The Pennsylvania State University

The Graduate School

PLANTS AS ILLEGITIMATE RECEIVERS OF INSECT SIGNALS: INSIGHT FROM

MAIZE AND TEOSINTE

A Thesis in

Entomology

by

Julianne Golinski

© 2020 Julianne Golinski

Submitted in Partial Fulfillment of the Requirements for the Degree of

Master of Science

August 2020

ii The thesis of Julianne Golinski was reviewed and approved by the following:

John F. Tooker Professor of Entomology and Extension Specialist Thesis Advisor

Jared G. Ali Assistant Professor of Entomology

Ben McGraw Associate Professor of Turfgrass Science

Gary W. Felton Professor of Entomology Head of Department of Entomology

iii ABSTRACT

Plants have evolved to be sensitive to a range of insect-associated cues to detect the presence of herbivorous species and mount effective defense responses against future attack.

Insect pheromones have recently been added to this library of cues as some plant species can act as “illegitimate receivers” that eavesdrop on the pheromones of insect herbivores, which act as reliable indicators of future larval damage. My thesis begins with a review that explores the perceptive capabilities of plants and how the ability to eavesdrop on insect pheromone cues may have evolved. I also cover the three known examples of plant illegitimate receivers and use shared traits in these systems to build criteria for finding other host plant species that may also utilize this strategy; native host plants species with (1) native, co-evolved insect herbivore species

(2) that release abundant amounts of chemicals on or near the host plant, and (3) substantially reduce host plant fitness.

My second chapter highlights two agriculturally relevant plant species that fit the criteria, maize (Zea mays ssp. mays; variety B73) and Balsas teosinte (Zea mays ssp. parviglumis). I investigated whether maize and its progenitor, teosinte, can detect the pheromone cues of a native co-evolved herbivore and important pest species, fall armyworm (FAW), and prime their defenses against future attack. To determine if maize or teosinte cue into FAW pheromone emissions, I exposed plants to FAW pheromones or the pheromones of sunflower head moth (SHM), an unassociated lepidopteran that acted as a chemical control. Following pheromone exposure, I allowed FAW caterpillars to feed on the plants. I found that feeding damage was reduced on maize plants that were exposed to FAW and SHM pheromone emissions and teosinte plants that were exposed to only FAW pheromone emissions. The levels of jasmonic acid in the leaves indicated that defenses were induced after feeding by FAW, but they were highly variable and did not predictably align with the expectation that FAW pheromone-exposed plants would have

iv stronger, primed defense responses compared to controls. These results suggest that B73 line maize appears to respond to a broad spectrum of volatile chemicals and Balsas teosinte may have more targeted responses to FAW pheromones.

v TABLE OF CONTENTS

LIST OF FIGURES ...... vi

LIST OF TABLES ...... viii

ACKNOWLEDGEMENTS ...... ix

Chapter 1 Plants as illegitimate receivers: how did it evolve and how common is it? ...... 1

Perceptive Abilities of Plants ...... 2 Plant Detection of Volatiles ...... 5 Plant Detection of Airborne Insect Cues ...... 7 Pheromones Derived from Plants ...... 9 Criteria for Finding Other Examples of Plant Illegitimate Receivers ...... 10 Known Examples of Plants as Illegitimate Receivers of Insect Pheromones ...... 12 Benefits of Understanding this Phenomenon ...... 13 Conclusion ...... 13 References ...... 15

Chapter 2 The effects of fall armyworm sex pheromones on defenses in maize and its wild ancestor teosinte ...... 23

Introduction ...... 23 Materials and Methods ...... 29 Plants and Insects ...... 29 Pheromone Lures ...... 30 Pheromone Lure Emissions ...... 31 Experimental Setup ...... 31 Pheromone Exposure ...... 32 Herbivore Feeding Assays ...... 33 Measuring Leaf Damage Area ...... 34 Phytohormone Extractions ...... 34 Quantification of Jasmonic Acid ...... 35 Statistical Analyses ...... 36 Results ...... 37 Pheromone Lure Emissions ...... 37 Fall Armyworm Feeding Damage and Weight Gain ...... 39 Phytohormone Analyses ...... 42 Discussion ...... 47 Conclusion ...... 54 References ...... 56

vi LIST OF FIGURES

Figure 1-1: Criteria for finding host plants with the capacity to eavesdrop on insect- produced volatile compounds: (1) native, co-evolved insect herbivore species (2) that release abundant amounts of chemicals on or near the host plant, and (3) substantially reduce host plant fitness ...... 11

Figure 2-1: Images of leaf damage after 48 h of feeding by second instar fall armyworm larvae (A) before and (B) after editing. Damage area was colored in to properly process leaf damage area in ImageJ ...... 34

Figure 2-2: Linear regression of mass gain of fall armyworm larvae that fed on maize and teosinte in relation to damage area (Mass Gain = 2.016 + 2.329*damage area, R2 = 0.58) ...... 37

Figure 2-3: Weight gain of fall armyworm larvae that fed on maize plants exposed to low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones (Tukey’s HSD P < 0.05). The bars shown are standard error bars and groups with different letters are statistically different ...... 40

Figure 2-4: Leaf damage area caused by fall armyworm larvae that fed for 48 h on maize plants exposed to low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones (Tukey’s HSD P < 0.05). The bars shown are standard error bars and groups with different letters are statistically different. The data shown are not transformed, but statistical analyses were carried out on square root-transformed data ...... 40

Figure 2-5: Weight gain of fall armyworm larvae that fed on teosinte plants exposed to low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones (Tukey’s HSD P < 0.05). The bars shown are standard error bars and groups with different letters are statistically different ...... 41

Figure 2-6: Leaf damage area caused by fall armyworm larvae that fed for 48 h on teosinte plants exposed to low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones (Tukey’s HSD P < 0.05). The bars shown are standard error bars and groups with different letters are statistically different. The data shown are not transformed, but statistical analyses were carried out on square root-transformed data ...... 41

Figure 2-7: Total salicylic acid in both maize and teosinte leaf tissues as related to damage area (Linear regression P > 0.05) ...... 43

Figure 2-8: Amount of cis-jasmonic acid present in maize plants exposed to pheromones (low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones) for 24 h and fall armyworm caterpillars for 48 h. Damage area did not have a significant effect on cis-JA levels (P > 0.5). Comparisons were made among pheromone-exposed and caterpillar-exposed plants

vii separately (Tukey’s HSD P > 0.05). N.S denotes no significant differences among the treatment groups and the bars represent standard error. The data shown are not transformed, but the statistical analyses were carried out on log-transformed data ...... 43

Figure 2-9: Amount of trans-jasmonic acid present in maize plants exposed to pheromones (low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones) for 24 h and fall armyworm caterpillars for 48 h. Damage area did not have a significant effect on trans-JA levels (P > 0.5). Comparisons were made among pheromone-exposed and caterpillar-exposed plants separately (Tukey’s HSD P > 0.05). N.S denotes no significant differences among the treatment groups and the bars represent standard error. The data shown are not transformed, but the statistical analyses were carried out on log-transformed data ...... 44

Figure 2-10: Total cis-jasmonic acid in maize leaf tissues as related to damage area (Linear regression P > 0.05) ...... 44

Figure 2-11: Total trans-jasmonic acid in maize leaf tissues as related to damage area (Linear regression P > 0.05) ...... 45

Figure 2-12: Amount of cis-jasmonic acid present in teosinte plants exposed to pheromones (low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones) for 24 h and fall armyworm caterpillars for 48 h. Damage area did not have a significant effect on cis-JA levels (P > 0.5). Comparisons were made among pheromone-exposed and caterpillar-exposed plants separately (Tukey’s HSD P > 0.05). N.S denotes no significant differences among the treatment groups and the bars represent standard error. The data shown are not transformed, but the statistical analyses were carried out on log-transformed data ...... 45

Figure 2-13: Amount of trans-jasmonic acid present in teosinte plants exposed to pheromones (low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones) for 24 h and fall armyworm caterpillars for 48 h. Damage area did not have a significant effect on trans-JA levels (P > 0.5). Comparisons were made among pheromone-exposed and caterpillar-exposed plants separately (Tukey’s HSD P > 0.05). N.S denotes no significant differences among the treatment groups and the bars represent standard error. The data shown are not transformed, but the statistical analyses were carried out on log-transformed data ...... 46

Figure 2-14: Total cis-jasmonic acid in teosinte leaf tissues as related to damage area (Linear regression P > 0.05) ...... 46

Figure 2-15: Total trans-jasmonic acid in teosinte leaf tissues as related to damage area (Linear regression P > 0.05) ...... 47

viii LIST OF TABLES

Table 2-1: Observed retention times for compounds detected in fall armyworm pheromone lure emissions. Collected fall armyworm pheromone lure emissions were run through an HP-1 GC column ...... 38

Table 2-2: Observed retentions times for compounds detected in sunflower head moth pheromone lure emissions. Collected sunflower head moth lure emissions were run on a HP-1 GC column ...... 38

ix ACKNOWLEDGEMENTS

I want to thank everyone in the Tooker lab, including Elizabeth Rowen, Kirsten Pearsons,

Eric Yip, Julie Baniszewski, and Andrew Aschwanden for their willingness to give advice, provide constructive feedback, and help with my experiments, but also for being people I can always laugh with and open up to. Special thanks go to Elizabeth and Eric for taking the time to have many sit-down statistics sessions. I also want to thank Kirsten who is always willing to have discussions about research, grad school, and experimental design. I especially want to thank my advisor, John Tooker, for taking me on as a student and for always finding time to bounce ideas around, effortlessly put everything into perspective, fix machines when they inevitably break, and always have a good laugh. I am so grateful to have worked with such a fun, tight-knit, talented group of people.

I also want to thank my committee members, Jared Ali and Ben McGraw, for being so flexible and supportive of my projects. Your input was deeply appreciated. Thank you to the Ali lab for providing B73 maize line seeds and access to their lab space, along with offering guidance about cleaning glass domes and running volatile headspace collections. I also want to thank

Michelle Peiffer, of the Felton lab, for providing fall armyworm larvae and being willing to answer all my questions.

Thank you to Shawn Steffan, our collaborator at the USDA-ARS and the University of

Wisconsin-Madison who gave guidance about pheromone mating disruption in systems with lepidopteran pests. Your input was incredibly helpful and played a central role in how I designed my experiments. Thank you to Mark Millard, a geneticist/maize curator with the USDA-ARS

(Ames, Iowa), who provided Balsas teosinte seeds and offered guidance about how to properly grow teosinte in a greenhouse. I would also like to thank Jim Tumlinson and Tom Baker for their invaluable guidance about lepidopteran pheromone emissions and experimental design.

x Lastly, I want to acknowledge the people in my support system who played a huge role in getting me to graduate school and supporting me through it. Special thanks go to Hannah

Greenberg and Rachel McLaughlin, my best friends at Penn State, who have been a constant source of laughter and support. I look up to both of you as people and scientists. Thank you to

Jennie Carr who first introduced me to research back in 2015, helped me get into graduate school, and has supported me during my time at Penn State. I am grateful to Rosemary Ford, the professor who introduced me to the wonderful world of plants and guided me through the undergraduate thesis that ultimately led me to the Tooker lab. I also want to give a huge thank you to my parents, family, and Cyle who have always embraced my love of plants and insects and have been my number one supporters since the beginning. I am forever grateful for your love and encouragement. For funding my thesis, I also want to thank the USDA.

This material is based upon work supported by the United States Department of

Agriculture under Award No. 2017-67013-26258. Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the author and do not necessarily reflect the views of the United States Department of Agriculture.

Chapter 1

Plants as illegitimate receivers: how did it evolve and how common is it?

During times of war, to anticipate attacks armies have relied on code breakers to decipher enemy communications. Remarkably, and unknown to most generals, these same tactics of espionage first evolved in nature, and insects are recognized among the most accomplished code breakers, but recent research has increasingly revealed that plants are also adept eavesdroppers on environmental signals. Plant and herbivorous insect species are engaged in co-evolutionary “arms races” and members of both taxa have adapted to detect and exploit cues from their prey or enemies. Organisms that utilize this strategy have been labeled unintended or illegitimate receivers (Otte 1974). For example, insect predators or parasitoids are often illegitimate receivers, and many species have evolved to eavesdrop on signals (e.g., visual, acoustic, or olfactory) that their prey species use to attract mates (Otte 1974, Zuk and Kolluru 1998). For insects, sex pheromones are commonly targeted cues because they are often species-specific and critical for communication. When illegitimate receivers exploit pheromonal cues, targeted prey species are forced to balance between attracting mates and avoiding natural enemies (Zuk and

Kolluru 1998). Thus, illegitimate receivers can strongly influence the pheromone signals that their prey use because emitters of signals must develop counter-adaptations to minimize detection

(Raffa and Klepzig 1989, Tumlinson et al. 1993).

Until recently, insects and other animals were viewed as the primary illegitimate receivers (Otte 1974, Zuk and Kolluru, 1998) but plants too can eavesdrop on chemical communications, often from nearby plants. New evidence suggests that plants can also detect and respond to the chemical communication of insects. This review will cover known examples of plants as illegitimate receivers and discuss how this trait likely evolved. The ability of plant

2 species to eavesdrop on cues from other plants was suspected decades ago, but was definitively confirmed more recently. This review will also describe the recent findings that plants can also eavesdrop on insect-produced cues, such as pheromones, and discuss scenarios under which this trait is likely to have evolved. This review will end by considering the opportunities that plant perception of insect cues provides for improving control of herbivorous insects and developing novel pest-management strategies.

Perceptive Abilities of Plants

In the ongoing battle with herbivores, plant species have had to evolve different strategies to detect and defend against herbivorous species. This ability to adapt requires sensitivity to a variety of insect-associated mechanical and chemical cues. Once detected, these cues can elicit indirect or direct defenses. Indirect defenses involve the emission of volatile blends that signal to natural enemies of attacking herbivores (Aljbory and Chen 2018) or enhanced production of nectar by extrafloral nectaries, whereas direct defenses include changes in host plant physiology to limit herbivore feeding such as physical barriers, (i.e. thorns, hardened cell walls, etc.), repellents, toxins, and enzymes that inhibit digestion (Chen 2008). Importantly, plants can exhibit these defense responses through induction or priming. Induced defenses can occur in response to attack by an herbivore (Frost et al. 2008, Heil and Ton 2008); however, this response requires time to take effect. In comparison, defense priming can occur when external cues indicate that an attack is likely or imminent, allowing plants that receive the cue to prepare and then react quickly when an attack occurs (Frost et al. 2008). If a plant has the advantage of priming defenses with a reliable cue prior to attack, induced defenses can be deployed more swiftly and powerfully once herbivory takes place. This allows plants to respond at the

3 appropriate time and optimally use resources while minimizing tissue loss and increasing plant fitness.

Plants can detect a variety of mechanical indicators of insects and their feeding damage.

For instance, insects walking on plants can create a signal that quickly causes plants to develop resistance to insect feeding (Bown et al. 2002, Hall et al. 2004) and broken trichomes can trigger defense responses that prepare plants for subsequent herbivory (Peiffer et al. 2009, Tooker et al.

2010). Vibrations produced during caterpillar feeding have also shown to induce chemical defenses in plants (Appel and Cocroft 2014). Each mechanical cue signals to a host plant that a threat is present, allowing for defense induction and a reduction in herbivore damage.

In addition to mechanical cues, plants can also detect chemical cues from herbivores when they contact plants. For example, insect oral secretions and regurgitants are indicators of herbivore feeding. These substances contain elicitors that differentiate herbivory from mechanical damage, trigger herbivore-induced defenses, and help identify the herbivore (De Moraes et al.

1998, Mescher and De Moraes 2015). For example, volicitin, an elicitor originally isolated from the oral secretions of beet armyworm (Spodoptera exigua Hübner), evokes release of volatiles that attract natural enemies of beet armyworm caterpillars (Alborn et al. 1997). Elicitors, like volicitin, can initiate volatile distress signals that are attractive to foraging natural enemies.

Notably, different plant species have also evolved to detect the chemical cues of non- insect herbivores such as snails (Orrock 2013, Orrock et al. 2018) and slugs (Falk et al. 2014).

Tomato (Solanum lycopersicum L.), for example, can detect the locomotion mucus of herbivorous snails (Helix aspersa Müller) leading to the induction of chemical defenses that subsequently reduced insect herbivore (Spodoptera exigua) growth and feeding damage (Orrock et al. 2018). Evidently, plants can be receptive to cues from a broad range of herbivore species, including generalist mollusks.

4 Although chemical cues like oral secretions and mucus are important for triggering plant defense responses, many plant species have also evolved to counteract herbivore damage before feeding starts. Oviposition fluids and insect eggs elicit plant responses (Hilker and Meiners,

2006; Reymond 2013) that include tissue necrosis, neoplasm formation, parasitoid-attracting volatile emissions, and generally unsuitable conditions for developing larvae (Hilker and Meiners

2011, Beyaert et al. 2012, Fatouros et al. 2012, Kim et al. 2012). Neoplasms create a barrier between insect eggs and the plant surface, which can cause eggs to detach and fall off (Doss et al.

2000, Hilker and Fatouros 2015). Similarly, necrotic plant tissue can cause eggs to fall off

(Balbyshev and Lorenzen 1997) or desiccate (Shapiro and DeVay 1987). Oviposition can also indicate future larval damage, which was first found in Scots pine (Pinus sylvestris L.) (Beyaert et al. 2012). Sawfly (Diprion dipi L.) larvae that fed on pine foliage that they developed on weighed less and suffered higher mortality in comparison to larvae that fed upon non-egg containing foliage. The resulting females also had lower fecundity compared to females that fed on egg-free pine (Beyaert et al. 2012). Oviposition cues from egg-laying sawflies triggered Scots pine to create inhospitable conditions for eggs and subsequent larvae, which had negative consequences for later generations of sawflies, demonstrating the potential outcomes of detecting herbivore-associated chemical cues before damage.

Plants must find a balance between allocating resources towards growth and defense against a diversity of environmental threats. In addition to insect-associated mechanical and chemical cues, plants have evolved to detect plant volatile cues that provide information about herbivores on the same plant or neighboring plants.

5 Plant Detection of Volatiles

Olfaction is a poorly understood sensory ability in plants that can help pinpoint insect herbivores in the environment. Airborne chemicals provide important insights on the presence and proximity of insect herbivores; however, most research on plant olfaction has focused on detection of volatile compounds associated with microbes and neighboring plants (Heil &

Karban, 2010, Sharifi & Ryu, 2018, Sharifi et al. 2018). Some plant species develop disease resistance when exposed to bacterial volatiles (Ryu et al. 2004) or the volatiles of pathogen- infected plants (Shulaev et al. 1997, Kishimoto et al. 2006, Yi et al. 2009, Das et al. 2013,

Castelyn et al. 2015, Quintana-Rodriguez et al. 2015). Plants likely evolved this ability because volatiles from pathogens indicate an elevated risk of infection and eavesdropping on volatile cues allows plants to elevate their defenses to match the higher risk.

Many plant species can detect volatiles emanating from their own tissues or from neighboring plants of the same species or different species. The topic of plant communication was initially seen as controversial, but it is now a widely accepted ecological phenomenon

(Karban et al. 2014), with over 48 studies alone that support the hypothesis that plants develop resistance to herbivores when exposed to the volatiles of damaged neighbors (Karban et al. 2014).

Although there are many studies supporting the idea of plants “eavesdropping” on their neighbors, there is also strong evidence that plants may have initially evolved the ability to release volatiles and detect volatiles as a way to signal to other parts of the same plant (Orians

2005, Frost et al. 2007). This idea makes sense because volatiles dissipate the further they are from an emitting plant (Orians 2005) and plants over 20-cm away are often not responsive to volatile emissions (Karban et al. 2003). Therefore, the signals appear to be intended as a within- plant signal. This signaling tactic has clear benefits because overcoming vascular constraints allows them to quickly induce resistance to undamaged areas to limit herbivore feeding. For

6 example, after receiving mechanical damage, sagebrush (Artemisia tridentata Nutt.) plants required airflow to send damage-signaling volatiles to plant sections that lacked vascular connections (Karban et al. 2006). This mechanism allows for a more efficient warning system that limits further herbivore damage across entire plants. Preceding studies tested this within- plant volatile-signaling strategy using herbivore damage instead of mechanical damage on hybrid poplar (Populus deltoides x nigra) (Frost et al. 2007), lima bean (Phaseolus lunatus L.) (Heil and

Silva Bueno 2007), and highbush blueberry (Vaccinium corymbosum L.) (Rodriguez-Saona et al.

2009). In the case of hybrid poplar, foliage damaged by gypsy moth (Lymantria dispar L.) emitted volatiles that cause a defense response in undamaged foliage sharing no vascular connections to damaged leaves (Frost et al. 2007). Like sagebrush, the poplar trees in this system also appear to rely on volatile cues when signaling via vascular networks is lacking (Frost et al.

2007). The evolved ability to signal herbivore presence to the rest of a plant has significant implications for plant survival. These airborne warnings allow plants to prepare but delay defense responses until a threat reaches the plant (Frost et al. 2007).

Although within-plant signaling is crucial for conveying warning signals, plants have also evolved to “eavesdrop” on these signals in their neighbors. This tactic gives an advantage to eavesdropping plants because they can detect cues from damaged plants, providing time to prime their tissues for visits by the same herbivores. Wild tobacco (Nicotiana attenuate Torr.), for example, eavesdropped on neighboring sagebrush plants that were clipped and releasing methyl jasmonate (Karban et al. 2000), a volatile that commonly causes plants to turn on defense genes against herbivores (Farmer and Ryan 1990), resulting in wild tobacco plants with induced herbivore resistance, higher levels of defense chemicals, and reduced herbivore damage (Karban et al. 2000). Lima bean plants also exhibited a defense response when exposed to volatiles of other lima bean leaves that were infested with spider mites. In this instance, undamaged, volatile- exposed leaves used the cue to prepare for herbivory by turning on five defense genes (Arimura et

7 al. 2000). Alder trees showed a similar response as undamaged trees developed resistance to alder leaf beetle (Agelastica alni L.) and had reduced subsequent herbivory when located near damaged trees. This induced resistance response likely reduced herbivory across entire alder tree stands (Dolch and Tscharntke 2000). Similarly, damaged maize (Zea mays L.) plants released indole, a commonly emitted volatile compound, that primed the release of herbivore-induced volatiles in emitting plants and neighboring maize plants, which ultimately reduced herbivore damage (Erb et al. 2015). These plant species are utilizing neighboring warning signals to save energy and prepare for future attack, instead of waiting for herbivore contact and time-consuming defense induction. They have essentially evolved to take advantage of other plants doing the defensive “leg work.”

Eavesdropping on neighboring plant volatiles has shown to be an effective strategy for many plant species, however research has been less focused on the detection of volatiles emanating directly from the causative agent of damage. In the next section, I will address recent evidence that tall goldenrod (Solidago altissima L.), Scots pine (Pinus sylvestris L.), and cotton

(Gossypium hirsutum L.) can eavesdrop on the reliable pheromone cues of insect threats and prepare their defenses for future attack (Helms et al. 2013, Bittner et al. 2019, Magalhães et al.

2019).

Plant Detection of Airborne Insect Cues

Plants clearly benefit from detecting insect cues (i.e., mechanical damage, footprints, oral secretions, oviposition) and plant volatile cues, but recent studies suggest that some plant species can also benefit from detecting volatile cues directly from insects. As mentioned above, insect pheromones are a volatile cue commonly exploited by insect predators and parasitoids because they are reliable cues indicating the presence of their prey. Similarly, insect pheromones of

8 herbivorous insects would seem to be reliable indicators of increased risk from herbivore species that attack regularly, and plants may benefit from being able to detect the cue and then prepare for the greater threat of attack.

In 2013, the first study documented a plant species that can eavesdrop on insect- associated olfactory cues. Tall goldenrod (Solidago altissima L.) was shown to detect the putative sex pheromones of its insect herbivore, the goldenrod gall fly Eurosta solidaginis Fitch, and prime defenses in anticipation of future attack (Helms et al. 2013). Decades of research have been completed in the Solidago/E. solidaginis system (see Abrahamson and Weis, 1997) but the presence of the fly’s putative sex pheromones were not reported until 2013. When exposed to the putative sex pheromone blend of E. solidaginis, S. altissima primed its defense responses and reduced the amount of feeding damage when attacked by the co-occurring beetles species

Trirhabda virgata LeConte and the community of herbivores that colonized S. altissima in the field (Helms et al. 2013, Yip et al. 2017). Following herbivory, plants exposed to E. solidaginis emissions had higher levels of jasmonic acid, a phytohormone that regulates plant defenses against many species of chewing insects (Helms et al. 2013). Beyond responding to the full emission of E. solidaginis, S. altissima is also responsive to E,S-conophthorin, the main component of the fly emission, indicating that this compound is among the few volatile elicitors of defense responses that has been identified (Erb et al. 2015, Helms et al. 2017). Work in this system revealed an entirely new class of volatile-mediated interactions between plants and chemical emissions of their herbivorous insects. Moreover, the work revealed that, much like insect predators and parasitoids, plants can be illegitimate receivers of volatile cues from insects.

9 Pheromones Derived from Plants

To better understand how this type of relationship formed, it is necessary to recognize that plants and plant compounds are tightly linked to the formation of insect pheromones. This connection originated early in the interaction between plants and insects; primitive moth and caddisfly pheromones, in particular, are thought to have been very similar to plant volatiles

(Visser 1986), and some insect pheromones are comprised of volatile plant chemicals (Stokl and

Steiger 2017).

To synthesize pheromones, some insects must obtain precursors from their diet, and then utilize plant compounds directly or transform diet-acquired components into signals (Yew and

Chung 2015). Orchid bees (Euglossini), for example, collect fragrant compounds from flowers without receiving a reward (Vogel 1966). These charismatic bees are an example of insects with plant-derived pheromones because they build fragrance profiles from volatiles present in plant material (flowers, rotting logs, etc.). They use these “perfumes” to attract females (Pokorny et al.

2017), so the harvested plant odors essentially act as sex pheromones. Similarly, female

Antistrophus rufus Gillette gall wasps use plant volatiles as a proxy for sex pheromones. In this system, female wasps reside inside the tissues of Silphium laciniatum L. and alter the volatile host plant chemicals to attract males (Tooker et al. 2002). In comparison, males of the arctiid moth

Utetheisa ornatrix L. produce a sex pheromone that is derived from host compounds they sequester as larvae. In this case, the larvae sequester pyrrolizdine alkaloids (PA) from host plants and produce a PA-derived sex pheromone to attract females. The more they sequester, the more attractive they are to females. These attractive males give the alkaloid to females through sperm packets and females then expose their eggs to PA, which protects them from predation (Eisner and Meinwald 1995). Female moths, like their eggs, also get protection from predation when gifted with PA (Gonzalez et al. 1999).

10 Some pheromones appear to have arisen from recycled plant compounds, but later evolved into more complex blends that can be synthesized from plant-derived precursors (Stokl and Steiger 2017). Because of the ability of plants to eavesdrop on cues from their neighbors

(Karban et al. 2014), it is easy to imagine that plants were predisposed to detecting these early pheromones. Additionally, if the scents are strongly associated with herbivory, selection would seem to favor plants that could detect and prepare defense responses to these sorts of compounds.

Moreover, as pheromone blends evolved with more complex compounds and blends, some plant species appear to have been able to keep pace and can detect complex compounds emitted by insects (e.g., E,S-conophthorin; Helms et al. 2017). Recent evidence suggests pheromones, whether comprised of plant compounds or are insect-derived, are reliable indicators of the presence of herbivore and insect attack, so it would be adaptive for plants to have evolved to detect them.

Criteria for Finding Other Examples of Plant Illegitimate Receivers

There are several conditions that increase the likelihood of finding plants that evolved a capacity to eavesdrop on insect-produced volatile compounds, including various types of pheromones and defense emissions. We hypothesize that these conditions include native plant species with (1) native, co-evolved insect herbivore species (2) that release abundant amounts of chemicals on or near the host plant, and (3) substantially reduce host plant fitness (Figure 1-1).

These criteria would appear to create conditions necessary for a host-plant species to evolve to exploit insect pheromones as cues for increased risk of attack. To find more examples of plant species that can detect insect-produced volatile compounds, we hypothesize that it would be most profitable to explore systems with insect species like tephritid flies, cerambycids beetles, and bark beetles that release copious amounts of compounds. The tephritid species mentioned above (E.

11 solidaginis) can emit approximately 70 µg of pheromones in 24 h (Helms et al. 2013). A good candidate, for example, could be the cerambycid Xylotrechus nauticus Mannerheim, a host of oak, walnut, and willow trees (Solomon 1995), that has males of which can release over 100 μg of aggregation pheromones per day (Hanks et al. 2007). By comparison, moth species release nanogram amounts of pheromones and it may be more challenging for plants to detect such low concentrations of chemicals. It seems possible, however, that plants could have been agents of selection, helping to encourage some groups of insects to produce lower amounts of pheromones.

If the amounts of chemicals emitted by animals, like moths, has decreased with co-evolutionary time, perhaps plants as illegitimate receivers were among the selection pressures that helped drive down the amounts of chemicals that such animals produced. To determine if this scenario is plausible, research would be necessary to clarify whether plants are sensitive to high and low doses of some lepidopteran pheromones.

Figure 1-1: Criteria for finding host plants with the capacity to eavesdrop on insect-produced volatile compounds: (1) native, co-evolved insect herbivore species (2) that release abundant amounts of chemicals on or near the host plant, and (3) substantially reduce host plant fitness.

12 Known Examples of Plants as Illegitimate Receivers of Insect Pheromones

In addition to the Solidago-Eurosta system, there are two other known examples of plant illegitimate receivers. The first example expands upon a well-studied evolutionary relationship between cotton (Gossypium hirsutum L.) and cotton boll weevil (Anthonomus grandis Boheman;

Magalhães et al. 2019). When exposed to a full blend of cotton boll weevil aggregation pheromones, cotton plants release volatile chemicals that attract the parasitic wasp Bracon vulgaris Ashmead, which uses larvae of A. grandis as hosts for its own larvae. So, domesticated cotton appears to detect boll weevil pheromones, use that as a reliable cue for impending herbivory, and recruit parasitic wasps to decrease the threat. This study system fits the criteria well: (1) G. hirsutum is native to central America and the Caribbean (Wendel et al. 2010) with a co-evolved herbivore (Resh and Cardé 2003); (2) A. grandis releases relatively high amounts of pheromones (~ 2 µg per day) while on their host plants; and (3) extensive boll weevil damage reduces cotton plant fitness.

The most recently discovered example focuses on the effects of sawfly (Diprion pini L.) sex pheromones on Scots pine (Pinus sylvestris L.) defenses and the survival of sawfly eggs

(Bittner et al. 2019). Scots pine increases expression of defense genes and hydrogen peroxide content in pine needles when exposed to sawfly sex pheromones. Sawfly egg survival is also reduced after exposure. Ultimately, Scots pine appears to associate sawfly sex pheromones with future egg deposition and, in response, utilizes the cue to prepare for future colonization and damage by larvae. This study also fits the criteria well: (1) Scots pine, a widely distributed plant that is native to Eurasia (Burns and Honkala 1990) is likely co-evolved with D. pini, an insect found across Europe; and (3) in Europe the sawfly larvae can cause major damage by defoliating trees likely decreasing plant reproductive output (Möller et al. 2017), thus reducing plant fitness.

Notably, D. pini produce about 24 ng of sex pheromone per day, indicating that host-plant species

13 may be able to detect even low amounts of pheromones if the cue still reliably predicts impending herbivory.

Benefits of Understanding this Phenomenon

New pest management strategies are an important potential benefit to dissecting and understanding the interactions between pheromone-emitting herbivore species and their eavesdropping host plant species. There is a need for pest management practices that could limit our reliance on synthetic insecticides, which can negatively influence human and environmental health. New pheromone-based management strategies may be less difficult to implement because pheromones are already a commonly utilized tool for management in terms of trapping, monitoring, and mating disruption (Silverstein 1981). They are also generally non-toxic and can have highly targeted effects because they are species-specific (Yew and Chung 2015). Therefore, prior to infestation growers could preemptively expose crops to the pheromones of a pest species, allowing plants to prime defenses and respond only once the threat is imminent. This tactic would require knowledge of pest cycles, pest pheromones, and timing of crop defenses, but could be a path to follow to develop plausible alternatives to insecticide use.

Conclusion

Based on the work completed thus far, eavesdropping on insect pheromones might be a common ability of plants. Many plant species can detect and respond to airborne chemicals from other plants of the same species or even within species, insect pheromones are often derived from host-plant compounds, and three unrelated plant species acting as illegitimate receivers have already been identified. So, it is likely that there are undiscovered plant species that fit the

14 criteria. This ability of plants to be illegitimate receivers adds an entirely new layer to our understanding of plant-insect interactions in ecological communities and it should be explored in more systems because of the implications for plant defenses, plant-insect coevolution, and pest management. Therefore, future studies should explore other plant species that fit the criteria, especially those of economic or agricultural importance. Studying these relationships could reveal insights about insect pheromone origins, the evolution of plant perception, and ultimately help us begin to decipher the code war between plants and insects.

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Chapter 2

The effects of fall armyworm sex pheromones on defenses in maize and its wild ancestor teosinte

Introduction

Plant species rely on a wide variety of insect-associated environmental cues, ranging from footprints to oral secretions, to initiate well-timed and effective anti-herbivore defenses

(Mescher and De Moraes, 2015). After detecting these cues, plants can respond by inducing defenses immediately or readying, or priming, defenses to prepare for an attack by an insect herbivore (Karban and Baldwin 1997, Conrath et al. 2006, Frost et al. 2008). These induced defenses are energetically more efficient than defenses that are always expressed (i.e., constitutive defenses) because when not threatened by herbivores, plants can allocate resources to other functions (e.g., growth or reproduction; Frost et al. 2008). Defense priming is a state in which plants can prepare to mount defenses without altering gene expression (Engelberth et al.

2004, Karban 2020) and using up precious resources (Medina et al. 2016, Karban 2020). Priming allows for a stronger, quicker, and longer defense response when an attack occurs (Hilker et al.

2016, Conrath et al. 2015, Karban 2020).

Recent studies showed that some plant species can detect the pheromones of co-evolved insect herbivore species and these pheromones can induce or prime anti-herbivore defenses

(Helms et al. 2013, Bittner et al. 2019, Magalhães et al., 2019). These discoveries suggest that plant perception of pheromones, or other insect chemical signals, is a broader phenomenon that warrants further study, particularly if this research can help develop novel approaches to pest management for plant species that are economically or agriculturally significant. To find other

24 examples of eavesdropping host plants, we looked for native plant species with co-evolved native herbivores that release pheromones on or near a host plant and substantially reduce plant fitness.

Maize and teosinte fit this criteria and therefore are good candidates to study.

Maize (Zea mays L. ssp. mays) is a grass native to North America that was domesticated

~ 9000 years ago in southern Mexico (Matsuoka et al. 2002). The movement of maize can be mapped through Mexico into what is now the southwestern United States ~4300 years ago, then into the eastern United States ~2000 years ago (Matsuoka et al. 2002, Merrill et al. 2009, Hart and Lovis 2013). Teosinte is the label that encompasses all taxa in the genus Zea except maize

(De Lange et al. 2014). Zea mays L. ssp. parviglumis, or Balsas teosinte, is native to southwest

Mexico in the Balsas river basin and was recently found to be the direct ancestor of modern maize (Matsuoka et al. 2002, Doebley 2004). Balsas teosinte will be referred to as teosinte for the rest of this paper.

Although teosinte and modern maize are genetically similar, they have very different phenotypes. For instance, teosinte plants have numerous branching sections with ears that bear 5-

12 kernels, which are encased in a hardened shell (Doebley 2004, De Lange et al. 2014). In contrast, maize plants comprise single stalks with ears that bear up to 500 exposed kernels that are fused to the central portion of the ear (De Lang et al. 2014). These differences highlight the consequences for maize of domestication; the hardened shell on teosinte seeds provides protection from pathogens and herbivores, without it maize seeds are more vulnerable to biotic and abiotic threats (Doebley 2004). Additionally, the lack of attachment allows teosinte seeds to disperse easily, whereas maize seeds require human intervention for dispersal (Doebley 2004, De

Lang et al. 2014). Even with different traits, maize and teosinte host similar communities of herbivorous insect species, including fall armyworm (FAW; Spodoptera frugiperda Smith), corn earworm (Helicoverpa spp.), and various species of thrips, aphids, grasshoppers, and weevils (De

Lange 2014).

25 Overall, maize and teosinte are strong candidates for eavesdropping on insect pheromones because they fit well with the criteria described above. For instance, teosinte is native to North America and has evolved with native insect herbivores, such as FAW, likely for millions of years. Likewise, maize is a native crop that hosted FAW since the beginning of its domestication (Gaillard et al. 2018). Fall armyworm, a prolific pest of both maize and teosinte, releases pheromones on or near the plants; female FAW moths climb to the tops of plant canopies and release sex pheromones once a night during their mating phase (Sparks 1979). Once mated, females tend to lay eggs on the undersides of leaves, but when fall armyworm populations are unusually high, they will lay eggs almost anywhere on plants (Sparks 1979). Lastly, FAW larvae are known to inflict extensive damage on maize and teosinte, reducing their fitness (Sparks 1979,

Andrews 1980, Takahashi et al. 2012, Gaillard et al. 2018). Overall, it seems plausible that maize and teosinte could have evolved to use FAW sex pheromones as reliable cues of future damage because the moths consistently release sex pheromones on or near plants and their offspring can strongly and negatively influence plants, with young plants being particularly vulnerable.

One challenge posed by FAW and other moth species is the small amounts of pheromones that moths tend to release. In two of the three systems in which pheromone-induced priming has been discovered, the insect species involved released relatively large amounts of pheromones (2 – 70 µg over 24 h; Helms et al. 2013, Magalhães et al. 2019). In the third system, groups of hundreds of sawflies released less pheromone (~ 500 ng over 24 h) (Bittner et al. 2019).

These amounts are considerably larger than the 3 ng of pheromone that FAW releases in an hour, which is typical of female released lepidopteran pheromones. Female lepidopterans release such low amounts of pheromones to potentially save energy (Greenfield 1981), avoid detection from predators and parasitoids, and/or actively test and filter for suitable males, assuming male sensitivity to pheromone emissions is linked to viability (Greenfield 1981, Cardé and Baker

1984). These hypotheses are not necessarily mutually exclusive. Alternatively, plant illegitimate

26 receivers may have also played a role in selecting for moths that release miniscule amounts of pheromones. Presumably, moths may have once released higher amounts of pheromones that triggered defense responses in eavesdropping host plants. Therefore, selective pressures from the plants may have forced moths to emit lower, undetectable concentrations of pheromones.

Domestication can reduce defensive traits in crops, suggesting that teosinte plants may be more likely than maize to effectively prime their defenses upon detecting lepidopteran pheromones. Crop domestication is a process in which, over time, humans artificially select plants with characteristics that are desirable because of how well they fit standards for taste, yield, and cultivation (Chen et al. 2015). Generally, artificial selection of crop species has led to a “domestication syndrome” in which many crop species share a suite of similar characteristics such as increased size and biomass, compact growth area, faster development, photoperiod insensitivity, and a loss of toxic compounds (Hammer 1984, Chen et al. 2015). Maize is a prime example of the effects of “domestication syndrome” because, as stated earlier, modern maize varieties have an excess of large, exposed kernels, compact, non-branching stalks, faster development, and a loss of chemical defenses. Compared to their wild ancestors, weakening of defenses, in particular, has been found across many domesticated crop species (Lindig-Cisneros et al. 1997, Rosenthal and Dirzo 1997, Gols et al. 2011, Rodriguez-Saona et al. 2011, Davila-

Flores et al. 2013). Maize-focused studies have found that herbivores tend to be more successful on maize than teosinte (Rosenthal and Dirzo 1997, Szczepaniec et al. 2012, Takahashi et al. 2012,

De Lange et al. 2014, Gaillard et al. 2018). Furthermore, studies comparing maize and teosinte have consistently shown a decrease in direct (Davila-Flores et al. 2013) and indirect defenses

(Rasmann et al. 2005, Köllner et al. 2008) in maize lines versus teosinte populations. In the latter case, researchers found that European maize lines and teosinte release (E)-β-caryophyllene, a compound that recruits entomopathogenic nematodes and parasitoids in response to threats from insects in soil or foliage, respectively, whereas North American maize lines appear to have lost

27 this ability some time during domestication (Rasmann et al. 2005, Köllner et al. 2008). This example illustrates that domestication and plant breeding can have important ramifications when plant defenses are not considered in the selection process. These outcomes appear to result from a focus of selection on yield traits that inadvertently render domesticated varieties less resistant to herbivore species (Chen et al. 2015).

Research focusing on gradients of domesticated crops and their wild counterparts have effectively illustrated consequences of yield selection on crop defenses. For example, to understand the effects of high yield on defenses against insect pests, a study focused on herbivore performance on a wild perennial teosinte, annual teosinte, land-race maize cultivar, and modern maize cultivar (Rosenthal and Dirzo 1997). This study found that feeding damage followed the gradient with the perennial teosinte having the least damage and the modern maize cultivar having the greatest amount of damage, indicating that domestication of maize neglected defenses, in favor of traits like fast growth and high yield. Perennial teosinte plants that must devote more resources to defense, due to a greater probability of encountering insect herbivores, grow the slowest and have the lowest yield but are the best defended (Rosenthal and Dirzo 1997, Chen et al. 2015). Similar to other studies on maize, teosinte, and FAW (Szczepaniec et al. 2013,

Takahashi et al. 2012), we focused on the defensive capabilities of a modern maize line and ancestral teosinte subspecies to observe any potential effects of domestication, but also factored in FAW pheromone cues as a potential trigger for defense priming.

Research on perception of insect herbivores by maize and teosinte has broader implications for developing novel methods of pest control. If maize lines were found to detect insect pheromones and prime defenses, then growers could potentially preemptively expose plants to pheromone blends, prime chemical defenses, and allow plants to align defense responses with the timing of infestations. Notably, there already is a market for compounds that prime crop defenses against pests and pathogens (Aranega-Bou et al. 2014, Conrath et al. 2015). β-

28 aminobutyric acid (BABA), for example, is a chemical that can prime plant defenses against pathogens, nematodes, and insects (Conrath et al. 2006, Conrath et al. 2015). Natural compounds such as vitamins, carboxylic acids, and aromatics that can prime plant defenses are desirable because they are typically non-toxic and versatile (Aranega-Bou et al. 2014). Insect pheromones are a newly discovered priming cue that would fit well into this market because they are target- specific, non-toxic alternatives to synthetic pesticides. Alternatively, if teosinte populations are found to have heightened defense responses with pheromone exposure, future work could investigate the genes or mechanisms involved to help improve modern maize lines. This is important because maize is a staple crop worldwide, especially in places like Africa, where small farmers rely heavily on the crop. Research on antiherbivore defenses in maize can contribute to a global movement that is already focused on the control of FAW, a pest which has spread to Asia and Africa and has the potential to cause devastating losses (FAO, 2020).

The goal of this study was to explore if maize and teosinte have evolved to eavesdrop on

FAW sex pheromones and prime their defenses against future larval damage. Furthermore, we explored if domestication has affected these defense responses in maize, compared to its wild ancestor, teosinte. We hypothesized that maize and teosinte plants exposed to a high, mating disruption-like dose of FAW sex pheromones would have less damage and enhanced defenses. In contrast, we hypothesized that plants exposed to a low dose, typical of the amount emitted from a female FAW moth, would have more damage and less pronounced defenses because eavesdropping host plants may have selected for moths that release minute amounts of pheromones to avoid detection. We also hypothesized that exposed teosinte plants would have stronger defense responses than exposed maize plants. Lastly, maize and teosinte would be unresponsive to the sex pheromones of sunflower head moth (SHM; Homeosoma electellum

Hulst), an unassociated lepidopteran that does not attack grasses.

29 To address these questions and hypotheses, we exposed maize and teosinte plants to high and low doses of sex pheromones of FAW along with the additional chemical control of SHM sex pheromones. We observed defense induction by assessing larval feeding damage and measuring levels of jasmonic acid and salicylic acid, phytohormones linked to plant defenses.

Materials and Methods

Plants and Insects

Maize (Zea mays L. ssp. mays) inbred line B73, a standard line in maize research, and

Balsas teosinte (Zea mays L. ssp. parviglumis) were used in the study. B73 seeds were obtained from Dr. Jared Ali’s lab at Pennsylvania State University and the teosinte seeds, which were originally collected in Guerrero Mexico (locality: El Salado/Mazatlan), from the USDA National

Germplasm System (accession Ames 21803).

Plants were grown in a Penn State greenhouse (Scent Mediation Ecology Lab) from

January 2020 to February 2020. The temperature in the greenhouse rooms was held at 21° C during the day and 24° C at night. The lights (Philips 400 W high pressure sodium bulbs) were set to turn on when light levels dropped below 240 watts per square meter. Seeds were potted in

6-in plastic pots with potting soil (Pro-Mix Mycorrhizae BX Mycorrhizae) and a slow-release fertilizer (Osmocote® Time Release Fertilizer; N:P:K = 14:14:14) and the plants were watered through trays. Additional nutrients were provided by adding dissolved fertilizer (Miracle Gro®

All Purpose Plant Food N:P:K = 24:8:16) to the trays every two weeks, along with chelated iron

(Grow More Organic Iron Chelate Concentrate) when iron deficiencies were evident (yellow striping on leaves). Due to differences in growth rate, maize plants were grown for two weeks and teosinte plants were grown for three weeks (three to four fully extended leaves).

30 FAW (Spodoptera frugiperda Smith) were obtained from Benzon Research (Carlisle, PA

USA). Fifty to sixty second instar FAW larvae were shipped in single containers of artificial diet for each trial and were used soon after arrival. Larvae of similar size and weight were chosen for each trial.

Pheromone Lures

Plants were exposed to moth sex pheromones via Pherocon® rubber septa (Trécé®

Incorporated, Adair, Oklahoma) that were loaded by Trécé with requested levels of moth pheromone blends. Four pheromone treatments: 3 ng/h (low FAW), 450 ng/h (high FAW), 450 ng/h (SHM (Homeosoma electellum)), and no pheromone (control) were applied. Blank rubber septa (VWR International, Radnor, Pennsylvania) were used for the control. Sunflower head moth sex pheromones were included as a chemical control to test if the plants had targeted responses to FAW or generalized responses to high amounts of volatile chemicals.

Rubber septa emission rates were based on female moth emissions and mating disruption doses. The low treatment is based on female FAW moths, which release around 3 ng of sex pheromones per hour (Tumlinson et al. 1986). The emission rate of 450 ng/h for the high FAW and SHM treatments was calculated by scaling down moth mating disruption levels provided by

Shawn Steffan, USDA-ARS (Madison, WI), a collaborator who studies mating disruption of lepidopteran pests of cranberries (S. Steffan personal communication); 75 g of active ingredient per hectare of field was calculated down to one square foot. Plants were exposed to low and high amounts of FAW sex pheromones to test their level of sensitivity to the emissions and test the hypothesis that host plants have selected for moths that release such low amounts to avoid detection and defense responses of eavesdropping host plants.

31 Pheromone Lure Emissions

Pheromone emissions from the pre-loaded rubber septa were verified via headspace collections and gas chromatography. To collect emissions from the rubber septa (one per 4 L glass dome), each rubber septum was placed on a two-piece aluminum base platform with openings plugged with cotton and covered with a glass dome. Using an in-house air system, air was forced (6 L per min [LPM]) into domes and used a house vacuum system to suck air out of the glass domes and through an attached filter containing 45 mg of SuperQ (Alltech Associates, location) (1 LPM). After 24 h, the filters were eluted with 150 µL of dichloromethane and analyzed using gas chromatography-flame ionizing detection (GC-FID; Agilent Technologies

7890A) The gas chromatogram had splitless injectors (7683B Series Injector) which were fitted with HP-1 columns (15 m x 0.25 mm x 0.25 µm film thickness; J&W Scientific, Folsom, CA)

(Yip et al. 2019).

To confirm that the pheromone emissions did not degrade over 24 hours, emissions were also collected for one hour at different time points (0 h, 6 h, 12 h, and 24 h) using the steps detailed above. Filters for each time point were eluted with dichloromethane and analyzed with

GC-FID. The peaks in each chromatogram were compared to the FAW and SHM sex pheromone retention times reported in the literature (Underhill et al. 1979, Tumlinson et al. 1986) and the areas under each peak were manually measured to compare the relative amounts of each component over time.

Experimental Setup

The trials took place in greenhouse rooms that were climate controlled and had sufficient natural light. The pheromone-treated plants were spaced as far apart as possible (about 0.6 m)

32 and control plants (no pheromone) were placed in a separate greenhouse room. To maintain similar conditions between the rooms, the lights were turned off and the temperature was held at the same range (21-24° C).

Three- to four-leaf plants (2-wk old maize, 3-wk old teosinte) of each species were randomly selected for pheromone treatment (low, high, SHM, none) and larval presence (FAW larvae, no FAW larvae) in a 2×4 factorial design (n = 24 plants per treatment). For pheromone exposure for each trial, treatment groups were organized into plastic bins without lids (Sterilite 90 qt. Storage Box, 30" L x 19 1/8" W x 13 3/8" H) by species and pheromone treatment (i.e. teosinte low FAW, teosinte high FAW, teosinte SHM, teosinte none, maize low FAW, maize high FAW, maize SHM, maize none). Each bin was permanently assigned to a treatment group and given a random position in the greenhouse each time (six positions in hallway, two positions for controls in greenhouse room). For each trial, four plants from each pheromone treatment group were placed into a corresponding bin. Six trials were performed in total.

Pheromone Exposure

To expose plants to the pheromone treatments, a single rubber septum was placed on top of the soil at the base of each pot. Sheets of organdy cotton cloth were fastened to the tops of each bin with binder clips to allow for airflow and avoid exposing each plant to an exaggerated dose of pheromones (combined emissions from eight rubber septa). Organdy also served to keep pests out of the bins. The rubber septa remained on the plants for 24 h.

After the 24 h exposure period, the rubber septa and harvested leaf samples that would be used in phytohormone extractions to show baseline levels of defense-related phytohormones before herbivory were removed. Therefore, one 3-inch and one 4-inch section along with one 2- inch and one 3-inch section starting from the tip of the second youngest expanded leaves of

33 teosinte and maize, respectively, were clipped. The differing lengths accounted for the changing width of the leaves and each clipped portion weighed roughly 100 mg. All the leaf samples were placed in labeled 2 mL Fast Prep screw top vials, immediately flash frozen in liquid nitrogen, and stored the samples at -80° C.

Herbivore Feeding Assays

After the first tissue collection (0 h), a fine-bristle paint brush was used to add a single, pre-weighed second instar FAW larva to half of the plants. Larvae were placed into the whorl of each plant and covered, including the other half of plants that did not receive larvae, with mesh fabric bags (55.9 cm x 19 cm) that were fastened on with a twist-tie. Larvae were allowed to feed freely for 48 h.

After 48 h, the larvae were removed and weighed. For FAW-free teosinte, one 4-inch and one 3-inch section were clipped from the same youngest expanded leaf. For FAW-free maize, one 3-inch and one 2-inch section were clipped from the same youngest expanded leaf, placed in Fast Prep 2 mL vials, and flash frozen in liquid nitrogen. The full length of the whorl and youngest expanded leaves of FAW-containing plants were photographed on a white sheet with a ruler (Figure 2-1A). After photographing the damaged leaves, one 4-inch and one 3-inch section of the same damaged teosinte leaves were clipped, along with one 3-inch and one 2-inch section from the same damaged maize leaves. As stated earlier, all clipped leaf tissues weighed roughly 100 mg and were placed in Fast Prep tubes, then flash frozen. During the first trial, the liquid nitrogen evaporated, so for the remaining trials, a combination of liquid nitrogen and dry ice was used to ensure that the samples remained frozen. The remaining portions of each plant

(not frozen) were placed in labeled envelopes and dried for 36 h in drying ovens (55° C). All frozen leaf tissue samples were stored at -80° C.

34 Measuring Leaf Damage Area

To quantify the amount of leaf damage by caterpillars, ImageJ (ImageJ 1.52a) software was used. First, the damaged portions of the photographed leaves were colored in using a sketching app (Tayasiu Sketches; Figure 2-1B). The edited images were opened in ImageJ and the area of damage was calculated (8-bit>adjusted threshold>make binary>analyze particles).

Figure 2-1: Images of leaf damage after 48 h of feeding by second instar fall armyworm larvae (A) before and (B) after editing. Damage area was colored in to properly process leaf damage area in ImageJ.

Phytohormone Extractions

To quantify levels of jasmonic (JA) and salicylic acids (SA) in leaves, a subsample of frozen leaf tissues were chosen and phytohormones were extracted using a published protocol

(Schmelz et al. 2003, Schmelz et al. 2004) with some minor alterations. Ten sets of leaf samples were randomly selected from each treatment group for both maize and teosinte (n = 10, two leaf

35 samples per plant [0 and 48 h]; total samples processed = 160 [10 plants x 4 treatments x 2 time points = 80 per species]).

After grinding down the frozen leaf samples, a series of steps were carried out to derivatize the carboxylic acid groups present on the targeted phytohormones and 10 µL of internal standard (10 ng/µL) containing dihydrojasmonic acid, indole-d5-3-acetic acid, d6- salicylic acid, d6-abscisic acid, γ-linolenic acid (Sigma-Aldrich, Saint Louis, Missouri) was added to each vial except the buffer vial, to help determine the quantity of each phytohormone in later steps. Using vapor phase extraction, the methyl esters were isolated and the final product was collected by eluting SuperQ filters with 150 µL of dichloromethane. Each sample was analyzed using GC-MS with isobutane chemical ionization with ion-monitoring (Agilent

Technologies 7890A GC, 5975C inert XL EI/CI MSD with triple axis detector) (Helms et al.

2013).

Quantification of Jasmonic Acid

Each phytohormone sample was analyzed with GC-MS and isotope-labeled internal standards and the quantity of each phytohormone was calculated. To identify JA and SA, GC-

MS with electron ionization was analyzed and retention times were compared with the pure labeled compounds. One hundred ng of methyl jasmonate was also run to pinpoint the correct retention times for cis-JA and trans-JA. The areas under the labeled standard peaks and unlabeled peaks for cis-JA, trans-JA, and SA were manually found to quantify the amount (ng) of each compound per gram of leaf tissue (Yip et al. 2019).

36 Statistical Analyses

The effect of pheromone treatment on FAW feeding damage area and weight gain was analyzed by one-way ANOVA and Tukey post hoc tests at α = 0.05. The effect of pheromone treatment on plant defense responses (total and induced cis-JA, trans-JA, SA) was analyzed with one-way ANOVA and Tukey post hoc tests at α = 0.05. Additionally, linear regression was used to determine the effects of total JA and SA levels (h 48) on FAW feeding damage area and weight gain. To normalize the data, the JA data was transformed. QQ plots indicated that a log transformation resulted in the most normally distributed data, therefore cis and trans-JA were log transformed for each analysis. Simple linear models and tobit models were tested on the JA data.

Unexpectedly, many plants had no detectable levels of cis or trans-JA, so to account for the high number of plants with no induction, generalized linear models (GLM) with binomial distributions were used. All collected variables, including trial, were put into the GLM models to determine the cause of the lack of induction. Total and induced cis and trans-JA values that were zero were excluded from the analyses. Ultimately a linear mixed effects model with random effects included was used to remove variance. The model was refined down to significant variables, resulting in a formula of damage area (or weight gain) = pheromone + (1|trial). Weight gain and damage area could not be included in the same formula because weight gain and damage area are

2 correlated (F1,174 = 122.7, p < 0.0001, RSE = 1.705, R = 0.5824; Figure 2-2). Bin arrangement in the greenhouse was examined to determine if pheromone treatments affected plants in neighboring bins. Categorical variables were assigned to plants based on placement next to low, high, and SHM pheromone treatments and feeding damage area and JA means were compared between categorized plants. The assumptions for linear models (linearity, homoscedasticity, independence, and multicollinearity) were checked while running the analyses. RStudio (version

1.1.463, © 2009-2018 RStudio, Inc.) was used for all statistical analyses.

37

Figure 2-2: Linear regression of mass gain of fall armyworm larvae that fed on maize and teosinte in relation to damage area (Mass Gain = 2.016 + 2.329*damage area, R2 = 0.58).

Results

Pheromone Lure Emissions

Chromatograms for each pheromone lure displayed peaks that corresponded with the main sex pheromone components (Table 2-1; Tumlinson et al. 1986). Both low and high FAW lures (n = 2) had five distinct peaks that correspond with dodecan-1-ol acetate, (Z)-7-dodecen-1- ol acetate, 11-dodecen-1-ol acetate, (Z)-9-tetradecen-1-ol acetate, and (Z)-11-hexadecen-1-ol acetate. The SHM lures (n = 2) had three peaks that correspond with tetradecan-1-ol, Z9- tetradecenol, and Z9, E12-tetradecadienol, or the components of previously described sex pheromone (Underhill et al. 1979) (Table 2-2). The relative ratios of the components in each blend were as expected (Underhill et al. 1979, Tumlinson et al. 1986).

38 The FAW and SHM pheromone emissions collected over the time points of 0 h, 6 h, 12 h, and 24 h displayed the same peaks as the previously mentioned pheromone lure collections and did not degrade over time.

Table 2-1: Observed retention times for compounds detected in fall armyworm pheromone lure emissions. Collected fall armyworm pheromone lure emissions were run through an HP-1 GC column.

Table 2-2: Observed retentions time for compounds detected in sunflower head moth pheromone lure emissions. Collected sunflower head moth lure emissions were run on a HP-1 GC column.

39 Fall Armyworm Feeding Damage and Weight Gain

Caterpillars generally ate less and weighed less on pheromone-exposed maize plants and

FAW pheromone-exposed teosinte plants, but not SHM pheromone-exposed teosinte plants.

Caterpillars on maize plants gained significantly less weight (X2 = 23.8 P < 0.01) and caused less damage (X2 = 22.05, P < 0.01) on pheromone-treated plants. All pheromone-exposed maize plants had caterpillars with significantly lower weight gain with a 35-39% drop in average caterpillar weight compared to the caterpillars on unexposed control plants (Figure 2-3).

Similarly, all pheromone-exposed maize plants had significantly less feeding damage compared to the control plants (Figure 2-4); the low FAW and SHM pheromone-treated plants reduced damage by 36-44% compared to the untreated control, while the damage area of high FAW pheromone-treated plants, though highly significant (P = 0.007), only reduced damage by 26%.

Caterpillars on teosinte plants gained significantly less weight (X 2 = 11.9, P < 0.01) and caused less damage (X 2 = 11.7, P < 0.01) among pheromone treatments as well. The caterpillars on low

FAW pheromone-treated teosinte plants gained significantly less weight compared to caterpillars on the control plants (Figure 2-5) with a 34.3% drop in average weight gain. In comparison, the high FAW and SHM pheromone-treated teosinte plants only had a 22-24% drop in weight gain compared to caterpillars on the control plants and were intermediate to the low FAW and control treatments (P > 0.05). The caterpillars on low and high FAW pheromone-exposed plants caused significantly less damage compared to caterpillars on the control plants (Figure 2-6) with a 37% and 35% drop in damage area, respectively. Caterpillars on SHM pheromone-treated plants only had a 22.6% reduction in damage area compared to the control caterpillars and experienced damage that was intermediate to FAW treatments and the control (P > 0.05).

40

Figure 2-3: Weight gain of fall armyworm larvae that fed on maize plants exposed to low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones (Tukey’s HSD P < 0.05). The bars shown are standard error bars and groups with different letters are statistically different.

Figure 2-4: Leaf damage area caused by fall armyworm larvae that fed for 48 h on maize plants exposed to low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones (Tukey’s HSD P < 0.05). The bars shown are standard error bars and groups with different letters are statistically different. The data shown are not transformed, but statistical analyses were carried out on square root-transformed data.

41

Figure 2-5: Weight gain of fall armyworm larvae that fed on teosinte plants exposed to low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones (Tukey’s HSD P < 0.05). The bars shown are standard error bars and groups with different letters are statistically different.

Figure 2-6: Leaf damage area caused by fall armyworm larvae that fed for 48 h on teosinte plants exposed to low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones (Tukey’s HSD P < 0.05). The bars shown are standard error bars and groups with different letters are statistically different. The data shown are not transformed, but statistical analyses were carried out on square root-transformed data.

42 Phytohormone Analyses

The JA and SA levels in the leaves could not explain the changes in caterpillar feeding or weight gain and were not related to pheromone treatment. After testing every variable, there was no indication as to why there was an absence of induction. The total SA levels varied widely, and did not differ among pheromone treatments (F1,78 = 0.33, P = 0.57). Salicylic acid was detected at low levels in every tissue sample, but there was no evidence that SA increased following caterpillar feeding, nor did SA appear to have an effect on caterpillar feeding (F3 = 0.33, P =

0.57, RSE = 73.42, R2 = 0; Figure 2-7). The levels of JA also did not appear to have an effect on caterpillar feeding. In maize, the levels of cis-JA and trans-JA varied significantly among pheromone treatments (cis: F3,30 = 0.68, P = 0.58; trans: F3,36 = 1.87, P = 0.15) (Figures 2-8, 2-9).

Cis-JA and trans-JA were also not related to feeding damage area (cis: R2 = 0.018; trans: R2 =

0.005) (Figures 2-10, 2-11) or caterpillar weight gain (cis: R2 = 0.01; trans: R2 = 0.001).

Similarly, in teosinte, the levels of cis-JA and trans-JA varied significantly among the pheromone treatments (cis: F3,21 = 1.67, P = 0.20; trans: F3,36 = 2.13, P = 0.11) (Figures 2-12, 2-13). Cis-JA and trans-JA were also not related to feeding damage area (cis: R2 = 0.057; trans: R2 = 0.034)

(Figures 2-14, 2-15) or caterpillar weight gain (cis: R2 = 0.08; trans: R2 = 0.0002). Jasmonic acid levels consistently varied among the pheromone treatments and did not align with reductions in leaf damage and weight gain, suggesting that there was no detectable relationship between JA and caterpillar feeding.

There were not enough replicates to detect any effects of bin placement on defense responses in maize and teosinte. However, the strongest pheromone cues were present within the bins, so it is unlikely that bin placement altered the results in a meaningful way.

43

Figure 2-7: Total salicylic acid in both maize and teosinte leaf tissues as related to damage area (Linear regression P > 0.05).

Figure 2-8: Amount of cis-jasmonic acid present in maize plants exposed to pheromones (low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones) for 24 h and fall armyworm caterpillars for 48 h. Damage area did not have a significant effect on cis-JA levels (P > 0.5). Comparisons were made among pheromone-exposed and caterpillar- exposed plants separately (Tukey’s HSD P < 0.05). N.S denotes no significant differences among the treatment groups and the bars represent standard error. The data shown are not transformed, but the statistical analyses were carried out on log-transformed data.

44

Figure 2-9: Amount of trans-jasmonic acid present in maize plants exposed to pheromones (low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones) for 24 h and fall armyworm caterpillars for 48 h. Damage area did not have a significant effect on trans-JA levels (P > 0.5). Comparisons were made among pheromone-exposed and caterpillar- exposed plants separately (Tukey’s HSD P < 0.05). N.S denotes no significant differences among the treatment groups and the bars represent standard error. The data shown are not transformed, but the statistical analyses were carried out on log-transformed data.

Figure 2-10: Total cis-jasmonic acid in maize leaf tissues as related to damage area (Linear regression P > 0.05).

45

Figure 2-11: Total trans-jasmonic acid in maize leaf tissues as related to damage area (Linear regression P > 0.05).

Figure 2-12: Amount of cis-jasmonic acid present in teosinte plants exposed to pheromones (low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones) for 24 h and fall armyworm caterpillars for 48 h. Damage area did not have a significant effect on cis-JA levels (P > 0.5). Comparisons were made among pheromone-exposed and caterpillar- exposed plants separately (Tukey’s HSD P < 0.05). N.S denotes no significant differences among the treatment groups and the bars represent standard error. The data shown are not transformed, but the statistical analyses were carried out on log-transformed data.

46

Figure 2-13: Amount of trans-jasmonic acid present in teosinte plants exposed to pheromones (low or high doses of fall armyworm pheromones, sunflower head moth pheromones, or no pheromones) for 24 h and fall armyworm caterpillars for 48 h. Damage area did not have a significant effect on trans-JA levels (P > 0.5). Comparisons were made among pheromone-exposed and caterpillar- exposed plants separately (Tukey’s HSD P > 0.05). N.S denotes no significant differences among the treatment groups and the bars represent standard error. The data shown are not transformed, but the statistical analyses were carried out on log-transformed data.

Figure 2-14: Total cis-jasmonic acid in teosinte leaf tissues as related to damage area (Linear regression P > 0.05).

47

Figure 2-15: Total trans-jasmonic acid in teosinte leaf tissues as related to damage area (Linear regression P > 0.05).

Discussion

Maize and teosinte plants were exposed to the pheromone cues of a co-evolved herbivore species to determine if the plant species perceive the cue as an indicator of future herbivory and prime their defenses in response. Because maize responded equally to pheromones from FAW and SHM, it appears that maize is generally sensitive to volatile chemical cues. In contrast, teosinte, the wild ancestor of maize, appeared to only detect and respond to FAW sex pheromones, though there was significant variation in the system despite the large sample size that was used (24 plants per treatment). As expected, JA was strongly inducible in both plant species, but JA concentrations did not align well with the levels of feeding damage that were measured.

For both plant species, caterpillar feeding damage and weight gain were positively correlated, suggesting that FAW larvae are well-adapted to maize and teosinte defenses or that neither species mounted effective defenses against FAW. Among the different direct chemical

48 defense strategies in plants, some plant species have evolved to reduce nutritional value of plant tissues, hinder insect digestion, or degrade structures within the insect digestive system (Chen

2008). For example, plant enzymes can degrade essential amino acids in the midgut of insect herbivores (Chen et al. 2005), certain inhibitory plant proteins can hinder insect digestion (Green and Ryan 1972), and compounds, such as proteases, can digest proteins found in the insect midgut lining (Pechan et al. 2000). Each strategy can be effective at reducing larval growth and development. For instance, a cysteine protease found in maize caused a 60-80% reduction of growth in FAW larvae that fed on the tissues (Pechan et al. 2000). Other studies have shown that

FAW larvae can degrade defensive compounds found in maize (Glauser et al. 2011), therefore

FAW larvae may have counteracted defense compounds in maize tissues. The FAW in the study consistently gained weight as more plant tissue was consumed, indicating that FAW larvae were able to manage any defenses that the plants may have deployed. The absence of variation in the effectiveness in chemical defenses against FAW larvae does not support the hypothesis that pheromone-exposed maize and teosinte plants would have heightened defenses against FAW.

Nevertheless, significant differences in the amount of feeding damage among treatments suggests that FAW larvae were sensitive to maize and teosinte defenses, but these defenses may not have been strongly associated with levels of jasmonic acid, which is only a proxy for a limited portion of plant defense responses.

Weight gain (Fig. 2-3) and larval damage (Fig. 2-4) was reduced on maize plants exposed to pheromones from both FAW and SHM, suggesting that the B73 maize line may have a generalized response to an excess of volatile chemicals or may be sensitive to a broad-spectrum of lepidopteran pheromones. It is possible that the maize plants may have been detecting lepidopteran pheromones that share a similar structure. About 75% of all lepidopteran sex pheromones are considered type I, meaning they are composed of a 10-18 carbon chain with mainly alcohol, or aldehyde functional groups (Ando et al. 2004). The main components of the

49 FAW and SHM sex pheromones fit under this category but differ from each other in the number of carbons, double bond placement, and functional group identity (Underhill et al. 1979,

Tumlinson et al. 1986; Tables 2-1, 2-2). Fall armyworm (family: Noctuidae) and SHM (family:

Pyralidae) share a common ancestor that arose in the mid-cretaceous period approximately 100 million years ago (Kawahara et al. 2019). Although there are differences between the pheromone blend components of these moth species, they still consist of long 14-16 carbon chains with an alcohol or alcohol derivative and 1-2 double bonds, so the receptor or mechanism that binds volatile compounds in maize may use those components to recognized that a general lepidopteran threat is near, leading to a response that affects caterpillar feeding. We do know that maize is sensitive to herbivore-induced plant volatiles (HIPVs) from neighboring maize plants (Engelberth et al. 2004, Ton et al. 2006, Ramadan et al. 2011, Ali et al. 2013), but the responses of maize to insect volatiles is uncertain and the machinery responsible for detecting volatiles is unknown. It should also be noted that domesticated maize has likely come in contact with SHM because of the widespread distribution of wild sunflower (Helianthus annus L.) and cultivated sunflower, a crop that was domesticated ~4,000 years ago in the eastern United States (Harter et al. 2004). Maize, however, is not known to host SHMs, which are mainly found on plants in the family Asteraceae

(Royer and Knodel 2019), so interactions with SHM are unlikely to influence maize fitness. This generalized response of maize did not support the hypothesis that maize plants would respond specifically to the pheromones of its co-evolved herbivore species.

In contrast to larvae on B73 maize plants, the larvae that fed on teosinte plants had reduced weight gain (Fig. 2-5) and feeding (Fig. 2-6) only when exposed to FAW pheromones, indicating that teosinte may have evolved a more targeted detection and response to FAW, although differences among treatments were small. The distribution of teosinte overlaps with

SHM because the moth species is native to North America and is found on many common plants in the family Asteraceae that could easily grow among teosinte populations (Royer and Knodel

50 2019). Like maize, teosinte likely has had some exposure to SHM, but because SHM caterpillars do not attack teosinte there is unlikely to have been any selection pressure for teosinte to detect and response to SHM pheromones. On the other hand, these results suggest that teosinte may have evolved to target and defensively respond to a greater threat (e.g. FAW), supporting the hypothesis that teosinte would detect and respond to the pheromone of its co-evolved herbivore species.

Maize and teosinte plants appeared to detect both low and high amounts of FAW pheromones, as larval feeding and weight gain was lowered in some capacity when plants of both species were exposed to mating disruption levels and natural levels of FAW pheromones. It is important to note that female moths typically release sex pheromones once per night during their mating period (Sparks 1979), so it is assumed that FAW moths would emit pheromones for a relatively short period of time compared to the 24 hour exposure period. However, plants could in theory encounter the collective emissions of moths over each night of mating. Overall, maize and teosinte may recognize FAW as a threat, and show a greater sensitivity and defensive responsiveness to their pheromone cues. The sensitivity to low pheromone levels does not align with the hypothesis that the plants would detect only high amounts of FAW pheromone. Our hypothesis considers that eavesdropping host plants may have helped select for moths that release minute amounts of pheromones, meaning the moths could go undetected by a host plant. If this were an evolved trait in moths, like FAW, then the plants would likely be unresponsive to the small amounts of pheromones emitted by female moths, but sensitive to high concentrations of pheromones.

Salicylic acid, the phytohormone linked to defenses against biotrophic pathogens and some phloem-feeding insects (Pieterse et al. 2012, Thaler et al. 2012), was only detected at low amounts that did not change after larval feeding, therefore it was not responsible for the reduction in damage. This is expected because jasmonic acid is the phytohormone that typically indicates

51 defense induction by chewing insect herbivores (Walling 2000). Studies on maize and teosinte defenses against insect herbivores have also focused on jasmonic acid-related defenses (Qi et al.

2018).

Although larval feeding and weight gain were influenced by pheromone treatments, the levels of jasmonic acid, a phytohormone which signals direct or indirect defensive action (Howe and Jander 2008), varied and did not predictably align with the reduction in feeding damage.

This was an unexpected finding because we hypothesized that maize and teosinte would have primed JA-mediated defense responses in FAW pheromone-exposed plants and would have heightened defenses after larval damage, compared to controls. We also hypothesized that this response would be greater in teosinte plants.

It should be noted that some of the plants with the greatest amount of damage showed no induction of jasmonic acid, indicating that there could be an issue with the mass spectrometry equipment, photoperiod, nutrient balance, and/or the amount of time points collected. We found that the baseline levels on the GC-MS chromatograms were abnormally high when manually calculating the areas under jasmonic acid peaks. A methyl-jasmonate standard was used to indicate the appropriate retention time range, however, trans-jasmonic acid peaks were often overshadowed by a larger, wider peak making it difficult to determine the presence of trans- jasmonic acid. Cis-jasmonic acid peaks were easier to pinpoint, but the baseline was high at times as well.

The maize and teosinte plants were grown outside of their typical growing season and they may not have received appropriate light cues. The plants were provided additional light to simulate summer because each plant was grown in January or February, which is an important step because phytohormones, like jasmonic acid, are affected by photoperiod and jasmonic acid- dependent defenses can be enhanced by longer day lengths (Cagnola et al. 2018). It is possible

52 that jasmonic-acid levels were affected because during winter, many plants in our greenhouse tend to have stunted growth even when lights are kept on longer (personal observation).

Additionally, it is possible that we did not collect enough time points to capture the induction of jasmonic acid for some plants. Other studies used multiple times points that varied anywhere from 4 and 24 h (Chuang et al. 2014) to 24, 48, 72, and 96 h (Acevedo et al. 2019).

However, jasmonic acid induction was detected at each time point. In one study, jasmonic acid induction was the highest 30 minutes after artificial herbivory on maize took place (Bosak et al.

2013).

Iron deficiencies could have also affected jasmonic acid levels; many of the plants displayed evidence of minor iron deficiencies across the growing period (indicated by yellow stripes on foliage (M. Millard personal communication)) and nutritional deficiencies can affect plant defenses. Iron is often involved in phytohormone biosynthesis (jasmonic acid, ethylene)

(Hänsch and Mendel 2009), therefore this could have affected jasmonic acid induction.

It is important to also consider the biological relevance of this interaction. The three other examples of eavesdropping host plants involve an emitting herbivore that remains on a host plant, this is not necessarily true for FAW; female FAW moths have been observed emitting pheromones from maize canopies (Spark 1979) but we do not know whether moths remain on the same host plant and, furthermore, there is no evidence that the moths lay eggs on the plants they emit on. In comparison, male boll weevils (Anthonomus grandis Boheman) likely find suitable host plants and attract females which subsequently mate and lay eggs in the same location

(Magalhães et al. 2019). Goldenrod gall flies (Eurosta solidaginis Fitch) perch on a host plant, release pheromones to attract females, and after mating, females will often lay eggs on the stem of the same plant (Helms et al. 2013). Lastly, sawflies (Diprion pini L.) may also lay eggs where pheromones are released after attracting males and mating (Novak 1976). So, it is possible that female FAW moths do not reliably emit pheromones on the plants on which they lay eggs,

53 meaning that the sex pheromones would not be reliable indicators of future herbivory and the plants would not have evolved to eavesdrop on these cues.

Although there is no support for primed defense induction from the jasmonic acid data, we found a pattern in the defense responses of maize and teosinte against FAW. It appears that maize is broadly sensitive to volatile cues. In comparison, the evidence suggests that teosinte may be more specifically tuned into the sex pheromone cues of its specialist, co-evolved herbivore species. It is surprising that maize plants responded to SHM cues because SHMs do not affect maize fitness, so we expected that maize plants would not waste resources on detecting and responding to the non-threat. Therefore, it is more likely that maize plants are broadly sensitive to volatile chemicals, which may be reflective of an evolved cautiousness against foreign volatile cues. The sensitivity of teosinte to only FAW pheromones is plausible because

FAW is a co-evolved herbivore that poses a serious threat to teosinte plant fitness, therefore host plants would invest resources into targeting and responding to FAW-associated cues.

The observed differences between maize and teosinte responses could be attributed to the length of time each plant species has been exposed to FAW. Teosinte has evolved with FAW for millions of years, which likely allowed time for teosinte to evolve to detect the threat. In comparison, maize has only interacted with FAW for 9000 years. Additionally, targeted defenses against FAW may not have been selected for in maize due to domestication. Studies have provided evidence that humans have selected for high-yielding crops with lower defenses compared to wild ancestors (Chen et al. 2015), however, this is not a consistent result across all studies. It should be noted that other studies have not been able to pinpoint the causes of reduced resistance in crops (Turcotte et al. 2014, Whitehead et al. 2016) and defense induction in some plant varieties is unaffected by domestication (Ballhorn et al. 2008, Rodriguez-Saona et al. 2011,

Chen et al. 2015). A meta-analysis also showed that volatiles were more often strongly induced in domesticated crops than wild ancestors (Rowen and Kaplan, 2016). More generally, the

54 observed differences between responses in maize and teosinte could be related to the tradeoff between plant growth and defense. Growth and defense tradeoffs are widely referenced in plant- insect studies and generally suggest that plants have a limited set of resources that can go towards growth or defense (Coley et al. 1985, Simms and Rausher 1987, Herms and Mattson 1992).

While there are studies that support this tradeoff, there are many other studies that do not (Yip and Tooker, in review). Due to the lack of empirical support and potential oversimplification of growth/defense processes in plants, we cannot attribute the differences observed between maize and teosinte to a selection for yield/growth over defenses.

Conclusion

Future studies are needed to further explore plant illegitimate receivers and compare defense responses between modern crop lines and wild ancestors. Building comparisons between domesticated and wild ancestral plant species in these studies is useful for understanding the evolution of plant defenses, and how humans have altered defenses in modern crops. These studies should include multiple genotypes of domesticated lines and wild populations, specialist and generalist herbivores, and should be conducted in native environments to accurately depict how plants respond to co-evolved insect threats (Chen et al. 2015). Thorough investigations of plant responses to common insect cues, like pheromones, could eventually help improve pest management strategies by utilizing pheromones to preemptively prime plant defenses and influence the development of future crop lines that could be selected for genes involved in detecting and defensively responding to insect pheromones. Developing pest management strategies that utilize manufactured, non-toxic, target-specific resources, like pheromones, and producing crop lines that could effectively detect and respond to insect pheromone cues could be

55 important in the fight against damaging pests like FAW, which has spread globally and has the potential to cause massive losses in crops such as maize, a staple in many countries.

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