LEPIDOPTERA: NOCTUIDAE) with ITS HOST PLANT TOMATO Solanum Lycopersicum and the EGG PARASITOID Trichogramma Pretiosum (HYMENOPTERA: TRICHOGRAMMATIDAE

Home , Adomerus, Codling moth, Helicoverpa, Helicoverpa zea, Trophic egg

The Pennsylvania State University

The Graduate School

Department of Entomology

OVIPOSITION-MEDIATED INTERACTIONS OF TOMATO FRUITWORM MOTH Helicoverpa zea (LEPIDOPTERA: NOCTUIDAE) WITH ITS HOST PLANT TOMATO Solanum lycopersicum AND THE EGG PARASITOID Trichogramma pretiosum (HYMENOPTERA: TRICHOGRAMMATIDAE)

A Dissertation in

Entomology

Jinwon Kim

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

May 2013

The Dissertation of Jinwon Kim was reviewed and approved* by the following:

Gary W. Felton, Ph.D.

Professor and Department Head of Entomology

Dissertation Advisor

Chair of Committee

John F. Tooker, Ph.D.

Assistant Professor of Entomology

James H. Tumlinson, Ph.D.

Professor of Entomology

Dawn S. Luthe, Ph.D.

Professor of Plant Stress Biology

*Signatures are on file in the Graduate School

ABSTRACT

An increasing number of reports document that, upon deposition of insect eggs, plants induce a

variety of defenses to remove the eggs from plant tissue using plant toxic compounds or

lending a hand of egg predators and egg parasitoids. In this research, I explored the interactions of tomato fruitworm Helicoverpa zea Boddie (Lepidoptera: Noctuidae) with its host plant tomato Solanum lycopersicum L. (Solanales: Solanaceae) and its egg parasitoid Trichogramma pretiosum (Hymenoptera: Trichogrammatidae) mediated via H. zea eggs laid on tomato plants.

In Chapters 2 and 3, tomato’s defensive response to H. zea oviposition was investigated, and in

Chapter 4, a novel defense mechanism of H. zea eggs against the egg parasitoid T. pretiosum was explored. The tomato fruitworm moth, H. zea, lays eggs on tomato plants and the larvae emerging from the eggs consume leaves first and then fruit to cause serious loss in the plant fitness. However, little is known about the oviposition-inducible defenses in tomato. When tomato plants were exposed to H. zea eggs, hydrogen peroxide (H2O2) was produced and pin2 expression was induced in the leaf tissue beneath H. zea eggs. H2O2 functions as a secondary messenger compound between early responses (e.g. activation of wound signaling pathway) and late responses (e.g. expression of defense traits such as protease inhibitors) in tomato, and pin2 is the gene encoding a well-studied induced defense trait of tomato, of which the expression indicates the level of induced defense in this plant. I also found that pin2 expression at the H. zea oviposition sites reached the highest right before the emergence of larvae from

the eggs. More importantly, H. zea oviposition primed tomato antiherbivore defensive responses. Jasmonic acid (JA), the plant hormone responsible for the activation of defenses against insect herbivores, is quickly and transiently produced in plant tissue when plants are challenged by chewing insects or mechanical damage. The level of JA accumulation in plant tissue represents the level of antiherbivore defenses in the plant. Tomato plants previously exposed to H. zea oviposition induced higher levels of both pin2 expression and JA accumulation upon mechanical damage than when without oviposition pretreatment. These results suggest that tomato antiherbivore defenses are primed by H. zea oviposition in preparation for the future herbivory by neonates that emerge from the eggs. Unfertilized eggs of H. zea also elicited pin2 expression at the oviposition site, but did not prime the defensive gene expression, indicating that tomato is able to distinguish the real future threat (i.e. viable fertile eggs) from the false alarm (i.e. inviable infertile eggs). Tomato also showed a varietal variation in the oviposition priming. The tomato cultivar Better Boy that was used throughout this research primed pin2 expression by H. zea oviposition as stated above, while another tomato cultivar Castlemart failed to prime pin2 expression upon H. zea oviposition. More interestingly, in the JA-deficient mutant of Castlemart, def-1, H. zea eggs suppressed pin2 induction upon the following wound treatment. The effect of priming of defenses by H. zea oviposition on the performance of H. zea neonates was dynamic. In one experiment, H. zea showed decreased performance on tomato plants pretreated with H. zea oviposition, but in the other experiment, previous H. zea oviposition treatment did not influence the growth and survival of H. zea neonates. Interestingly, some neonates were found feeding inside of rachises

iii

of tomato plants, and they apparently grew faster than other leaf-eaters. This rachis-boring behavior of H. zea neonates might be one of the reasons of the inconsistent results and an adaptation of H. zea neonates to cope with the decreased quality of food plant by induced defense in tomato. In Chapter 4, I tested the hypothesis that H. zea unfertilized eggs may function as a lethal trap of T. pretiosum. H. zea virgin females laid significantly fewer unfertilized eggs than fertilized eggs laid by mated females in the absence of tomato plants.

However, when tomato plants are present, H. zea virgin females laid as many unfertilized eggs on tomato plants as mated females lay fertilized eggs. It was also found that, when the population density is high, H. zea females may remain unmated in the presence of males and lay unfertilized eggs on the host plants, implying male mate choice. T. pretiosum egg parasitoids not only parasitized H. zea unfertilized eggs but also preferred them as the host to the fertilized eggs. Many of H. zea unfertilized eggs desiccated in a few days after parasitization by T. pretiosum, and the undesiccated eggs were almost completely parasitized, meaning the parasitization rate of T. pretiosum on H. zea unfertilized eggs is almost 100%. While T. pretiosum successfully emerged from 90% of H. zea fertilized eggs, only half of H. zea unfertilized eggs allowed successful development and emergence of T. pretiosum, mainly because of desiccation of the unfertilized eggs. These results demonstrate that H. zea unfertilized eggs can function as a lethal trap of T. pretiosum egg parasitoids. The results of this dissertation provide valuable insight into the nature of the interactions between tomato and H. zea and between H. zea and T. pretiosum mediated by H. zea eggs deposited on tomato plants.

TABLE OF CONTENTS

LIST OF FIGURES ·································································································································ix

LIST OF TABLES ···································································································································xi

ACKNOWLEDGEMENTS ······················································································································xii

CHAPTER 1: Introduction ······················································································ 1

PLANTS AND INSECTS ·························································································································2

PLANT DEFENSES AGAINST INSECT HERBIVORES ··············································································4

JASMONATE SIGNALING PATHWAY ···································································································5

PLANT EARLY RESPONSE TO FUTURE HERBIVORY ·············································································6

PLANT EGG-INDUCIBLE DEFENSIVE RESPONSES ················································································7

UNFERTILIZED EGGS OF INSECTS ·······································································································8

CHAPTERS ···········································································································································10

PRIMING OF ANTIHERBIVORE DEFENSIVE RESPONSES IN PLANTS ···················································12

Abstract ········································································································································13

Introduction ·································································································································14

HIPV-Mediated Priming of Defense ·····························································································16

Non-HIPV-Mediated Priming of Defense ·····················································································18

Transgeneration priming of defense ·····················································································19

Priming of defense by insect oviposition ···············································································20

Priming of defense by seed treatment ··················································································22

Priming of defense by heavy metal stress ·············································································23

Molecular Mechanisms of Defense Priming ················································································24

Specificity of Primed Defenses ····································································································28

Summary ······································································································································30

REFERENCES ·······································································································································31

TABLES ················································································································································51

FIGURES ··············································································································································56

CHAPTER 2: Insect eggs can enhance wound response in plants: A study system

of tomato Solanum lycopersicum L. and Helicoverpa zea Boddie ·························· 58

ABSTRACT ···········································································································································59

INTRODUCTION ··································································································································60

RESULTS··············································································································································63

Tomato perceives H. zea oviposition and induces defensive responses at the oviposition Site 63

Helicoverpa zea oviposition elicits H2O2 accumulation at the oviposition site on tomato

foliage ·······························································································································63

Pin2 is expressed at the H. zea oviposition site and the level of expression decreased

with distance from the egg ·····························································································63

Pin2 expression at the oviposition site was highest just before the emergence of

neonates ··························································································································64

Unfertilized eggs induced pin2 as well ··················································································64

Induction of pin2 and accumulation of JA were primed by H. zea oviposition for subsequent

simulated H. zea herbivory ····································································································65

DISCUSSION ········································································································································67

MATERIALS AND METHODS ···············································································································73

REFERENCES ·······································································································································79

FIGURES ··············································································································································86

CHAPTER 3: Varied responses of tomato to Helicoverpa zea oviposition ··············· 93

ABSTRACT ···········································································································································94

INTRODUCTION ··································································································································95

RESULTS··············································································································································97

Helicoverpa zea oviposition does not prime pin2 induction in Castlemart, and suppresses

pin2 response in the JA-deficient mutant def-1 ······························································97

Unfertilized eggs of H. zea do not prime pin2 induction in tomato plants ···························98

Extract of the accessory glands of H. zea adults do not induce pin2 in tomato plants ·········99

vii

The effect of H. zea egg-induced defense priming on the performance of H. zea

neonates ··························································································································99

H. zea Neonates perform better on rachises than on leaves of tomato ·······························100

DISCUSSION ········································································································································102

MATERIALS AND METHODS ···············································································································107

REFERENCES ·······································································································································111

FIGURES ··············································································································································114

CHAPTER 4: Why do Helicoverpa zea lay unfertilized eggs?: Defensive function of unfertilized eggs laid by Helicoverpa zea virgin females against Trichogramma pretiosum egg parasitization ················································································· 120

ABSTRACT ···········································································································································121

INTRODUCTION ··································································································································122

RESULTS··············································································································································124

Under What Conditions H. zea Virgin and Mated Females Lay Fertilized and Unfertilized Eggs?

················································································································································124

Fecundity and fertility of the eggs laid of virgin and mated females of H. zea ·····················124

The presence of H. zea male moths does not stimulate the deposition of unfertilized

eggs by virgin females ······································································································125

viii

Tomato plants strongly stimulate deposition of unfertilized eggs by H. zea virgin females

··········································································································································125

Can H. zea Unfertilized Eggs Function as a Lethal Trap of T. Pretiosum Parasites? ····················126

Unfertilized eggs laid by H. zea virgin females are not preferred as a food source by

neonates ··························································································································126

T. pretiosum not only parasitizes unfertilized eggs of H. zea but also prefers them to

fertilized eggs ···················································································································127

T. pretiosum is trapped and die in the desiccating unfertilized eggs of H. zea ·····················128

H. zea females remain unmated and lay unfertilized eggs in the presence of males when

the population density is high ·························································································129

DISCUSSION ········································································································································131

MATERIALS AND METHODS ···············································································································136

REFERENCES ·······································································································································144

CHAPTER 5: Conclusions························································································ 160

LIST OF FIGURES

Fig 1-1. Priming of antiherbivore defensive responses ····································································56

Fig 2-1. Response of tomato leaves to H. zea eggs at the oviposition site ·······································86

Fig 2-2. Intensity of pin2 induction with distance from eggs ····························································87

Fig 2-3. Temporal fluctuation of transcriptional level of tomato pin2 at the H. zea oviposition

site ········································································································································88

Fig 2-4. Effect of the egg fertility on tomato pin2 expression at the H. zea position site ·················89

Fig 2-5. Priming effect of H. zea oviposition on tomato pin2 expression ·········································90

Fig 2-6. Priming effect of H. zea oviposition on JA levels in tomato leaves ······································91

Fig 2-S1. Effect of trichome disruption on the level of pin2 expression upon the subsequent

mechanical wounding and application of H. zea OS ····························································92

Fig 3-1. Expression of pin2 in tomato cultivar Better Boy in response to H. zea oviposition upon

the following wound treatment ···························································································114

Fig 3-2. Expression of pin2 in tomato cultivar Castlemart (CM) and its JA-defective mutant, def-1,

in response to H. zea oviposition upon the following wound treatment ····························115

Fig 3-3. Tomato response to deposition of H. zea unfertilized eggs and the following wound

treatment in the cultivar Better Boy ····················································································116

Fig 3-4. Effect of H. zea accessory gland extracts on tomato pin2 expression in tomato cultivar

Better Boy ·····························································································································117

Fig 3-5. Effect of H. zea oviposition on the performance of H. zea neonates on tomato cultivar

Better Boy ·····························································································································118

Fig 3-6. Neonatal growth and food consumption efficiency of H. zea on tomato leaflets and

rachises of tomato cultivar Better Boy ·················································································119

Fig 4-1. Lethal trap hypothesis ···········································································································152

Fig 4-2. Fecundity and fertility of H. zea females mated with a different number of males ············153

Fig 4-3. Effect of male presence on the the number of eggs laid by H. zea virgin females ··············154

Fig 4-4. Effect of tomato foliage on the number of unfertilized eggs laid by H. zea virgin females. 155

Fig 4-5. The size of fertilized and unfertilized eggs of H. zea ····························································156

Fig 4-6. Initial feeding behavior of H. zea neonates ··········································································157

Fig 4-7. Parasitization of H. zea eggs by T. pretiosum ·······································································158

Fig 4-8. Effect of the number of H. zea male-female pairs on H. zea egg fertility ····························159

LIST OF TABLES

Table 1-1. Chemical structure of HAMPs and the original insect species used for isolation ············51

Table 1-2. HIPV-mediated priming of antiherbivore defensive responses ·······································52

Table 1-3. Non-HIPV-mediated priming of antiherbivore defensive responses ·······························54

Table 1-4. Specificity of effect ···········································································································55

Table 4-1. T. pretiosum choice test between fertilized and unfertilized eggs of H. zea ···················151

Table 4-2. Mortality of T. pretiosum in fertilized and unfertilized eggs of H. zea ·····························152

xii

Acknowledgement

At the ESA meeting at Indianapolis in 2006, I had a chance to have lunch with my advisor Dr.

Gary W. Felton and someone who used to be his Ph.D. student. At one point, out of the blue,

his old student said, “Thank you, Gary, for everything. I am indebted” and Gary said smiling,

“For what?” … It did not take me long to understand what made him so grateful to Dr. Felton. I have been here at Penn State for more than 6 years, and many things happened, but he has

been nothing but supportive and supportive and supportive, and always wished the best to his

students. About a month ago, I told him “Thank you for everything,” and he said smiling “For

what?” So, I told him, “I knew that you would say that.” I am deeply indebted to my advisor, Dr.

Felton.

I would also like to thank my Dissertation Committee members, Drs. Luthe, Tooker and

Tumlinson, for their support, advice and guidance. It was always my great pleasure to discuss my results with the best scientists in the area of plant-insect interactions. Many faces are

floating in my mind; all the faculty members in this wonderful Entomology program who taught

me a lot with passion in the class, Drs. Kelly Hoover, Nancy Ostiguy, Diana Cox-Foster, James

Frazier, Shelby Fleischer, Mike Saunders, K.C. Kim, Chris Mullin, Ottar Bjørnstad, Consuelo De

Moraes, Mark Mescher, Christina Grotzinger, Harland Patch, Tom Baker and James Marden,

and Dr. Dietmar Schwartz who is now in West Washington State University; all the angel-like

staff members LuAnn, Ellen, Nick, Thelma, Karen, Greg, Maryann Steve, Dave, Scott, Marcia,

xiii

and Roxie. I miss Roxie a lot; and all the blessed Entomology graduate students, especially the year 2006 members, Christy, Alexis, Raj, Kerry and Shaun, Tracy, and my smoking crew David (I am sorry David doesn’t smoke any more). I am grateful to the Penn State Entomology who made me from a chemist into an Entomologist, an ecologist, and a scientist.

I am thankful to my lovely Felton lab members, Michelle, Joe, Gloria, Auzzie, Loren and Flor (say hi to Sophia for me!), and Luthe lab members, Casper, Torrence, and “Everybody Loves” Ray. I would especially like to thank Seungho and his wife Hyojung who have been my best friends as well as best scientists, and their lovely kids Kyle Jihoon and Amelia Jane, for the great time we shared. And, Vanessa and her husband Edgar, and their son Giovani, oh, I am going to miss them so much. I miss Raul “The Great” Ruiz and his Queen Oralia, the nicest people on the earth. And, I thank my best friends Min-Cheol and Hee-Jong for their ever-lasting support and friendship.

For my parents, their love, support, and sacrifice, I can’t even find the right words to thank them but I love them. I am most deeply indebted. I am also thankful to my brother Jin-Beom and his wife Song-A for their bottomless support and for being the eldest son and daughter-in- law in the family while I am absent. Most of all, I am deeply grateful to my dearest daughter,

Irene, for growing so well.

I thank all of them I named above and others I don’t remember right now. Without them, it would never have been impossible. I am grateful for everything that has happened to me, good or bad, that has made me what I am today.

CHAPTER 1:

Introduction

Plants and herbivorous insects have been engaged in the ever-lasting arms race since the first

herbivory on terrestrial plants about 400 million years ago (Futuyma and Agrawal, 2009;

Labandeira, 2007). Plants and herbivorous insects in the present time possess a variety of

mechanisms to defend against, and to disarm the defense of, their opponents as a consequence of reciprocal adaptations and evolution (Ehrlich and Raven, 1964; Futuyma and Agrawal, 2009).

Recently, a growing body of literature indicates that plants recognize insect eggs deposited by herbivorous insects as an impending threat to the plant fitness and induce a variety of defenses before herbivorous damage occurs (Hilker and Meiners, 2006). Considering many herbivorous insects deposit eggs on their host plants, egg-inducible plant defenses may be widespread.

In this research, I studied interactions between tomato fruitworm moth, Helicoverpa zea, and

its host plant tomato, Solanum lycopersicum, and between H. zea and its egg parasitoid,

Trichogramma pretiosum, mediated by H. zea eggs laid on tomato plants. Egg deposition by H. zea on tomato plants is an important event for all of these organisms. In the interactions between tomato and H. zea, careful selection of oviposition site by H. zea mother can be critical for the fitness of her offspring because the mortality rate of insect eggs and neonates is usually very high (Gripenberg et al, 2010; Zalucki et al, 2002; Srivastava et al, 2005). For tomato plants,

H. zea eggs on plant tissue are the prelude of the impending herbivory by the hatchlings, and

induction of proper defense mechanisms to lower the fitness of insect eggs or neonates should

be beneficial. In the interactions between H. zea and T. pretiosum, the egg parasitoid is a serious threat to the survival of H. zea eggs, but no defense of H. zea eggs against T. pretiosum

parasitoids was reported yet. For, T. pretiosum, the mother has a small window of time to

parasitize H. zea eggs, i.e. after egg deposition before larval emergence, which is usually 2-3

days, and thus the fast and efficient location and parasitization of the host eggs is essential for the fitness of the offspring.

This research comprises two main parts: tomato’s response to H. zea oviposition and the defense of H. zea against T. pretiosum. Chapters 2 and 3 aim to understand tomato responses to H. zea oviposition. The main hypothesis of Chapter 2 is that H. zea oviposition primes antiherbivore defensive responses in tomato plants. In Chapter 3, various aspects of tomato response to H. zea oviposition were studied as a follow-up of the Chapter 2. In Chapter 4, I investigated the possible role of unfertilized eggs laid by virgin H. zea females on tomato plants as a defense mechanism against T. pretiosum. The hypothesis of the Chapter 4 is that H. zea unfertilized eggs may function as a lethal trap of the parasites of T. pretiosum.

Plants and Insects

Tomato is one of the most economically important crop plants ranked 10th in the annual

production with over 150 billion kg and 4th in the net production value with over $55 billion

worldwide in 2010 (FAOSTAT, 2010). Tomato hosts 100-200 species of arthropod pests

worldwide (Lange and Bronson, 1981), and among the most serious pests is tomato fruitworm.

Tomato fruitworm moth, H. zea, is a lepidopteran insect in the family Noctuidae. H. zea inhabits

in the New World, and its closest relative species in the Old World and Australia is Helicoverpa armigera. The phylogenetic analysis of H. zea revealed that H. zea in North and South America is a single species and was established via inter-continental founder event from H. armigera

(Behere et al, 2007). The tomato fruitworm is a generalist phytophagous insect, and its host plants include many important agricultural crops such as tomato, maize, cotton, soybean, sorghum, sugar cane, and strawberry (Matthews, 1991). This insect is called by a few common names, tomato fruitworm, corn earworm, and cotton bollworm, according to what host plants

H. zea is eating. On tomato plants, H. zea adults prefer leaflets adjacent to flower blossoms for oviposition, and tomato plants with more open flowers are more attractive to H. zea adults for oviposition (Snodderly and Lambdin, 1982; Alvarado-Rodriguez et al, 1982; Fitt, 1991).

T. pretiosum is an egg parasitoid that is important for control of H. zea (Oatman and Platner,

1971; Martin et al, 1976; Hoffmann et al, 1990) and can be mass-produced (King and Coleman,

1989). Parasitization rate of T. pretiosum on H. zea eggs is about 95% in laboratory conditions

(Nogueira de Sá and Parra, 1994), while it was varied in the field experiments approximately between 50-85% (Oatman and Platner, 1971; Martin et al, 1976; Hoffmann et al, 1990; Romeis et al, 2005). The egg parasitoid lays 2-3 eggs per H. zea egg (Nogueira de Sá and Parra, 1994) and one egg per Sitotriga cereallela egg (Brower JH, 1983). It appears that T. pretiosum adults regulate the number of eggs laid per host egg depending on the size of the host egg (Bai et al,

1992). T. pretiosum was found to be attracted by H. zea sex pheromone (Noldus, 1988). The

hexane extract of tomato plants stimulates T. pretiosum parasitization on H. zea eggs, but

maize extract does not change the egg parasitization behavior (Nordlund et al, 1985). T.

pretiosum-parasitized eggs of Manduca sexta lose water and emit CO2 passing through the egg

shell. So do unparasitized eggs, but at different rates (Potter and Woods, 2012).

Plant Defenses against Insect Herbivores

Plant defenses to cope with insect herbivores can be categorized into direct defenses and indirect defenses. Direct defenses include repellents and toxic compounds produced by the host plant (Howe and Jander, 2008). Indirect defenses allow host plants to be protected by the natural enemies of herbivorous insects (e.g. parasitoids, predators, entopathogenic nematodes)

(McCormick et al, 2012). Plant defenses also can be grouped into constitutive and induced defense. The defensive traits whose expression is consistent irrespective of insect herbivory stimulation are called constitutive defenses, while the defensive traits whose expression level increases in response to herbivory are called induced defenses. Increasing evidence indicate that induced defense may be more favored than constitutive defenses with minimized cost and enhanced defense efficiency (Agrawal and Karban, 1999; Zangerl, 2003). Upon insect herbivory, plants recognize insect-derived cues (e.g. continuous feeding damage, herbivore-associated molecular patterns or HAMPs; Table 1-1) (Felton and Tumlinson, 2008) and induce a specific set

of defenses depending on a given insect species, which is critical for the evolution of induced defense in plants (Agrawal and Karban, 1999; Zangerl, 2003).

Jasmonate Signaling Pathway

JA is a plant hormone that is responsible for plant antiherbivore defenses or wound response

(Howe and Jander, 2008). The JA biosynthetic pathway starts α-linolenic acid released from the chloroplast membrane lipids. α-linolenic acid is processed into 12-OPDA (12-oxo-phytodienoic acid) via lipoxygenase (LOX), allene oxide synthase (AOS) and allene oxide cyclase (AOC) in chloroplast. Then, 12-OPDA is transported from chloroplast to peroxisome and is catalyzed by

OPDA reductase and subject to three consecutive ẞ-oxidations, producing the final product JA

(Howe, 2010). JA is accumulated in the plant tissue quickly and transiently after mechanical

wounding, herbivory, or defense elicitor treatment. Accumulated JA is transformed into the

active form JA-Ile conjugates. In unstressed plants, jasmonate ZIM-domain (JAZ) protein binds

to and repress MYC2 transcription factor, which is responsible for the activation of JA-response

genes in the downstream. Upon wounding, herbivory, or elicitor treatment, increased JA-Ile

conjugates form a complex with JAZ and SCFCOI1, releasing the JAZ repressor from the MYC2

transcription factor. The JAZ repressor is then marked by ubiquinylation by the SCF moiety of

the complex to be degraded by 26S proteosome. The MYC2 transcription factor liberated from

JAZ protein activates JA-response genes (Thines et al, 2007; Howe, 2010).

Plant Early Response to Future Herbivory

Many reports describe early perception by plants of signs of future herbivory followed by

induction of defensive response (Hilker and Meiners, 2010). These early-induced defenses are

considered more effective and beneficial because plants may avoid or reduce initial feeding

damage by eliminating future enemies or by preparing appropriate defenses before the herbivory occurs. The three early events that plants take as possible future herbivory reported up to now are insect walking on plant tissue, herbivore-induced plant volatiles released from

neighboring plants suffering herbivore attack, and insect oviposition on the host plant (Kim et al,

2011). Some plants induce defensive responses on leaf tissue in response to insect walking, a

common behavior in searching for sites suitable for feeding or oviposition (Bown et al, 2002;

Peiffer et al, 2009). Some plants induce or prime defenses after exposed to the herbivory volatile signals from the neighbors (Heil and Karban, 2009). In the so called plant-plant communication, the receiver plant appear to take the volatile signals from the emitter as the increased risk of herbivory in the near future (Engelberth et al, 2004; Ton et al, 2007; Heil and

Silva Bueno, 2007; Heil and Karban, 2009). Priming of defense is defined as increased readiness of induced defense, and primed plants induce defenses more quickly and strongly upon the anticipated stresses (Conrath, 2009; Conrath, 2011; Kim et al, 2012). Priming of antiherbivore

defensive responses in plants is comprehensively reviewed at the end of this chapter. Plant

defenses induced by insect oviposition are discussed below.

Plant Egg-Inducible Defensive Responses

Deposition of insect eggs induces various responses in the host plants. Oviposition may change

plant volatile profiles (Colazza et al, 2004a; Bruce et al, 2010; Tamiru et al, 2011; Peñaflor et al,

2011; Fatouros et al, 2012), gene expression (Köpke et al, 2008; Fatouros et al, 2008; Beyaert et al, 2012), photosynthetic rate (Schröder et al, 2005; Velikova et al, 2010), and epicuticular chemistry on the leaf surface (Blenn et al, 2012). Insect oviposition may induce indirect defenses in the host plant. Many reports documented that oviposition-treated plants attract or arrest either egg parasitoid (Hilker et al, 2002; Colazza et al, 2004a,b; Fatouros et al, 2005;

Fatouros et al, 2008; Büchel et al, 2011) or larval parasitoid (Bruce et al, 2010) or egg-larval parasitoid (i.e. parasitization in the host egg- adult emergence from the host larva) (Deshpande and Kainoh, 2012), or both of egg and larval parasitoids (Tamiru et al, 2011; Fatouros et al,

2012). It was reported that egg parasitoids are not attracted to the host plants once eggs hatch

(Colazza et al, 2004b). Egg parasitoid females usually use olfactory information to locate egg- laden plants (Hilker et al, 2002; Colazza et al, 2004a,b; Fatouros et al, 2008; Bruce et al, 2010;

Büchel et al, 2011; Tamiru et al, 2011), but sometimes information gathered by contact chemoreception is critical (Fatouros et al, 2005; Deshpande and Kaino, 2012).

Host plants may show different responses to the eggs of generalist and specialist herbivorous insects. Brassica nigra is the host plant of a specialist Pieris brassicae and a generalist Mamestra brassicae. In response to the specialist oviposition, the host plant attracts both the egg parasitoid Trichogramma brassicae and the larval parasitoid Cotesia glomerata, whereas oviposition by the generalist on the host plant did not catch the interest of either of two

parasitoids (Fatouros et al, 2012). Some herbivorous insects show adaptations to the host

plant’s egg-induced defenses. The adults of Pieris brassicae and Spodoptera frugiperda were

found to suppress the defensive responses of their host plants, Arabidopsis thaliana and maize, respectively, via oviposition (Little et al, 2007; Bruessow et al, 2010; Peñaflor et al, 2011). A leaf beetle, Pyrrhalta viburni, shows aggregative oviposition behavior and prefers to oviposit on the plants with conspecific eggs previously laid to decrease egg mortality rate caused by the egg crushing defense of Viburnum spp., implying the Allee effect (Desurmont and Weston, 2011).

Unfertilized Eggs of Insects

Insects in many orders lay unfertilized eggs for asexual reproduction, for nutritional purposes, or for unknown reasons. Unfertilized eggs laid by parthenogenetic insects are viable and develop into sexually mature individuals. Parthenogenesis is taxonomically widespread among insect orders (Chapman, 1995). Some insects lay unfertilized eggs, so called trophic eggs, to

feed larvae hatching from viable fertilized eggs (Perry and Roitberg, 2005). Trophic eggs are

found in many eusocial and subsocial insect groups and rarely among non-eusocial insects, but

have not been reported in Lepidoptera (Perry and Roitberg, 2006). Trophic eggs are often

different in shape, color, size, surface structure, deposition location, or laying order, from fertile

eggs (West and Alexander, 1963; Henry, 1972; Nakahira, 1994; Kudo and Nakahira, 2005; Kudo

et al, 2006; Ento et al, 2008; Filippi et al, 2009; Baba et al, 2011), and are selectively consumed

by neonates as their first food. Insect larvae provisioned with trophic eggs show enhanced

performance compared to those that grew without trophic eggs (Mockford, 1957; West and

Alexander, 1963; Henry, 1972; Crespi, 1992; Kudo and Nakahira, 2004; Hironaka et al., 2005;

Kudo and Nakahira, 2005; Perry and Roitberg, 2006; Kudo et al., 2006; Ento et al., 2008; Filippi

et al., 2009, Baba et al., 2011). The mother of insects producing trophic eggs may regulate the ratio of trophic eggs over fertile eggs according to the nutritional conditions. For example, when a lady beetle mother is provided a sufficient number of aphids, she lays more fertile eggs and less trophic eggs than when she is starved (Perry and Roitberg, 2005). For this reason, trophic eggs are considered an adaptive phenotype of the mother (Crispi, 1992). In the cases other than parthenogenesis and trophic eggs, unfertilized eggs are mostly laid by mated females in a small number probably due to e.g. insufficient sperm supply (Perry and Roitberg,

2005; Witzgall et al., 2005; Wallace et al., 2004; Vickers, 1997).

Some reports describe deposition of unfertilized eggs by virgin females of some lepidopteran

species: six species in Noctuidae (Fehrenbach et al., 1987; Adler et al., 1991; Wang and Dong,

2001; Gemeno et al., 1998), two species in Tortricidae (Rivet and Albert, 1990; Wallace et al.,

2004; Fehrenbach et al., 1987), and the gypsy moth in Lymantriidae (Richerson et al., 1976).

Virgin females of H. zea and Spodoptera ornithogalli are known to lay unfertilized eggs. While mated females lay more fertilized eggs in a few days after mating, virgin females of these two noctuid moths lay fewer unfertilized eggs somewhat consistently for many days (Adler et al,

1991). Oviposition behavior of mated and unmated females was investigated with a tortricid moth, Choristoneura fumiferana. Almost all the behaviors examined were different between mated and unmated females, and while mated females laid fertilized eggs in masses of 20-30

eggs, virgin females laid unfertilized eggs singly in small scattered batches of 1-5 eggs (Wallace

et al, 2004; Rivet and Albert, 1990). Deposition of unfertilized eggs by virgin females of the

gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae), has long been known as “spewing”

(Richerson et al, 1976). Gypsy moth females spew eggs before they start to attract males, but little else is known. Why virgin females of some lepidopteran species lay unfertilized eggs is

unknown.

Chapters

In the Chapter 2, I investigated tomato response to H. zea oviposition. Different from other oviposition-induced plant defenses reported up to now, whether H. zea oviposition influences the quality of host plants as a food source of the conspecific neonates that hatch from the eggs.

Recently, Beyaert et al (2012) demonstrated that insect oviposition by pine sawfly, Diprion pini, may reduce the quality of the host plant Scots pin, Pinus sylvestris. The larvae of D. pini that hatched and were grown on the same twigs of P. sylvestris showed reduced performance than those grown on the oviposition-free twigs. However, the underlying mechanism was not elucidated. The results in the Chapter 2 suggest that tomato plants are primed by H. zea oviposition, which allows enhanced defensive responses upon the following simulated herbivory (Kim et al, 2012).

In the Chapter 3, various aspects of H. zea egg-induced defense priming in tomato plants were explored as a follow-up of the Chapter 2 including (1) varietal variation of tomato response to H.

zea oviposition, (2) the response of the jasmonic acid (JA)-deficient mutant of the cultivar

Castlemart, def-1, to H. zea oviposition, (3) whether tomato plants distinguish between fertilized and unfertilized eggs of H. zea in oviposition-mediated defensive priming, (4) the effect of the extract of H. zea accessory glands of on the defensive response of tomato, and (5) the effect of H. zea oviposition on the host plant quality.

In Chapter 4, the possible defensive role of unfertilized eggs laid by virgin females of H. zea against T. pretiosum was investigated. The temporal patterns of unfertilized egg deposition, the fertility of the eggs laid by mated females, and the conditions under which virgin females lay unfertilized eggs were determined. Then, whether unfertilized eggs of H. zea are parasitized by

T. pretiosum, whether T. pretiosum shows differential preference between fertilized and unfertilized eggs of H. zea for parasitization, and the difference in the mortality of T. pretiosum egg parasites in fertilized and unfertilized eggs of H. zea were investigated.

PRIMING OF ANTIHERBIVORE DEFENSIVE RESPONSES IN PLANTS

(Accepted in Insect Science)

Jinwon Kim, Gary W. Felton*

Department of Entomology and Center for Chemical Ecology

Pennsylvania State University, University Park, PA, USA

Abstract

Defense priming is defined as increased readiness of defense induction. A growing body of literature indicates that plants (or intact parts of a plant) are primed in anticipation of impending environmental stresses, both biotic and abiotic, and upon the following stimulus, induce defenses more quickly and strongly. For instance, some plants previously exposed to herbivore-inducible plant volatiles (HIPVs) from neighboring plants under herbivore attack show faster or stronger defense activation and enhanced insect resistance when challenged with secondary insect feeding. Research on priming of antiherbivore defense has been limited to the HIPV-mediated mechanism until recently, but significant advances were made in the past three years, including non-HIPV-mediated defense priming, epigenetic modifications as the molecular mechanism of priming, and others. It is timely to consider the advances in research on defense priming in the plant-insect interactions.

Introduction

Plants have evolved a variety of antiherbivore defenses through the interactions with

herbivorous insects over evolutionary time (Futuyma and Agrawal, 2009). Plant defenses can be

categorized into either constitutive or induced defense by whether a given environmental

stress elevates the basal level of resistance to the stress (Cipollini et al., 2003; Zangerl, 2003;

Karban, 2011). With induced defenses, plants are allowed to manage resources flexibly between defense and growth by eliciting antiherbivore defense only when necessary, although

following initial damage there is a lag time from the start of defense activation to the point

when the defense is fully activated (Zangerl, 2003; Karban, 2011).

Priming of defense means increased readiness of induced defense (Conrath, 2009; Karban, 2011;

Kim et al., 2011). When plants anticipate herbivory in the future through the perception of indicative signal cues or the experience of herbivory at their parental generation, plants are physiologically prepared and induce stronger and faster defenses upon the anticipated herbivory (Fig. 1). In this way, plants are better defended against the insect herbivores with enhanced resistance and/or with reduced lag time (Karban, 2011; Kim et al., 2011). When the expected herbivory does not ensue, plants would not waste resources because the cost of priming itself is considered moderate (van Hulten et al., 2006); this may allow the primed state to remain effective for a longer time (Frost et al., 2008a). Increased plasticity of induced defense by priming may also reduce the possibility of development of counteradaptive strategies by insect herbivores (Zheng and Dicke, 2008).

Priming of defense against biotic stresses is better understood in the plant-pathogen interactions (Conrath, 2009). In the plant-insect interactions, since the first report of defense in maize (Engelberth et al., 2004), priming research had been limited until recently to the priming of defense mediated by herbivore-inducible plant volatiles (HIPVs) that are produced and released by the neighboring plants (or plant parts) under herbivore attack. However, significant advances in understanding of priming of antiherbivore defenses have been made since the last review of defense priming dedicated to plant-insect interactions (Frost et al., 2008a). Now we have reports of priming of defenses across generations (Rasmann et al., 2012), by insect oviposition (Kim et al., 2012), by seed treatment with plant defense elicitors (Worrall et al.,

2011), and by heavy metal stress (Winter et al., 2012). Recently, the molecular mechanisms underlying the priming of defense are being unraveled (Beckers et al., 2009; Conrath, 2011;

Jaskiewicz et al, 2011; Rasmann et al., 2012; Luna et al., 2012; Luna and Ton, 2012).

We present a comprehensive review on various aspects of defense priming in plant-insect interactions. We summarize reports regarding HIPV-mediated and non-HIPV-mediated priming of defense and discuss their ecological implications, introduce the most recent advances in the study of molecular mechanisms underlying defense priming, and close with agricultural applications of defense priming.

HIPV- Mediated Priming of Defense

It is well understood that plants upon damage by herbivorous arthropods release a mixture of

HIPVs (green leaf volatiles (GLVs), terpenoids, and others) to attract natural enemies of the herbivores (McCormick et al., 2012). HIPVs also function as between- and within-plant signaling cues and induce or prime defensive responses in neighboring intact plants or intact plant parts on the same plant (Table 1) (Engelberth et al., 2004; Heil and Kost, 2006; Choh and Takabayashi,

2006; Kessler et al., 2006; Heil and Silva Bueno, 2007; Ton et al., 2007; Frost et al., 2007;

Rodriguez-Saona et al., 2009; Peng et al., 2011; Muroi et al., 2011; Hirao et al., 2012; Li et al.,

2012). Volatiles produced upon mechanical damage and insect feeding and several GLVs among them are capable of priming defenses. Plants prime a variety of defensive responses to HIPVs.

These include accumulation of jasmonic acid (JA; a plant hormone that regulates induction of antiherbivore defenses) (Engelberth et al., 2004; Frost et al., 2008b), linolenic acid (precursor of

JA and GLVs) (Frost et al., 2008b), plant secondary metabolites (Kessler et al., 2006; Hirao et al.,

2012), increased protease inhibitor activity (Kessler et al., 2006), enhanced transcription of antiherbivore defense genes (Ton et al., 2007; Peng et al., 2011), emission of HIPVs (Engelberth et al., 2004; Ton et al., 2007; Frost et al., 2007; Frost et al., 2008b; Rodriguez-Saona et al., 2009;

Muroi et al., 2011; Li et al., 2012), secretion of extrafloral nectar (EFN; extra sugar source to attract general predators such as ants on plants) (Heil and Kost, 2006; Choh and Takabyshi,

2006; Heil and Silva Bueno, 2007), reduced herbivore performance (Kessler et al., 2006; Ton et al., 2007; Rodriguez-Saona et al., 2009; Peng et al., 2011; Muroi et al., 2011) and attraction of natural enemies of herbivores (Ton et al., 2007; Peng et al., 2011; Muroi et al., 2011). HIPV-

mediated priming was found in a various types of plants including herbaceous plant, tree, vine,

bush, and grass, whether annual or perennial, but not yet in non-flowering plants. HIPV-based

signal cues were effective in the laboratory, growth chamber, greenhouse, and natural

environment (Heil and Kost, 2006; Kessler et al., 2006; Heil and Silva Bueno, 2007).

The discovery of the within-plant systemic signaling function of HIPVs opened a new era in the

HIPV-mediated defense priming, resolving many issues that were not explained from the context of the between-plant signaling mechanism (Heil and Karban, 2009). Research on HIPV- mediated priming and/or induction of defense originates from so-called ‘talking trees’ or ‘plant- plant communication’ (Karban et al., 2000; Karban and Maron, 2002; Karban et al., 2004;

Karban et al., 2006; Heil and Karban, 2009). Despite continued reports of plant-plant communication, to what extent this plant-plant communication prevails in the nature and whether this phenomenon even exists has been in dispute for a long time. That is because this interaction is opposed to the anticipated behaviors of plants competing for limited resources in the same area. In other words, natural selection does not favor the plant that benefits its neighboring competitor (Karban and Maron, 2002; Heil and Karban, 2009). From the perspective of the receiver plant, why plants needed to ‘smell’ HIPVs was not clear, especially when the effective range of plant-plant communication via airborne volatiles is only about 50-

60 cm, which is not far enough to have, if any, influence on neighboring plants (Karban et al.,

2006; Heil and Adame-Άlvarez, 2010). Scientists working on the plant-insect interactions frequently do not observe significant effects of wounded plants on the neighboring intact plants in the greenhouse (Agrawal, 2000).

HIPVs’ role as systemic messengers clarified many of these problems (Karban et al., 2006; Heil and Siva Bueno, 2007; Frost et al., 2007; Rodriguez-Saona et al., 2009; Heil and Karban, 2009).

Volatiles have many advantages as a signal carrier (Heil and Ton, 2008). GLVs are released quickly after wounding or herbivory (e.g. (Z)-3 hexenal release peaked in a minute) (D’Auria et al., 2007). Volatiles are fast-moving in the atmosphere. And, most importantly, movement of

HIPVs is not limited by plant vasculature. Consistent reports of plant-plant communication of sagebrush (Artemisia tridentata) may be attributed to the sectoriality of sagebrush indicating limited vasculature between branches (Karban et al., 2006). In addition, the short effective range of volatile signals, 50-60 cm, roughly covers the radius of many plants, which supports the target of volatile signals as the self (Heil and Adame-Άlvarez, 2010). The role of HIPVs in the signaling in within-signaling of herbivory helps explain the evolution of the plants’ ability to release and perceive HIPVs.

Non-HIPV-Mediated Priming of Defense

Recent reports document different mechanisms of defense priming other than HIPVs. These include transgenerational priming of defense (Rasmann et al., 2012), priming of defense by insect oviposition (Kim et al., 2012), priming of defense by seed treatment with defense elicitors (Worrall et al., 2012), and priming of defense by heavy metal stress (Winter et al., 2012)

(Table 2). The former two probably evolved by repeated herbivory after priming stimuli (i.e.

herbivory at the parental generation and deposition of herbivorous insect egg on host plants,

respectively). Evolutionary explanation for the latter two is more elusive.

Transgenerational Priming of Defense

Humans have been aware of the effectiveness of crop rotation on pest management since the

Roman and Greek times (Harris, 1995). A significant number of insect pests have limited

dispersal and oviposit or overwinter on or around the host plant (e.g. Colorado potato beetles)

and may damage the same host plant or its progeny. Crop rotation is effective because the

‘resident’ insect species in the next generation encounters a different crop plant which is unsuitable for feeding (Zehnder et al., 2007). However, in the natural environment, some plants may have sustained feeding by the same insect species over generations. Thus, in these cases there may be strong selection pressure on plants evolve mechanisms by which they pass the parental memory of herbivory to their progeny for enhanced defense. A few reports describe enhanced antiherbivore resistance of plants whose parents experienced herbivores in wild radish (Raphanus raphnistrum), yellow monkeyflower (Mimulus guttatus), and apomictic dandelion (Taraxacum officinale) (Agrawal et al., 1999; Holeski, 2007; Verhoeven and van Gurp,

2012; Holeski et al., 2012). Priming appears a good strategy to express inherited defense traits in the progeny plants when there is a ‘good’ probability of herbivory in the progeny by the same insect species exists and a small chance that the expected herbivores do not occur.

Recently, Rasmann et al. (2012) reported transgenerational priming of defense in Arabidopsis

and tomato. Arabidopsis and tomato plants whose parents sustained feeding damage exhibited significantly enhanced resistance against herbivory by the same insect species.

Transgenerational primed responses of Arabidopsis included JA accumulation, JA-dependent

antiherbivore defense genes, and production of leaf glucosinolates. Primed defenses showed

specificity; on the plants whose parents were fed by Pieris rapae and Plutella xylostella, larvae

of P. rapae and Spodoptera exigua showed reduced performance, whereas larval growth of P.

xylostella and Trichoplusia ni was not influenced by the parental experience (Table 3). More

interestingly, Arabidopsis mutants defective in the RNA-directed DNA methylation (RdDM)

pathway did not show the inheritance of resistance, indicating involvement of DNA methylation

in transgenerational priming of defense. The molecular mechanisms of defense priming and

specificity of primed defense are discussed in more details in the Molecular Mechanism of

Defense Priming section and Specificity of Primed Defense section, respectively.

Priming of Defense by Insect Oviposition

Oviposition by herbivorous insects on the host plant results in herbivory by the hatchlings.

Induced defenses that displace or kill eggs before hatching should be advantageous because plants will not then sustain herbivore damage. A variety of egg-induced defenses that dislodge

or remove the eggs from host plants have been reported (Hilker and Meiners, 2006); some

plants directly kill eggs by producing ovicidal substances (Seino et al., 1996) or by changing the

physical structure or physiological conditions at the oviposition site (Shapiro and DeVay, 1987;

Balbyshev and Lorenzen, 1997; Doss et al., 2000; Videla and Valladares, 2007; Petzold-Maxwell

et al., 2011; Desurmont and Weston, 2011), and some plants release airborne signals to announce the existence of insect eggs to egg parasitoids and egg parasitoids (Hilker et al., 2002;

Colazza et al, 2004b). On the other hand, eggs deposited on plant tissue do not always hatch and result in herbivory. In Lepidoptera, egg mortality is about 60% on average (Zalucki et al.,

2002) mainly due to top-down effects (e.g. egg predation) and harsh abiotic environments (e.g. dryness) (Walker and Jones, 2001); if plants induce defense for every single egg, a significant amount of resources would be wasted, and thus priming as an intermediate step before the activation of defense may be a more efficient strategy than defense induced without priming.

The first report of priming of defense by insect oviposition was recently presented from the

system of tomato (Solanum lycopersicum) and its fruitworm moth Helicoverpa zea (Kim et al.

2012). Tomato plants pretreated with oviposition by H. zea adults showed stronger induction of

protease inhibitor2 (pin2) gene expression and higher accumulation of JA upon subsequent

mechanical wounding and application of H. zea larval oral secretion (OS; regurgitant + saliva).

Protease inhibitor2 (Pin2) inhibits protein digestion in the insect gut after ingestion; the target

of Pin2 should be larvae, not eggs. In another study with Scots pine (Pinus sylvestris) and pine

sawfly (Diprion pini), on pine needles whose twigs were exposed to pine sawfly oviposition,

pine sawfly showed reduced larval performance and lowered fecundity of the resulting adults

(Beyaert et al., 2011). Although priming of defense was not directly tested, pine sawfly oviposition may have caused primed or induced defenses against feeding by newly hatched

larvae. Considering many herbivorous insects start their life history from eggs laid directly on the host plants, more reports of priming by insect oviposition are expected.

Priming of Defense by Seed Treatment

Worrall et al. (2011) reported priming of defenses by seed treatment with JA and β- aminobutyric acid (BABA). Tomato plants from seeds treated with JA showed enhanced defensive gene responsiveness and increased resistance against tobacco hornworm (Manduca sexta), green peach aphids (Myzus persicae), and spider mites (Tetranychus urticae). When tomato seeds were treated with BABA, the resulting plants showed improved resistance against biotrophic fungal pathogen powdery mildew (Oidium neolycopersici). BABA is a non-protein- amino acid which is rarely found in nature, and has been known to induce or prime plant defenses against a wide range of tomato biotic stresses including microbial pathogens, nematodes, and aphids, and against abiotic stresses as well (Pieterse et al., 2006). It is generally recognized that induction of plant defense against arthropod herbivores and necrotrophic pathogens is dependent on JA biosynthesis pathway, and defense against biotrophic pathogens is regulated by salicylic acid (SA)-dependent defense pathway, and JA and SA often act antagonistically (Pieterse et al., 2009). Intriguingly, when seeds were treated with both JA and

BABA, the antagonistic effects between JA- and SA-regulated plant defense pathways were not significant (Worrall et al., 2011).

Maintenance of primed defensive state in the crop field could be a promising strategy for integrated pest management with enhanced defense activation and efficient resource management. Priming of plant defense by seed treatment with defense elicitors (e.g. JA, BA) is of the greatest importance among priming mechanisms from the perspective of agricultural applications with following reasons. First, the method (i.e. dipping seeds in the elicitor solutions) is exceptionally easy and industrially applicable (Worrall et al., 2011). Second, the antagonistic effects frequently found between JA-dependent plant defenses against insect herbivores and

SA-dependent plant defenses against biotrophic plant (Worrall et al., 2011). Last, JA-dependent plant defenses are well characterized from the molecular to ecological levels, and any toxicological or environmental problems have not been reported.

Priming of Defense by Heavy Metal Stress

Trace heavy metals are usually found at low concentrations in nature and many of them are necessary for plant growth, but are toxic above certain concentrations (Kopittke et al., 2010).

Recently, Winter et al. (2012) demonstrated priming of antiherbivore defensive responses in plants under copper (Cu) stress. Maize grown in hydroponic solution containing 80 μM of Cu ion showed increased JA accumulation and HIPV emission in response to feeding by Spodoptera frugiperda larvae. Priming of antiherbivore defenses by Cu treatment was accompanied with

H2O2 accumulation in roots. Treatment with another toxic heavy metal, cadmium (Cd), did not result in defense priming and H2O2 accumulation in roots.

The priming of antiherbivore responses by metal stress is not clear in the ecological and

evolutionary context, but the similarities of plant response to these two seemingly divergent

stresses have been recognized. Cu treatment at higher concentrations induced emission of

volatiles and the production of reactive oxygen species (ROS) (Mithöfer et al., 2004; Attaran et

al., 2008), both of which are often implicated with insect herbivory (Bi and Felton, 1995;

Orozco-Cárdenas et al., 2001; McCormick et al., 2012). In fact, most environmental stresses including biotic and abiotic stresses accompany ROS production, and on this account, the existence of a single universal mechanism was proposed that orchestrates plant responses to both abiotic and biotic stresses with ROS as a common signal mediator (Mithöfer et al., 2004;

Vickers et al., 2009; Winter et al., 2012). ROS responses in plants to environmental stimuli show considerable specificity, which supports this theory (Mittler et al., 2011). In addition, yet to be reported in plants, ROS was shown to induce epigenetic modifications in human liver cancer cells (Lim et al., 2008), which is considered a potent molecular mechanism of priming of defense (Conrath, 2011).

Molecular Mechanisms of Defense Priming

Mitogen-activated protein (MAP) kinase signal transduction pathway is one of the best studied cell signaling mechanisms (Hommes et al., 2002). Along a series of signaling proteins, extracellular signals are conveyed from the cell surface to the DNA in the nucleus. In the MAP kinase (MAPK) cascade, a part of MAP kinase signaling pathway, MAP kinase kinase kinases

(MAPKKKs or MEKKs) phosphorylates MAP kinase kinases (MAPKKs or MEKs), which in turn

phosphorylate and activate MAP kinases. Activated MAPKs phosphorylate many transcription factors, each of which regulates transcription of genes in the downstream.

The faster or stronger defensive response of primed plants requires upregulated transcription efficiency of defense-related genes (Xia, 1996), and hence accumulation of transcription factors or signaling proteins such as MAPKs was proposed as a potent molecular mechanism for

defense priming (Bruce et al., 2007). Soon after, it was reported that Arabidopsis plants primed

with benzothiadiazole (BTH) against virulent plant pathogenic microbes accumulated inactive

MAP kinases, MPK3 and MPK6, which were activated by secondary stimulus, resulting in the enhanced resistance of primed plants (Becker et al., 2009). However, Pastor et al. (2012) were not convinced, and argued that additional regulatory mechanisms are necessary to explain the molecular mechanism of defense priming because of the short turnover times of signaling proteins compared to long-lasting primed state.

Epigenetic modification of chromatin is currently the most promising candidate of molecular mechanism of defense priming. DNA methylation and histone post-translational modifications

induce changes in chromatin structure, which alters gene transcription (Berger, 2007;

Jaskiewicz et al., 2011). DNA methylation occurs at cytosine residues at the CG or CHG sites, or

asymmetrically at CHH sites (H= A, C, or T). Two copies of each of histone proteins, H2A, H2B,

H3, and H4, agglomerate into the core of the nucleosome, a unit of chromatin, with 147 bp

genomic DNA strands wrapped around the core (Conrath, 2011). Structure of histone proteins

are post-translationally modified at lysine (K), arginine (R), proline (P), and serine (S) residues of

the N- and C-terminal tails by methylation (mono-, di-, or trimethylation), acetylation, phosphorylation, ubiquitylation, and SUMOylation (SUMO, small ubiquitin-like modifier)

(Berger, 2007).

Recent papers provide evidence indicating that priming of defense against pathogens and herbivores accompanies epigenetic modifications to promote transcription efficiency of defense genes upon subsequent stimulus (Jaskiewicz et al., 2011; Rasmann et al., 2012; Luna et al., 2012; Luna and Ton, 2012). According to Jaskiewicz et al. (2011), in Arabidopsis plants primed by BTH treatment at moderate concentrations, several types of histone modifications were abundant on the promoter regions of SA-dependent transcription factors WRKY6,

WRKY29 and WRKY53. Trimethylation and dimethylation at lysine 4 of H3 (H3K4me3 and

H3K4me2, respectively) were abundant on the promoter regions of WRKY6 and WRKY53, and the promoter region of WRKY29 was labeled with acetylations at H3K9, H4K5, H4K8 and H4K12 as well as H3K4me3 and H3K4me2 (Jaskiewicz et al., 2011). Most of histone modifications found in BTH-primed plants are known to favor gene activation (Berger, 2007).

Rasmann et al. (2012) reported transgenerational priming of antiherbivore defenses in

Arabidopsis and tomato. One of the functions of siRNA is to induce de novo DNA methylation of cytosines in the DNA region whose sequence is homologous to small interference RNA (siRNA)

(Matzke et al., 2007). Arabidopsis mutants defective in the production of siRNA failed to exhibit transgenerational priming, implying that RNA-directed DNA methylation (RdDM) is critical in conveying environmental memories of parents to their progeny for augmented resistance

(Rasmann et al., 2012). DNA methylation generally inhibits transcription (Berger, 2007). The

reason that DNA methylation in the transgenerational priming of defense is specifically

important is that DNA methylation is epigenetically inherent, but histone modifications are lost

during the meiosis (Cedar and Bergman, 2009; Rasmann et al., 2012). Increasing evidence indicates that DNA methylation and histone modification may be complementary so that DNA methylation might guide restoration of histone marks after lost during meiosis (Cedar and

Bergman, 2009).

More reports support the role of DNA methylation in defense priming. Arabidopsis plants exposed to virulent Pseudomonas syringae pv tomato DC3000 (PstDC3000) at the parental generation displayed enhanced resistance in the offspring (Luna et al., 2012). The plants whose parents were infected with PstDC3000 were more resistant against the conspecific pathogen and (hemi) biotrophic pathogen Hyaloperonospora arabidopsidis than control plants. In addition, in the primed progeny, the promoter regions of SA-inducible defense gene

PATHOGENESIS-RELATED GENE1 (PR1) and transcription factorsWRKY6 and WRKY53 were marked with H3K9ac, whereas the promoter of JA-inducible promoter PLANT DEFENSIN1.2

(PD1.2) with H3K27me3 (Luna et al., 2012). H3K9ac generally instructs gene activation, and

H3K27me3 acts against transcription (Berger, 2007). Probably as a result, upon subsequent stimulus, PR1, WRKY6 and WRKY53 in the primed plants exhibited enhanced transcription levels, but transcription of JA-dependent PD1.2 was even more reduced in PstDC3000-primed plants than non-primed plants (Luna et al., 2012). Furthermore, the drm1drm2cmt3 triple mutant impaired in DNA methylation on CHG and CHH sites, failed to show transgenerational priming of defense (Luna et al., 2012), meaning hypomethylation at CHG and CHH sites is critical in

transgenerational defense priming. In the follow-up study, another RdDM pathway mutant

drm1drm2, which is impaired in DNA methylation on CHH sites, showed no impairment in

transgenerational priming defense, meaning hypomethylation at CHH sites is not required in

defense priming across generations. Therefore, the authors reached the conclusion that

hypomethylation on CHG sites is critical in transgenerational defense priming (Luna and Ton,

2012).

Specificity of Primed Defenses

Each defense trait of a plant impacts a specific spectrum of target herbivores (Zangerl, 2003). As a plant sustains damage by several insect herbivores (Strauss, 1991), identification of the given insect species is critical for the induction of a selective set of defenses effective on the performance of the given insect herbivores (Zangerl, 2003). Specificity of defense is parsed into

‘specificity of elicitation’ and ‘specificity of effect’ (van Zandt and Agrawal, 2004; Chung and

Felton, 2011). How distinct defenses are induced upon different insect species defines

‘specificity of elicitation’. Accumulated results indicate that plant induced defense in some cases is specific enough to show distinct responses to feeding by two closely related species of whitefly (Zarate et al., 2007). ‘Specificity of effect’ is defined by whether the induced defenses are effective on the performance of subsequent herbivores (van Zandt and Agrawal, 2004;

Chung and Felton, 2011) (Table 3A). Plant defenses induced by one insect species could be

effective, neutral, or counter-effective on the other, and the generalized pattern of specificity of effect has not been established yet.

Specificity is also found in the primed defenses. Between two GLVs, (Z)-3-hexenol and (E)-2- hexenal, reported to prime plant defensive responses in maize (Engelberth et al., 2004) and native tobacco (Kessler et al., 2006), respectively, only (E)-2-hexenal primed defensive response of Arabidopsis for subsequent MJ treatment (Hirao et al., 2012), implying plant’s perception of specific volatiles and specificity of elicitation of primed defense. Among HIPVs of native tobacco,

(E)-2-hexenal and methacrolein primed defense, whereas MJ did not (Kessler et al., 2006). In tomato, MJ primed pin2 expression in tomato at moderate concentrations (Kim and Felton, unpublished data). Specificity of effect of primed defense is also described in the report of transgenerational priming of defense of Arabidopsis. Larvae of Pieris rapae and Spodoptera exigua showed reduced performance on the plants whose parents suffered herbivory by P. rapae and Plutella xylostella, whereas larval herbivory by P. xylostella and Trichoplusia ni on the parent plants did not influence on the performance of P. rape on the progeny (Rasmann et al.,

2012) (Table 3B). Primed defense has one more dimension of specificity because, in response to a given stimulus, plants may prime some defense traits and induce others. In response to HIPVs from the herbivore-damaged neighboring plants, the receiver hybrid aspen induced EFN secretion, but primed HIPV emission for the secondary herbivory (Li et al., 2012). More specificity of primed defenses is found at the epigenetic level. As stated above, in transgenerationally primed Arabidopsis, the promoters of SA-inducible defense genes were labeled with transcription-activating marks, whereas the promoters of JA-dependent defense

genes were abundant with epigenetic modifications that instruct repression of transcription

(Luna et al., 2012).

Summary

Priming of antiherbivore defense provides enhanced resistance when the anticipated herbivory occurs, and more flexibility in resource management if no damage is done to the plant.

Research on priming has focused on so called plant-plant communication has extended to long- distance signaling and priming and/or induction of systemic defense within a plant using HIPVs, which resolved many issues in the evolution of plants’ ability to emit and perceive HIPVs.

Reports of defense priming without HIPV as a mediator started to emerge. Deposition of insect eggs on plant tissue likely results in herbivory, but there is a small chance that the eggs are removed, e.g. by egg predators, and thus priming of defense by insect oviposition may be advantageous to plants over egg-induced defenses that physically remove eggs. Defense priming by seed treatment with defense elicitors appears of great potential importance from the perspective of agricultural applications. Specificity of defense is also found in primed defense, which will result in enhanced induction of a set of effective defense traits upon subsequent biological stresses. Molecular mechanisms underlying defense priming started to be unraveled, and epigenetic modifications of the promoter regions of defense-related genes for enhanced or repressed transcription appears most promising as the molecular mechanism for defense priming.

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1 TABLES

2 Table 1-1. Chemical structure of HAMPs and the original insect species used for isolation.

HAMPs Structure Original Insect species References

Volicitin Spodoptera exigua Alborn et al., 1997

Caeliferin A16:1 Schistocerca americana Alborn et al., 2007

Inceptin Spodoptera frugiperda Schmelz et al., 2006

Bruchus pisorum Bruchin A Doss et al., 2000 Callosobruchus maculatus

4 Table 1-2. HIPV-mediated priming of antiherbivore defensive responses

Plant Priming treatments† Within- or Secondary stimuli Containment and delivery of Enhanced defensive References between-plants HIPVs/ Experimental venues responses†

Maize HIPVs from wounded and S. exigua OS Between plants Mechanical wounding Glass cylinders/ Greenhouse JA accumulation Engelberth et al., 2004 treated plants +S. exigua OS Sesquiterpene emission HIPVs from S. exigua infested plants

(Z)-3-hexenal,-ol, -yl acetate

Lima bean Synthetic HIPVs in lanolin paste NA Mechanical wounding Perforated plastic bags/ EFN secretion Heil and Kost, 2006 Natural environment

Lima bean HIPVs from spider mite infested plants Between plants Spider mite infestation Acrylic cages/ greenhouse EFN secretion Choh and Takabayashi, 2006

Native HIPVs from damaged sagebrush Between plants M. sexta larval feeding Close placement of damaged Chlorogenic acid accumulation Kessler et al., 2006 tobacco foliage sagebrush foliage (within 1- TPI activity cm)/ Natural environment (E)-2-hexenal Reduced leaf damage Methacrolein Larval mortality

Maize HIPVs from S. littoralis larvae infested Between plants S. littoralis larval Glass cylinders, Teflon tubing/ Defense gene expression Ton et al., 2007 plants feeding Laboratory Volatile emission

Attraction of larval parasitoid C. marginiventris

Reduced S. littoralis larval growth

Hybrid HIPVs of L. dispar larvae infested Within plant L. dispar larval feeding Glass-Teflon chambers, Teflon Terpenoid emission Frost et al., 2007 poplar leaves on vascularly connected and tubing/ Walk-in chambers

limited leaves

Lima bean HIPVs from mechanically damaged Within plant Mechanical wounding Plastic bags/ Natural EFN secretion Heil and Silva Bueno, and JA treated leaves on the same + JA application environment 2007 plant

Hybrid (Z)-3-hexenyl acetate Within plant L. dispar larval feeding Glass- Teflon chambers/ Terpenoid emission Frost et al., 2008b poplar Walk-in chambers JA, LNA accumulation

Antiherbivore gene expression

Highbush HIPVs from L. dispar larvae infested Within plant L. dispar larval feeding Polyester sleeves/ Volatile emission Rodriguez-Saona et al., blueberry neighboring branch on the same Greenhouse 2009 Reduced leaf damage plant

Brussels HIPVs from P. brassicae larvae Between plants P. brassicae or M. Glass jars, Teflon tubing/ LOX expression Peng et al., 2011 sprouts infested neighboring plants brassicae larval Greenhouse Attraction of larval parasitoid feeding C. glomerata

Reduced larval growth of P. brassicae and M. brassicae

Lima bean, HIPVs from the lima bean (E)-β- Between plants Spider mite infestation Polypropylene openflow Volatile emission Muroi et al., 2011 maize ocimene synthase-transformed tunnel/ Not specified M. separate larval Reduced spider mite tobacco plants feeding oviposition on lima bean

Reduced M. separata larval growth on maize

Attraction of predatory mite and larval parasitoid C. kariyai

Hybrid HIPVs from plants infested with larvae Between plants Feeding by larvae of E. Not specified/ Environmental Volatile emission Li et al., 2012 aspen of E. autumnata or P. plantaginis autumnata or P. chambers plantaginis

Arabidopsis (E)-2-hexenal NA MJ application Glass tubes/ Growth chamber Anthocyanin accumulation Hirao et al., 2012 5 †Treatments or responses showing no significant defense priming are not included. Plant species: Arabidopsis, Arabidopsis thaliana; Brussels sprouts, Brassica oleracea; highbush blueberry, 6 Vaccinium corymbosum; hybrid aspen, Populus tremula X Populus tremuloides; hybrid popular, Populus deltoides X Populus nigra; lima bean, Phaseolus lunatus; maize, Zea mays; tobacco, Nicotiana 7 tabacum. Insect species: Cotesia glomerata, C. kariyai, C. marginiventris (Hymenoptera: Braconidae); Epirrita autumnata (Lepidoptera: Geometridae); Lymantria dispar (Lepidoptera: Noctuidae); 8 Mamestra brassicae (Lepidoptera: Noctuidae); Manduca sexta (Lepidoptera: Sphingidae); Mythimna separata (Lepidoptera: Noctuidae); Parasemia plantaginis (Lepidoptera: Arctiidae); Pieris 9 brassicae (Lepidoptera: Pieridae); Spodoptera exigua, S. littoralis (Lepidoptera: Noctuidae). Mite species: spider mite, Tetranychus urticae; predatory mite, Phytoseiulus persimilis. Abbreviations: 10 EFN, extrafloral nectar; GLVs, green leaf volatiles; HIPV, herbivore-inducible plant volatiles; JA, jasmonic acid; LNA, linolenic acid; LOX, lipoxygenase; MJ, methyl jasmonate; NA, not applicable; OS, 11 oral secretion (regurgitant + saliva); TPI, trypsin protease inhibitor.

12 Table 1-3. Non-HIPV-mediated priming of antiherbivore defensive responses.

Priming type Plant Priming treatments† Secondary stimuli Experimental venues Enhanced defensive responses† References

Transgenerational Arabidopsis Arabidopsis: Arabidopsis: Arabidopsis: Arabidopsis: Rasmann et al., 2012

Tomato MJ application on parents Caterpillar feeding on progeny Growth chambers Leaf glucosinolate 1MI3M

Caterpillar feeding on parents‡ Tomato: Tomato: JA accumulation

Tomato: H. zea larval feeding on progeny Greenhouse LOX2, AOS expression

Mechanical wounding on Reduced caterpillar growth‡ parents Tomato: MJ application on parents Reduced H. zea larval growth H. zea larval feeding on parents

Oviposition Tomato Oviposition by H. zea adults Mechanical wounding + H. zea OS Greenhouse pin2 gene expression Kim et al., 2012

JA accumulation

Seed treatment Tomato JA seed treatment Feeding by arthropod herbivores Greenhouse Reduced performance of spider Worrall et al., 2011 mites, M. sexta larvae, and M. Inoculation with B. cinerea persicae

Increased resistance against necrotrophic fungus B. cinerea

pin2 gene expression by B. cinerea inoculation

Heavy metal stress Maize Hydroponic solution containing S. frugiperda larval feeding Climate chambers JA accumulation Winter et al., 2012 80 μM Cu HIPV emission

13 †Treatments or responses showing no significant defense priming are not included. ‡Detailed combinations of herbivore insects on parent and progeny plants are listed in Table 3. Plant species: 14 Arabidopsis, Arabidopsis thaliana; maize, Zea mays; tomato, Solanum lycopersicum. Insect species: Helicoverpa zea (Lepidoptera: Noctuidae); Manduca sexta (Lepidoptera: Sphingidae); Myzus 15 persicae (Hemiptera: Apididae); Spodoptera frugiperda (Lepidoptera: Noctuidae). Mite species: spider mite, Tetranychus urticae. Fungal species: Botrytis cinerea (necrotrophic fungal pathogen). 16 Abbreviations: 1MI3M,1-methoxy-indol-3-ylmethyl glucosinolate; AOS, allene oxide synthase; Cu, copper; HIPVs, herbivore-inducible plant volatiles; JA, jasmonic acid; LOX2, lipoxygenase2; MJ, 17 methyl jasmonate; OS, oral secretion (regurgitant + saliva); pin2, protease inhibitor2; ROS, reactive oxygen species

Table 1-4. Specificity of effect. (A) Specificity of effect of induced defense of wild radish. (B) Specificity of effect of transgenerationally primed defense of Arabidopsis.

A. Within a generation of wild radish (Agrawal, 2000)

Primary herbivore Secondary herbivore P. rapae P. xylostella T. ni S. exigua

P. rapae ↓† ↓ ― ― P. xylostella ― ↓ ― ― T. ni ― ― ― ↓ S. exigua ― ― ― ↓

B. Across generations of Arabidopsis (Rasmann et al., 2012)

Secondary Primary herbivore on the parents herbivore on the progeny P. rapae P. xylostella

P. rapae ↓ ↓ P. xylostella ― NA T. ni ― NA S. exigua ↓ ↓

†Effect of the primary herbivory on the performance of the secondary herbivores was designated as “↓” for reduced growth and “―” for no effect.

FIGURES

Fig 1-1. Priming of antiherbivore defensive responses. (A) HIPV-mediated priming of antiherbivore response in maize. Effect of previous exposure to HIPVs from neighboring maize plants on JA accumulation upon following simulated S. exigua herbivory (wounding plus S. exigua OS treatment) (Engelberth et al., 2004 © National Academy of Sciences, USA). Symbols:

triangle, control (no previous exposure to HIPVs, no simulated herbivory; squares, no previous exposure to HIPVs, simulated herbivory; diamond, pretreatment with HIPVs, simulated herbivory; **, significant difference (n = 4). (B) Non-HIPV-mediated priming of antiherbivore response in tomato. Effect of previous H. zea oviposition on the induction of tomato pin2 upon following simulated H. zea herbivory (wounding plus H. zea OS treatment) (Kim et al., 2012).

Symbols: closed circle (Control), intact plants without H. zea oviposition treatment; open circle

(Oviposition), plants treated only with H. zea oviposition; closed triangle (Wounding), plants mechanically damaged and H. zea OS-applied without oviposition pretreatment; open triangle

(Ovi+Wnd), plants pretreated with oviposition followed by mechanical wounding and H. zea OS application. Without mechanical damage, there are only Control and Oviposition at time 0h. At times 8h and 1d, closed circles (control) are hidden behind open circles (oviposition). Relative pin2 expression is presented in the graph. Letters next to data spots in the graph, significant difference (n = 4 or 5).

CHAPTER 2:

Insect Eggs Can Enhance Wound Response in Plants: a Study System of

Tomato Solanum lycopersicum L. and Helicoverpa zea Boddie

(Published in PLoS One 7: e37420)

Jinwon Kim1,3, John F. Tooker1,3, Dawn S. Luthe2,3, Consuelo M. De Moraes1,3, and Gary W.

Felton1,3*

1Department of Entomology, 2 Department of Soil and Crop Science, and 3Center for Chemical

Ecology, Pennsylvania State University, University Park, Pennsylvania, USA

ABSTRACT

Insect oviposition on plants frequently precedes herbivory. Accumulating evidence indicates that plants recognize insect oviposition and elicit direct or indirect defenses to reduce the pressure of future herbivory. Most of the oviposition-triggered plant defenses described thus far remove eggs or keep them away from the host plant or their desirable feeding sites. Here, we report induction of antiherbivore defense by insect oviposition which targets newly hatched larvae, not the eggs, in the system of tomato Solanum lycopersicum L., and tomato fruitworm moth Helicoverpa zea Boddie. When tomato plants were oviposited by H. zea moths, pin2, a highly inducible gene encoding protease inhibitor2, which is a representative defense protein against herbivorous arthropods, was expressed at significantly higher level at the oviposition site than surrounding tissues, and expression decreased with distance away from the site of oviposition. Moreover, more eggs resulted in higher pin2 expression in leaves, and both fertilized and unfertilized eggs induced pin2 expression. Notably, when quantified daily following deposition of eggs, pin2 expression at the oviposition site was highest just before the emergence of larvae. Furthermore, H. zea oviposition primed the wound-induced increase of pin2 transcription and a burst of jasmonic acid (JA); tomato plants previously exposed to H. zea oviposition showed significantly stronger induction of pin2 and higher production of JA upon subsequent simulated herbivory than without oviposition. Our results suggest that tomato plants recognize H. zea oviposition as a signal of impending future herbivory and induce defenses to prepare for this herbivory by newly hatched neonate larvae.

INTRODUCTION

Upon herbivory, plants induce a variety of defenses that developed via coevolution with herbivorous arthropods, especially insects (Howe and Jander, 2008; Zhu-Salzman et al, 2008;

Futuyma and Agrawal, 2009). With intensive study during the past few decades, it is now

generally understood that upon insect herbivory plants perceive insect-derived cues (e.g.

continuous feeding damage, herbivore-associated molecular patterns or HAMPs) and initiate a

set of defenses tailored to given herbivore species (Wasternack et al, 2006; Felton and

Tumlinson, 2008; Howe and Jander G, 2008; Farmer and Dubugnon, 2009). Compared to

constitutive defenses, which are continuously expressed irrespective of herbivory, induced

defenses are considered more flexible and efficient (Agrawal and Karban, 1999; Karban, 2011).

Recently, increasing research interest has focused on the deployment of plant defense traits

prior to herbivory (Hilker and Meiners, 2006; Kim et al, 2011). The basic premise is that early-

induced defenses could be even more effective and adaptive than defenses induced after

herbivores start feeding. By perceiving reliable cues of impending herbivory and initiating

appropriate defenses in advance, plants may be able to totally avoid or significantly reduce

herbivory even before a full-induced defense is activated (Hilker and Meiners, 2006; Kim et al,

2011). Thus far, plants appear to recognize at least three events as indicators of future

herbivory. First, some plants increase resistance against insects when a neighboring plant

suffers insect herbivory (Karban and Maron, 2002; Heil and Karban, 2009). In this case, plants

appear to “eavesdrop” on volatile organic compounds released by the neighboring plant under

herbivory and elicit their defenses. Moreover, the volatile-receiving plants showed priming of

defenses, meaning the receiver plants activated faster or stronger defenses upon the

anticipated herbivory (Karban and Maron, 2002; Engelberth et al, 2004; Ton et al, 2007; Heil

and Siva Bueno, 2007; Frost et al, 2008). Second, insect footsteps can induce defensive responses in plants either by caterpillars breaking cells when crochettes dig into leaves (Bown et al, 2002) or when caterpillars or moths break trichomes (Peiffer et al, 2009). Third, oviposition, one of the most common events preceding insect larval herbivory, can induce a variety of direct and indirect defenses of plants (Hilker and Meiners, 2006; Hilker and Meiners,

2011). Mechanisms of oviposition-induced defenses include production of ovicides (seino et al,

1996), a hypersensitive response or necrosis leading to drying or dropping of eggs (Shapiro and

DeVay, 1987; Balbyshev and Lorenzen, 1997; Petzold-Maxwell et al, 2011), excessive growth of hard tissue (neoplasm) under the eggs to force neonates to hatch outside and be exposed to harsh environment (Doss et al, 2000; Petzold-Maxwell et al, 2011), egg crushing (Desurmont and Weston, 2011), egg extrusion (Videla and Valladares, 2007), and calling in egg or larval parasitoids by the host plant (Hilker et al, 2002; Colazza et al, 2004; Tamiru et al, 2011).

While most of previous studies of oviposition-induced plant defense have focused on defenses that remove or kill insect eggs from the host (Hilker and Meiners, 2006; Hilker and Meiners,

2011), there have been only two reports of the effect of insect oviposition on the quality of the host plant as food source and thus on the performance of emerging neonates. Pieris brassicae L. oviposition on Arabidopsis thaliana L. appeared to suppress antiherbivore defenses and the application of P. brassicae egg extract resulted in improved growth of Spodoptera littoralis larvae on the host plant (Bruessow et al, 2010). More recently, preceding oviposition treatment with pine sawfly (Diprion pini L.) was shown to reduce the performance of the conspecifics on

Scots pine (Pinus sylvestris L.) branches, although pine sawfly oviposition on pine needles

involves mechanical damage by ovipositors and deposition of eggs inside the wound (Beyaert et

al, 2012).

In this study, we hypothesized that tomato plants recognize H. zea oviposition as an indicator of

future herbivory and induce or prime defenses targeting neonates to hatch. To test the

hypothesis, we first investigated whether tomato plants reacted to H. zea oviposition and

elicited defensive responses at the oviposition site. We examined hydrogen peroxide (H2O2) production on tomato leaves under H. zea eggs, as reactive oxygen species including H2O2 are

often related to antiherbivore plant defenses (Orozco-Cárdenas et al, 2001; Little et al, 2007).

Then, we measured the transcriptional level of pin2, a gene encoding protease inhibitor2 (Pin2),

at the oviposition site, assessed the effect of H. zea oviposition on the induction of pin2 at the

oviposition site, and determined the spatial and temporal dynamics of pin2 expression pattern.

The level of pin2 expression was selected as a defense index because the induction of pin2 by mechanical wounding and arthropod herbivory is well understood in tomato (Green and Ryan,

1972; Felton and Tumlinson, 2008; Fowler et al, 2009) and because Pin2 is a defensive protein that targets insects under active feeding, not eggs. We also tested whether H. zea oviposition primed antiherbivore defense of tomato plants, i.e. whether oviposition-treated tomato

showed intensified defense induction upon subsequent herbivory by measuring pin2 expression and jasmonic acid (JA) concentration in tomato leaves. JA is a plant hormone that orchestrates the induction of antiherbivore defenses (Pieterse et al, 2009), and its concentrations in leaves are a good marker of the plant defense level and were successfully used to indicate priming in a previous report (Hilker and Meiners, 2010).

RESULTS

Tomato Perceives H. zea Oviposition and Induces Defensive Responses at the Oviposition Site

Helicoverpa zea oviposition elicits H2O2 accumulation at the oviposition site on tomato foliage

It has been proposed that H2O2 plays a role as a second messenger between early response genes (e.g. genes involved in the biosynthesis of JA) and late response genes (e.g. genes whose

products function as defensive traits such as protease inhibitors) (Orozco-Cárdenas et al, 2001;

Fowler et al, 2009). In addition, accumulation of H2O2 and other reactive oxygen species at the

oviposition site was previously reported (Little et al, 2007). Production of H2O2 at the

oviposition site was also detected in the interaction between tomato and H. zea. When H. zea

egg-laden tomato leaves were stained with 3,3’-diaminobenzidine (DAB) solution, H2O2

production was clearly visualized right under the eggs (Fig 2-1A).

Pin2 is expressed at the H. zea oviposition site and the level of expression decreased with

distance from the egg

Leaf tissue sampled at the H. zea oviposition site showed significantly higher level of pin2

expression (Fig 2-1B; Non-parametric GLM; Chi-square= 6.8182, p = 0.009, n = 5). The area of

pin2 expression was more extensive than expected. Transcriptional levels of pin2 at 0, 10, and

20-mm away from eggs were significantly higher than that of intact plants, and the intensity

decreased with distance from (Fig 2-2; Non-parametric GLM; Chi-square = 14.4695, p = 0.0023,

n = 4 or 5).

Pin2 expression at the oviposition site was highest just before the emergence of neonates

To understand its temporal dynamics following oviposition, we tracked levels of pin2 expression

at the oviposition site over three days after oviposition and before the emergence of neonates

(Fig 2-3). Pin2 expression after 1d was significantly higher at the oviposition site than that of

intact plants (Non-parametric GLM; Chi-Square = 3.9382, p = 0.0472, n = 5). There was no

difference in levels of pin2 expression between the two groups on day 2 (Non-parametric GLM;

Chi-Square = 0.2400, p = 0.6242, n = 4 or 5). However, levels of pin2 expression dramatically

increased on day 3 (Non-parametric GLM; Chi-Square = 6.0000, p = 0.0143, n = 4 or 5), the final

day before the emergence of neonates.

Unfertilized eggs induced pin2 as well

A considerable portion of females of many insect species fail to mate in the field (Rhainds,

2010). Many female moths of H. zea caged with males were found unmated, but they laid

about half as many unfertilized eggs as fertilized eggs deposited by mated females (Adler et al,

1991). As only fertilized eggs produce neonates and result in herbivory, we examined whether tomato plants would respond to infertile eggs as well as fertile ones. In this experiment, plants

were caged with no moth, with male moths only, with virgin female moths only, and with male

and female moths together. From now on, we will refer to the female moths that were caged with male moths as ‘mated’ female moths whether they are virgin or mated, although not all females in the group of ‘mated female moths’ are mated. Virgin female moths laid seemingly as many eggs on tomato plants as mated females. Infertility of the eggs laid by virgin female moths was confirmed as the eggs desiccated on tomato leaves in a few days, while caterpillars hatched from the eggs from mated female moths. No significant transcriptional difference in pin2 was observed between intact plants and plants caged with male moths only. The eggs from mated female moths induced pin2, consistent with the results stated above. Interestingly, significant induction of pin2 was elicited at the oviposition site of unfertilized eggs. Although the mean of pin2 expression of tomato leaf tissue under unfertilized eggs appeared lower than that of fertilized ones, the difference was not statistically significant (Fig 2-4; Proc GLM; F3,15 =

22.99, p < 0.0001, n = 4 or 5).

Induction of pin2 and Accumulation of JA Were Primed by H. zea Oviposition for Subsequent

Simulated H. zea Herbivory

Our results thus far strongly suggest that tomato plants perceive H. zea eggs and elicit a

defensive response. We further hypothesized that H. zea oviposition may prime antiherbivore

defenses of tomato in anticipation of herbivory by neonates hatching from eggs. To test this

hypothesis, we exposed tomato plants to egg-laying H. zea moths, and then mechanically

wounded the terminal leaflet and applied fresh oral secretion (OS; a mixture of regurgitant and

saliva) of H. zea larvae to simulate insect herbivory. Compared to the typical pattern of pin2

expression, which increases and then decreases within 24 hr after wounding, tomato plants

previously exposed to H. zea oviposition showed much stronger induction of pin2 following mechanical wounding (Fig 2-5; Non-parametric GLM; at 0h, Chi-Square = 21.00, p = 0.0025, n =

4; at 3h, Chi-Square = 6.3240, p = 0.0969, n = 4 or 5; at 8h, Chi-Square = 13.2857, n = 5, p =

0.0024; at 1d, Chi-Square = 14.3843, n = 4 or 5, p = 0.0024). Simple disruption of glandular trichomes, which had been recently reported to induce pin2 expression (Peiffer et al, 2009) did not prime pin2 expression (Fig 2-S1).

In addition to gene expression data, we also investigated the influence of insect oviposition on

JA production after simulated herbivory. We found that oviposition did not change the basal JA levels in leaf tissue (Fig 2-6A; Proc GLM; F1,8 = 0.03, p = 0.8600, n = 5). However, when plants

were mechanically wounded and treated with OS of H. zea 5th instars to simulate herbivory, JA

levels were significantly higher in oviposition-treated plants than in intact plants (Fig 2-6B; Non-

parametric Proc GLM; at 30 min, Chi-Square = 11.2604, p = 0.0036, n = 5; at 1 hr, Chi-Square =

11.18, p = 0.0037, n = 5; at 3 hr, Chi-Square = 9.7582, p = 0.0076, n = 4 or 5). Enhanced level of pin2 expression and JA burst strongly indicate that tomato defenses are primed by H. zea oviposition.

DISCUSSION

Our results are consistent with the hypothesis that host plants can perceive cues associated

with oviposition and then induce and prime defensive responses that can be effective against

soon-to-emerge neonates. The response of tomato to H. zea oviposition was comprehensively explored using a suite of defensive responses that are reliable indicators of tomato defense against feeding by insect herbivores. As Pin2, the end product of pin2, acts as a defensive trait only after ingested into the insect digestive system, induction or priming of pin2 by insect oviposition suggests tomato plants recognized the eggs as a future danger and became prepared for herbivory by the neonates, not the eggs. It was demonstrated that the local production of H2O2 under H. zea eggs, the coincidence between the distribution of eggs and

transcriptional map of pin2 on tomato leaves, and the coincidence between the time of the

highest pin2 expression and the larval hatching time. All of these results indicate that the induction of tomato defense by H. zea oviposition was caused not by other factors such as trichome disruption, but by the eggs.

There have been two reports showing that insect oviposition can influence the quality of the host plant as food source (Bruessow et al, 2010; Beyaert et al, 2012). The eggs of P. brassicae

accumulated salicylic acid, a plant hormone acting antagonistically against JA (Pieterse et al,

2009), at the oviposition site and suppressed antiherbivore defenses in A. thaliana (Bruessow et

al, 2010). As a result, Spodoptera littoralis Boidsduval caterpillars (but not P. brassicae larvae)

performed better on the A. thaliana previously treated with the extract of P. brassicae eggs

(Bruessow et al, 2010). More recently, oviposition by pine sawfly adults on the Scots pine was

shown to reduce the performance of the conspecific larvae, although the relevant defense

mechanism of the host plant was not elucidated (Beyaert et al, 2012). In the present study, we

showed that insect oviposition can induce defenses that are known to inhibit the growth of

feeding insects and that plant defenses can be primed by insect oviposition. Besides egg

deposition, there are other factors associated with oviposition that may induce defensive

responses from tomato. For example, disruption of glandular trichomes by moth walking on leaves has been found to induce tomato pin2 (Peiffer et al, 2009). However, the focused nature of our results just around the oviposition site strongly suggests that cues associated with egg deposition or the egg itself are at least the primary factor that tomato plants perceive to trigger defense induction.

Hydrogen peroxide molecules were clearly visualized under eggs laid singly on leaf surface (Fig

2-1A). Hydrogen peroxide and other reactive oxygen species function as key cellular signaling molecules (Mittler et al, 2011), and in tomato H2O2 has been demonstrated to mediate early

defense response genes (e.g. genes involved in JA biosynthesis) and late defense response genes such as pin2 (Orozco-Cárdenas et al, 2001; Fowler et al, 2009). Hydrogen peroxide production has also been detected beneath eggs of the specialist lepidopteran P. brassicae on leaves of A. thaliana (Little et al, 2007), although in this case, H2O2 production appears a part of elicitation of hypersensitive response and suppression of plant antiherbivore defense by insect

oviposition (Bruessow et al, 2010). Another recent paper documented H2O2 production, JA and

JA-regulated wound responses in tomato by the oviposition of Orius laevigatus (De Puysseleyr

et al, 2011). Orius oviposition accompanies mechanical damage because its eggs are not laid, but thrusted into leaf tissue, which probably elicited wound response in tomato.

The expression of pin2 was found upregulated in a broad area on leaves around the egg deposition site, including 20-mm away (Fig 2-2). The induced area was large enough that eggs laid a few centimeters apart would activate pin2 transcription in a whole tomato leaflet. The non-uniform expression of pin2 on leaves that was highest at the oviposition site might be important as a defense trait. After emerging, neonates wander searching for suitable feeding sites within or between host plants (Zalucki et al, 2002). Neonates of H. zea will hatch where pin2 expression is highest and move away to find more desirable feeding sites. Because predation is one of the main mortality factors for neonates (Zalucki et al, 2002) and larval movement increases predation risk (Bernays, 1997), increased expression of pin2 at the oviposition sites might contribute to elevated predation risk of neonates. Assessment of the movement of neonates on oviposition-treated tomato plants will provide a more detailed understanding of uneven expression of pin2 around the oviposition site.

Our results suggest that pin2 expression coincided with the emergence of neonates (Fig 2-3).

Although pin2 is considered one of late response genes induced between 4 to 24 hr following herbivory (Fowler et al, 2009), the observed pin2 expression three days after oviposition is well beyond the ordinary time frame of pin2 expression induced by mechanical wounding or insect herbivory. This delayed culmination of defense suggests that the induction of defense may be synchronized to the time of emergence of neonates. In this way, plants may be able to produce defensive compounds without wasting resources by premature expression of defense traits.

Plants might be able to trace air temperature, which is most important for hatching time (Howe,

1967; Davidson, 1944), or perceive egg-derived HAMPs that indicate larvae are about to emerge. Synchronicity of defense gene induction following insect oviposition with larval emergence was recently reported (Beyaert et al, 2012). The transcription of sesquiterpene synthase genes of Scots pine (P. sylvestris L.) was found to be the most intense 14 days following pine sawfly (D. pini L.) oviposition on pine branches, just prior to emergence of pine sawfly larvae.

Notably, unfertilized eggs also induced tomato pin2 expression (Fig 2-4). Many females of insects fail to mate in the field (Rhainds, 2010). A large portion of H. zea females were also found unmated even after spending two nights individually with a male, and virgin female moths laid unfertilized eggs for unknown reasons (Adler et al, 1991). Brussels sprouts (Brassica oleracea L. var. gemmifera) respond to an antiaphrodisiac of its herbivorous butterfly, P. brassicae (Fatouros et al, 2008) and this compound is delivered from males to females with seminal fluid during copulation and reduces the interest of females in further mating

(Andersson et al, 2003). Brussels sprouts may recognize insect oviposition through detection of this compound on leaves and may be able to even distinguish between fertilized and unfertilized eggs to save resources. However, our results indicate that tomato plants respond to eggs irrespective of egg fertility. It is interesting that tomato responded to infertile eggs, which would not lead to any feeding damage on the host plant in the future. We conjecture that in the interactions between tomato and H. zea, unfertilized eggs laid together with fertilized eggs

might increase the “alertness” on the host plant. This is the first report of the induction of plant

defensive response by deposition of unfertilized eggs.

Our results indicate that insect oviposition can prime plant defenses (Figs 2-5,6). Generally,

induced defense is considered more advantageous with priming (Karban, 2011). Priming may

reduce the possibility of development of a strategy to suppress plant defensive traits by

herbivorous arthropods (van Hulten et al, 2006), and the cost of priming is considered relatively

low (Zheng and Dicke, 2008). Priming by oviposition should benefit plants with induction of

more powerful defense upon anticipated herbivory as well as with minimized waste of

resources if eggs fail to hatch or if they are removed by predators. Due to the advantages of

priming and the frequency of oviposition by herbivorous insects on the host plant in the field

(Hilker and Meiners, 2011), priming of defenses by insect oviposition might be a common but

overlooked defense strategy of plants against future herbivory by neonates. Indeed,

suppression of antiherbivore defenses by insect oviposition found recently in Arabidopsis

(Bruessow et al, 2010) might be a counterploy by insects against this defensive strategy induced by insect oviposition. Interestingly, priming of plant defenses by insect oviposition was predicted (Hilker and Meiners, 2010).

In summary, we presented a series of results that indicate eggs deposited on tomato foliage by adult H. zea moths elicited a suite of defensive responses, including accumulation of H2O2,

expression of pin2, a defense gene aiming actively feeding insects, and elevated levels of the

defense hormone JA. Moreover, the spatial and temporal patterns of pin2 expression at the oviposition site were also determined. Our results indicate that oviposition primed plant

defense for impending herbivory. Taken together, the results presented here suggest that, upon H. zea oviposition, tomato plants perceive insect eggs and induce defense directed towards larvae that will soon hatch and inflict damage on plant tissue. A former study showed egg-induced plant effects on larval performance, but did not detect the chemical or molecular causes of these effects (Beyaert et al, 2012); in contrast, the present study detected egg- induced changes of JA-levels and transcript levels of a plant defense gene, but did not yet prove that these changes affect herbivore performance. In the future, it will be valuable to examine whether induction of defenses targeting neonates by insect oviposition is common in the field and how effective oviposition-induced defenses are. Characterization of potential elicitors of plant defenses may be useful for pest control as well as understanding of molecular mechanisms of oviposition-induced defense.

MATERIALS AND METHODS

Plants and Insects

Seeds of tomato (Solanum lycopersicum L. cv. Better Boy) were purchased commercially. Plants

were fertilized once with Osmocote Plus (15-9-12, Scotts, Marysville, OH, USA) 7-10 days after

seedlings were transferred to individual pots with Pro-Mix potting soil (Premier Horticulture,

Quakertown, PA, USA). Plants were grown in the greenhouse at the Pennsylvania State

University (University Park, PA) on a cycle of 16-hr day: 8-hr night at 24-28oC. Tomato plants

between the 4- to 5-leaf stages were used for oviposition treatment.

Eggs, larvae, and adults of H. zea were kept in an incubator on a cycle of 16-hr day: 8-hr night at

26oC. The eggs of H. zea were supplied from BioServ (Frenchtown, NJ, USA), and larvae were reared on artificial diet (Chippendale, 1970) in a 30-mL diet cup. The ingredients of artificial diet

were purchased from BioServ (Frenchtown, NJ, USA) and Sigma-Aldrich (St. Louis, MO, USA).

After pupation, each pupa was transferred to a new diet cup until the emergence of adults.

Oviposition Treatment

Five to six tomato plants were caged with 20 - 30 females and 10 - 15 males of 1-3 day old H.

zea moths in a cage (W x L x H = 75 x 63 x 88 cm) for 1.5 - 2 days with two scotophases in the experiments for Figs 2-1, 2-2, 2-5, and 2-6. Each moth in a cup was provided with several squirts

of 10% sugar solution for 2 - 4 hr. Moths laid different numbers of eggs per plant from dozens

to hundreds, and plants with at least 5 eggs on the distal leaflet of the 4th compound leaf were

used for further treatments.

In the experiment for Fig 2-3 where pin2 expression at the oviposition site was traced for 3 days,

moths were kept in a mating jar for 24 hr with 10% sugar solution on the bottom and squirted

on the wall before they were released into cages with tomato plants inside in order to reduce

the time of oviposition treatment to 1 day. In the experiment for Fig 2-4 to see the effect of

mating on the pin2 expression, three groups of moths of 30 virgin females, 20 virgin females

with 10 virgin males, and 30 virgin males, were kept in separate mating jars with sugar solution

as stated above for 24 hr, then each group of moths was released into a cage with 6 tomato

plants inside.

H2O2 Detection by DAB Staining

H.zea oviposition-treated tomato leaves were excised and the petioles had been dipped

overnight in 1 mg mL-1 solution (pH 3.8) of 3,3’-diaminobenzidine (DAB) under light at the room temperature. Then, chlorophyll of leaves was removed in double-boiling ethanol and H2O2

production was visualized as brown spots. Leaves were photographed before and after

dechlorophyllization (Orozco-Cárdenas et al, 2001).

Collection of Leaf Tissue

Each leaf tissue sample was collected from an individual plant. In the experiments where pin2

expression was measured at the oviposition site (results for Figs 2-1b, 2-2, 2-3, and 2-4), 15-20

egg-laid leaf disks of 5-mm diameter were punched off, eggs on leaf disks were removed, leaf

disks were put in a 2-mL tube with a metal milling ball, frozen in liquid nitrogen, and stored at -

80oC until RNA extraction. Leaf disks were sampled from the distal leaflet, and if necessary also from the medial and proximal leaflets, of the 4th compound leaf. For priming tests with pin2

(Figs 2-5 and 2-S1), 50-100 mg of leaf tissue from the distal leaflet of the 4th compound leaf was

taken after eggs were removed, frozen with a milling ball in liquid nitrogen, and stored at -80oC

until RNA extraction.

RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

RNA extraction was executed as previously described (Peiffer et al, 2009). Leaf tissue sampled

as described above was powdered with a metal milling ball in a 2 mL sample tube using

GenoGrinder 2000 (Spex SamplePrep, Metuchen, NJ, USA) at 1200 strokes per min, and RNA

was extracted with an RNeasy Plus Mini-kit (Qiagen, Valencia, CA, USA) following the

manufacturer’s instruction. cDNA was synthesized from 1 mg of RNA with High Capacity cDNA

Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and was used as template

for qRT-PCR after 10 times dilution. The sequences of the forward and reverse primers of pin2

(Gene Bank Accession number K03291) for qRT-PCR were 5`-GGA TTT AGC GGA CTT CCT TCT G-

3` and 5`- ATG CCA AGG CTT GTA CTA GAG AAT G- 3`, respectively. PCR product was amplified

with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and the

relative expression of pin2 was analyzed with 7500 Fast Real-Time PCR System (Applied

Biosystems, Foster City, CA, USA). Tomato ubiquitin gene was used as a reference gene (Gene

Bank Accession number X58253) and the sequences of the forward and reverse primers were

5’-GCC AAG ATC CAG GAC AAG GA-3’ and 5’-GCT GCT TTC CGG CGA AA-3’, respectively

(Rotenberg et al, 2006).

Test of priming by oviposition and trichome disruption

To test whether expression of tomato pin2 is primed by H. zea oviposition (Fig 2-S1), the

terminal leaflet of the 4th compound leaf of tomato plants treated with H. zea oviposition was

damaged by rolling a pattern wheel 25-mm long twice paralleled with the mid vein, and 20 μL

of 5-time diluted OS collected fresh from the 5th instar larvae of H. zea was applied on the

wound immediately. Leaf tissue was collected 0, 3, 8, and 24 hr after wounding treatment for

RNA extraction and qRT-PCR.

To test the possibility of priming by trichome disruption, a distal leaflet of the 4th compound

leaf was gently rubbed with a latex-gloved finger to break down leaf glandular trichomes.

Twenty four hr after disruption of trichomes, the leaflet was wounded and applied with 5 μL of

H. zea OS to mimic H. zea herbivory as described above. Leaf tissue was sampled after another

24 hr for RNA and qRT-PCR. The level of pin2 transcription was compared among intact plants,

trichome-disrupted plants, wounded plants, and plants treated with both trichome breakdown

and herbivory mimicry.

Quantification of JA

The amount of JA was quantified based on the method described by Tooker and De Moraes

(Tooker and De Moraes, 2005). After H. zea oviposition and wounding treatment and the eggs

were gently removed, 100 mg of leaf tissue was sampled under liquid nitrogen into a FastPrep

tubes (Qbiogene, Carlsbad, CA, USA) containing 1 g of Zirmil beads (1.1 mm; Saint-Gobain ZirPro,

Mountainside, NJ, USA), 400 µL of extraction buffer (1-PrOH : H2O : HCl = 2 : 1 : 0.002, v/v), and

100 ng of dihydrojasmonic acid (diH-JA) as an internal standard. DiH-JA was obtained by alkaline hydrolysis of methyl dihydrojasmonate (Bedoukian Research Inc., Danbury, CT, USA).

Leaf tissue was sampled 0, 30, 60, and 180 min after treatment with wounding and H. zea OS treatment and stored at -80oC until necessary.

Plant leaf tissue was shredded in FastPrep FP120 (ThermoSavant, Holbrook, NY) for 40 sec at

5.5 unit speed at the room temperature. After 1 mL of CH2Cl2 was added, FastPrep tubes were shaken against in FastPrep FP120 for 40 sec at 5.5 unit speed at the room temperature. After centrifugation at 10,000 g for 1 min (Heraeus Biofuge Pico, Thermo Fisher Scientific, Waltham,

MA), the organic layer was transferred to a 4-mL screw-capped glass vial with a glass syringe

(Hamilton Company, Reno, NV) and dried up under gentle air flow at the room temperature. JA

in the dried samples were methylesterificated into methyl jasmonate (MJ) with 2.3 µL of

trimethylsilyl diazomethane (TMS-CH2N2; 2M in hexane; Sigma-Aldrich, St. Louis, MO, USA) in

100 µl of MeOH / diethyl ether (1 : 9, v/v) for 25 min at the room temperature. The remaining

TMS-CH2N2 was neutralized by addition of 2.3 µL of hexane / AcOH (88 : 12, v/v) for additional

25 min at the room temperature. MJ was evaporated at 200oC into a SuperQ (80/100 mesh;

Alltech, Deerfield, IL) trap for 2 min and recovered with 150 µL of CH2Cl2 into a glass insert in a

GC vial for GC-MS analysis.

MJ was chemically ionized with isobutene and analyzed on the selected ion monitoring mode

by GC/MS (6890 Plus / 5973N, Agilent, Santa Clara, CA) equipped with HP-1MS column (length

30 m, inner diameter 0.25 mm, film thickness 0.25 µm; Agilent, Santa Clara, CA). The injection

port was maintained at 250oC and the oven temperature was kept on 40oC for 1 min, increased

by the rate of 15oC min-1, and maintained at 250oC for 7 min.

Statistics

All the data were subject to Grubb’s test to statistically remove outliers (p < 0.05; Graphpad

Software). When log transformed data satisfied the assumptions of normality and equal variances, significant difference of data was determined with Proc GLM, and when the assumptions were not satisfied, non-parametric GLM was used instead. Multiple comparisons of data were carried out with Tukey’s test (SAS 9.3, SAS Inc.).

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FIGURES

Fig 2-1. Response of tomato leaves to H. zea eggs at the oviposition site. (A) H2O2 production under eggs of H. zea was visualized by DAB staining on an oviposition-treated tomato leaf. Left panels, the upper surface of a leaf; right panels, the lower surface of a leaf; upper panels, before DAB staining; down panels, after DAB staining. (B) Induction of pin2 expression at the H. zea oviposition site. Relative pin2 expression is presented in the graph. Data were analyzed for significance with non-parametric Proc GLM (Mean ± SE; ** above bars indicate significant difference; Chi-square = 6.8182, p = 0.009, n = 5).

Fig 2-2. Intensity of pin2 induction with distance from eggs. Relative pin2 expression is presented in the graph. Data were analyzed for significance with non-parametric Proc GLM and

compared with Tukey’s test (Mean ± SE; letters above bars indicate significant difference; Chi- square = 14.4695, p = 0.0023, n = 4 or 5).

Fig 2-3. Temporal fluctuation of transcriptional level of tomato pin2 at the H. zea oviposition site. Relative pin2 expression is presented in the graph. Data were collected for 3 days from the oviposition treatment to the emergence of neonates and analyzed for significance with non- parametric Proc GLM (Mean ± SE; letters above bars indicate significant difference; Day 1, Chi-

Square = 3.9382, p = 0.0472, n = 5; Day 2, Chi-Square = 0.2400, p = 0.6242, n = 4 or 5; Day 3,

Chi-Square = 6.0000, p = 0.0143, n = 4 or 5).

Fig 2-4. Effect of the egg fertility on tomato pin2 expression at the H. zea position site. Relative pin2 expression is presented in the graph. Data were analyzed for significance with Proc GLM

and compared with Tukey’s test (Mean ± SE; letters above bars indicate significant difference;

F3,15 = 22.99, p < 0.0001, n = 4 or 5).

Fig 2-5. Priming effect of H. zea oviposition on tomato pin2 expression. Effect of previous H. zea oviposition on the induction of tomato pin2 upon following simulated herbivory was investigated. Control, intact plants without oviposition treatment (closed circle); Oviposition, plants treated only with oviposition (open circle); Wounding, plants mechanically damaged and

OS-applied without oviposition treatment (closed triangle); Ovi+Wnd, plants treated with oviposition followed by mechanical wounding and OS application (open triangle). Without mechanical damage, there are only Control and Oviposition at time 0h. At times 8h and 1d, closed circles (control) are hidden behind open circles (oviposition). Relative pin2 expression is presented in the graph. Data were analyzed for significance with non-parametric Proc GLM and compared with Tukey’s test (Mean ± SE; letters next to spots indicate significant difference; n.s., data not significantly different; at 0h, Chi-Square = 21.00, p = 0.0025, n = 4; at 3h, Chi-Square =

6.3240, p = 0.0969, n = 4 or 5; at 8h, Chi-Square = 13.2857, n = 5, p = 0.0024; at 1d, Chi-Square =

14.3843, n = 4 or 5, p = 0.0024).

A 8 B 200 C C 6 150 n.s. 4 100 B B JA (ng/g leaf) JA (ng/g JA (ng/g leaf) JA (ng/g 50 2 B B A A A 0 0 intact v w v/w v w v/w v w v/w CON OVI 30 min 1 hr 3 hr Time After Wounding

Fig 2-6. Priming effect of H. zea oviposition on JA levels in tomato leaves. (A) Effect of H. zea oviposition on basal JA levels. Data were analyzed for significance with Proc GLM (Mean ± SE; n.s., data not significantly different; Proc GLM; F1,8 = 0.03, p = 0.8600, n = 5). (B) Effect of previous H. zea oviposition on the induction of JA production by mechanical wounding and application of H. zea OS. Data were analyzed for significance with non-parametric Proc GLM and compared with Tukey’s test (Mean ± SE; letters above bars indicate significant difference; n.s., data not significantly different; at 30 min, Chi-Square = 11.2604, p = 0.0036, n = 5; at 1 hr,

Chi-Square = 11.18, p = 0.0037, n = 5; at 3 hr, Chi-Square = 9.7582, p = 0.0076, n = 4 or 5).

Abbreviations: v, tomato plants treated with H. zea oviposition; w, tomato plants treated with mechanical wounding and application of H. zea OS; v/w, tomato plants treated with H. zea oviposition followed by mechanical wounding and application of H. zea OS.

250

C 200 C

150

100 Expression of pin2 of Expression 50 B A 0 CON TRI WND TRI+WND

Fig 2-S1. Effect of trichome disruption on the level of pin2 expression upon subsequent

mechanical wounding and application of H. zea OS. Trichome disruption did not influence the level of pin2 expression upon subsequent simulated herbivory. Data were analyzed for significance with non-parametric Proc GLM and compared with Tukey’s test (Mean ± SE; Chi-

Square = 17.3945, p = 0.006, N = 5 or 6; letters above bars indicate significant difference).

Chapter 3:

Varied responses of tomato to Helicoverpa zea oviposition

Jinwon Kim and Gary W. Felton*

Department of Entomology and Center for Chemical Ecology, Pennsylvania State University,

University Park, Pennsylvania, USA

ABSTRACT

The eggs of herbivorous insects laid on the host plants are a bad omen for the plants because

the larvae will hatch soon from the eggs and cause serious damage on the plant fitness until

they leave or pupate. Many plants are now known to possess a variety of defense mechanisms to remove or kill the insect eggs laid on the host plant. Recently I reported that tomato plants are primed by Helicoverpa zea oviposition and induce stronger defensive responses to the following mechanical wounding. In this study I investigated various aspects of H. zea oviposition-induced priming of defensive responses in tomato, including (1) varietal variation of tomato response to H. zea oviposition, (2) the response of the jasmonic acid (JA)-deficient mutant of the cultivar Castlemart, def-1, to H. zea oviposition, (3) whether tomato plants distinguish between fertilized and unfertilized eggs of H. zea in the oviposition-mediated defensive priming, (4) the effect of the extract of H. zea accessory glands of on the defensive response of tomato, and (5) the effect of H. zea oviposition on the host plant quality. I found that (1) different tomato varieties show different responses to H. zea oviposition, (2) H. zea oviposition suppressed wound response in the def-1 mutant, (3) tomato was not primed by H. zea unfertilized eggs, (4) the application of the extract of H. zea accessory glands on tomato foliage did not elicit significant defensive response in tomato, and (5) H. zea oviposition on tomato plants inconsistently influenced on the performance of the conspecific neonates on the host plant. The results presented in this study indicate that the interactions between H. zea and tomato plants mediated by H. zea oviposition are more complicated than they appear.

INTRODUCTION

Eggs deposited by insect herbivores on the host plant may turn into a disaster in the near future

because larvae will hatch from the eggs and consume a considerable amount of plant tissue,

causing serious loss to the plant fitness. At the same time, eggs are sessile and a developmental

stage vulnerable to predation and environmental stresses. Thus, if plants perceive the insect

eggs and induce proper defenses, it could be easier and more efficient for plants to remove the

eggs before the ambulatory larvae hatch and start feeding on the plant tissue. Many plant

defense mechanisms to kill insect eggs are known (Hilker and Meiners, 2006).

I recently showed that the eggs of tomato fruitworm moth, Helicoverpa zea Boddie

(Lepidoptera: Noctuidae), laid on tomato leaves prime and induce stronger defensive responses

upon the following wound treatment (Kim et al, 2012). As a follow-up, I investigated other

aspects of H. zea oviposition-induced defense priming in tomato plants. First, I examined

varietal variation in tomato’s response to H. zea oviposition between the tomato cultivar Better

Boy that showed oviposition-induced defense priming and another tomato cultivar Castlemart.

Second, I explored the effect of H. zea oviposition on the wound-induced pin2 induction in the

jasmonic acid (JA)-deficient mutant, def-1 (Howe et al, 1996). JA is a plant hormone responsible

for defense induction against herbivory (Pieterse et al, 2009), and pin2 is the gene encoding

protease inhibitor2, a representative induced defense trait of tomato. Pin2 induction was found to be primed by H. zea oviposition in the previous report (Kim et al, 2012). Due to the lack of the JA-dependent signaling pathway, the level of pin2 induction in the def-1 mutant is

significantly attenuated compared to its wild type cultivar Castlemart. I conducted experiments to understand how tomato plants respond to H. zea oviposition in the def-1 mutant. Third, unfertilized H. zea eggs were demonstrated to induce pin2 expression at the oviposition site on tomato plants in the previous report (Kim et al., 2012). Here I tested whether H. zea unfertilized eggs can prime tomato pin2 expression as the fertilized eggs do. Fourth, the effect of the extract of H. zea accessory glands on the tomato defensive response was measured. Brussels sprouts are known to induce indirect defense and attract egg parasitoids when Pieris brassicae butterflies deposit eggs on the leaf tissue. This indirect defense of the host plant was found to be induced by benzyl cyanide, an antiaphrodisiac that is produced in the male accessory glands and transferred to the female accessory glands during mating to prevent further mating

(Fatouros et al, 2008). I investigated whether the accessory glands of H. zea adults are responsible for the tomato response to H. zea eggs. Last, I explored whether H. zea oviposition priming of defensive responses in tomato plants reduces the performance of H. zea neonates.

RESULTS

Helicoverpa zea Oviposition Does Not Prime pin2 Induction in Castlemart, and Suppresses

pin2 Response in the JA-Deficient Mutant def-1

In the previous study, I reported priming of pin2 expression by H. zea oviposition in the tomato cultivar Better Boy (Kim et al, 2012). To simplify the experiment design, I collected leaf tissue 24 hr after the wound treatment on the oviposition-pretreated tomato plants instead of executing a time course experiment, and I obtained the results indicating priming of pin2 expression by H. zea oviposition (Fig 3-1: Non-parametric Proc GLM; Chi-Square = 12.2426; p = 0.0066; n = 4). H. zea eggs increased the resting pin2 expression level and primed pin2 expression resulting in enhanced pin2 expression level upon the following wound treatment accompanied with the application of H. zea oral secretion. I will simply use ‘wound treatment’ instead of ‘wound treatment accompanied with the application of H. zea oral secretion’ from now on.

Then, I tested whether H. zea oviposition causes priming of defense in a different tomato cultivar Castlemart and its JA-deficient mutant def-1. The experiment was repeated twice. In the first experiment, H. zea egg deposition induced the resting level of pin2 expression in the

Castlemart, but did not prime pin2 induction (Fig 3-2A: Castlemart; Non-parametric Proc GLM;

Chi-Square = 16.5965; p = 0.0009; n = 4-6). In the JA-deficient def-1 mutant, oviposition did not

increase the resting pin2 expression level, nor primed pin2 expression. Instead, H. zea

oviposition-pretreated def-1 mutants exhibited the attenuated level of pin2 expression upon the following wound treatment (Fig 3-2B: def-1; Proc GLM after log transformation; F3,16 = 27.1; p < 0.0001; n = 5).

In the second experiment, H. zea oviposition treatment on Castlemart did not increase the resting pin2 expression level, nor prime pin2 induction. H. zea eggs even suppressed pin2 expression upon the secondary wound treatment (Fig 3-2C: Castlemart; Non-parametric Proc

GLM; Chi-Square = 13.4137; p = 0.0038; n = 4 or 5). In the def-1 mutant, the suppressive effect

of H. zea oviposition on tomato pin2 expression was more pronounced than the first

experiment (Fig 3-2D: def-1; Proc GLM after log transformation; F3,16 = 6.26; p = 0.0045; n = 5).

It is noteworthy that the pin2 expression level induced by mechanical wounding in Castlemart

was much higher than that of Better Boy (Fig 3-1, Fig 2A,C).

Unfertilized Eggs of H. zea Do Not Prime pin2 Induction in Tomato Plants

In the previous study, I showed tomato plants upregulate the transcriptional level of pin2 at the

oviposition site of unfertilized eggs H. zea. I tested whether H. zea unfertilized eggs also prime

pin2 expression. Different from the previous report, H. zea unfertilized eggs did not increase

the resting level of pin2 expression in this experiment. This is because leaf tissue was collected

from a wider area of leaflets, instead of leaf disks right beneath the unfertilized eggs, and thus

the number of unfertilized eggs per unit tomato leaf area was much lower in this experiment.

The unfertilized eggs of H. zea did not prime pin2 induction, either. The pin2 expression level in response to wound treatment was somewhat higher with H. zea unfertilized egg pretreatment than without H. zea unfertilized eggs on average, but the difference was not statistically significant (Fig 3-3: Non-parametric Proc GLM; Chi-Square = 15.2743; p = 0.0016; n = 5), indicating tomato plants distinguish between fertilized and unfertilized eggs of H. zea.

Extract of the Accessory Glands of H. zea Adults Do Not Induce pin2 in Tomato Plants

H. zea eggs elevate the resting level of pin2 expression at the oviposition site and resulted in

stronger and faster accumulation of pin2 transcripts in the primed leaf tissue upon the

following wound treatment (Kim et al, 2012). However, it is unknown what causes egg-induced

responses in tomato plants. I tested whether H. zea accessory glands are involved in the

oviposition-induced tomato response. Accessory glands were taken out by dissection from

virgin females, virgin males, mated females, and mated males, and applied on tomato leaves.

Dissection of H. zea adults for accessory glands were carried out next to the test tomato plants in the greenhouse because the materials in the female accessory glands polymerize within seconds after detached from H. zea abdomen. One day after the application, tomato pin2 mRNA was extracted and quantified. The extract of H. zea accessory glands did not induce the pin2 expression level in tomato leaves (Fig 3-4: Proc GLM after log transformation; F4,24 = 2.37; p = 0.0809; n = 5 or 6).

The Effect of H. zea Egg-Induced Defense Priming on the Performance of H. zea Neonates.

I investigated the effect of H. zea oviposition on the performance of H. zea neonates. The experiment was repeated twice. In the first experiment, tomato plants were previously treated with H. zea oviposition for 2 days. All the eggs were gently removed from tomato plants, and twenty neonates of H. zea were gently placed with a camel’s hair brush on the distal leaflets of

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the 3rd and 4th compound leaves, ten on each distal leaflet. The number of neonates surviving on tomato plants was counted for 3 days, and surviving larvae were weighed on day 3. H. zea larvae showed decreased growth and survival on H. zea oviposition-pretreated tomato plants. H. zea larvae on control plants gained about 40% more weight (Fig 3-5A: Proc GLM; F1,10 = 44.27; p

< 0.0001; n = 6) and showed about 25% more survival (Fig 3-5B: Day 1: Non-parametric Proc

GLM; Chi-Square = 0.5824; p = 0.4454; Day 2: Proc GLM; F1,10 = 11.21; p = 0.0074; Day 3: Proc

GLM; F1,10 = 15.32; p = 0.029; n = 6) than those on H. zea oviposition-primed plants. However, in

the second experiment, previous H. zea oviposition on tomato plants did not influence on the performance of H. zea. Growth of H. zea neonates was lower on H. zea oviposition-pretreated plants, but the difference was marginally not significant (Fig 3-5C: F1,10 = 4.22; p = 0.067; n = 6),

and survival of H. zea neonates was not different between the two plant groups (Fig 3-5D: F1,10

= 0.06; p = 0.8059; n = 6). The effect of H. zea oviposition pretreatment on the performance of conspecific neonates was inconclusive.

However, it was noticed that in the second experiment, some neonates were found boring

inside the rachises and looked bigger than leaf-eaters, which might have diminished the

statistic difference. The performance of H. zea neonates on leaves and on rachises was

compared and the results are discussed below.

H. zea Neonates Perform Better on Rachises than on Leaves of Tomato

Some of H. zea neonates were found to consume the inside of the rachises and appeared to

grow faster than leaf-eaters. I compared the performance of H. zea neonates on leaves and on

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rachises. Leaflets and rachises were prepared from the 4th compound leaf of the tomato plants

with four fully expanded leaves. Neonates were provided with the distal leaflet and the rachises of the 4th compound leaf and grown for 5 days at 26oC. Surviving neonates were counted and weighed. While the survival of neonates either on leaves or on rachises after 5 day feeding was not statistically different (20 and 17 out of 20 neonates, respectively; Exact binomial test; p =

0.7428), H. zea neonates grew more than two times faster on rachises than on leaflets (Fig 3-6A:

Non-parametric Proc GLM; Chi-Square = 15.9579; p < 0.0001; n = 20 and 17, respectively). The neonates consumed some 20% more rachis tissue than leaf tissue (Fig 3-6B: Non-parametric

Proc GLM; Chi-Square = 10.8502; p = 0.0010; n = 20 and 17), and more interestingly, the efficiency of conversion of ingested plant tissue into larval body weight (weight gain / plant tissue loss) was over 2 times higher on rachises than on leaves (Fig 3-6C: Non-parametric Proc

GLM; Chi-Square = 13.3762; p = 0.0003; n = 20 and 17, respectively). The results indicate that

the rachis tissue is more suitable food source with higher nutritional values or lower level of

antiherbivore defenses than leaf tissue and implies the possibility that the rachis-boring

behavior of some H. zea neonates may be an adaptation to the decreased food quality by

induced defense in leaves. In addition, the outer dermal tissue of the rachises is hard, and some

shavings of hard dermal tissue were found at the entrance hole of the tunnels formed by

neonatal rachis-boring. While feeding the soft and nutritive vascular tissue and the protection

from predation inside rachises are the benefits, and the drilling of the hard dermal tissue of the

rachises appears the cost of the rachis-boring behavior of H. zea neonates on tomato plants.

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DISCUSSION

In the previous study, I reported priming of defensive responses by H. zea oviposition in tomato

cultivar Better Boy (Kim et al, 2012), which was confirmed in this study with Better Boy (Fig 3-1).

Priming by oviposition is considered beneficial with induction of more powerful defenses against the anticipated insect herbivore and with flexible resource management for the cases when the expected herbivory does not occur, e.g. when the eggs are removed by egg predators

(Kim and Felton, 2013). However, defense priming was not observed in a different tomato cultivar Castlemart (Fig 3-2A,C). Castlemart is a favored cultivar in the research of induced defense of tomato with its sensitivity in the defense activation. In this research, the relative pin2 expression was measured using qRT-PCR and thus the pin2 expression levels are expected to be somewhat varied among experiments. However, the range of the pin2 expression level induced by mechanical wounding in Better Boy is much lower than that in Castlemart (hundreds vs. hundreds of thousands, respectively) (Fig 3-1, Fig 3-2A,C; and see the data in Kim et al, 2012;

Peiffer et al, 2011; Chung and Felton, 2011). Hence, it could be possible that pin2 expression level induced by mechanical wounding in Castlemart was already too high for defense priming to be evident, whereas in Better Boy, the pin2 induction by wound treatment was moderate and thus the oviposition priming resulted in significantly enhanced induction of pin2.

Tomato def-1 mutant is defective in JA biosynthetic pathway and susceptible to insect herbivores (Howe et al, 1996). I investigated how the def-1 mutant responds to H. zea oviposition and following mechanical wounding. Mechanical wounding induced pin2 expression

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in the def-1 mutant but on a small scale, compared to its wild-type counterpart Castlemart.

However, when the def-1 mutant was previously exposed to H. zea oviposition, the pin2 induction by mechanical wounding was significantly suppressed, and in the second experiment, the suppressive effect of H. zea oviposition was more pronounced (Fig 3-2B,D). Considering H. zea oviposition sensitizes wound-inducible pin2 induction in the cultivar Better Boy, suppression of defense induction was unexpected. Suppression of plant defensive responses by insect oviposition was previously reported in the two different study systems, Arabidopsis thaliana and Pieris brassicae (Bruessow et al, 2010), and Zea mays and Spodoptera frugiperda

(Peñaflor et al, 2011). Salicylic acid (SA) is the hormone organizing the defense against biotrophic pathogens in plants, and acts antagonistically against JA-signaling pathway (Pieterse et al, 2009). Herbivorous insects are often found to activate SA-signaling pathway to suppress

JA-dependent antiherbivore defenses (Pieterse et al, 2009), and the manipulation of the plant hormonal crosstalk between JA and SA was proposed as the underlying mechanism of the suppression of defense in A. thaliana by P. brassicae oviposition in the former reports

(Bruessow et al, 2010). In the interactions between tomato and H. zea, it might be possible that

H. zea utilizes some effectors to suppress JA-dependent antiherbivore defenses in tomato, for example, via induction of the SA signaling pathway, as shown in the def-1 mutant, but that the tomato possesses a defense mechanism triggered by the perception of the egg-associated effectors, which results in defense priming in the presence of the intact JA-dependent defense

pathway, as shown in Better Boy. It is warranted to investigate whether JA and SA signaling

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pathways are induced or suppressed at the hormonal and transcriptional levels in tomato in

response to H. zea oviposition followed by herbivory.

Whereas tomato induced pin2 in leaf tissue beneath unfertilized H. zea eggs (Kim et al, 2012), the infertile eggs did not prime tomato pin2 expression (Fig 3-3), indicating that tomato plants,

at least Better Boy, distinguish between fertile and infertile eggs. The results make sense because unfertilized eggs laid by H. zea virgin females pose no threat to the plant fitness. It is probable that induction of pin2 right on tomato leaves right beneath unfertilized eggs (Kim et al,

2012) might have been caused by disruption of tomato leaf trichomes, which was reported to

induce, but not prime, pin2 expression (Peiffer et al, 2010; Kim et al, 2012). In the system of the

Brussels sprouts (Brassica oleracea L.), the cabbage great white butterfly (Pieris brassicae L.)

and the egg parasitoid Trichogramma brassicae Bezdenko, the Brussels sprouts appears to

perceive the deposition of the butterfly eggs by sensing a chemical in the egg glue, benzyl

cyanide. Benzyl cyanide is an antiaphrodisiac produced in the accessory glands of the male

butterfly and transferred to the female accessory glands during mating to prevent further

mating (Fatouros et al, 2008). I investigated whether any components in the accessory glands of

H. zea are involved in the egg-inducible tomato response, but the accessory glands from males

or females, whether mated or virgin, failed to induce pin2 expression on tomato leaves (Fig 3-4).

As tomato appeared to distinguish between fertilized and unfertilized eggs, it was expected

that the accessory glands would secrete egg elicitors to induce the defensive responses in

tomato, but it was not the case. Tomato plants might utilize some chemical cues that are

formed on fertilization (i.e. some chemicals that might be formed after eggs and sperm meet

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before the eggs are laid) or that are slowly released from the eggs after deposition onto plant

surface.

Plants are considered to induce a selective set of defensive traits that are most effective against

the given insect species (Agrawal and Karban, 1999; Zangerl, 2003). Considering a plant species

usually host multiple herbivore species(Strauss, 1991), the effect of the defensive traits induced by an early insect species on the next herbivore, so called specificity of effect, will be critical in understanding plant-insect interactions at the community level. Specificity of effect was reported in plants being attacked by herbivorous insects (van Zhant and Agrawal, 2004; Chung

and Felton, 2011), and is even found in the transgenerationally primed defenses (Rasmann et al,

2012; Kim and Felton

, 2013). The effect of H. zea oviposition on the performance of conspecific neonates was

investigated in the repeated experiments, but the results were not conclusive. In the first experiment, H. zea neonates showed significantly lower performance on the H. zea oviposition- pretreated tomato plants (Fig 3-5A,B), an expected results caused by the oviposition-priming effect. In the second experiment, however, there was no difference in the larval performance

(Fig 3-5C,D).

It was noticed that some H. zea neonates were found feeding inside of the rachises of both the control and oviposition-treated plants, and they looked bigger than other leaf-eating neonates.

In a non-choice feeding test, the rachises were shown to be more suitable food for the larval growth than leaflets (Fig 3-6A). The neonates consumed about 20% more rachis tissue (Fig 3-

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6B), and more importantly, the efficiency of conversion of ingested food to body substance of

the rachis-borers was more than two-fold higher than the leaf-eaters (Fig 3-6C). In addition, H.

zea neonates were completely concealed in the rachises and well protected from predators and

dryness. However, the rachises are also well protected with the hard dermal tissue. Thus,

drilling through the hard shell into the soft vascular tissue should be the cost of the rachis-

boring behavior by H. zea neonates. Despite all the benefits, not all neonates showed the

rachis-boring behavior. It might be the results of the variation in the structure or hardness of the mouthparts, in the sclerotization time, or in the feeding behavior, of H. zea neonates. Some insect larvae move around are ambulatory are known to manipulate the level of plant defense in their favor using the defense-suppressing effectors (Musser et al, 2002; Wu et al, 2012). In addition to physiological adaptations, larvae may move away or balloon off from the unsuitable feeding site, searching for a new feeding site. The rachis-boring by H. zea neonates also may be a behavioral adaptation of H. zea neonates against induced defense of tomato plants.

In this study, I showed different responses of the tomato cultivar Castlemart from Better Boy upon H. zea oviposition followed by the following mechanical damage, and also the suppression of pin2 expression in the JA-deficient Castlemart mutant, def-1. Different from viable fertilized eggs, tomato did not prime defensive response upon the deposition of H. zea unfertilized eggs, and different from the system of the Brussels sprouts and pierid butterflies, the accessory glands were not responsible for the oviposition-inducible response in tomato. The effect of defense priming by H. zea oviposition on the performance of the conspecific neonates was dynamic. The results presented in this chapter indicate that the interactions between tomato plants and H. zea surrounding H. zea eggs are more complicated than they appear.

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MATERIALS AND METHODS

Plants and Insects

Seeds of the tomato cultivar Better Boy (Solanum lycopersicum L.) were purchased commercially. Seeds of the cultivar Castlemart and its JA-deficient mutant, def-1, were generously provided by Dr. Gregg Howe (Michigan State University). Tomato seeds were planted and germinated in masses and the seedlings were transferred individually to pots with

Pro-Mix potting soil (Premier Horticulture, Quakertown, PA, USA). Tomato plants were fertilized once with Osmocote Plus (15-9-12, Scotts, Marysville, OH, USA) 7-10 days after seedlings were transferred. Plants were grown in the greenhouse at the Pennsylvania State University

(University Park, PA) on a 16-hr photoperiod cycle at 24-30oC. Tomato plants with four fully

expanded leaves were used for all the experiments unless stated otherwise.

H. zea was grown in the incubator on a 16-hr photoperiod at 26oC. Eggs were supplied from

BioServ (Frenchtown, NJ, USA). Larvae were grown on wheat-germ-based artificial diet in 30-mL

diet cups (Chippendale, 1970). The ingredients of artificial diet were purchased from BioServ

(Frenchtown, NJ, USA) and Sigma-Aldrich (St. Louis, MO, USA). After pupation, each pupa was

transferred to a new diet cup. After adults emerged, they were sexed every day by their wing

color (male, greenish brown; female, brown).

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Oviposition and Wound Treatment

The 6-L mating jars were prepared for each of cages. A mating jar was bedded with 2 sheets of paper towel, and 10% sugar solution (w/v) was poured enough to soak the paper towel sheets and squirted fully on the wall until sugar solution drops started to run down. 15 - 20 of 1-3 day old males and females were thrown in a 6-L mating jar and allowed to feed to sugar solution for about 3 hr. Satiated moths in a mating jar were released into a cage (W x L x H = 75 x 63 x 88 cm) and mated and laid eggs on tomato plants for 1.5 - 2 days (two scotophases). Moths laid a varied number of eggs per plant from dozens to two hundred. Plants whose distal leaflet of the

4th compound leaf received at least 5 eggs were used for further treatments or feeding

experiments. For the unfertilized egg treatment on tomato plants, 20 virgin females were used

without males and other conditions were the same.

Wound Treatment and Sampling of Leaf Tissue

H. zea oral secretion (OS) means the mixture of regurgitant and saliva. Regurgitant and saliva

are unavoidably mixed when collected because of the structure of mouthparts and spinneret of

H. zea caterpillars. Fresh H. zea OS was collected from 1-2 day old 5th instars of H. zea by

physically stimulating their mouthparts and 5-time diluted with deionized H2O. The distal leaflet

of the 4th compound leaf of tomato plants was mechanically wounded by rolling a pattern

wheel twice in parallel to the midvein, and 20 μL of diluted H. zea OS was applied on the line-

puncture wound. 50-100 mg of leaf tissue was harvested 24 hr after wounding treatment,

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deep-frozen with liquid nitrogen, and stored at -80oC until RNA extraction. H. zea eggs laid on leaf tissue were manually removed before sampling.

RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Frozen leaf tissue was pulverized with a metal milling ball in a 2 mL sample tube using

GenoGrinder 2000 (Spex SamplePrep, Metuchen, NJ, USA) for 30 sec at 1200 strokes per min.

RNA was extracted using an RNeasy Plus Mini-kit (Qiagen, Valencia, CA, USA) following the