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 iv © 2012 Julie A. Kemarly-Hopkins All Rights Reserved 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 nitrile- producing 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, 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. 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. 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. vii TABLE OF CONTENTS Page INTRODUCTION….. ........................................................................................................... 1 MATERIALS AND METHODS………………………………………………………….. 5 Arabidopsis thaliana genetic lines and growth conditions ........................................ 5 Insect species… ......................................................................................................... 7 No-choice tests ........................................................................................................... 8 Choice tests…. ........................................................................................................... 9 Data analyses.... ......................................................................................................... 9 RESULTS………....... ........................................................................................................... 10 No-choice experiments .............................................................................................. 10 Choice experiments .................................................................................................... 11 DISCUSSION ………............................................................................................................ 12 REFERENCES ...................................................................................................................... 17 viii LIST OF FIGURES/TABLES Figure Page 1 Effects of distinct glucosinolate profiles on P. rapae and S. exigua ......................... 24 2 Effects of glucosinolate hydrolysis products on P. rapae and S. exigua ................... 25 3 Choice trials of P. rapae and S. exigua ...................................................................... 26 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 plant- insect 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 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 3 generalist herbivorous insects, while simultaneously acting as oviposition and feeding stimulants for specialist