Preferences of Plutella Xylostella Oviposition for Mechanically Damaged, Herbivore Damaged, and Plant-Plant Primed Arabidopsis Thaliana

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PREFERENCES OF PLUTELLA XYLOSTELLA OVIPOSITION FOR MECHANICALLY DAMAGED, HERBIVORE DAMAGED, AND PLANT-PLANT PRIMED ARABIDOPSIS THALIANA

Tyler Thompson

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

December 2014

Committee:

M. Gabriela Bidart-Bouzat Advisor

Helen Michaels

Daniel Wiegmann

ii ABSTRACT

M. Gabriela Bidart-Bouzat, Advisor

Previous studies have shown that herbivore-induced plant volatiles can affect responses of damaged plants, neighboring plants, and insect herbivores. However, little is known about how different types of plant induction affect the oviposition behavior of insect herbivores. In this study, the effects of inducing plants by exposing them to an insect herbivore, mechanical damage or a damaged neighboring plant was evaluated on the oviposition preferences of the diamondback moth Plutella xylostella. The potential role of plant genotypes differing in their glucosinolate hydrolysis profiles was also evaluated by using a wild ecotype (Col-0) and a genetically modified (GM) line(tgg1tgg2) of the model plant Arabidopsis thaliana.. While the wild type Col-0 line has normal production of hydrolysis products (mainly isothiocyanates in its leaf tissues), the double myrosinase knockout (tgg1 tgg2) is defective in the production of these volatiles. Dual choice oviposition assays were performed using naïve females of Plutella xylostella and the two A. thaliana lines differing in their hydrolysis profiles, which were exposed to the three different types of induction treatments. Female oviposition preferences were significantly influenced by the type of induction to which a plant was exposed as well as by the plant genotype, which differed in their glucosinolate hydrolysis profiles. For example, P. xylostella females significantly preferred to oviposit on herbivore damaged plants (versus undamaged controls) when Col-0 plants were used, but chose control plants when presented with the double myrosinase knockout tgg1tgg2. However, plant genotype did not influence oviposition choices between plant-plant primed or mechanically damaged plants and undamaged controls. Results from this study showed that the type of plant induction and genotype influenced insect oviposition preferences and suggest that these factors may be important to iii consider as part of the management strategies used to control specialist insect pests such as the diamondback moth P. xylostella. iv

ACKNOWLEDGMENTS

I would like to firstly thank my advisor, Dr. Gabriela Bidart-Bouzat for all of the direction and encouragement she has given me. Without her help and expertise this work would not have been possible. I would also like to thank my committee members, Dr. Helen Michaels for the mentoring and support, as well as Dr. Daniel Wiegmann for his dedication and recommendations.

I am also very grateful for the support of my lab members for their team effort in helping in many stages of this work, in particular Amanda Curtis; her dedication to helping others is incredible. I would also like to thank my friends and family for their support. Lastly, I would like to specifically thank my parents, whom have been invaluable with their guidance and belief in my ability to reach my goals. v

TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 5

Study Organisms and Growth Conditions ...... 5

Experimental Design ...... 6

Data Analyses ...... 8

RESULTS ...... 9

DISCUSSION ...... 11

CONCLUSION ...... 15

LITERATURE CITED ...... 20 vi

LIST OF FIGURES/TABLES

Figure/Table Page

Figure 1 COL-0 Genotype Treatments and Oviposition Index ...... 17

Figure 2 tgg1 tgg2 Genotype Treatments and Oviposition Index ...... 18

Table 1 ANOVA results showing the effects of treatment, trial, and genotype on an

oviposition preference index ...... 19 1

INTRODUCTION

As a specialist of crucifer crops, P. xylostella has evolved the ability to breakdown toxic glucosinolates (GSLs) and their hydrolysis products, which are secondary compounds involved in the defense response of plants belonging to the Brassicaceae (Fahey et al. 2001; Ratzka et al.

2002). Glucosinolates and myrosinases (the enzymes that break down GSLs) are stored separately within the plant cell. Upon herbivore damage these chemicals come into contact and release a variety of glucosinolate hydrolysis products, such as isothiocyanates or nitriles

(Kliebenstein et al. 2005; Barth & Jander 2006). These hydrolysis products are also known as

“herbivore induced plant volatiles” (HIPVs), since they are produced as a result of the interaction between damaged plant tissues and insect saliva (Dicke & van Loon 2000). In addition to intact

GSLs, these HIPVs can act as either deterrents or ovipostion attractants to crucifer insect pests and associated predators/parasitoids, as well as have a role in inducing or priming neighboring plants (Dicke & van Loon 2000; Godard et al. 2008; Puente et al. 2008; Heil & Karban 2009;

Sun et al. 2009; Badenes-Perez et al. 2012). Even though the role of GSL hydrolysis products on insect oviposition has been previously studied, it is not known how different types of plant induction influence oviposition preferences of insects associated with the Brassicaceae .

As noted above, GSL hydrolysis products are released when plants are damaged, for example, by insect herbivores; and the presence of myrosinase enzymes are critical for the production of these products. Different natural ecotypes of the model plant Arabidopsis thaliana are known to have distinct GSL hydrolysis profiles. For example, while the well-known wild type Col-0 ecotype produces mainly isothiocyanates in its leaves and the Landsberg (Ler) ecotype produces nitriles as products of hydrolysis (Lambrix et al. 2001). In contrast, genetically modified lines, which have decreased or null production of myrosinase enzymes, fail

2 to form GSL hydrolysis products upon tissue damage (Barth & Jander, 2006). Therefore,

Arabidopsis thaliana genotypes with variation in GSL hydrolysis profiles are useful to make predictions about the potential role of these chemicals on insect oviposition responses.

Glucosinolate hydrolysis products are known to be involved in plant responses to both specialist and generalist insect herbivores (Lambrix et al. 2001; Kliebenstein et al. 2002;

Beekwilder et al. 2008; Sarosh et al. 2010; Verhage et al. 2011). However, different types of hydrolysis products appear to have different effects on different insect herbivore species. For example, isothiocyanates (ITCs) appear to be more toxic to generalist than to specialist crucifer insects (Kos et al. 2012; Ryan & Bidart-Bouzat, 2014). Often ingestion of this type of hydrolysis product by generalist insects such as Trichoplusia ni or Spodoptera exigua results in slower insect developmental time, decreased growth, and increased mortality, as well as lower plant herbivore damage levels (Bidart-Bouzat & Kliebenstein, 2011; Ryan & Bidart-Bouzat,

2014). Conversely, previous evidence suggests that specialists such as P. xylostella females actually prefer to oviposit on crucifer plants with higher levels of GSLs and hydrolysis products such as the wild plant Barbarea vulgaris (Badenez-Perez et al. 2009) or the wild type Col-0 ecotype of Arabidopsis thaliana (Ryan & Bidart-Bouzat 2014) rather than on crop varieties or mutants devoid of these chemicals. It is known that P. xylostella uses these chemicals as oviposition cues and that the larvae are able to detoxify these compounds by using a specific enzyme (i.e., GSL sulfatase) present in their guts (Ratzka et al. 2002; Bones & Rossiter 2006).

In contrast to ITCs, nitriles are another type of GSL hydrolysis product that seems to be better tolerated by insects despite their degree of ecological specialization (Lambrix et al. 2001;

Wittstock et al. 2004). For example the generalist Trichoplusia ni preferred the nitrile producing

A. thaliana lines (e.g., Ler) instead of the more toxic isothiocyanates (Lambrix et al. 2001).

Therefore, insect responses to GSL hydrolysis products seem to be tightly linked to the evolutionary history of the plant and insect species involved in an interaction.

It is well known that insect specialists use plant chemicals as cues to locate plant hosts as well as for oviposition purposes. For example, females of the specialist P. xylostella can use aliphatic or indole GSL as well as their hydrolysis products (e.g., isothiocyanates or nitriles) as pre and post-alighting signals for host location and oviposition (Sun et al. 2009). Aliphatic GSL- derived hydrolysis products, in the absence of indole GSLs, have been shown to be oviposition stimulants for P. xylostella females (Renwick et al. 2006). For example, sulforaphane and iberin are isothiocyanates (ITCs) believed to be pre-alighting stimulants for gravid P. xylostella (Sun et al. 2009). Other post-alighting chemical signals may be used once a host has been located, such as intact indole GSLs that can be detected by the insect at the plant surface (Sun et al. 2009).

More information on the chemicals associated with plant host location by insects are important in furthering the understanding of oviposition choices in P. xylostella and other crucifer insect pests.

Plants, as well as insects, are capable of perceiving herbivore induced plant volatile

(HIPV) signals emitted by damaged neighboring plants (Karban et al. 2010). Responses to

HIPV signals from a damaged plant to an intact neighbor have been referred to as plant-plant signaling. As HIPVs are produced, they travel through the air and can be received by neighboring plants, which can then become induced/primed. This form of plant induction is known as priming, and can possibly reduce herbivore attacks, which in turn can minimize tissue damage to the plant (Karban et al. 2004). In addition, the primed state has been shown to be associated with an up-regulation of genes related to plant defenses (Godard et al. 2008). Priming may allow plants to respond faster and in a more precise way to target the specific herbivore

4 attacker (Conrath et al. 2006). Previous studies using Arabidopsis thaliana, willow (Salix sitchensis), cotton (Gossypium hirsutm), lima bean (Phaseoulus lunatus), corn (Zea mays), sagebrush (Artemisia tridentata), and bean (Vicia faba) have found evidence of this type of plant-plant signaling (Godard et al. 2008; Heil et al. 2009; Karban et al. 2010). In one such study, sagebrush plants that were primed by plant volatiles from a damaged focal plant received significantly less herbivory compared to plants not exposed to the volatile treatment (Karban et al. 2010). This plant-plant priming effect has been studied in relation to herbivory, but little research has been done to understand the effects priming may have on insect oviposition preferences.

The main goal of this study was to evaluate the effects of three different types of plant induction; that is, mechanical damage, herbivore damage and plant-plant priming, on oviposition preferences of the specialist Plutella xylostella. There is evidence that different forms of plant induction, such as herbivore damage by leaf chewers versus phloem feeders or plant-plant priming, can result in the up-regulation of different plant defense pathways (Godard et al. 2008;

Bidart-Bouzat and Kliebenstein 2011; Schweiger et al. 2014). However, the comparative effects of different types of plant induction on insect oviposition behavior remain unknown; especially, regarding the potential role of GSL hydrolysis products on insect oviposition when plants are induced by other plants rather than by insect herbivory. Results from this study may have implications for the management of insect pests, such as the diamondback moth, which are difficult to control with conventional pest control strategies.

MATERIALS AND METHODS

Study Organisms and Growth Conditions:

Two genotypes of A. thaliana were used in this study: the wild type Col-0 and the double myrosinase knockout tgg1tgg2. The Col-0 genotype is a wild type of A. thaliana, which produces isothiocyanates as the main hydrolysis product released by damaged leaves (Panthee et al. 2011). In contrast, tgg1tgg2 is a double myrosinase knockout, which results in impaired production of GSL hydrolysis products in damaged leaf tissue (Barth and Jander 2006). These two A. thaliana lines were selected to evaluate oviposition responses to different forms of plant induction, which may also be dependent on the plant genotype used.

All A. thaliana plants used in this experiment were grown from seed in germination flats

(24 x 45 x 7 cm deep) using a commercial soil mix Pro-Mix BX (Premier Tech Horticulture,

Quakertown, PA). Plants were placed in a cold treatment at 4°C in dark conditions for five days to ensure uniform germination and eliminate dormancy. After the cold treatment, plants were moved to growth chambers set at 21°C with a photoperiod of 16 h light: 8 h dark. Plants were grown for four weeks before being used in the experiments.

Plutella xylostella larvae used in this experiment were purchased from Benzon’s

Research (Carlisle, Pennsylvania, USA) and housed in a sealed growth chamber at 21°C with a photoperiod of 16 h light: 8 h dark. Larvae were reared on artificial diet also supplied by

Benzon’s Research and allowed to eclose in plastic containers inside a growth chamber without plants. After eclosion, adult moths were transferred to 30 cm3 Bug Dorm-1 Rearing Cages

(BioQuip, California, USA) and reared on a 10% honey solution. Adults were allowed to mate freely for at least 24 h to ensure that females used were gravid prior to use in oviposition assays.

Experimental Design:

Arabidopsis thaliana plants were randomly assigned to the following treatment groups: control (no induction), mechanically damaged, herbivore damaged, and plant-plant primed.

Control treatment plants were housed in a sealed growth chamber separate from exposure to any other treatment group. Randomly selected plants were clipped twice with scissors on the same leaf (avoiding the mid-vein of the leaf) for the mechanical damage treatment. This treatment was designed to mimic the damage caused by herbivore feeding. For the herbivore damage treatment, two 2nd instar P. xylostella larvae were placed on each plant 24 h prior to use in the oviposition assay. Plants were then covered with a fine mesh (Svenson LS econet M, Hummert

International, USA) to avoid larval movement between plants while permitting light and gas transmittance. These plants were housed in a sealed growth chamber separate from the control treatment and not allowed to come into physical contact with other plants. Mesh covering and P. xylostella larvae were removed prior to use in the oviposition assays. Plant-plant primed treatment plants were placed in the same growth chamber as those from the herbivore damage treatment 24 h before the assay. While these plants were held in close proximity to herbivore- induced plants, they were not permitted to physically touch any other plant.

Oviposition trials used 30.5 x 30.5 x 43.18 cm styrofoam coolers, lined with aluminum foil prior to trials to minimize non-target oviposition (Ryan and Bidart-Bouzat, 2014). Within each cooler, one control plant was placed 10 cm apart from a treatment plant. A single gravid female was collected from the breeding cage and introduced to the center of the cooler. Then, the cooler was sealed. Since P. xylostella oviposits at night, a heavy-duty black plastic garbage bag was placed over the cooler to further ensure darkness inside the cooler. After five hours, moths and plants were removed from coolers. Using a dissecting microscope, the total number of

7 eggs laid on each plant was scored. Female P. xylostella moths were only used once and not allowed to re-integrate into the breeding population after exposure to plants; thus, ensuring all females used in the experiments were naïve to the plant chemicals.

For each genotype, the dual choice oviposition assays were conducted using a single plant from each induction treatment versus a control plant. Plants and P. xylostella females were used only once in each choice oviposition trial. A total of 174 trials using Col-0 plants (348 Col-

0 plants) and 253 trials using tgg1tgg2 plants (506 tgg1tgg2 plants) were used in the dual choice oviposition assays. Specifically, two separate rounds of experiments using each of the two plant genotypes were performed (repetition of the experiments). For the first round using the wild type Col-0, 32 control vs. mechanical damage, 21 control vs. herbivore damage, and 23 control vs. plant-plant primed trials were performed. For the second round using this genotype, 24 control vs. mechanical damage, 44 control vs. herbivore damage, and 30 control vs. plant-plant primed trials were performed. Likewise, using the tgg1tgg2 genotype, 60 control vs. mechanical damage, 44 control vs. herbivore damage, and 23 control vs. plant-plant primed trials were completed for the first round of dual choice experiments; and 36 control vs. mechanical damage;

28 control vs. herbivore damage; and 62 control vs. plant-plant primed trials were completed for the second round. All previous counts refer to trials that resulted in females ovipositing on plants. Trials that resulted in no oviposition on either the control or the treatment plant were excluded from the dataset.

Data Analyses:

To evaluate oviposition choices of P. xylostella for plants with different induction treatments versus control plants, an oviposition index was calculated as follows: OPI = (X-

Z)/(X+Z), where X represents eggs laid on the control plant, and Z represent eggs laid on the treatment plant (Ryan & Bidart-Bouzat, 2014). The index values ranged from -1 to +1, where negative values represent female oviposition preferences for the induction treatment plant and positive values indicate preferences for the control plant. All statistical tests were performed using SAS Version 9.2 (SAS Institute, 1999). An ANOVA on rank-transformed data was performed to test for the effects of the different dual oviposition choice assays (i.e., control vs. each plant induction treatment), genotype, and repetition of the full experiment (2 rounds of replicated oviposition choice assays at different times). Means of the OPI associated to the three oviposition choice assays were pairwise compared by performing a non-parametric CI test on ranked transformed data. Confidence intervals (99%) were estimated as well as two tailed t-tests were performed to determine whether the OPIs were equal or significantly different from zero for each dual oviposition choice assay (i.e., plants from each induction treatment versus control).

RESULTS

The oviposition preference of P. xylostella was significantly impacted by the treatment to which the individual plants were exposed. As shown by the results from an Analysis of

Variance, the type of dual oviposition assay (i.e., control versus each of the plant induction treatments) significantly influenced the oviposition preference index (P = 0.0318; Table 1).

There were no significant differences between the 2 repetitions of the full experiment (replicated oviposition choice assays) at different times (P = 0.6971, Table 1). In addition, even though the effect of plant genotype per se was not significant (P=0.2162; Table 1), the interaction between the type of oviposition assay and genotype was found to be significant (P = 0.0018, Table 1).

This significant result indicates that oviposition choices of females in the different assays were influenced by the plant genotype used (i.e., wild type Col-0 or the double knockout tgg1tgg2).

For example, in the “control vs. herbivore damage” assay, females significantly preferred to oviposit on herbivore damaged plants when Col-0 plants were used (Figure 1), but chose control plants when the assay was done with tgg1tgg2 plants (Figure 2).

Female oviposition choices (i.e., treatments vs. the control groups) were corroborated by estimating 99% confidence intervals (Figures 1 and 2). When Col-0 plants were used in the assays, females chose to oviposit on herbivore damaged vs. control plants but preferred control over plant-plant primed plants (Figure 1). Conversely, females preferred to oviposit on control rather than on herbivore damaged plants when tgg1tgg2 mutant plants were used (Figure 2).

However, oviposition responses to plant-plant priming were the same for both genotypes; i.e., female moths preferred to oviposit on control rather than on plant-plant primed plants when using either Col-0 or tgg1tgg2 plant genotypes (Figures 1 and 2).

Results from oviposition assays using the mutant tgg1tgg2 showed different female oviposition responses compared to those using the wild type Col-0. Multiple comparisons among OPI means indicated that these indices differed among the different oviposition assays

(i.e., control versus plants exposed to the three different induction treatments). Results using the wild type Col-0 showed the OPI mean for the herbivore damage vs. control assay was significantly different from those of both the mechanical damage versus control assay and the plant-plant priming vs. control assay (Figure 1). On the other hand, results from experiments using tgg1tgg2 mutants showed that only OPIs of herbivore damage or mechanically damaged vs. control assays were significantly different from each other (Figure 2).

DISCUSSION

This study provides evidence that the type of plant induction (i.e., mechanical or herbivore damage, or plant-plant priming) can significantly influence oviposition preferences of a specialist insect pest. Insect oviposition preferences were influenced not only by the type of plant induction, but also by the plant genotype used in the dual oviposition choice assays. For example, female oviposition preferences for herbivore damaged Col-0 plants (over undamaged control Col-0 plants) were reversed when tgg1tgg2 plants were used in the assays. This result suggests that GSL hydrolysis products released by Col-0 plants (mostly isothiocyanates following plant tissue damage), but absent in the tgg1tgg2 double knockout, may be important in determining oviposition choices of P. xylostella females. On the other hand, the absence of oviposition choices when the plants were mechanically damaged suggests that this form of induction does not have the same effect as when plants are actually damaged by a conspecific, regardless of the plant genotype used in the assays. These results are consistent with by previous findings by Reddy et al. (2004), which showed that P. xylostella prefers to oviposit on cabbage plants that have been previously damaged by conspecific larvae rather than on undamaged plants. Finally, it is worth noting that insect oviposition was influenced by plant-plant priming in a different manner than when plants were exposed to actual herbivory. An oviposition avoidance of plants that have been induced/primed by other plants, regardless of the GSL hydrolysis profile of the two plant genotypes used, suggests that other unknown volatiles may be involved in this type of plant-plant induction.

Results from dual oviposition choice assays including plants damaged by P. xylostella larvae suggested that the type of plant genotype used in the assays was important in determining the outcome of the experiment. Since the genetically modified tgg1tgg2 A. thaliana line shares

12 the same genetic background of the wild type Col-0 (from which it was derived), the two lines used in this study only differed in their GSL hydrolysis profiles (Barth & Jander, 2006).

Therefore, the presence of myrosinase activity, which is needed for the production of GSL hydrolysis products (i.e., ITCs in Col-0) and is impaired in tgg1tgg2 plants, may explain the difference in P. xylostella oviposition responses when either the wild type Col-0 or the double myrosinase mutant is used. Previous studies have shown that hydrolysis products released by A. thaliana Col-0 plants (i.e., ITCs) act as oviposition stimulants for the specialist P. xylostella

(Renwick et al. 2006; Vos et al. 2008; Sun et al. 2009; Ryan & Bidart-Bouzat, 2014). Two of these studies found that P. xylostella females had an innate preference for the ITC-producing line

Col-0 over another genetically modified A. thaliana (35S:ESP) line that produces nitriles, which is a different type of hydrolysis product (Vos et al. 2008; Bidart-Bouzat & Ryan 2014). In addition, P. xylostella developed an oviposition preference for Col-0 over tgg1tgg2 plants when the insects were reared on Col-0 plants; that is, when the insect experienced Col-0 volatiles in its natal environment (Ryan & Bidart-Bouzat, 2014). These previous results are in accordance with those of the present study, in terms of emphasizing that GSL hydrolysis products, and in particular ITCs, seem to have an important role in the oviposition choices of the specialist P. xylostella.

In contrast to findings related to the herbivore-damaged plant induction treatment, female oviposition preferences for primed versus control plants were not influenced by the plant genotype used. When using either the wild type Col-0 or the tgg1tgg2 genotype, there was an oviposition avoidance of plants that had been previously exposed to damaged plants (plant-plant priming treatment) and a preference for control plants. It is known that a variety of herbivore- induced plant volatiles (HIPVs) are released upon damage, and that these HIPVs, in turn can

13 induce a response in the receiving plant, commonly known as “priming” (Dicke & Baldwin,

2009). There is evidence that HIPVs can play a role in plant defense and insect oviposition choices (Conrath et al. 2006; Godard et al. 2008; Karban et al. 2010). In fact, a previous study by Goddard et al. (2009) has shown that plant-plant priming induces an up-regulation of some defense-related genes in the plant. However, the potential role of these volatiles in plant-plant signaling, and the identity of the volatiles responsible for this type of communication are less understood. It is also unknown if there is a role of GSL hydrolysis products in plant-plant priming. Results from this study suggest that these hydrolysis products (or at least ITCs) may not have a major role in plant-plant communication, as oviposition choices were not dependent on the genetic line used. If ITCs would have a role in plant-plant priming, then the same oviposition choice outcome detected in the herbivore damage vs. control assay when using either Col-0 or tgg1tgg2 plants should have been observed. In other words, females should have preferred to oviposit on primed Col-0 plants in the same way that they preferred herbivore-induced Col-0 plants. Further research to evaluate the specific volatiles that are involved in plant-plant communication is underway.

Previous studies suggest that plant signaling chemicals such as jasmonic acid (JA) or ethylene may be involved in plant-plant communicaton (Conrath et al. 2006; Heil & Karban,

2009; Karban et al. 2010; Balunra & Ninkovic, 2010). It is well known that JA is one of the most important signaling molecules involved in the early steps of induction of defense-related pathways (Howe, 2004; Tian et al. 2013), and probably one of the key compounds involved in plant-plant signaling (Baldwin et al. 2006; Conrath et al. 2006). In a study by Engelberth et al.

(2004), mechanically and herbivore damaged plants induced the production of JA and other volatile organic compounds (VOCs) in undamaged plants. Other VOCs such as monoterpenes,

14 which are emitted by A. thaliana damaged plants, were also found to induce the production of methyl jasmonate. Likewise, Peng et al. (2011) found that VOCs released by cabbage plants were involved in priming of neighboring plants. Specifically, plants exposed to volatiles emitted by plants damaged by Pieris brassicae larvae had increased accumulation of a key enzyme (i.e., lypoxygenase) involved in the synthesis of JA. These plant-plant primed plants also attracted more parasitoids than control plants not exposed to damaged plants. Furthering the understanding of the role of JA and other VOCs in plant-plant communication can be important for the management of insect pests, especially in strict monocultures found in modern agriculture.

CONCLUSION

This study provides evidence that the oviposition preferences of P. xylostella can be significantly altered by the type of induction to which a plant is exposed (i.e., herbivore or mechanical damage, or plant-plant priming). This information may be helpful when considering alternative management strategies for insect pests such as P. xylostella, which can be difficult to control with conventional techniques. In addition, differences in oviposition preferences when using different genetic lines of A. thaliana suggest that these responses may be affected by genetic modification of crop plants, which is also important for integrated pest management. It is worth noting that the effect of the genotype was most pronounced in oviposition assays including herbivore-damaged plants. Since selected genotypes differed in glucosinolate hydrolysis profiles, these results corroborate previous findings related to the role of these chemicals on insect oviposition choices (Renwick et al. 2006; Badenez-Perez et al. 2009; Sun et al. 2009). Conversely, the presence or absence of glucosinolate hydrolysis products did not seem to impact the oviposition behavior of P. xylostella females; that is, moths preferred control and avoided primed plants in both genotype treatments. This result suggests that glucosinolate hydrolysis products may not be directly involved in plant-plant signalling; however, it was not possible to elucidate in this study what other phytochemicals were responsible for the observed responses. Further research to identify the specific volatiles involved in plant-plant priming is underway. This information is important because these unknown chemicals induced by plant- plant priming have been shown to have the potential to reduce oviposition by common agricultural pests such as Spodoptera littoralis (Zakir et al. 2013), Heliothis virescens (De

Moraes et al. 2001) and P. xylostella (this study). More studies are needed to identify what chemicals are involved in plant-plant signaling in crucifers and other economically important

16 plant species. Ultimately, this study contributes to the further understanding of insect oviposition preferences on plant hosts that are exposed to different types of induction, which maybe an important aspect to be considered by integrated pest management.

FIGURES AND TABLES

Figure 1. Effects of different types of induction of Arabidopsis thaliana Col-0 plants on P. xylostella oviposition preferences. Means of an oviposition preference index (OPI) associated to three different oviposition choice assays were compared using a non-parametric 99% CI test. Different letters indicate significant differences among OPI means (P<0.05). Negative values on the Y-axis represent a preference for plants exposed to an induction treatment vs. control plants, and positive values indicate preference for control plants in each choice assay. Asterisks next to the letters indicate that the OPI mean is significantly different than zero based on 99% confidence intervals.

Figure 2. Effects of different types of induction of Arabidopsis thaliana tgg1tgg2 plants on P. xylostella oviposition preferences. Means of an oviposition preference index (OPI) associated to three different oviposition choice assays were compared using a non-parametric 99% CI test. Different letters indicate significant differences among OPI means (P<0.05). Negative values on the Y-axis represent a preference for plants exposed to an induction treatment vs. control plants, and positive values indicate preference for control plants in each choice assay. Asterisks next to the letters indicate that the OPI mean is significantly different than zero based on 99% confidence intervals.

Table 1. ANOVA results showing the effects of replicated P. xylostella oviposition choice assays (using plants exposed to different induction treatments vs. control plants), two A. thaliana genotypes, and repetition of the experiments on an oviposition preference index.

Source DF F-value P-value

Oviposition Assay 2, 420 3.14 0.0442

Genotype 1, 420 1.53 0.2162 Assay × Genotype 2, 420 6.39 0.0018

Repetition 1, 420 0.15 0.6971

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