EVALUATION OF NATURAL PLANT CHEMICAL DEFENSES IN COMPARISON TO BACILLUS THURINGIENSIS ON THE SURVIVAL AND FEEDING PREFERENCES OF OSTRINIA NUBILALIS

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1 EVALUATION OF NATURAL PLANT CHEMICAL DEFENSES IN COMPARISON TO BACILLUS THURINGIENSIS ON THE SURVIVAL AND FEEDING PREFERENCES OF OSTRINIA NUBILALIS by Kelsey E. Fisher A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Entomology Fall Kelsey E. Fisher All Rights Reserved

2 ProQuest Number: All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. ProQuest Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI

3 EVALUATION OF NATURAL PLANT CHEMICAL DEFENSES IN COMPARISON TO BACILLUS THURINGIENSIS ON THE SURVIVAL AND FEEDING PREFERENCES OF OSTRINIA NUBILALIS by Kelsey E. Fisher Approved: Charles E. Mason, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: Jacob L. Bowman, Ph.D. Chair of the Department of Entomology and Wildlife Ecology Approved: Mark W. Rieger, Ph.D. Dean of the College of Agriculture and Natural Resources Approved: Ann L. Ardis, Ph.D. Interim Vice Provost for Graduate and Professional Education

4 ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Charles Mason, for his on-going support and encouragement through the duration of my degree. I truly enjoyed the many conversations that jumpstarted a majority of the ideas for this thesis. I would like to acknowledge Dr. Lindsey Flexner, for his constant interest and enthusiasm. Without him, this project would not have been possible. I would also like to thank Dr. Judith Hough- Goldstein for her knowledge and comments that improved this thesis. Furthermore, I would like to acknowledge Dr. John McDonald and Zaiqi Pan for their abundant knowledge and aid with my statistical analyses. I would like to thank a few people for technical and logistical support. First, Keith Bidne at Iowa State University, for maintaining a colony of Z-race European corn borers and sending weekly shipments of eggs and 3 rd instars. Additionally, I would like to thank Bill Bartz, Rodney Dempsey, and the University of Delaware greenhouse staff for making it possible for me to conduct experiments in the greenhouse and providing the necessary materials. I would like to thank Scott Hopkins, Bill Cissel, and the University of Delaware farm staff for helping set up my field experiments and teaching me about irrigation. Next, I would like to thank Holly Walker, David Ingber, Maddie Chura, Jessica Faucher, and Michael Palmer for help executing my many experiments. Last, but certainly not least, I would like to thank all of my family and friends for their understanding and encouragement.

5 TABLE OF CONTENTS LIST OF TABLES... vi LIST OF FIGURES... vii ABSTRACT... ix Chapter 1 LITERATURE REVIEW...1 Feeding Behavior Overview...1 Agriculture Pest Behavior...5 The European Corn Borer SURVIVORSHIP OF THE EUROPEAN CORN BORER ON A RANGE OF HOST PLANTS VARYING IN DEFENSIVE CHEMISTRY...15 Introduction...15 Materials and Methods...17 Insects and Plants...17 Growth Chamber...20 Neonate Infestation...20 Third Instar Infested...20 Greenhouse...21 Neonate Infested...21 Third Instar Infested...21 Field...22 Neonate Infested...22 Third Instar Infested...23 Statistical Analysis...24 Results...25 Growth Chamber...25

6 Neonate Infestation...25 Third Instar Infestation...26 Greenhouse...26 Neonate Infestation...26 Third Instar Infestation...27 Field...27 Discussion EUROPEAN CORN BORER PLANT CONSUMPTION RATES AND PREFERENCE AMONG A RANGE OF AGRICULTURAL HOST CROP SPECIES...43 Introduction...43 Materials and Methods...45 Insects and Plants...45 Consumption Rates and Preference...47 Statistical Analysis...49 Results...50 Discussion...50 REFERENCES...58 Appendix A TEST FOR INDUCED DEFENSES IN CUCUMBER...66

7 LIST OF TABLES Table 1 Average weight of mid-development larvae or pupae under all experimental designs. Letters indicate significantly different weights analyzed with two-way ANOVAs (P < 0.05)...36

8 LIST OF FIGURES Figure 1 Figure 2 Figure 3 Survival curve for neonate-infested growth ghamber feeding trials. Survival curves based on the longevity of larvae fed various host plants determined by degree-days accumulated until death prior to pupation. Lines for hosts followed by the same letter did not differ in survival time (Tukey-Kramer mean separation test (P < 0.05)...37 Growth chamber survival through the first stadium of treatment.(a) Neonates or (B) third instars fed one of various host plants (N = near isoline, SC = sweet corn, C = cucumber, F = Cry1F, GB = green bean, T = tomato) through the duration of development. Bars represent the average percent of the individuals that survived to the subsequent instar based on degree-day accumulation (106 degree-days = 2 nd instar; 132 degree-days = 4 th instar). Bars within each graph with different letters are significantly different (Tukey-Kramer test, P < 0.05). Error bars represent SEM...38 Survival curve for third instar-infested growth chamber feeding trials. Survival curves based on the longevity of larvae fed various host plants determined by degree-days accumulated until death prior to pupation. Lines for hosts followed by the same letter did not differ in survival time (Tukey-Kramer mean separation test (P < 0.05)...39 Figure 4 Greenhouse recovery. (A) Neonate infested plants (N = near isoline, SC = sweet corn, C = cucumber, F = Cry1F, GB = green bean, SQ = squash, T = tomato) were dissected after 355 degree-days for mid-development recovery or (B) after 790 degree-days for pupal recovery. (C) Plants infested with 3 rd instars were dissected after 567 degree-days for pupal recovery. Bars represent the number of individuals recovered at each dissection with SEM. Different letters within one graph are significantly different (P < 0.05)...40 Figure 5 Figure 6 Field recovery. (A) Neonate infested plant groupings (N = near isoline, SC = sweet corn, C = cucumber, F = Cry1F, GB = green bean, SQ = squash, T = tomato) were dissected after 355 degree-days for a mid-development recovery or (B) after 790 degree-days for pupal recovery. (C) Plant groupings infested with 3 rd instars were dissected after 567 degree-days for pupal recovery. Overall, recovery was very low; none of the experiments yielded significant results...41 Pupal weight comparison from third instar infested plants in the

9 field. Average weight of pupae recovered from 3 rd instar infested field experiments from non-bt corn (sweet corn and near isoline) (n = 7) and tomato (n = 8). Significant difference represented by different letters (P < 0.01). Error bars represent SEM...42 Figure 7 Dry biomass consumed. Third instars fed one of the various hosts (N = near isoline, SC = sweet corn, C = cucumber, F = Cry1F, GB = green bean, SQ = squash, T = tomato) for 48 hours. Bars represent the estimated amount of dry biomass consumed. Different letters are significantly different (P < 0.05). Error bars show SEM...55 Figure 8 Figure 9 Figure 10 Third instar larval weight change over 48 hours. Third instars fed one of the various hosts (N = near isoline, SC = sweet corn, C = cucumber, F = Cry1F, GB = green bean, SQ = squash, T = tomato) for 48 hours. Bars represent either gained (+) or lost (-) weight based on host. Different letters are significantly different (P < 0.05). Error bars show SEM...56 Preference determined by dry biomass consumed. Third instars given a choice between 2 host plants (near isoline (N) and either sweet corn (SC), Cry1F (F), tomato (T), green bean (GB), or cucumber (C), which are presented in figure 3A, or Cry1F (F) and tomato (T), presented in figure 3B) for 48 hours. Bars represent the estimated dry biomass consumed. Significant differences, within one group are distinguished with an asterisk (*). Error bars show SEM...57 Cucumber induced defenses. 3 rd instars given a choice between leaf tissue from an infested or non-infested plant for 4 hours (Figure 4A), 24 hours (Figure 4B), and 48 hours (Figure 4C). Bars represent the estimated amount of dry biomass consumed. Error bars show SEM; no significance was detected (P > 0.05)...69

10 ABSTRACT The European corn borer (ECB), Ostrinia nubialis (Hübner), is an herbivorous pest of over 200 documented hosts, especially corn (Zea mays L.), in the United States and Canada, and had been labeled a generalist herbivore. The feeding behavior of insects, including ECB, depends on perception of multiple physical and chemical features presented by the plant and the insect s response to these cues. Recently, in the United States there has been little evidence of ECB presence outside of cornfields. Generalist insect species have the ability to evolve over time into specialists when the focal plant is common, predictable and abundant. All of these criteria are met by corn in the United States. This study investigated the growth and survivorship of ECB on a range of hosts that vary in defensive chemistries and their preferences among these hosts (sweet corn, Cry1F Bt corn (maize), non-bt near isoline maize, cucumber, squash, tomato, and green bean). Experiments were conducted in the growth chamber, greenhouse, and field to determine survival under different conditions. Survival was significantly higher on non- Bt corn hosts than any other host provided. Choice feeding assays were conducted to determine preference based on biomass consumption. Results generally supported the expected outcome with greater consumption of non-bt corn hosts. From the results on survival and preference, it can be concluded that non-bt corn is the most suitable host plant for ECB, thus strongly supporting ECB corn specialization.

11 Chapter 1 LITERATURE REVIEW Feeding Behavior Overview There are complex ecological and evolutionary relationships between herbivorous insects and the plants they consume. Many factors affect the feeding behaviors in a plantinsect relationship. Plants defend vegetative tissues against herbivorous predators by mechanical and chemical mechanisms (Ehrlich and Raven 1964; Mooney et al. 2009). Insects must overcome these defenses in order to grow and reproduce. In turn, damage from herbivores affects photosynthesis and growth of plants, causing impacts on plant reproduction (Futuyma and Gould 1979; Niesenbaum 1996). Most herbivorous insects have host plant ranges. These are plants the insects are capable of consuming to obtain adequate nutrition for growth and reproduction. Additionally, these plants often provide a protective habitat when the herbivorous insect is able to overcome the plant defenses (Ehrlich and Raven 1964). Typically, when referring to a host plant, it is either a plant family or genus, not usually a single species (Forister et al. 2015). A topic that has long been debated pertaining to herbivorous insects host range is the generalist versus specialist argument including monophagy, oligophagy, and polyphagy. Monophagous insects have one or few closely related acceptable host plants. Many of these scenarios occur within Lepidoptera, Hemiptera, and Coleoptera (Schoonhoven et al. 2005). An example of monophagy is the Monarch Butterfly larvae s ability to only feed on milkweed species (Van Zandt and Agrawal 2004). Oligophagous insects can feed on species limited to one family or genus, and polyphagous insects can feed on many plants represented across several families. 1

12 Polyphagous insects will accept many host plants and rarely exercise choice (Schoonhoven et al. 2005). An argument comes in when claims are made about generalist and specialist herbivores. Generalists, typically oligophagous or polyphagous, can feed on a wide array of plant types and are able to metabolize a multitude of plant defenses (Schoonhoven et al. 2005). Specialist insect herbivores have the ability to tolerate specific plant defenses and manipulate hosts to their benefit to reduce predation and parasitism (Jaenike 1990). It has been documented that less than 10% of herbivores feed on more than three different plant families (Bernays and Graham 1988). Based on these definitions, specialists are much more common than generalists. According to a metaanalysis by Ali and Agrawal (2012), the identification of generalist or specialist is still unclear because of experimental limitations. Often researchers place insects into selfdetermined categories based on their own definitions of generalist and specialist. There is often more complexity in the plant-insect world, and the answer may not be as simple as two categories. Host plants can provide an assortment of nutrients and defenses in a number of ways. Due to this variation, the host species may affect larval success by influencing rates of growth and development (Singer et al. 2002). Nitrogen is a major component of proteins and amino acids, which are sources for arthropod growth (Chen et al. 2009). Higher plant nitrogen content correlates with greater growth, higher conversion of ingested food into energy, and shorter developmental time (Slansky and Feeny 1977; Mattson 1980; Woods 1999; Chen et al. 2008, 2009). Greater growth is adaptive because it has been shown that larger insects perform better (Kingsolver and Huey 2008). Higher conversion of ingested food into energy means that the insect would be able to consume 2

13 smaller quantities of food in order to obtain optimal energy, leaving more time to be allocated to other activities and less time exposed to predators and parasitoids. In a study on tobacco hornworm larvae, it was shown that when a diet is less nutrient rich, the larvae feed longer in order to maintain their intake of nutrients (Timmins et al. 1988). Furthermore, a shorter development time would cause earlier pupation, allowing less opportunity for predation or parasitism (Benrey and Denno 1997) Additionally, a shorter developmental time can increase fitness by increasing the number of generations per year. In addition to host plants having a direct impact on larval fitness based on nutrition, there is an impact due to plant defenses. Plants have two types of biochemistry that occur within their tissues. The basic chemical processes required for day-to-day functions are classified as primary plant metabolism. This chemistry is involved in the formation and breakdown of a limited set of chemicals to create proteins, carbohydrates, lipids, and nucleic acids (Buchanan et al. 2000). Secondary chemistry is involved in plant reproduction, insect attractants, and insect deterrents (Ehrlich and Raven 1964). These, along with physical barriers like trichomes and leaf toughness, play a key role in manipulating insect behavior (Wink 1988). Secondary chemistry develops as derivatives from primary biosynthesis. Plant chemical attractants and deterrents can be present in glandular trichomes, and in epicuticular waxes as volatile compounds, or stored in the plant cells. Attractants play a small roll in host selection for herbivorous insects, as continual feeding is highly influenced by deterrent chemistries (Jermy 1976). Some deterrents are always present in the plant tissues, while others can be induced by an insect behavior such as herbivory or oviposition (Schoonhoven et al. 2005). 3

14 An abundance of studies have been conducted to determine host plant preference. Many analyze host plant range (Kogan and Goeden 1970), preference in the presence of additive toxins (Davis and Coleman 1997; Gore et al. 2005), or pest preference for other crops or weeds when the major host is not present (Losey et al. 2001; Tate et al. 2006). Others have been conducted for specific analyses like allelochemical or nitrogen abundance s affect on feeding behavior (Glendinning and Slansky 1994; Elasyed 2011). It has also been shown that herbivorous insects can detect host plant quality and feed or oviposit preferentially on plants containing more nitrogen (White 1984; Fox et al. 1990; Prudic et al. 2005; Chen et al. 2008). There is a basic overview of insect feeding behavior that is widely accepted for most chewing, herbivorous insects. First, the insect is hungry, which causes search behavior as a random walk until the insect detects a plant cue. The term walk is used for simplicity purposes. Most insects can use multiple modes of mobility including jumping and flying, which are actions by insects with wings or strong legs like grasshoppers or beetles. Other insects are less mobile, such as larval Lepidoptera. To search for plant cues, less mobile insects use walking, crawling, or ballooning: a form of movement where Lepidoptera larvae produce silk and hang from the plant resulting in dropping below or floating away with a gust of wind (Zalucki et al. 2002; Goldstein et al. 2010). For Lepidoptera larvae, some of the searching behavior is shortened because these individuals are placed on an acceptable host plant by adult oviposition. Nevertheless, sometimes the larvae reject their natal plant in search of a new one. Insect mobility provides potential for insects to switch host plant species one or more times (Singer et al. 2002). Once the insect detects a plant cue the walk becomes directional toward the 4

15 stimulus. Eventually there is contact between the plant and the insect, initiating selection behavior. These behaviors include physical and chemical evaluation of the plant that could not occur from a distance. Some examples are scratching, drumming with tarsi, palpating, ovipositor dragging, and test biting. Test bites are often smaller than a regular bite. The plant material may be kept longer in the pre-oral cavity than during regular food intake due to gathering sensory information. Finally, continual feeding or discontinuation of feeding and leaving the plant confirms host plant acceptance or rejection. (Elsayed 2011). It is possible for generalist insect species to evolve over time into specialists (Forister et al. 2015). Host plant specialization typically occurs where plants are common, predictable, and abundant, making mating and oviposition site location easier (Jaenike 1990). Host specialization allows the insect to gain ability to tolerate plant defenses, manipulate hosts to their benefit, and evolve ways to reduce predation and parasitism (Ali and Agrawal 2012). This also typically occurs when one host plant is superior in many ways to other host plants in terms of nutrient availability, abundance, and predation risk (Ehrlich and Murphy 1988). Agriculture Pest Behavior The agriculture industry produces the majority of food sources for humans. According to the USDA National Agricultural Statistics Service for crop production, in 2014, United States farmers produced 149 billion dollars worth of crops. One of the most abundant crops was corn (Zea mays L.), producing 84 million acres worth 63.9 billion dollars. Stable monocultures of crop plants are grown in the United States because it is a 5

16 profitable platform for farmers and easy to make control decisions. A monoculture consists of a single plant species over a wide area of land. However, monocultures can be difficult to manage due to herbivorous agricultural pests exploiting the crop resources. Monocultures are easy for herbivores to locate because of the amplification of plant cues released. Pests are attracted to monocultures for reasons that can be explained by the resource concentration hypothesis and the enemy free hypothesis. The resource concentration hypothesis refers to the abundance of food sources available for an insect and its future offspring. All of the plants in the area are healthy host plants (Grez and Gonzalez 1995). The enemy free hypothesis states that due to the lowered plant diversity the insect diversity is decreased as well. Typically, there are fewer natural enemies present in agriculture fields to feed on or parasitize pest species (Letourneau 1987). In addition to the enemy free and resource concentration hypotheses, plant breeding plays a large role in herbivorous insects attraction to crops grown in monoculture. Over time, plant breeders have selected for the highest yielding, largest, and most palatable varieties. In order to do this, plants energy is directed toward growth rather than defense (Rosenthal and Dirzo 1997). While selecting for large, high yielding fruit, breeders often selectively reduced plant defensive chemistry (Wink 1988; Rosenthal and Dirzo 1997; Rasmann and Agrawal 2009). This is exemplified in a study comparing cultivated and wild Brassica (Gols et al. 2008). An additional example is with lupine seeds. Lupine seeds are similar to soybeans containing 40% protein and 20% lipids, however, lupine seeds naturally contain 5% quinolizidine alkaloids that are toxic to vertebrates. In order to provide these seeds safely for human consumption, plant breeders selected varieties lacking alkaloids. Lupine seeds are widely grown in France, but cannot 6

17 be cultivated without pesticide application. Unlike its wild relative, the sweet variety of lupine seed is susceptible to a number of pathogens and herbivores (Wink 1988). Plant breeding to reduce secondary metabolites is widely practiced all over the world, making plants more palatable for humans, but also unintentionally, for all other herbivores. Plant breeding is also beneficial for increasing defensive strategies. Tougher leaves and stronger stalks have been bred to reduce pest abundance, however, subpopulations of insects can evolve to overcome these resistances, so they alone are not enough to eliminate pest populations (Rausher 2001). When no pest control measures are implemented, insect herbivores have the greatest impact on crops, with a recorded 10% to 100% decrease in crop yield (Schoonhoven et al. 2005). According to the world census, the world population is 7.2 billion people. It is expected to reach 8.9 billion people by the year 2050 (Cohen 2003). Not only is the world population growing, but also there are many people currently starving. As of the food insecurity census in 2012, 11.3% of the world is populated with people who are starving or hungry. While trying to feed the current population and preparing for the future world population, it is imperative that crops yield the most that they possibly can. Drastic yield losses due to pests cannot be tolerated. In order to feed the ever-increasing human population, crop production must continue with high yields, but problems with compounding pesticide use should be reduced. The relationships between plants and insects must be better understood in order to advance insect pest control in agriculture (Schoonhoven et al. 2005). 7

18 The European Corn Borer The European corn borer (ECB), Ostrinia nubialis (Hübner), is a Lepidopteran in the family Crambidae, whose larvae are typically stem borers. ECB originated in Europe and was introduced to North America in the early 1900s with the importation of broomcorn (Sorghum technicum L.) from Italy and Hungary. The preferred and most important host plant for ECB is corn (Zea mays L.) (Hodgson 1928; Beck 1987). Since, corn is native to Central or South America and ECB originated in Europe, it is likely that the original hosts were millet (Panicum miliaceum L.) and hops (Humulus spp.). However, ECB is known as a generalist herbivore able to succeed on over 200 crop and weedy species, so the original host cannot be known for certain (Hodgson 1928; Hüber et al. 1928; Beck 1987). The largest effect of ECB is observed in corn agriculture where it is documented to cause more than 1 billion US dollars in annual yield loss and management/prevention costs (Mason et al. 1996; Hutchinson et al. 2010). However, when ECB has large populations, there is often spill over from cornfields into surrounding weeds and crop fields, as ECB are opportunistic generalist feeders (Hodgson 1928). Also, if corn is less attractive or not available, ECB will feed on alternate hosts like tomato and green bean (Brindley and Dicke 1963). In Delaware, and most of the Corn Belt, ECB has two generations per season. ECB can have as few as one generation per year further north and as many as four generations per year further south (Mason et al. 1996). ECB overwinter as larvae within plant stalks or stems. In the spring, the larvae pupate. Around late May, the adults emerge for their first flight. Adults find each other via pheromones in order to mate. After 8

19 mating, the females lay multiple egg masses of eggs on the underside of corn leaves or on other available host plants. In early June, the eggs hatch and neonates remain on or reject their natal plant. Those that reject the plant can move (balloon) to another plant via silk that gets caught in the wind (Goldstein et al. 2010). Early instars feed externally on leaf tissue leaving windowpane damage. In corn, young ECB feed through the soft leaf tissue of whorl stage corn creating shot hole damage, which is indicative of ECB presence. Larvae are capable of plant-to-plant movement until approximately 3 rd instar when they bore into the stalk or stem. The duration of their larval development occurs within the stalks or stems where they will pupate or overwinter depending on generation (Mason et al. 1996). ECB typically oviposit on corn plants that are V5 stage (5 th leaf fully exposed) to R1 (first reproductive stage) (Mason et al. 1996) because prior to this stage corn plants contain the chemical defense 2,4-dihydroxyl-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA) in the leaf tissues (Guthrie et al. 1986). The presence of DIMBOA causes the pre-v5 stages of the plant to be resistant to first generation ECB because it is toxic to larvae that feed on the plant (Guthrie et al. 1986). If V5-R1 corn is not available by the time adult ECB are ready to oviposit, they will oviposit on other host plants that are available (Mason et al. 1996). There are two types of pheromone races of ECB, E and Z. The E pheromone race tends to feed on alternate host plants like weedy plants and hops in the United States and Europe, after breaking hibernal diapause because they tend to eclose as adults prior to the presence of corn. The Z pheromone race tends to feed and oviposit mostly on corn. This is because their first generation life cycle is more synchronized with the availability of 9

20 corn in V5-R1. In the United States, these races can breed to create hybrids, however, this is not the case for Europe. In Europe, the races do not hybridize and tend to stay specialized on their particular host species (Calcagno et al. 2007). All plants have natural secondary plant compounds often used for defense, like DIMBOA found in corn. Some examples include, but are not limited to, alkaloids, phenolic acids, hydroxybenzoic acids, hydroxycinnamic acids, and flavonoids (Buhr et al. 1958; Thorsteinson 1960; Boppre 1989; Isman 2002; Abu-Reidah et al. 2013; Wink 2013). Recent observations suggest that ECB do not feed on tomato like they once did (personal observations, C. E. Mason), which indicates that tomato is now an unfavorable plant. This could be due to the presence of tomatine, an alkaloid that is found in the green parts of the tomato plant including the leaves, stem, and unripe fruit. Studies on the effects of tomatine on insect feeding behavior have shown that tomatine may be toxic and deter feeding activities for beetles (Buhr et al. 1958; Thorsteinson 1960; Boppre 1989; Isman 2002). Cucumber and squash have been documented as host plants, but ECB are rarely, if ever found on them. These plants contain cucurbitacin (a very bitter compound that has a reputation as a defensive compound against herbivory) in the vegetative parts of the plant leading to the belief that both are unfavorable plants. In a study conducted by Tallamy et al. (1997), cucurbitacin was observed as a feeding deterrent for mandibulate insects, and specifically detected and avoided by ovipositing ECB. Green bean plants contain a wide array of plant secondary chemistries including phenolic acids, hydroxybenzoic acids, hydroxycinnamic acids, and flavonoids. The compounds present vary across green bean variety (Abu-Reidah et al. 2013; Wink 2013). There were many attempts through history to battle ECB including release of 24 10

21 parasitoid species following the establishment of the European Parasite Laboratory in France (Bartlett et al. 1978), pesticide development (Pepper and Carruth 1945), and selection for host plants able to withstand insect attack (Maxwell and Jennings 1980). However, the most successful solution to control ECB was the introduction of genetically modified crops (Koziel 1996). These crops contain encoded insecticidal crystalline (Cry) proteins from the soil bacterial species Bacillus thuringiensis (Bt) to protect the crops from damage. This type of genetically modified corn expresses the Bt toxin through many plant tissues, forcing the insects to come in contact with the toxin upon feeding. The proteins bind to specific receptors in the larva s gut causing ulceration and blocked nutrient absorption resulting in starvation and sepsis (Gill et al. 1992). Genetically modified crops containing Bt are particularly effective because neonates come in contact with the toxins in their first bite of food. Due to the successful control measures of Bt corn against ECB, it has become widely adopted, accounting for 90% of the corn planted in the United States (Fernandez-Cornejo et al. 2014). Currently, ECB is controlled with Bt corn without the evolution of resistance (Fernandez-Cornejo et al. 2014); however, the cost of control is still present (i.e. seed costs) to prevent ECB from feeding on and damaging corn (Hutchinson et al. 2010). As part of an insect resistance management strategy developed by the EPA, any field planted with Bt corn requires a sizable separate refuge patch of non-bt corn in order to prevent the evolution of resistance (Gould 2000). The refuge patch can take many shapes including alongside of the cornfield, certain row strips in the field, around the perimeter the field, or across a roadway from the Bt field (Goldstein et al. 2010). The majority of insects from the Bt field will die, but some could have a mutation for 11

22 resistance and survive. Those that survive will most likely mate with the large number of individuals from the refuge plot that are still susceptible to Bt toxins. All of the offspring will be heterozygous for susceptibility and therefore die when they come in contact with Bt through feeding (Gould 2000). Refuge planting can be expensive and inconvenient for farmers leading to noncompliance of refuge planting. To increase compliance, a refuge in the bag system was developed that would combine the non-bt refuge corn seed with Bt corn seed, providing a mixed planting of non-bt corn integrated into the Bt cornfield (Goldstein et al. 2010). The refuge in a bag system has great potential of facilitating insect resistance, specifically with single toxins while pyramided traits are of less concern. Since Bt and non-bt plants are integrated together, larval movement may lead to increased survival by sub-lethal doses of Bt toxin allowing partially resistant insects to remain (Mallet and Porter 1992). It has been shown that neonate ECB abandon their natal host plants at very high rates. Approximately 75% of neonates abandon Bt natal plant, whereas only 42% of neonates abandon non-bt natal host plants (Razze et al. 2011). Young larvae move around a great deal prior to plant establishment, thus potentially obtaining sub-lethal doses of Bt toxin, such as when neonates start on non-bt corn and move to Bt corn as older larvae. Individuals that encounter Bt as older larvae can survive to adulthood and reproduce (Secchi et al 2006). Davis and Coleman (1997) observed feeding behavior of ECB on Cry1Ab Bt corn and found that neonates and 4 th instars fed much more on non- Bt leaf tissue than on Bt leaf tissue, but the 4 th instars were able to feed preferentially more on Bt tissue than the neonates. This phenomenon has also been shown with older diamondback moth larvae (Liu et al. 1995). These and similar studies lead to questions 12

23 concerning whether later instar larvae can feed more on other host plants with defensive chemistries with effectiveness similar to Bt corn. The older instars may be more equipped to overcome plant defenses than neonates. Corn agriculture in the United States has provided ideal conditions for host plant specialization due to corn reliability and abundance. Recently, there has been little evidence of ECB presence outside of cornfields due to the wide adoption of Bt corn decreasing population size (Hutchison et al. 2010). During several decades of the traditional plant breeding process, less defensive plants were selected in order to make the crop more palatable for human consumption (Wink 1988). This minimization of defensive chemistry leaves domesticated crops open to easy attack from herbivorous insects. Furthermore, corn monocultures provide low diversity of predators and parasitoids due to the enemies hypothesis. Crop species like tomato and cucumber still have strong deterrent chemistries in their leaves and stems (tomatine and cucurbitacin) despite breeding, providing challenges for herbivorous insects to overcome in order to feed. With a hundred years of ECB feeding most abundantly on corn in the United States, defensive chemistry of other crops and weeds may have pressured selection of ECB populations as corn specialists with few members of the population capable of surviving on non-corn hosts. The main objective of this research is to investigate the phenology of larval host choice behavior and comparative survivorship of Z-race ECB on a range of host plants that vary in defensive chemistries. ECB has had an interesting history over the past 100 years. ECB were once abundant and overwhelming in many cropping systems as well as their weeds (Hutchison et al. 2010). V5 or older corn was an easy target because it lacks 13

24 the strong defensive chemistries found in other hosts, has minimal natural enemies, and has an abundance of stem tissue for boring/tunneling. To combat infestations, stronger plants were selected by plant breeders to withstand ECB infestations. Now, genetically modified corn containing Bacillus thuringiensis has driven populations to the point that natural ECB are difficult to locate (field observations). It is pertinent to determine how human manipulations of ECB food sources have affected ECB ability to survive and select various hosts that they once infested. Studying the current feeding behavior, as well as survival characteristics of ECB on a range of host plants (Cry1F Bt corn, non-bt near isoline corn, sweet corn, tomato, cucumber, and green bean), allows for comparisons to be made among quality, utilization, and preference of various hosts. This research addresses the following objectives through laboratory, greenhouse, and field studies: 1. Evaluate the ability of ECB to utilize a range of host plants with varying defensive chemistries in comparison with Bt corn. 2. Compare neonate and larger larvae s ability to survive on different hosts containing various plant chemical defenses. 3. Evaluate amount of feeding on each host plant based on leaf tissue consumption in a 48-hour period by 3 rd instar ECB. 4. Determine feeding preferences based on leaf tissue consumption by 3 rd instar ECB when presented with host choices. 14

25 Chapter 2 SURVIVORSHIP OF THE EUROPEAN CORN BORER ON A RANGE OF HOST PLANTS VARYING IN DEFENSIVE CHEMISTRY Introduction Many factors affect the feeding behaviors in a plant-insect relationship, particularly plant defenses and insect adaptations to these defenses. Host plant ranges consist of all of the plants insects are capable of consuming to obtain adequate nutrition for growth and reproduction (Ehrlich and Raven 1964). Host plants provide an assortment of nutrients and defenses, which affect the success of the organism during the larval stage (Singer et al. 2002). For example, higher plant nitrogen content is associated with greater growth, higher conversion of ingested food into energy, and shorter developmental time for the insect herbivore (Mattson 1980; Woods 1999; Chen et al. 2008, 2009). Chemical feeding deterrents play a key role in manipulating insect behavior (Wink 1988). For chewing herbivores, continual feeding is highly influenced by deterrent chemistries (Jermy 1976). Agricultural crop plant breeders have unintentionally made it easy for herbivores to take advantage of crops by selecting for the highest yielding, largest, and tastiest varieties through reduced plant defensive chemistry (Wink 1988; Rosenthal and Dirzo 1997; Gols et al. 2008; Rasmann and Agrawal 2009). The European corn borer (ECB), Ostrinia nubialis (Hübner), was introduced to North America in the early 1900s with the importation of broomcorn (Sorghum technicum L.) from Italy and Hungary. ECB is a known herbivorous pest on multiple hosts in North America. At introduction, ECB was documented to succeed on 223 crop and weedy plant species, earning the label of generalist herbivore, able to overcome a 15

26 multitude of plant defenses (Hodgson 1928; Hüber et al. 1928). The preferred and most important host of ECB is corn (Zea mays L.). However, when ECB has large populations, there is often spill over from cornfields into surrounding weeds and crop fields, as ECB are opportunistic generalist feeders (Hodgson 1928). Also, if corn is less attractive or not available, ECB will feed on alternate hosts like tomato and green bean (Brindley and Dicke 1963). Through the 1920s, 1930s, and 1940s, ECB devastated the corn industry due to late instar feeding damage. Many control attempts were made, but few were successful until the introduction of genetically modified corn containing genes from Bacillius thuringensis (Bt) in 1996, causing larval death upon feeding (Gill et al. 1992). Bt corn is highly effective; it is widely adopted for its success and accounts for 90% of corn planted in the United States (Fernandez-Cornejo et al. 2014). Evolution of Bt resistance is managed by planting a refuge of non-bt corn, thus maintaining Bt susceptible individuals in the population (Gould 2000). To increase refuge compliance, mixed plantings of Bt and non-bt corn were introduced as a refuge option (Goldstein et al. 2010), however, larval movement in mixed plantings potentially leads to increased survival (Mallet and Porter 1992; Secchi et al. 2006). For example, older instars have better survival than neonates in the presence of Bt toxin (Davis and Coleman 1997). Larvae that move from a non-bt plant to a Bt plant will survive and obtain a sub-lethal dose of Bt toxin. It is possible for generalist insect species to evolve over time into specialists (Forister et al. 2015) when host plants are common, predictable, and abundant (Jaenike 1990). In the United States corn plantings are reliable and abundant, especially within the Corn Belt. ECB has been feeding most abundantly on corn in the United States for

27 years. Some crops, even though more palatable than wild relatives, still contain strong deterrent chemistries (i.e. tomato and cucurbits), providing challenges for some herbivorous insects to overcome in order to oviposit or feed (Thorsteinson 1960; Metcalf et al. 1982; Tallamy et al. 1997). Unpalatable defensive chemistry of some crops may have become a challenge for ECB larvae. The observed decline of ECB presence in crop systems other than corn (Hutchison et al. 2010) is possibly due to lack of neonate survival resulting from defensive chemistry. This research addresses the following objectives through laboratory, greenhouse, and field studies: (1) evaluate the ability of ECB to utilize a range of host plants with varying defensive chemistries in comparison with Bt corn and (2) compare neonate and larger larvae s ability to survive on different hosts containing various plant chemical defenses. Materials and Methods Insects and Plants Z-race European corn borer (ECB) eggs and third instars were received from the USDA-ARS Corn Insects and Crop Genetics Research Unit (Ames, IA). Each year, field captured ECB are incorporated into the colony to maintain field relevance and reduce inbreeding. Third instars were shipped to the University of Delaware (Newark, DE) gregariously, feeding on ECB diet. Eggs were kept in plastic bags filled with air until blackhead stage. Both eggs and larvae were housed in a Percival Scientific (Perry, IA, USA) growth chamber and maintained at 25 C, 40-60% humidity, and 16:8 L:D. All subsequent experiments in the growth chamber were maintained at these same conditions. 17

28 Tested host plants were chosen based on early host plant literature documenting presence of ECB eggs and larval feeding a few years after ECB introduction to North America (Hodgson 1928; Hüber et al. 1928) and from more recent literature on ECB oviposition deterrents (Tallamy et al. 1997). To determine ECB s ability to overcome natural plant chemical defenses, specific crop types were selected to encompass a range of hosts based on known deterrent/antifeedant chemistry or minimal defenses. The crops tested include corn encoded with Cry1F Bt toxin along with its near isoline (DuPont Pioneer, Wilmington, DE, USA), Silver Queen sweet corn, Bronco green beans, Mountain Fresh Plus VFFN hybrid tomato, Fancipak pickling cucumber, and Buttercup squash. Non-corn varieties selected for the experiments were listed in the 2013 Delaware Commercial Vegetable Production Recommendations as suggested varieties for commercial farmers in the Delaware area. Unfavorable host plants were classified as those containing Bt toxin or chemical defenses expected to limit ECB development. These plants included Cry1F Bt corn, tomato, cucumber and squash. Cry1F Bt corn is known to kill ECB because of the presence of Bt toxin (Koziel et al. 1996). Tomato contains tomatine, an alkaloid found in the green plant parts, and is known to deter many insects (Buhr et al. 1958; Thorsteinson 1960; Boppre 1989; Isman 2002). Cucumber and squash contain varying concentrations of cucurbitacin, a bitter deterrent that is specifically detected and avoided by ovipositing ECB (Tallamy et al. 1997). Cucurbitacin content of squash is 0.02mg/g of fresh weight in the leaves, while cucumber contains trace amounts (Metcalf et al. 1982). Cucurbit plants were tested for larval feeding to see if results would coincide with results from ECB oviposition studies where cucurbitacin was avoided (Tallamy et al. 1997). 18

29 Favorable host plants were classified as those with minimal or lacking known defensive characteristics. These included non-bt near isoline corn, sweet corn, and green bean. Seedling corn contains high concentrations of DIMBOA (2,4-Dihydroxy-7- methoxy-1,4-benzoxazin-3-one), a compound that prevents feeding and is toxic to ECB neonates until corn reaches vegetative stage 5 (V5) (Guthrie et al. 1986). Vegetative stage 6 (V6) corn was utilized for experiments to avoid effects of DIMBOA. Near isoline corn is genetically identical to Cry1F, except without the Bt toxin, and it is used commonly as refuge corn. Sweet corn was tested to determine if it has greater suitability for ECB compared to near isoline. Green bean does not provide many toxins that directly affect ECB (Webb et al. 1987). ECB adults have been associated with green bean because it offers a proper environment for rest during the day and mating at night, and adults are known to oviposit on green bean (Mason et al. 1996). In addition, ECB even in low densities can have a large economic impact on the green bean industry causing entire fields to be rejected if one larva is found in the harvested beans. Plants used in growth chamber and greenhouse experiments were grown in the greenhouse maintained at C, 40-60% humidity, and 16:8 L:D supplemented during short day lengths. Plants for field experiments were seeded into the ground, except for tomato, which consisted of two-week old seedlings. All plants were grown to approximately 1000 accumulated degree-days to correspond with corn at V6. At this point in development, plants were used to initiate experiments or their leaves were excised to feed ECB. 19

30 Growth Chamber Neonate Infestation Neonate ECB hatched from egg masses in a plastic bag and were moved via their silk using a fine brush into individual 37 ml Solo plastic cups (Lake Forest, IL, USA). Each cup contained approximately five cm 2 of leaf tissue of one of the host plants and a dry cotton wick to collect excess moisture. Both leaf material and cotton wicks were changed daily. Seventy-five replicates were grouped into three blocks of 25 by date. Cups were maintained in the growth chamber at the same growth chamber conditions described previously. Each day, survival was recorded. At mid-development (355 degree days since hatch), larvae were weighed. Individuals were monitored for degree-day accumulation to pupation and pupal weight was recorded. Degree-day accumulation at death was recorded for those that did not survive to pupation. Third Instar Infestation Third instar larvae were moved from the gregarious, ECB diet container into individual 37 ml Solo plastic cups. Each cup contained approximately five cm 2 of leaf tissue of one of the host plants and a dry cotton wick to collect excess moisture. Both leaf material and cotton wicks were changed daily. Seventy-five replicates were grouped into three blocks of 25 by date. Cups were maintained in the growth chamber at the same conditions described previously. Each day, survival was recorded. Individuals were monitored for degree-day accumulation to pupation and pupal weight was recorded. Degree-day accumulation to death was recorded for individuals that did not survive to pupation. 20

31 Greenhouse Neonate Infestation To infest whole plants with neonates, two blackhead stage egg masses were placed in 1.5 ml microcentrifuge tubes (Fisher Scientific, Waltham, WA, USA) and attached to the plants with a paper twist tie (Staples, Framingham, MA, USA). Additional sweet corn plants were infested with neonates to serve as larval developmental monitors after infestation. Plants were covered with a cage created from thin mesh fabric (JoAnn Fabric, Hudson, OH, USA) and either 84 cm or 107 cm galvanized steel wire round tomato cages (Lowes, Mooresville, NC, USA) based on host plant size to prevent larval movement away from the plant. Using a split design, infested plants were either dissected at mid-development (355 degree-days of development) or after estimated pupation (790 degree-days of development). Ten plants were dissected over 5 dates for mid-development larvae and eight plants were dissected over 4 dates for pupae on each host type. The number of individuals collected per plant at dissection and their weights were recorded. If larvae were recovered from plants at 790 degree-days, they were kept with stalk/stem tissue in the growth chamber until pupation. Third Instar Infestation For 3 rd instar experiments, five individuals were placed on different leaves of the plant to prevent crowding effects. Additional sweet corn plants were infested with 3 rd instars to be dissected prior to experimental dissections to observe ECB development. Subsequent to infestation, plants were covered with a cage created from thin mesh fabric and either 84 cm or 107 cm galvanized steel wire round tomato cages based on host plant size to prevent larval movement. Eight plants were dissected over 4 dates for pupae (567 21

32 degree-days of development after infestation) on each host type. The number of individuals collected per plant at dissection and their weights were recorded. If larvae were recovered, they were kept with stalk/stem tissue in the growth chamber until pupation. Field The design for field experiments corresponded with the greenhouse experiments. Field plots were created from small three-row clusters of plants of the same species with three plants in each row (nine plants total). Neighboring plants were spaced 30 cm apart. Plants were seeded into the ground, except for tomato, which consisted of two-week old seedlings. If seeds failed to germinate in the field, back-up seedlings were transplanted into empty spaces. If those failed, plants were transplanted with similar degree-day accumulation from weekly greenhouse plantings. Groupings were arranged in a complete random block design for the seven host plants with 45cm of space between groupings. There were a total of twelve groupings planted for each host plant and each experimental design. Plantings occurred in mid-may and late-may to stagger infestations and dissections into more manageable numbers. Black plastic (Rain-Flo Irrigation, East Earl, PA, USA) was laid between groupings to prevent weeds. Weeds were manually removed within row middles and with Roundup (Monsanto Company, St. Louis, MO, USA) outside of research plots. Neonate Infestation In neonate experiments, the center plant of a grouping was infested with a 1.5 ml microcentrifuge tube containing two blackhead stage egg masses. Tubes were attached to 22

33 the plants with a paper twist tie. The surrounding eight plants were left uninfested to receive only individuals moving off of the infested plant. Additional sweet corn groupings were planted and infested with neonates to serve as developmental monitors. Using a split plot design, neonate infested plants were either dissected at middevelopment (4 th instar; 355 degree-days) or at estimated pupation (790 degree-days). Both mid-development and pupal dissections consisted of 12 groupings split evenly over the two plant dates for each host plant type. All nine plants in a grouping were inspected; the number of individuals collected per grouping and their weights were recorded. In addition, injury on plants without individuals recovered was documented. If larvae were recovered at the later dissection, they were kept on ECB diet in the growth chamber until pupation. Third Instar Infestation For 3 rd instar experiments, the center plant of a grouping was infested with five 3 rd instars placed on separate leaves to minimize density dependent effects. The surrounding eight plants were left uninfested to receive only individuals moving off the infested plant. There were two replicates of six plant clusters per host. Additional sweet corn groupings were planted and infested with 3 rd instars as developmental monitors. All nine plants in a grouping were inspected at estimated pupation (567 degree-days after infestation). The number of individuals collected per grouping and their weights were recorded. In addition, injury on plants without individuals recovered was documented. If larvae were recovered, they were kept with ECB diet in the growth chamber until pupation. 23

34 Statistical Analysis The mid-development and pupal weights in all experiments were analyzed using two-way ANOVAs followed by Tukey-Kramer s mean separation test in the program R 2.14 (R Development Core Team) to determine an interaction between the two independent variables on the dependent variable. The nominal and independent variables were date of experiment and host plant. The measurement/dependent variables were middevelopment or pupal weight. Growth chamber survival data was analyzed using the Kaplan-Meier Method for survival analysis to compare the longevity of insects reared on different hosts until pupation followed by the Wilcoxon test to adjust for multiple comparisons and Tukey-Kramer s mean separation test in the program SAS 9.3 (SAS Institute Inc., Cary, NC). Overall length of survival was measured in degree-days accumulated until death. Individuals that pupated were censored data points and removed from the analysis, since very few survived to pupation. Percent survival after the one stadium of treatment (neonate to 2 nd instar; 3 rd to 4 th instar) based on degree-day accumulation (2 nd = 106 degree-days; 4 th = 132 degree-days) was analyzed using a oneway ANOVA in the program R based on survival percentages of the 3 replications. The number of individuals recovered per plant (greenhouse) or plant grouping (field) was analyzed with two-way ANOVAs followed by Tukey-Kramer s mean separation test in the program R. The nominal variables were date of experiment and host plant. The measurement variable was the number of individuals recovered. For all analyses, P- values of 0.05 or less were considered statistically significant. 24

35 Results Growth Chamber Within the growth chamber statistical analyses, it should be noted that there were significant interactions regarding experiment replication (Table 1). There were occasions when one date was significantly different from the other two when measuring weight or survival The survival analyses conducted did not take this effect into consideration. Neonate Infestation Only neonates fed near isoline or sweet corn survived to the mid-development (355 degree-days) and pupal weight observed points. Individuals were significantly heavier at mid-development when fed near isoline than those fed sweet corn (Table 1). There were no significant differences in pupal weight when larvae were fed near isoline or sweet corn (Table 1). Survival curves show that larvae fed sweet corn or near isoline survived approximately the same length of time, and significantly longer than those fed the other four host plants (Figure 1). In addition, these were the only hosts that allowed individuals to reach pupation. Larvae fed Cry1F, cucumber, or tomato survived the same length of time, but shorter than the other host plants. Larvae fed green bean survived for an intermediate period of time significantly different from larvae fed the other host plants (Figure 1). Percentage of survival through the first stadium of treatment was consistent with the overall survival (Figure 2A). Here, survival to 2 nd instar is presented based on degree-day accumulation. The largest percentage of individuals survived 106 degree-days when fed sweet corn, near isoline, or green bean (Figure 2A). Individuals fed cucumber, tomato, and Cry1F had the lowest survival (Figure 2A). 25

36 Third Instar Infestation Third instars only survived to pupation on near isoline and sweet corn. Pupae were not significantly heavier when fed near isoline than those fed sweet corn (Table 1). Survival curves show third instars fed sweet corn or near isoline survived statistically the same amount of time, and significantly longer than larvae fed any other host plant tested (Figure 3). Larvae fed cucumber survived an intermediate length of time, while larvae fed Cry1F, green bean, or tomato survived the shortest (Figure 3). Percentage of survival through the first stadium of treatment was consistent with the overall survival (Figure 2B). The largest percentages of individuals to survive 132 degree-days were fed sweet corn or near isoline, cucumber had intermediate survival, and individuals fed Cry1F, green bean, or tomato had the lowest survival. Greenhouse Neonate Infestation In the neonate experiments, only enough individuals were recovered from near isoline or sweet corn to collect larval mid-development (355 degree-days) and pupal, weights. Mid-development individuals were significantly heavier when fed sweet corn than those fed near isoline (Table 1). There was no significant difference in pupal weights when fed sweet corn or near isoline (Table 1). At mid-development dissection, corn plants were in late vegetative stages and all other plants were beginning to flower. There were significantly more individuals found on sweet corn and near isoline than any other host tested (Figure 4A). There was an average of 18 individuals recovered from sweet corn or near isoline and fewer than 1 26

37 individual per squash, cucumber, tomato, green bean, or Cry1F plant. At pupal dissection, corn plants were in early reproductive stages and all other plants were flowering. There were significantly more individuals recovered from sweet corn than from near isoline, but still more individuals from near isoline than cucumber, Cry1F, green bean, tomato, or squash (Figure 4B). No individuals were recovered from Cry1F or tomato. For both dissections, there was rarely injury observed on tomato or Cry1F, minimal injury on green bean, cucumber, and squash, and heavy injury on sweet corn and near isoline. Third Instar Infestation In the 3 rd instar experiments, only enough individuals were recovered from near isoline or sweet corn to collect pupal weights. At pupal dissection, corn plants were in late vegetative stages and all other plants were beginning to flower. There was no significant difference in pupal weights when fed sweet corn or near isoline (Table 1). The highest pupal recovery was from non-bt corn hosts, which were significantly higher than all other hosts (Figure 4C). Of five individuals placed on each plant, on average two to three were recovered from near isoline and sweet corn, and none from cucumber, Cry1F, green bean, and tomato. There was heavy damage on near isoline and sweet corn; there was minimal damage observed on tomato, green bean, cucumber, squash, and Cry1F. Field All of the plants grown in the field were noticeably larger than those grown in the greenhouse. There were no pots to restrict growth and the plants were open to natural sunlight. At plant dissection plants were much more developed than in greenhouse experiments. Corn was typically in late vegetative stages for mid-development dissection 27

38 and early reproductive stages for pupal dissection. All of the other plants were fruiting at both dissections. Additionally, plants were open to other herbivorous pests, while ECB larvae were exposed to predators (especially ladybird beetles) and parasitoids. Very few individuals were recovered from field experiments on any host plant. There were no differences detected in recovery for any of the field experiments, neonate or third instar infested plants (Figure 5A: P > 0.23; Figure 5B: P > 0.25; Figure 5C: P > 0.07). Of the two egg masses (~ 40 neonates) released in the 9-plant clusters, on average 0.25 or fewer were recovered per plant at mid-development (Figure 5A), with even fewer at pupation (Figure 5B). Of the five third instars placed in the clusters of nine plants, fewer than one per plant were recovered (Figure 5C). However, ECB injury was observed on all plants. Minimal injury was found on Cry1F, squash, and cucumber. Moderate injury was observed on sweet corn, near isoline, green bean, and tomato. More injury was observed on green bean and tomato than expected based on growth chamber and greenhouse results. Many empty galleries were observed in sweet corn, near isoline, and green bean. There were only enough individuals recovered from the plants that were infested at third instar and dissected at pupation to analyze weight. In this case, to increase the sample size, individuals from sweet corn and near isoline were pooled into one category ( non-bt corn ) since no significant differences were found in the growth chamber or greenhouse trials. The average pupal weight recovered from non-bt corn was significantly higher than pupae recovered from tomato (Figure 6). 28

39 Discussion Growth chamber trials yielded minimal survival to pupation. When the larvae were fed plant tissue in the growth chamber, it was only leaf tissue without stalks or stems. Since ECB are stem-boring insects, the larvae may have been stressed by being limited to leaf tissue, resulting in a high amount of death. However, there were still meaningful trends present in the growth chamber survival data (Figure 1 and 2). In both experiments (neonate or 3 rd instar infestation), larvae survived significantly longer on non-bt corn hosts (near isoline and sweet corn) than any of the other hosts, suggesting that non-bt corn is the most acceptable host among those tested. In turn, larvae survived the shortest period (approximately 2-6 days) on Cry1F and tomato, suggesting that these are very poor hosts for larval growth and development. Green bean was an intermediate host for neonates and a poor host for 3 rd instars. Cucumber was a poor host for neonates and an intermediate host for 3 rd instar larvae. Because of this, it is difficult to classify either cucumber or green bean as a favorable or unfavorable host species. Consequently, it is appropriate to classify them as intermediate in host suitability. In the greenhouse, the greatest survival was on non-bt corn hosts, with little to no survival on any other plant species in all experiments (neonate and 3 rd instar). Sweet corn and near isoline were expected to provide optimal survival because of relatively reduced host defenses (Wink 1988; Rosenthal and Dirzo 1997). When neonates remained and survived on the plants until pupation sweet corn was able to sustain more individuals, supporting the idea that sweet corn is a more suitable host than near isoline. Few individuals were recovered and minimal plant injury was observed from ECB on squash, green bean, and cucumber, but the differences in growth, development, and survival were 29

40 not enough to be statistically significant. The small ECB recovery numbers could be because these are not sufficient hosts for complete ECB development, but also because these plants offer less plant tissue and stem diameter than the corn hosts, providing insufficient habitat and food resources for ECB development (Ehrlich and Raven 1964; Losey et al. 2002). The average dry weight of the corn plants provided at infestation was 22.3 mg and 4.5 mg for non-corn hosts. It has been found that when the stem diameters of non-corn hosts are too small, ECB will abandon the plant and search for a new host (Losey et al. 2002). There was minimal injury and no individuals were recovered from Cry1F. Rarely was any injury observed on tomato plants in the greenhouse; only one individual survived to pupation from a 3 rd instar infestation. These results suggest that Cry1F and tomato are unsuitable host plants for ECB used in this experiment. In the field, there were no significant trends observed in any experiment. Recovery was so low, with most plants recovering zero individuals, that it is difficult to draw any conclusions. Most unfailingly, one individual on average was recovered from sweet corn groupings, but this did not occur frequently enough to cause a significant difference. Strangely, there were a relatively large number of individuals recovered as larvae and pupae from tomato plants. This does not coincide with the experiments conducted in the incubator or greenhouse. There is potential that these individuals moved from other host plants in the field. If a host plant is unsuitable, ECB are capable of abandoning the host and surviving three weeks in search of a more suitable host (Losey et al. 2002). There were never individuals recovered from Cry1F, cucumber, or squash in the field. 30

41 There are a few external reasons that could have caused the low survival on all host species in the field. While in the field, I noticed an abundance of generalist predators, specifically ladybird beetles. It is likely that generalist predators consumed many of the eggs and larvae since ladybird beetles provide biological control as general predators (Dixon 2000). In addition, many of the larvae that we recovered and reared in the laboratory were parasitized, which was another source of mortality in the study. Even though degree-day accumulation was closely followed and monitor plants were frequently checked, occasionally empty galleries were observed that appeared to have housed pupae that had already eclosed as adults. The majority of pupae recovered were female leading to the belief a portion of the males may have escaped. Female ECB eclose a few days after males (Mason et al. 1996). If I were to conduct this study again in the future, I would plan to dissect the host plants a few days earlier and rear the recovered larvae on diet until pupation occurred instead of hoping to recover pupae. Weight data was collected from all of the experiments in the growth chamber, greenhouse, and field. In most experiments, only individuals from non-bt corn hosts survived to the point of weight data collection. It was not expected that there would be a difference in weight or degree- day accumulation between individuals fed sweet corn or near isoline since they are both non-bt corn hosts providing relatively the same nutrition and plant defenses. In some parts of the study, there was a significant difference between the two, but which larvae were was heavier varied between the two host plants and was not consistent. Sometimes sweet corn had insects heavier and other times near isoline produced insects with greater weights. This variation was due to some other factor, not the host itself. 31

42 Pupae recovered from tomato plants in the field were significantly lighter than those recovered from non-bt corn hosts (Figure 6). Larger pupae yield larger adults, which have been shown to have greater fitness and are more successful (Kingsolver and Huey 2008). This suggests that when ECB larvae successfully develop on tomato, there is a reduction in their potential fitness. In the growth chamber and greenhouse experiments, individuals fed tomato did not survive to pupation. The tomatine present in the green leaf tissue of tomato plants may be initially acting as an antifeedant when larvae first encounter it (Isman 2002). If there are no other host options, larvae will either starve to death or eventually feed on tomato. However, those that do feed end up significantly smaller, leading to reduced fitness when they become adults. Components of host plant quality, such as nitrogen and defensive metabolite concentrations, have been studied in depth. Host plant quality is a strong determining factor in female fecundity. Individuals that were fed a better quality host plant have greater reproductive potential and are, therefore, more fit (Awmack and Leather 2002). When neonates remained on the plants in the greenhouse until pupation, sweet corn was able to sustain more individuals, possibly because sweet corn contains greater nutritional value than near isoline. Digestible protein was suspected as one nutritional difference, however, we were unable to determine a difference when near isoline (n = 6; = 13.25%; SE = 0.45) and sweet corn (n = 6; = 13.55%; SE = 1.28) tissue samples were sent to AgriAnalysis in Leola, PA for a basic forage test (P = 0.83). It is likely that the presence of plant deterrents and toxins caused the large significant difference in length of survival in the growth chamber and recovery from the greenhouse between the non-bt corn hosts and all other plants. First, it is known that 32

43 Cry1F will kill ECB larvae upon feeding (Gill et al. 1992). Additionally, it has repeatedly been shown that alkaloids are deterrents or toxins (Wink 1988; Thorsteinson 1960; Bentley et al. 1984). There are some insect species that can detoxify alkaloids, but these insects are plant specialists that utilize alkaloids to their defensive benefit, like the tobacco hornworm (Boppre 1989). However, there is no evidence of ECB utilizing alkaloids, which helps to explain the lack of survival on tomato plants. It has been shown that cucurbitacin is an oviposition deterrent specifically for ECB (Tallamy et al. 1997). This compound may be the cause for reduced larval survival on cucurbits. Reduced survival on green bean may be explained by plant chemistry, as well. Some legumes produce a high diversity of secondary metabolites used for defense against herbivores (Wink 2013). It is important to remember that Z-race ECB from a laboratory colony were utilized for all of the experiments in this study. The Z pheromone race tends to feed and oviposit mostly on corn because their first generation life cycle is more synchronized with the availability of corn in V5-R1, while E pheromone race ECB have been known to feed on alternative host plants because they eclose prior to corn availability (Clacagno et al. 2007). If the same experiments were conducted with E-race ECB instead of Z-race ECB, the results may show differing survival and host utilization. All of the data presented here from the growth chamber and greenhouse experiments support the idea that there is a tendency toward host plant specialization on corn, or ECB was never as general of a pest as once documented. The criteria for host specialization include: (1) the hosts provided should always be accepted or rejected, (2) the hosts in an expanded diet would be of low suitability, and (3) the most suitable host is 33

44 easy to find (Jaenike 1990; Charnov and Stephens 1988; Futuyma 1983; Levins and MacArthur 1969; Rausher 1983). Supporting the first and second criteria, less suitable hosts are only acceptable occasionally when a more suitable host is not available, and never consistently. In the growth chamber and greenhouse the vast majority of survival was on non-bt corn hosts with little to no survival to pupation on any other host. Further supporting this, Cry1F and tomato were consistently rejected in the growth chamber and greenhouse. Not only were there no survivors, but also there was no feeding damage. The third criterion above is easily met by the agricultural corn system. Bt Corn is one of the most abundant crops grown in the United States. Every Bt field requires a refuge of non- Bt corn, the most suitable host for ECB, making it easy for ECB to find. Corn also is much more abundant than any other potential host plant in the Corn Belt (the abundant soybeans are not a preferred ECB host), where historically the worst ECB infestations have occurred (Brindley and Dicke 1963). In addition, since corn is grown in monoculture, both the resource concentration and reduced enemies may play a role in host specialization. The resource concentration hypothesis refers to the abundance of food sources an insect will have for itself and its future offspring. All of the plants in the area are healthy host plants (Grez and Gonzalez 1995). The enemy free hypothesis states that due to the lowered plant diversity the insect diversity is decreased as well. There are fewer natural enemies present in agriculture fields to feed on or parasitize pest species (Letourneau 1987). Since ECB is a stem borer, they have the capability of hiding from potential enemies in corn s strong stalks. Finally, as stated previously, corn provides minimal defenses because plants have been selected for yield, size, and palatability. 34

45 Plants energy is directed more toward growth rather than defense (Rosenthal and Dirzo 1997). In summation, sweet corn and near isoline are the only consistent favorable host plants for Z-race ECB, supporting the idea of host plant specialization. As expected, Cry1F was consistently a poor host, yielding no survival or feeding damage in any experiment. Green bean, cucumber, and tomato are unsuitable hosts that occasionally yield feeding damage and survival. They are not consistently adequate hosts, but there is potential that ECB can survive on these plant species. However, when they survive on tomato, they are less fit in comparison to larvae fed non-bt corn. It appears very unlikely that there will be an ECB infestation in any of these crops because survival was very rare. Based on the history of many non-corn hosts in the early literature (Hodgson 1928; Hüber et al. 1928), it appears that Z-race ECB has evolved a host plant specialization for corn. Continual monitoring for Bt resistance, participation in resistance management strategies, and high abundances of general predators and parasitoids (as was seen in the field experiments) will continue to reduce ECB populations. Most ECB die because of Bt corn, and those that do survive, have a high likelihood of predation or parasitism (field observations). If it continues in this trend, Z-race ECB eventually may become rare in the United States, or it may evolve back toward selecting other crops or weeds as hosts. 35

46 Table 1 Average weight of mid-development larvae or pupae under all experimental designs. Letters indicate significantly different weights analyzed with two-way ANOVAs (P < 0.05). Host Plant Mean ± SEM Weight (mg) Growth Chamber: Neonate Infested, Mid-Development Weight a Near Isoline ± 3.07 a Sweet Corn 9.95 ± 0.95 b Growth Chamber: Neonate Infested, Pupal Weight b Near Isoline 41.0 ± 3.41 a Sweet Corn ± 2.26 a Growth Chamber: Third Instar Infested, Pupal Weight c Near Isoline ± 1.72 a Sweet Corn ± 1.45 a Greenhouse: Neonate Infested, Mid-Development Weight d Near Isoline 9.77 ± 0.63 a Sweet Corn ± 1.27 b Greenhouse: Neonate Infested, Pupal Weight e Near Isoline ± 3.11 a Sweet Corn ± 2.23 a Greenhouse: Third Instar Infested, Pupal Weight f Near Isoline ± 6.82 a Sweet Corn ± 9.01 a Field: Third Instar Infested, Pupal Weight g Non-Bt Corn ± 8.82 a Tomato ± 3.56 b a Date: F = 15.1, df = 2, P < 0.01; Host: F = 13.1, df = 1, P < 0.01; Date*Host: F = 14.7, df = 2, P < 0.01 (two-way ANOVA). b Date: F = 2.2, df = 1, P = 0.17; Host: F = 1.9, df = 1, P =0.20 (two-way ANOVA). c Date: F = 7.8, df = 2, P < 0.01; Host: F = 3.1, df = 1, P = 0.09; Date*Host: F = 0.5, df = 1, P = 0.49 (two- way ANOVA). d Date: F = 36.4, df = 4, P < 0.01; Host: F = 31.8, df = 1, P < 0.01; Date*Host: F = 4.1, df = 1, P < 0.01 (two-way ANOVA). e Date: F = 1.0, df = 3, P = 0.39; Host: F = 0.4, df = 1, P = 0.53; Date*Host: F = 0.5, df = 3, P = 0.69 (two- way ANOVA). f Date: F = 2.7, df = 3, P = 0.07; Host: F = 3.6, df = 1, P = 0.07; Date*Host: F = 2.2, df = 3, P = 0.11 (two- way ANOVA). g Date: F = 2.2, df = 1, P = 0.17; Host: F = 11.0, df = 1, P < 0.01; Date*Host: F = 0.02, df = 1, P = 0.89 (two-way ANOVA). 36

47 1 Near Isoline (a) Sweet Corn (a) Survival Probability Cucumber (c) Cry1F (c) Green Bean (b) Tomato (c) Degree-Day Accumulation Figure 1 Survival curve for neonate-infested growth chamber feeding trials. Survival curves based on the longevity of larvae fed various host plants determined by degree-days accumulated until death prior to pupation. Lines for hosts followed by the same letter did not differ in survival time (Tukey-Kramer mean separation test (P < 0.05). 37

48 A. Average % Survival ab a bc c abc bc 0 N SC C F GB T B. 100 a ab Average % Survival bc c c c 0 N SC C F GB T Host Plant Figure 2 Growth chamber survival through the first stadium of treatment. (A) Neonates or (B) third instars fed one of various host plants (N = near isoline, SC = sweet corn, C = cucumber, F = Cry1F, GB = green bean, T = tomato) through the duration of development. Bars represent the average percent of the individuals that survived to the subsequent instar based on degree-day accumulation (106 degree-days = 2 nd instar; 132 degree-days = 4 th instar). Bars within each graph with different letters are significantly different (Tukey-Kramer test, P < 0.05). Error bars represent SEM. 38

49 1 Near Isoline (a) Sweet Corn (a) Survival Probability Cucumber (b) Cry1F (c) Green Bean (c) Tomato (c) Degree-Day Accumulation Figure 3 Survival curve for third instar infested growth chamber feeding trials. Survival curves based on the longevity of larvae fed various host plants determined by degree-days accumulated until death prior to pupation. Lines for hosts followed by the same letter did not differ in survival time (Tukey-Kramer mean separation test (P < 0.05). 39

50 A. Average # Recovered a a b b b b b N SC C F GB SQ T B. C. Average # Recovered Average # Recovered a b c c c c c N SC C F GB SQ T a a b b b b b N SC C F GB SQ T Host Plant A: Date: F = 0.3, df = 4, P = 0.85; Host: F = 41.2, df = 6, P < 0.01; Date*Host: F = 1.3, df = 24, P = 0.36 (two-way ANOVA). B: Date: F = 2.4, df = 3, P = 0.09; Host: F = 107.9, df = 6, P < 0.01; Date*Host: F = 5.1, df = 18, P < 0.01 (two-way ANOVA). C: Date: F = 1.9, df = 3, P = 0.15; Host: F = 26.0, df = 6, P < 0.01; Date*Host: F = 1.3, df = 18, P = 0.26 (two-way ANOVA). Figure 4 Greenhouse recovery. (A) Neonate infested plants (N = near isoline, SC = sweet corn, C = cucumber, F = Cry1F, GB = green bean, SQ = squash, T = tomato) were dissected after 355 degree-days for mid-development recovery or (B) after 790 degree-days for pupal recovery. (C) Plants infested with 3 rd instars were dissected after 567 degree-days for pupal recovery. Bars with represent the number of individuals recovered at each dissection with SEM. Bars with different letters within one graph are significantly different (P < 0.05).. 40

51 A. B. Average # Recovered N SC C F GB SQ T Average # Recovered C. Average # Recovered N SC C F GB SQ T N SC C F GB SQ T Host Plant A: Date: F = 0.7, df = 1, P = 0.40; Host: F = 1.4, df = 6, P =0.22; Date*Host: F = 0.7, df = 6, P = 0.64 (twoway ANOVA). B: Date: F = 3.5, df = 1, P = 0.07; Host: F = 1.7, df = 6, P = 0.14; Date*Host: F = 1.7, df = 6, P = 0.14 (two-way ANOVA). C: Date: F = 3.5, df = 1, P = 0.06; Host: F = 3.2, df = 6, P < 0.01; Date*Host: F = 1.1, df = 6, P = 0.38 (two-way ANOVA). Figure 5 Field recovery. (A) Neonate infested plant groupings (N = near isoline, SC = sweet corn, C = cucumber, F = Cry1F, GB = green bean, SQ = squash, T = tomato) were dissected after 355 degree-days for a mid-development recovery or (B) after 790 degree-days for pupal recovery. (C) Plant groupings infested with 3 rd instars were dissected after 567 degree-days for pupal recovery. Numbers did not differ for any trial (P > 0.05). 41

52 Average Pupal Weight (mg) Non Bt Corn Host Plant Tomato Date: F = 2.2, df = 1, P = 0.17; Host: F = 11.0, df = 1, P < 0.01; Date*Host: F = 0.02, df = 1, P = 0.89 (twoway ANOVA). Figure 6 Pupal weight comparison from third instar infested plants in the field.average weight of pupae recovered from 3 rd instar infested field experiments from non-bt corn (sweet corn and near isoline) (n = 7) and tomato (n = 8). There was a significant difference between the two bars (P < 0.01). Error bars represent SEM. 42

53 Chapter 3 EUROPEAN CORN BORER PLANT CONSUMPTION RATES AND PREFERENCE AMONG A RANGE OF AGRICULTURAL HOST CROP SPECIES Introduction The European corn borer (ECB), Ostrinia nubialis (Hübner), was introduced to North America in the early 1900s with the importation of broomcorn (Sorghum technicum L.) from Italy and Hungary. At introduction, ECB had a preference for corn (Zea mays L.), but was known to feed opportunistically on 223 crop and weedy host plant species, earning the label of generalist herbivore (Hodgson 1928; Hüber et al. 1928). Some common alternative hosts include bean, beat, cauliflower, celery, eggplant, pepper, and tomato (Hodgson 1928). The largest impact of ECB has been observed in corn agriculture where it is documented to cause more than 1 billion US dollars in annual yield loss and management/prevention costs (Mason et al. 1996). Currently, ECB is controlled without resistance by corn genetically modified with insecticidal crystalline (Cry) proteins from the soil bacterial species Bacillus thuringensis (Fernandez-Cornejo et al. 2014). Adult oviposition is the primary influence of host selection for larval Lepidoptera. However, neonate ballooning and larval movement can also facilitate host selection (Ross and Ostlie 1990). Specifically, larval movement is a concern in resistance management with a mixed planting of Bt and non-bt refuge in a cornfield. It has been documented that 75% of ECB neonates abandon Bt natal plants and 42% of neonates abandon non-bt natal host plants (Razze et al. 2011). Young ECB larvae move around a great deal prior to boring into the plant in the 3 rd instar, potentially obtaining sub-lethal doses of Bt toxin as 43

54 older larvae and speeding up the development of Bt resistance. Larval movement also opens potential for movement to alternate non-corn hosts, most likely field weed species (Tate et al. 2006). Previously, survival was observed on various early documented hosts and oviposition deterrents (Hodgson 1928; Hüber et al. 1928; Tallamy et al. 1997), and it was found that ECB most consistently succeeded on non-bt corn hosts (non-bt near isoline corn and sweet corn), never succeeded on Cry1F Bt corn, and succeeded with variability on cucurbits, green bean, and tomato (Chapter 2). Based on assessment of growth and development on these hosts it was concluded that non-bt corn hosts were the most favorable host plants and all others tested are unfavorable or no longer acceptable hosts since survival to maturity did not occur under experimental conditions. No choice and choice tests are common methods for determining larval feeding preferences and help predict pest occurrence in the presence of host plant options (Barnes and Ratcliff 1967; Jackai 1991; Smith et al. 1994; Davis and Coleman 1997; Tate et al. 2006). In this part of the study, no choice and choice tests were conducted over the same range of host plants previously tested for survival to determine if ECB feed more and prefer host plants that yield better survival. Preference tests help to determine if ECB will preferentially switch hosts if other options become available. This research addressed the following objectives through laboratory studies: (1) evaluate amount of feeding on each host plant based on leaf tissue consumption in a no choice 48-hour period by 3 rd instar ECB, and (2) determine feeding preferences based on leaf tissue consumption by 3 rd instar ECB when presented with a choice. 44

55 Materials and Methods Insects and Plants Larval, Z-race European corn borers (ECB) were received from the USDA-ARS Corn Insects and Crop Genetics Research Unit (Ames, IA). Each year, field captured ECB are incorporated into the colony to maintain field relevance and reduce inbreeding. Third instars were shipped to the University of Delaware (Newark, DE) gregariously, feeding on ECB diet and maintained in a Percival Scientific (Perry, IA, USA) growth chamber and maintained at 25 C, 40-60% humidity, and 16:8 L:D. All subsequent experiments in the growth chamber were maintained at these same conditions. Tested host plants were chosen based on early host plant literature documenting presence of ECB eggs and larval feeding a few years after ECB introduction to North America (Hodgson 1928; Hüber et al. 1928), and from more recent literature on oviposition deterrents (Tallamy et al. 1997). To determine ECB s ability to overcome natural plant chemical defenses, specific crop types were selected to encompass a broad range of feeding hosts based on known deterrent/antifeedant chemistry or minimal defenses. The crops tested included corn encoded with Cry1F Bt toxin along with its near isoline (DuPont Pioneer, Wilmington, DE, USA), Silver Queen sweet corn, Bronco green beans, Mountain Fresh Plus VFFN hybrid tomato, Fancipak pickling cucumber, and Buttercup squash. Non-corn varieties selected for the experiments were listed in the 2013 Delaware Commercial Vegetable Production Recommendations as suggested varieties for commercial farmers in the Delaware area. Unfavorable host plants were identified as plants that yielded minimal survival in previous growth chamber and greenhouse studies due to Bt toxin or chemical defenses 45

56 (Chapter 2). These plants included Cry1F Bt corn and tomato. Cry1F Bt corn is known to kill ECB because of the presence of Bt toxin (Koziel et al. 1996). Tomato contains tomatine, an alkaloid found in green plant parts, and is known to deter many insects, and potentially other compounds (Buhr et al. 1958; Thorsteinson 1960; Boppre 1989; Isman 2002). Intermediate hosts were those that have defensive chemistry, but ECB were inconsistently able to survive under growth chamber and greenhouse conditions (Chapter 2). These hosts include cucumber, squash, and green bean. Cucumber and squash contain varying concentrations of cucurbitacin, a bitter deterrent that is also specifically detected and avoided by ovipositing ECB (Tallamy et al. 1997). Cucurbitacin content of squash is 0.02 mg/g of fresh weight in the leaves, while cucumber contains trace amounts (Metcalf et al. 1982). Green bean was originally classified as a favorable host plant because it offers a proper environment for ECB adults to rest during the day and mate at night, and adults are known to oviposit on green bean (Mason et al. 1996). However, legumes contain a diversity of secondary metabolites in the leaf tissues (Wink 2013; Abu-Reidah 2013). Cucumber, squash, and green bean provided inconsistent survival results (Chapter 2). Favorable host plants were classified as those with minimal or lacking defensive characteristics that previously provided the highest survival probability (Chapter 2). These included non-bt near isoline corn and sweet corn. Seedling corn contains high concentrations of DIMBOA (2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one), a compound that prevents feeding and is toxic to ECB neonates until corn reaches vegetative stage 5 (V5) (Guthrie et al. 1986). Vegetative stage 6 (V6) corn was utilized 46

57 for experiments to avoid effects of DIMBOA. Near isoline corn is genetically identical to Cry1F, except without the Bt toxin, and is used commonly as refuge corn. Sweet corn was tested to determine if it is a more preferred host for ECB than near isoline. Plants used were grown in the greenhouse maintained at C, 40-60% humidity, and 16:8 L:D supplemented during short day lengths. All plants were grown to approximately 1000 accumulated degree-days to correspond with corn at V6. At this point in development, plant leaf tissues were excised to feed ECB. Consumption Rates and Preference Choice and no choice tests are common methods for determining larval feeding preferences (Barnes and Ratcliff 1967; Jackai 1991; Smith et al. 1994; Davis and Coleman 1997; Tate et al. 2006). However, most previous experiments used an estimated percentage of leaf material consumed to determine preference. The most accurate measurement is taken by adjusting a known leaf area based on dry weight of the sample (Barnes and Ratcliff 1967). After feeding assays, remaining leaf area (cm 2 ) and dry weight of remaining leaf material (mg) were measured to determine the amount of dry biomass consumed (mg) based on original leaf area presented of each host type. ImageJ computer imaging software (Rasband 2015) was used to measure leaf area before and after feeding (Crawford et al. 2010; Cooney et al. 2012; Salazar and Marquis 2012). First, no choice experiments were conducted to measure the amount of leaf tissue consumed over a 48-hour period of exposure to one host plant at a time. A total of 25 replicates were conducted per host plant split evenly over five dates. Leaf disks (1.75 cm in diameter) were cut using a brass cork borer (Humboldt, Elgin, IL, USA). Four disks 47

58 of one host were photographed and placed in a 9 cm petri dish (Fisher Scientific, Waltham, WA, USA) containing 9 cm filter paper (Whatman, Buckinghamshire, UK) dampened with distilled water to retain moisture. One 3 rd instar larva was weighed and placed in the center of the four leaf disks. Each petri dish was wrapped in parafilm (American Can Company, Greenwich, CT, USA) to prevent escaping larvae as well as desiccation of larvae or leaf tissue. Petri dishes were maintained in the growth chamber for 48 hours. After 48 hours, the larva was removed and weighed. Leaf disks were photographed, dried, and weighed. Larval weight change was calculated by subtracting the weight before from the weight after. Using photographs and ImageJ software, leaf area before and after consumption were calculated. After drying, the leaf material not consumed of each leaf per host plant type was weighed. These measurements were used to calculate the amount of dry biomass consumed by the individual over 48 hours. To determine preference, leaf disks (1.75 cm in diameter) of two host species were cut using a cork borer. Host comparisons included near isoline compared with sweet corn, Cry1F, green bean, cucumber, or tomato and Cry1f compared with tomato. The sample size for each combination was 25 leaf disk comparisons. Four leaf disks total were photographed and placed in a 9 cm petri dish containing 9 cm filter paper dampened with distilled water to retain moisture. Two disks of the same host type were placed diagonally across from each other. One 3 rd instar larva was placed in the center of the four leaf disks. Each petri dish was wrapped in parafilm to prevent escaping larvae, as well as desiccation of larvae or leaf tissue. Petri dishes were maintained in the growth chamber for 48 hours. After 48 hours, the larva was removed. Leaf disks were photographed, dried, and weighed. Using photographs and ImageJ software, leaf areas 48

59 before and after consumption were calculated. Total area of leaf tissue consumed was calculated. After drying, the leaf material not consumed of each leaf per host plant was weighed. These measurements were used to calculate the amount of dry biomass consumed of each host by the individual over 48 hours. Statistical Analysis For the no choice experiments, the dry weight and the leaf area of material not consumed were converted into dry biomass per cm 2. Multiplying the dry biomass per cm 2 by the area of the leaf disk before feeding produced the estimated dry biomass of the leaf disk before feeding. Lastly, by subtracting the dry biomass after feeding from the estimates dry biomass before feeding, the dry biomass consumed was calculated. This is expressed in the following equations: Both larval weight change and dry biomass consumed were analyzed using two-way ANOVAs followed by the Tukey-Kramer post hoc test in the program R 2.14 (R Development Core Team). The nominal variables were date of experiment and host plant. The measurement variables were either dry leaf weight consumed or larval weight gain/loss. For choice tests, dry weights of leaf tissue not consumed and leaf areas were used to estimate the dry biomass of leaf tissue consumed of each host over a 48-hour period using the equations provided previously. Choice data was analyzed as a proportion of one 49

60 host against the other host offered. The dry biomasses consumed were analyzed using a one-sample t-test in Excel Results In no choice experiments, the most dry biomass was consumed of near isoline followed by sweet corn (Figure 7). Cucumber and squash were consumed in intermediate amounts, and very little Cry1F, green bean, or tomato tissue was consumed (Figure 7). On average, larvae gained the most weight on non-bt corn hosts (near isoline and sweet corn) in comparison to other hosts tested (Figure 8). Individuals that consumed squash or cucumber gained less weight while those that fed on green bean neither gained nor lost weight (Figure 8). Individuals provided with Cry1F or tomato lost weight (Figure 8). For choice trials, the comparisons that were not significantly different include near isoline with sweet corn (Figure 9A), near isoline with Cry1F (Figure 9A), and Cry1F with tomato (Figure 9B). Larvae consumed significantly more near isoline when provided with a choice between near isoline and tomato or near isoline with green bean (Figure 9A). Larvae ate significantly more cucumber than near isoline (Figure 9A). Discussion Leaf tissue consumption and weight gain show similar trends (Figures 7 and 8). Larvae gained the most weight and fed most when presented with only near isoline or sweet corn. From the results here, sweet corn and near isoline remain suitable hosts. In present-day agriculture, non-bt corn provides an abundant food source that has minimal 50

61 defensive chemistry (Rosenthal and Dirzo 1997) and a large diameter stalk for larvae to bore (Ehrlich and Raven 1964). As for the intermediate hosts, when larvae were presented with green bean, they consumed almost no plant material, but maintained their weight. Potentially, if given more than 48 hours, larvae may feed more abundantly on green bean, or they may lose weight. When larvae were presented with cucumber or squash, they consumed leaf tissue and gained some weight within 48 hours. In the experiments provided here, cucumber and squash were suitable hosts for third instars, providing weight gain and a moderate consumption rate. These data also suggest that it would take a longer amount of time for larvae on cucumber, squash, or green bean to develop than those consuming non-bt corn hosts. Slower developmental rate and obtaining greater resources for development are criteria of less than ideal host plants (Timmins et al. 1988). When larvae were presented with only Cry1F or tomato, there was little to no feeding resulting in weight loss. Cry1F and tomato remain categorized as unfavorable host plants. Cry1F corn is encoded with Bt toxin that is known to greatly reduce feeding (Koziel et al. 1996). From observations, it was assumed that tomato would be a poor host due to larval avoidance observed in preliminary studies. In addition, tomato is a highly defended plant, with very few insects capable of detoxification (Boppre 1989). Neither Cry1F nor tomato is a suitable host plant for ECB. By losing weight, larval growth is regressing which will ultimately lead to death. Preference was determined as a ratio of one host against the other host option. There was no preference observed when larvae were given near isoline with sweet corn. Sweet corn and near isoline were expected to be the same because they provide similar 51

62 nutrition value and minimal plant defenses (Chapter 2; Rosenthal and Dirzo 1997). When presented with a choice between Cry1F and near isoline, there was a preference for near isoline. A preference for near isoline was expected because it has been found that ECB have a preference for near isoline when given a choice between Cry1AB and its near isoline (Davis and Coleman 1997). No preference was detected when a choice was offered between Cry1F and tomato. This was expected because they are both very poor hosts causing the larvae to feed in very small amounts, lose weight, and have poor survival (Koziel et al. 1996; Thorsteinson 1960). As anticipated, there was a preference for near isoline when it was paired with tomato; tomato is a toxic host and near isoline is a suitable host plant for ECB. There was also a preference for near isoline, as expected, when it was paired with green bean. Based on the no choice data, green bean was an intermediate host with minimal feeding, resulting in no change in weight over 48 hours. An unexpected result from the choice experiments arose when a strong preference for cucumber over near isoline was observed (Figure 9A). Based on previous experiments, cucurbits provide poor survival for ECB (Chapter 2). Yet, from the results of the consumption analysis and preference tests, cucumber appears to provide moderate consumption (Figure 7) and moderate weight gain (Figure 8), as well as a preference over near isoline (Figure 9). Since larvae did not survive to pupation in the growth chamber, greenhouse, or field, there must be another explanation for the behavior. One potential difference in these experiments pertains to the leaf tissue present. In the previous greenhouse and field experiments, whole plants were utilized, while in the no choice/choice experiments, excised leaf disks were provided. This suggests that ECB may be inducing plant defenses when feeding on whole plants that are 52

63 not present with excised leaf tissue due to cutting off the connective elements to the leaf tissue. It has been shown that the squash beetle girdles cucurbit leaves, cutting off transfer of compounds in order to reduce interaction with induced defenses (Tallamy 1985). Additionally, when ECB larvae were fed cucumber leaf tissue throughout development in the growth chamber, there was intermediate survival from 3 rd to 4 th instar for individuals fed cucumber (Chapter 2). In the present experiment, leaf tissue was removed from the plants, potentially preventing induced defenses from a fully functioning plant resulting in greater survival on plant parts than on full plants. Induced defenses of cucumber were briefly explored, however, no significant differences were found (see Appendix A). In summary, this study shows that near isoline field corn and sweet corn are the only hosts of those tested that can be classified as favorable host plants based on high consumption rates, weight gain, and high preference of the hosts evaluated here. Based on low levels of the same criteria, Cry1F and tomato are classified as unsuitable host plants. Green bean, cucumber, and squash remain intermediate hosts, however, the role where cucurbits fit in as ECB host plants requires further investigation. Generalist insects will accept many host plants and rarely exercise choice (Schoonhoven et al. 2005), which is not the case for the Z-race ECB tested in this study. Aside from the cucumber data, all of the experiments presented here further support claims of host plant specialization made in Chapter 2. The criteria for host specialization include: (1) the hosts provided should always be accepted or rejected, (2) the hosts in an expanded diet would be of low suitability, and (3) the most suitable host is easy to find (Jaenike 1990; Charnov and Stephens 1988; Futuyma 1983; Levins and MacArthur 1969; Rausher 1983). In support 53

64 of the first criterion, ECB feed more abundantly on the host plants on which they succeed best (non-bt corn hosts) and ECB preferentially select non-bt corn as a larval host plant (except for excised cucumber leaves). The second criterion is met by previous survivorship studies presented in Chapter 2. Non-Bt corn hosts were found to be the most suitable of the plant types tested, and all of the other plant type resulted in decreased survival. The third criterion above is easily met by the agricultural corn system, despite the abundance of Bt corn grown. Corn, the most suitable host for ECB, is one of the most abundant crops grown in the United States making it easy for ECB to find. It also is much more abundant than any other potential host plant in the Corn Belt (the abundant soybeans are not a preferred ECB host), where historically the worst ECB infestations have occurred (Brindley and Dicke 1963). From the data presented here, it appears that ECB is becoming a corn specialist. 54

65 Dry Biomass Consumed (mg) a b c cd d d d N SC C F GB SQ T Host Plant Date: F = 1.6, df = 4, P = 0.18; Host: F = 55.2, df = 6, P < 0.01; Date*Host: F = 1.3, df = 24, P = 0.19 (twoway ANOVA). Figure 7 Dry biomass consumed. Third instars fed one of the various hosts (N = near isoline, SC = sweet corn, C = cucumber, F = Cry1F, GB = green bean, SQ = squash, T = tomato) for 48 hours. Bars represent the estimated amount of dry biomass consumed. Different letters are significantly different (P < 0.05). Error bars show SEM 55

66 8 a Average Weight Change (mg) a b d cd bc d -4 N SC C F GB SQ T Host Plant Date: F = 7.8, df = 4, P < 0.01; Host: F = 39.5, df = 6, P < 0.01; Date*Host: F = 1.5, df = 24, P = 0.07 (twoway ANOVA). Figure 8 Third instar larval weight change over 48 hours. Third instars fed one of the various hosts (N = near isoline, SC = sweet corn, C = cucumber, F = Cry1F, GB = green bean, SQ = squash, T = tomato) for 48 hours. Bars represent either gained (+) or lost (-) weight based on host. Different letters are significantly different (P < 0.05). Error bars show SEM. 56

67 A. Dry Biomass Consumed (mg) * * * * N Other B. Dry Biomass Consumed (mg) SC F T GB C 0 F T Host Plant Compared to Near Isoline Host Plant Figure 9 Preference determined by dry biomass consumed. Third instars given a choice between 2 host plants (near isoline (N) and either sweet corn (SC), Cry1F (F), tomato (T), green bean (GB), or cucumber (C), which are presented in figure 3A, or Cry1F (F) and tomato (T), presented in figure 3B) for 48 hours. Bars represent the estimated dry biomass consumed. Significant differences within one group are distinguished with an asterisk (*). Error bars show SEM. 57

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76 Appendix A TEST FOR INDUCED DEFENSES IN CUCUMBER After reviewing the results from the no choice/choice experiments in comparison to the survivorship experiments, it was noticed that there was an inconsistency with individuals fed cucumber. No individuals were recovered and there was very minimal feeding damage on cucumber plants in any of the greenhouse trials where the insect was provided with a full, intact plant (Figure 4), however, when provided with an excised leaf disk, 3 rd instar larvae fed abundantly (Figure 7). When larvae were given a choice between near isoline and cucumber leaf disks, they fed significantly more on the cucumber than corn (Figure 9A). One difference is that greenhouse experiments used the whole plant while no choice and choice experiments used a leaf disk punched from the plant. This led to hypothesize that ECB induces chemical defenses on full cucumber plants, therefore, preventing continual feeding. Punching out a leaf disk maybe artificially trenching the plant, preventing the flow of defenses to the leaf tissue. This would allow the insect to feed without harm. To test this hypothesis, the greenhouse and choice tests were combined into one experiment. Cucumber plants were induced by infesting the plants with larvae, and a choice test was conducted between leaf disks taken from infested and non-infested cucumber plants. Hypothetically, the leaf disks from the infested plant should have induced defenses to deter feeding while the leaf disks from the non-infested plants should represent artificially trenched leaf tissue, yielding no plant defenses. 66

77 In the greenhouse, twenty cucumber plants were raised until they accumulated degree-days after seeding in the greenhouse. Fifty third instars were placed in individual empty 37 ml Solo (Lake Forest, IL, USA) plastic cups to starve overnight (approximately 18 hours). The next morning, five of the starved third instars were placed on separate leaves of 10 of the cucumber plants to prevent crowding and initiate induced defenses. The other 10 cucumber plants were left uninfested. All 20 Plants were covered with a cage created from thin mesh fabric (JoAnn Fabric, Hudson, OH, USA) and an 84 cm galvanized steel wire round tomato cages (Lowes, Hebei, China) to keep conditions consistent between treatments and keep the larvae on the plant. Plants were placed on opposite sides of the greenhouse to prevent talking, communication via volatiles when a defense is induced (Baldwin et al., 2006). Meanwhile, forty new 3 rd instars were placed in empty cups in the growth chamber and starved for three hours. After three hours, four leaves were collected from each of the plants starting with the oldest leaf and the next three in order. Two leaf disks from each leaf (1.75 cm in diameter) were cut using a cork borer (Humboldt, Elgin, IL, USA). A total of four disks were photographed and placed in a 90 mm petri dish (Fisher Scientific, Waltham, WA, USA) containing 90 mm filter paper (Whatman, Buckinghamshire, UK) dampened with distilled water to retain moisture. Two disks from the same leaf were placed diagonally across from each other. Infested plants were compared to uninfested plants using leaves of the same age. One 3-hour starved 3 rd instar larva was placed in the center of the four leaf disks. Each petri dish was wrapped in parafilm (American Can Company, Greenwich, CT, USA) to prevent escaping larvae, as well as desiccation of larvae or leaf tissue. After four hours, the arenas were opened and leaf disks were photographed. The 67

78 same larva and disks were placed back into the arena. Twenty-four and 48 hours after the start of the experiment, the arenas were opened and photographed again. After 48 hours, the individual was removed. Using photographs and ImageJ software, total area of leaf tissue consumed was calculated. The leaf disks were dried and weighed. Weight for the leaf area not consumed per host plant species was calculated to estimate the dry biomass of leaf tissue consumed by the individual. Data were analyzed as a proportion of biomass consumed of infested compared with non-infested and analyzed using a one-sample t-test in the program Excel. To analyze this data set, feeding at different time points was compared among the leaf ages. All leaves of the same age were compared to one another. Since there was no significant difference based on leaf age (P < ), we compiled all of the data into one analysis to compare feeding abundance at each time point (Figure 10). After 4 hours, there was very minimal feeding on either infested or non-infested disks (Figure 10A). After 24 hours, there was a moderate amount of feeding overall (Figure 4B). After 48 hours, there was a good amount of feeding; some of the disks were % consumed (Figure 4C). None of the experiments yielded significant results. Based on this data, it was concluded that ECB is not inducing defenses in cucumber. 68

79 A B. Dry Biomass Consumed (mg) C. Dry Biomass Consumed (mg) Dry Biomass Consumed (mg) infested non-infested infested non-infested infested non-infested Host Plant Criteria Figure 10 Cucumber induced defenses. 3 rd instars given a choice between leaf tissue from an infested or non-infested plant for 4 hours (Figure 4A), 24 hours (Figure 4B), and 48 hours (Figure 4C). Bars represent the estimated amount of dry biomass consumed. Error bars show SEM; no significance was detected (P > 0.05). 69