DIFFERENTIAL EFFECTS OF GLUCOSINOLATE PROFILES AND HYDROLYSIS PRODUCTS IN ARABIDOPSIS THALIANA ON GENERALIST AND SPECIALIST INSECT HERBIVORES

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1 iii DIFFERENTIAL EFFECTS OF GLUCOSINOLATE PROFILES AND HYDROLYSIS PRODUCTS IN ARABIDOPSIS THALIANA ON GENERALIST AND SPECIALIST INSECT HERBIVORES Julie A. Kemarly-Hopkins 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 2012 Committee: M. Gabriela Bidart-Bouzat, Advisor Juan L. Bouzat Dan Wiegmann

2 iv 2012 Julie A. Kemarly-Hopkins All Rights Reserved

3 iii ABSTRACT M. Gabriela Bidart-Bouzat, Advisor Glucosinolates and their hydrolysis products, found in plants of the order Brassicales, are a widely studied group of secondary chemicals known for their defensive properties against insect herbivores. However, the specific effects of individual glucosinolates as well as their hydrolysis products are still largely unknown. Arabidopsis thaliana (Col-0) genetic lines with mutations that modify the type of glucosinolate, such as myb28myb29, which lacks aliphatic glucosinolates, and the line cyp79b2cyp79b3 that does not produce indolyl glucosinolates, make it possible to test the specific effects of these different glucosinolates on herbivores. Likewise, the effects of different glucosinolate hydrolysis products can be evaluated using genetically modified lines of Col-0 (which naturally produces isothiocyanates), including the nitrileproducing line 35S:ESP and the double knockout tgg1tgg2. In no-choice experiments with the crucifer specialist insect Pieris rapae, differences in hydrolysis products had no significant effect on insect fitness-related traits, while differences in glucosinolate products did significantly affect pupae weight as well as days to pupation. Pieris rapae larvae spent significantly less time on plants lacking camalexin, and the pupae reared on plants lacking aliphatic glucosinolates weighed significantly less than those reared on the other genetic lines. In contrast, the generalist insect Spodoptera exigua was significantly affected in all fitness-related traits measured, preferring those lines lacking hydrolysis products (tgg1tgg2) as well as those lacking either aliphatic or indolyl glucosinolates (myb28myb29 and cyp79b2cyp79b3, respectively). Spodoptera exigua larvae spent significantly less time on plants lacking aliphatic glucosinolates,

4 iv while the pupae and adults weighed more when reared on those lines lacking indolyl glucosinolates. Results from feeding choice trials showed that Pieris rapae had no particular preference for any of the genotypes. In contrast, Spodoptera exigua had a significant feeding preference for the double mutant tgg1tgg2, which virtually lacks hydrolysis products. Overall, this study provides evidence that variation in glucosinolate profiles and hydrolysis products can influence insect performance as well as feeding choices, although the responses are dependent on the insect species.

5 v I dedicate this thesis to my husband Sean Patrick Dowland. Never did I think I would get so lucky as to find a man like you. You are an O-class star in a field of class-m s.

6 vi ACKNOWLEDGMENTS I want to thank my advisor, Dr. Gabriela Bidart-Bouzat, for all her help and assistance during this project. It really hasn t been easy! I also want to thank my lab mate Debo for all of her wonderful advice and support. I want to thank Dr. Juan L. Bouzat and Dr. Dan Wiegmann for challenging me to improve and for the valued constructive criticism. And above all else, I want to thank the one man who believed in me more than anyone else, my wonderful husband. Thank you honey, for knowing I could do this before I did.

7 vii TABLE OF CONTENTS Page INTRODUCTION MATERIALS AND METHODS... 5 Arabidopsis thaliana genetic lines and growth conditions... 5 Insect species... 7 No-choice tests... 8 Choice tests Data analyses RESULTS No-choice experiments Choice experiments DISCUSSION REFERENCES... 17

8 viii LIST OF FIGURES/TABLES Figure Page 1 Effects of distinct glucosinolate profiles on P. rapae and S. exigua Effects of glucosinolate hydrolysis products on P. rapae and S. exigua Choice trials of P. rapae and S. exigua... 26

9 1 INTRODUCTION Plants serve as a food source for a wide variety of insect herbivores across multiple families and feeding guilds. Leaf chewer insects, such as beetles (Coleoptera) and caterpillars (Lepidoptera), make up the vast majority of phytophagous insects (Schoonhoven et al. 1998). The goal of these insect herbivores is to successfully derive nutrition from the plant, while the goal of the plant is to avoid being damaged by the insects. There are multiple strategies that plants use to avoid being eaten by insects, most of them mechanical or chemical in nature. In order to survive, the insect must overcome plant defenses. As a result, a co-evolutionary relationship is formed, where, for example, an insect species evolves adaptations to withstand plant defenses and the plant species evolves counter-adaptations to maintain fitness in the presence of insect herbivores. This evolutionary arms-race has resulted in a number of plants producing a vast array of volatile secondary chemicals, which usually play a crucial role in plantinsect interactions. It is now known that these chemicals can act as either plant defenses against herbivory by insects (Wittstock and Gershenzon 2002; Wittstock et al. 2003; Hopkins et al. 2009) or serve to attract other insect herbivores (Wittstock et al. 2003; Hopkins et al. 2009; Ikeura et al. 2010) and parasitoids (Wittstock et al. 2003; Hopkins et al. 2009). Some secondary chemicals can even be sequestered by herbivorous insects to be used in their own defense against predation (Malcolm 1995; Hopkins et al. 2009). Although secondary chemicals can be effective in deterring or repelling insect herbivores and thus, negatively influence their fitness, other insect species are unaffected by these phytochemicals. Some insects species, namely generalist feeders (i.e., insects with a wide host range), do suffer a decrease in fitness, including higher mortality, when they feed on plants with

10 2 toxic secondary chemicals (Li et al. 2000; Wittstock et al. 2003; Arany et al. 2008; Wu et al. 2010). However, specialist feeders (insects with a narrow host range) have not only adapted to these chemicals, but are often attracted to plants producing them (Wittstock et al. 2003; Hopkins et al. 2009; Ikeura et al. 2010). In such cases, these insects have evolved multiple strategies to mitigate potential negative effects from the toxins, including detoxification, sequestering of chemicals, and behavioral strategies (Krieger et al. 1971; Ratza et al. 2002; Agrawal and Kurashige 2003; Wittstock et al. 2004). The ability of specialist insect herbivores to tolerate secondary chemical defenses within their host plants, versus that of generalist insects is a key principle of the specialist-generalist paradigm (reviewed in Ali and Agrawal 2012). While it is assumed, according to the specialist-generalist paradigm, that specialist insects are relatively unaffected by secondary chemicals, this is not always the case, and there have been a number of recent studies suggesting contrary trends (Agrawal 2000; Van Dam et al. 2000; Kliebenstein et al. 2002; Agrawal and Kurashige 2003; Bidart-Bouzat and Kliebenstein 2011). Even so, there are obvious differences in the effects of secondary chemicals on generalist and specialist insect species, primarily due to the capacities of specialists to circumvent or detoxify plant toxic compounds (Li et al. 2000; Cornell and Hawkins 2003; Arany et al. 2008). Among the most well studied interactions between plants and insects is of the one between the Brassicaceae plant family and its specialist and generalist herbivores. The principal secondary metabolites found in this family are known as glucosinolates (Fahey et al. 2001; Halkier and Gershenzon 2006). Glucosinolates are hydrolyzed by enzymes called myrosinases when the plant is fed upon, producing, in some cases, toxic hydrolysis products called isothiocyanates. The glucosinolate/myrosinase system is also commonly known as the mustard oil bomb (Wittstock et al. 2003). Glucosinolates are considered to be feeding deterrents for

11 3 generalist herbivorous insects, while simultaneously acting as oviposition and feeding stimulants for specialist insects (Wittstock et al. 2003; Barth and Jander 2006; Arany et al. 2008; Bidart- Bouzat and Kliebenstein 2008; Ikeura et al. 2010). This dichotomy has led to differential ecological effects on crucifer plants, because to protect against one herbivore potentially makes it vulnerable to another (Bidart-Bouzat and Kliebenstein 2008). However, the specific effects of distinct glucosinolates and their hydrolysis products on specialist and generalist insect herbivores are still unclear. While some studies have supported the specialist-generalist paradigm (Van Der Meijden 1996; Burow et al. 2006a; Arany et al. 2008), others have contested it (Van Dam et al. 2000; Agrawal and Kurashige 2003; Bidart-Bouzat and Kliebenstein 2011; Ali and Agrawal 2012). Early studies on the effects of glucosinolates and their hydrolysis products were limited by the complexity of these chemicals and their interactions in experimental settings. For example, intact glucosinolates added to insect artificial diets cannot demonstrate the effects of the hydrolysis products on insects. In addition, glucosinolate hydrolysis products are volatiles and may even react with components of the artificial diet (Wittstock et al. 2003). However, the identification of specific genes used in plant defense (specifically related to the glucosinolatemyrosinase system) has led to the production of transgenic and mutant plants varying only in glucosinolate profiles or hydrolysis products, which in turn have provided an excellent tool for the study of the effects of these secondary chemicals on insect herbivores and the extent to which they may deter herbivory (Wittstock et al. 2003). While several studies have investigated the effects of glucosinolates on insect herbivores, few studies have evaluated the role of these compounds and their hydrolysis products on both generalist and specialist herbivores. In this experiment, genetically altered A. thaliana plants differing only in their glucosinolate profiles and hydrolysis products were used to assess the

12 4 effects of these secondary compounds on the specialist Pieris rapae and the generalist Spodoptera exigua. This study tested the hypothesis that the selected specialist and generalist species will likely differ in their performance and feeding preferences for A. thaliana lines differing in intact glucosinolate profiles and hydrolysis products as a result of their distinct physiological adaptations. Them it can be predicted that the specialist P. rapae (adapted to feed on glucosinolate containing plants) will perform equally well on all genetic lines in the no choice experiment and show no preference for any particular line in the choice experiment. Conversely, the generalist S. exigua, which is not adapted to glucosinolates, will likely perform better (in no choice experiments) and show preferences (in choice experiments) when reared or given the choice for lines lacking a type of intact glucosinolate or on lines releasing hydrolysis products considered to be less toxic to herbivores. The selected plant lines used in this study have the same genetic background as the wild type Col-0 plants from which they are derived. No-choice experiments, where larvae were placed on the different genetically modified plants to complete their growth cycle, were used to assess the effects of the different intact glucosinolates and hydrolysis products on fitness-related traits of the selected insect herbivores (traits related to growth and developmental). In addition to the no-choice experiments, choice experiments were conducted to assess preferences of each lepidopteran species for different A. thaliana glucosinolate hydrolysis profiles. These choice and no-choice experiments and the use of genetically modified A. thaliana lines with known differences in their glucosinolate profiles and hydrolysis products are important for evaluating ecological questions related to insect preferences and host plant use in the field of plant-insect interactions.

13 5 MATERIALS AND METHODS Plant Genetic Lines and Growth Conditions Arabidopsis thaliana is a self-fertilizing, annual plant of the Brassicaceae family, which includes broccoli, oilseed rape (canola), mustard and cabbage, as well as many other agriculturally important crops. Even though A. thaliana is not a commercially important plant, its relationship to other members of its family, as well as its life history attributes, make it an ideal model organism for the study of plant-insect interactions. Like other members of this plant family, A. thaliana produces glucosinolates, which are secondary chemicals that serve an important role in plant defense against herbivores and pathogens. About 120 different glucosinolates have been identified within the Brassicaceae family (Bones and Rossiter 1996; Fahey et al. 2001; Halkier and Gershenzon 2006). Distinct glucosinolates (β-thioglucoside-nhydroxysulfates) share the same basic chemical composition, a central carbon bound to a thioglucose group and a sulfate group, and a variable side chain derived from aminoacid precursors (Wittstock and Halkier 2002). When plant tissues are damaged, the enzyme myrosinase (β-thioglucoside glucohydrolase, TGG) comes into contact with the glucosinolates and hydrolyzes them, producing isothiocyanates, nitriles, thiocyanates, and other glucosinolate hydrolysis products (Wittstock and Halkier 2002; Halkier and Gershenzon 2006). The model plant A. thaliana is known to produce at least 34 distinct glucosinolates, the composition of which varies depending on the ecotype (Kliebenstein et al. 2001). The wild type Col-0 and five mutant lines were used in this study to evaluate the effects of the different glucosinolate profiles and hydrolysis products on the performance and feeding choice of two insect species. The ecotype Col-0 produces primarily isothiocyanates (Wittstock et al. 2003),

14 6 and each of the mutant lines used in this study (35S:ESP, tgg1tgg2, cyp79b2cyp79b3, pad3 and myb28myb29) contain the same genetic background as Col-0, differing only in their glucosinolate profiles or hydrolysis products (Barth and Jander 2006; Müller et al. 2010; Zhou et al. 1999). Both 35S:ESP and tgg1tgg2 contain the same glucosinolate profiles as Col-0, but differ in the type of hydrolysis product formed. The transgenic line 35S:ESP overexpresses the epithiospecifier protein (ESP) found in another Arabidopsis ecotype, Landsberg erecta (Ler), which causes the most abundant glucosinolate found in Col-0 leaves, 4- methylsulfinylbutylglucosinolate, to be hydrolyzed into simple nitriles instead of the corresponding isothiocyanate (Burow et al. 2006b). The double mutant tgg1tgg2 lacks the two major aboveground myrosinases that are responsible for the hydrolysis of leaf glucosinolates in A. thaliana. This deficiency prevents the production of isothiocyanates and greatly slows the production of nitriles (Barth and Jander 2006). The genetic lines cyp79b2cyp79b3 and myb28myb29 differ from Col-0 in their intact glucosinolate profiles. The double knockout cyp79b2cyp79b3 has mutations in two genes that lead to the production of indole-3-acetaldoxime and, therefore, blocks the production of indolyl glucosinolates (Zhao et al. 2002). The double mutant myb28myb29, in contrast, blocks the production of aliphatic glucosinolates (Beekwilder et al. 2008). Camalexin is an indole phytoalexin, which is also synthesized from indole-3-acetaldoxime, and is therefore, also absent in cyp79b2cyp79b3. Finally, the pad3 mutant blocks only the production of camalexin while leaving the pathway to indole production intact, thereby serving as a control for the effects of indolyl glucosinolates (Zhou et al. 1999; Zhao et al. 2002). Seeds of the wild type Col-0 were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA) and the genetically modified lines were

15 7 kindly supplied by Dr. Daniel Kliebenstein (University of California, Davis). Plants used for all experiments were stratified for five days at 5 C in complete darkness before planting in 50-well flats using a commercial soil mix (ProMix, Premier Horticulture Inc., Quakertown PA, USA). At two weeks old, plants were transferred to 10 cm pots and placed in growth chambers set at a 10/14 day/night photoperiod and a 23/20 C day/night temperature cycle. Experiments were conducted when plants were at the pre-bolting stage. Insect Species The generalist moth Spodoptera exigua (Lepidoptera: Noctuidae) and the specialist butterfly Pieris rapae (Lepidoptera: Pieridae) were used in both choice and no-choice experiments. Both S. exigua (commonly known as beet armyworm) and P. rapae (small cabbage white butterfly) are common agricultural pests of cruciferous plants. The moth S. exigua, which originated in Southeast Asia, was first identified in Oregon (USA) in 1876 and by 1924 it had reached Florida (Tingle and Mitchell 1977). Currently, S. exigua exists in 101 different countries (Zheng et al. 2011). The life cycle of this common insect pest can be completed in as few as 24 days, and during summer months this species can complete multiple life cycles (Wilson 1934). The specialist butterfly P. rapae, a Palearctic native that is widespread across Europe and Asia, was first reported in North America in 1860 in Quebec City, Canada, and like S. exigua quickly made its way south and was found on the gulf coast by 1886 (Scudder 1887). The number of generations this species completes in a year depends on climatic conditions. It can complete its life cycle in as few as three weeks in warmer climates or in as many as six weeks (Richards 1940). Both S. exigua and P. rapae are resistant to numerous pesticides (Zhou et al. 2011; Tang et al. 1988) and cause much damage to agriculturally important crops, like

16 8 cabbage and broccoli. However, while P. rapae feeds primarily on (and is adapted to) mustard oil containing crucifers, S. exigua larvae utilize a much broader food spectrum including vegetable, field and flower crops across 18 plant families (Pearson 1982). Naïve Spodoptera exigua and P. rapae larvae used in the no-choice experiments were obtained from Benzon s Research Inc. (Carlisle, PA) and Carolina Biological Supply Co. (Burlington, NC), respectively, as 1 st instars and were reared on artificial diet from the same supplier until they reached the 2 nd instar stage, when they were placed on experimental plants. In addition, naïve third-instar larvae of both insect species were purchased from the same commercial suppliers and immediately used in the choice experiments. All insects were maintained on artificial diet in growth chambers set at a 23/20 C day/night temperature cycle and 10/14 day/night photoperiod. No-Choice Experiments Twenty Arabidopsis thaliana plants from each of the selected genetic lines (i.e., Col-0, 35S:ESP, tgg1tgg2, cyp79b2cyp79b3, myb28myb29 and pad3) were used in the no-choice experiments performed using both the generalist (S. exigua) and the specialist (P. rapae) insect species. One second instar-larva of each species was placed on each of the experimental plants. Each pot was then covered with a plastic cage with a mesh lid for aeration and to prevent insects from moving to a different plant. During the course of the experiment, plants were kept in growth chambers set at a 23/20 C day/night temperature cycle and 10/14 day/night photoperiod. Once daily, plants were inspected to assess larval status or the initiation of the pupal stage. At pupation, insects were removed from the plant, weighed, placed in a petri dish, and returned to the growth chamber until adult eclosion. Each pupa was checked daily for eclosion. At

17 9 eclosion, adults were flash-frozen, weighed and sexed. Times to pupation and adult eclosion were recorded. Choice Experiments Three Arabidopsis thaliana genetic lines were used in choice tests: Col-0, 35S:ESP, and tgg1tgg2. For each insect species, 60 choice trials were performed. Larvae used were all in the late 3 rd instar stage and had been reared on artificial diet only. For the experiment, one leaf from each genotype was placed equidistantly in a 14 cm petri-dish. Selected leaves for the experiment were matched for size within and among different trials. One larva was placed in the center of the petri-dish and left for 1 hour to make a choice, which was determined by observed feeding on the selected leaf. If a larva failed to choose a plant leaf within one hour, it was removed from the experiment and a new petri-dish was set up for a fresh larva. Data Analyses For the no-choice experiments, a one-way ANOVA was performed to test whether different glucosinolate profiles or hydrolysis products affected fitness-related traits of S. exigua and P. rapae. Selected variables included time to pupation and eclosion, as well as pupae and adult weight. A non-parametric test (Kruskal-Wallis) was used for developmental variables because they were not normally distributed. For the choice experiments, Chi-square tests were used to determine whether there was a significant difference between the frequencies of each genotype chosen by each insect species. All statistical analyses were performed using SAS (version 9.2, SAS Institute 2008).

18 10 RESULTS No-Choice Experiments For P. rapae, variation in glucosinolate profiles did not have a significant effect on adult weight but did significantly influence pupae weight and days to pupation (Figure 1). Larvae of P. rapae reared on cyp79b2cyp79b3 plants, which lack indolyl glucosinolates, spent significantly more time as larvae than those reared on Col-0 or pad3 (Figure 1). Larvae reared on Pad3, which contains indolyl glucosinolates but produces no camalexin, spent significantly less time as larvae than insects raised on Col-0, cyp79b2cyp79b3 or myb28myb29, all of which produce camalexin (Figure 1). For S. exigua, variation in glucosinolate profiles had a significant effect on all measured fitness-related traits (Figure 1). Pupae and adult weights of insects reared on cyp79b2cyp79b3, which does not produce indolyl glucosinolates and camalexin, were significantly higher than those reared on Col-0 (which produces both types of chemicals) or pad3 (which produces indolyl glucosinolates but does not produce camalexin). Pupae reared on cyp79b2cyp79b3 (with aliphatic glucosinolates present, but indolyl ones absent) also weighed more than those raised on myb28myb29, which produces indolyl glucosinolates but lacks the aliphatic type. Spodoptera exigua spent more time as larvae on the wild type Col-0 and pad3 than on myb28myb29 or cyp79b2cyp79b3, spending significantly less time on the indolyl producing myb28myb29 than on the aliphatic producing cyp79b2cyp79b3. For P. rapae, variation in hydrolysis products did not significantly affect measured fitness-related traits (Figure 2). However, for S. exigua, variation in hydrolysis products did have a significant effect on all measured fitness-related traits (Figure 2). Pupae and adult weight

19 11 was higher for insects reared on the hydrolysis lines 35S:ESP and tgg1tgg2, which do not produce isothiocyanates, than on the wild type Col-0. Pupae weight for tgg1tgg2-reared insects, which did not encounter any toxic hydrolysis products, was significantly higher than weights for those reared on Col-0 or 35S:ESP. Larvae of S. exigua also spent significantly more time as larvae when reared on Col-0 plants than on 35S:ESP or tgg1tgg2 mutants, and had longer developmental times when reared on 35S:ESP than on the double mutant tgg1tgg2. Choice Experiments Variation in glucosinolate hydrolysis products affected the feeding choices of S. exigua but not those of P. rapae larvae. Larvae of S. exigua significantly preferred the double mutant tgg1tgg2 over the nitrile producing 35S:ESP and the wild type Col-0 (Figure 3).

20 12 DISCUSSION This study offers experimental evidence that variation in plant glucosinolate profiles and hydrolysis products can differentially affect fitness-related traits and feeding choices of insect pests, such as P. rapae and S. exigua. In accordance with the specialist/generalist paradigm, the specialist P. rapae was less affected by the secondary metabolites used in this study than was the generalist S. exigua. Performance of S. exigua was significantly affected by both intact glucosinolates and hydrolysis products. Specifically, this species showed a higher tolerance and preference for the double mutant tgg1tgg2, which virtually lacks hydrolysis products, and for myb28myb29, which lacks aliphatic glucosinolates. The specialist/generalist paradigm also predicts that specialist species like P. rapae would be virtually unaffected by differences in glucosinolate profiles and hydrolysis products. While in general this prediction was corroborated some minor effects were noted, which suggest some lag responses, or costs associated with detoxification or behavioral strategies leading to decreased glucosinolate induction (Wittstock et al. 2004; Burow et al. 2006; Mumm et al. 2008; Ali and Agrawal 2012). For example, no significant difference in the weight of P. rapae adults was detected; however, there was a significant difference in larval developmental time and a slight significant difference in pupae weight when reared on plants differing in glucosinolate profiles. Specifically, larval duration was much shorter on indolyl than on aliphatic producing lines and pupae weight was lower on lines lacking aliphatic glucosinolates. Longer developmental time of larvae is considered to be detrimental because it increases both their exposure to predators and their developmental time by reducing the number of generations per year. These results indicate that

21 13 while the specialist/generalist paradigm may generally apply, specialist insects can also be susceptible to plant intraspecific variation in secondary chemistry. Previous studies of the specialist/generalist paradigm have yielded some mixed results. Some studies demonstrate strong differences in how plant defenses affect generalist and specialist insect herbivores (Li et al. 2000; Voelckel and Baldwin 2004; Arany et al. 2008; Müller et al. 2010) while others, like this study, suggest that specialists are also affected, albeit to a lesser extent (Agrawal and Kurashige 2003; Barth and Jander 2006; Bidart-Bouzat and Kliebenstein 2011; Zheng et al. 2011). However, many studies tested only two or three mutant lines or have mainly focused on either hydrolysis products or glucosinolate profiles. For example, Müller et al. (2010) tested P. rapae on the mutant lines myb28myb29 and cyp79b2cyp79b3 and determined that there were no major changes in larval performance due to decreased aliphatic and/or indole glucosinolate content. In the study presented here, those mutant lines showed only a slight significant difference on P. rapae pupae weight (Figure 1). However, when larvae were also reared on Col-0 and pad3, it became apparent that P. rapae performed better on the pad3 mutant which contained both aliphatic and indolyl glucosinolates but, unlike Col-0, lacked camalexin. This difference in P. rapae performance between myb28myb29, cyp79b2cyp79b3, and Col-0 vs. pad3 was significant and warrants further study, as little is known about the effects of camalexin (assumed to be mainly induced by pathogen attack) on insect feeding choices and performance (Brader et al. 2001; Reviewed in Kliebenstein 2004; Pegadaraju et al. 2005). Variation in glucosinolate profiles in A. thaliana ecotypes has been attributed to the selection pressures exerted on individual plants and populations by distinct insect herbivores (Kliebenstein et al. 2001). For example, in a study on the impact of generalist and specialist

22 14 insects on the selection of A. thaliana populations for distinct glucosinolate profiles, Arany et al. (2008) hypothesized that if generalist and specialist herbivores exert a contrasting selection pressure on A. thaliana, they should perform differently on those plants according to their chemical composition. Indeed, they found that the insects did respond differently to secondary chemistry and concluded that insects differing in their feeding guilds and ecological specializations may impose selection on plant secondary chemistry. In this study, the distinct glucosinolate profiles differentially affected the specialist P. rapae and generalist S. exigua (Figure 1). Pieris rapae spent significantly less time as a larva on mutant plants producing both indolyl and aliphatic glucosinolates (i.e. Col-0 and pad3), preferring the pad3 mutant over all. Conversely, S. exigua larva preferred plants that do not produce indolyl glucosinolates to Col-0 or pad3, which produce both. An interesting finding includes the weight of S. exigua larvae reared on the cyp79b2cyp79b3 mutant (lacking indolyl glucosinolates), which was significantly higher than larvae reared on the aliphatic producing mutant myb28myb29. This result is in accordance with a previous study by Müller et al. (2010), where S. exigua experienced an increase in fitness on both the myb28myb29 and the cyp79b2cyp79b3 mutants when compared to Col-0. However, these moth species grew better on the double mutant cyp79b2cyp79b3 myb28myb29, which produced neither aliphatic nor indolyl glucosinolates, demonstrating an additive effect for these glucosinolates on this generalist herbivore. The specialist P. rapae was not significantly affected by variation in glucosinolate hydrolysis products. Conversely, the generalist S. exigua was significantly affected by variation in hydrolysis products in no-choice experiments, as predicted by the specialist/generalist paradigm. When this insect was reared on mutant lines differing in hydrolysis products, it demonstrated intolerance for isothiocyanates, which is known to be toxic to generalist insects

23 15 (Eckardt 2001; Lambrix et al. 2001; Barth and Jander 2006; Burow et al. 2006a). However, even though S. exigua performed significantly better on the 35S:ESP line than on the Col-0 wild type in the no-choice experiments, these insects did not significantly prefer 35S:ESP over Col-0 in choice trials (Figure 3). In fact, S. exigua larvae overwhelmingly preferred the tgg1tgg2 double mutant over lines producing glucosinolate hydrolysis products. These results suggest that while nitriles may be less toxic to this insect than isothiocyanates, they may still prefer to avoid these toxic compounds in a choice situation. The results of this and previous studies support the argument that variation in plant glucosinolate profiles and their hydrolysis products can lead to differential effects on generalist and specialist insect herbivores, likely as a result of their evolutionary history with plants producing these chemicals. In addition, this study has demonstrated that camalexin may be a more important player in plant chemical defense against specialists than expected. However, we cannot rule out the effects of other, as yet unidentified, chemical changes in the pad3 mutant, which could have caused the observed effects. More research is needed on the role of camalexin in insect fitness and herbivory, as well as potential synergistic effects of multiple glucosinolates and hydrolysis products on insects. Further studies on the effects of nitriles and indolyl glucosinolates on both specialist and generalist herbivores are also warranted. While isothiocyanates are probably one of the most frequently studied hydrolysis product of glucosinolates, this study provides evidence that nitriles could play a larger role than expected in direct defense against generalist herbivores. Overall, results from this study provide further evidence that diversity among glucosinolate profiles and hydrolysis products is necessary for crucifer plants like A. thaliana to thrive in environments that consist of both generalist and specialist insect herbivores. In addition, the ability to study the distinct effects of specific

24 16 glucosinolate profiles and hydrolysis products on generalist and specialist insect herbivores can provide valuable information about the evolutionary relationship and associated selection pressures that exist between these herbivores and their host plants.

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32 24 Pieris rapae Spodoptera exigua Pupae weight (g) F 3,65 = 3.35 P = AB B A A F 3,67 = P < A B C A Adult weight (g) F 3,61 = 0.86 P = 0.47 F 3,59 = 4.40 P = A AB B A Days to Pupation F3,65 = 6.23 P < A AB B A F 3,67 = P < A B C A Col-0 Myb28-29 Cyp79B2/B3 Pad3 Col-0 Myb28-29 Cyp79B2/B3 Pad3 Figure 1: Effects of distinct glucosinolate profiles on fitness-related traits of Pieris rapae and Spodoptera exigua.

33 25 Pieris rapae Spodoptera exigua Pupae weight (g) F 2,54 = 0.81 P = 0.45 F 2,52 = P < A B C Adult weight (g) F 2,53 = 1.11 P = 0.34 F 2,45 = 6.27 P = A B B Days to pupation F2,54 = 0.81 P = 0.45 F 2,52 = P < A B C Col-0 35S:ESP tgg1tgg2 Col-0 35S:ESP tgg1tgg2 Figure 2: Effects of glucosinolate hydrolysis products on fitness-related traits of Pieris rapae and Spodoptera exigua.

34 26 Pieris rapae Choice (%) Spodoptera exigua B Choice (%) A A Col-0 35S:ESP tgg1tgg2 Figure 3: Feeding choice of S. exigua and P. rapae larvae exposed to A. thaliana leaves differing in glucosinolate hydrolysis products.