will prepare and submit a manuscript on the results of the continuous-time model. PD will present modeling results at a national or international conference. We will begin analyzing a discrete-time version of the model (to represent annual or annually-cropped plant populations).

Impacts
What was accomplished under these goals? Objective 1. Measuring the effect of herbivore density on the plastic induction of defenses in plant populatuions through time. During summer 2022 we conducted afield experiment at the UTIA's East Tennessee Research and Education Center to characterize the effect of herbivore density on the plastic induction of plant defenses through time in upland cotton, Gossypium hirsutum. Plants used for the experiment were propagated from seedacquired from the USDA Cotton Germplasm and originally collected from wild populations in Mexico. We used 70 experimental populations of 10 plants each, split among four herbivore density treatments. We introduced second-instar beet armyworm larvae into caged experimental populations, then allowed continuous feeding by the caterpillars within the cages. Wecollected leaf samples from all plants in experimental populations 2, 4, 7, 12, or 21 days after introducing caterpillars. Data collected: We measured plant biomass and percent leaf damage across the growing season. We measured plant's resistance to herbivory at each timepoint as beet armyworm performance (i.e., relative growth rates) and leaf area consumed in no-choice bioassays. We are currently processing the collected leaf samples to measure plant defense phenotypes at each timepoint: trichome and terpenoid gland densities (on the leaf surface) and terpenoid aldehyde concentrations. Preliminary results: The mean resistance level of plant populations increased quickly upon herbivory, regardless of herbivore density: after 2 days, caterpillar growth was 2-3xlower when fed leaf material from herbivore addition treatments (compared to no-herbivore controls). Mean resistance level continued to increase through time (i.e., after 7-12 days), but only for populations with high herbivore density. These preliminary findings so far suggest that plant populations are highly responsive to damage from herbivory and thatgreater herbivore density corresponds to greater investment in defenses over time. Objective 2. Modeling the effect of defense plasticity on consumer-resource dynamics in annual and perennial plant populations. We developed a continuous-time model forchange through time of the total biomass of seedlings and mature plants, themean induced defense levels of seedlings and mature plants, and herbivore density. We used simulations to characterize the model'sbehavior at equilibrium for different parameter sets (i.e., under which conditions the system goes extinct, goes to a stable equilibrium, or results in cyclic dynamics). Specifically, we used simulations to investigate how parameters describing defense induction, effectiveness, and costs affect plant biomass, seed production, and herbivore densities, and to what extent these effects depend on plant demography. We are currently preparing a manuscript on the results of our model analyses.

Publications