EVALUATION OF NATURAL PLANT CHEMICAL DEFENSES IN ...

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 2015

© 2015 Kelsey E. Fisher All Rights Reserved

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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

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 3rd 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.

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 ............................................................................8

2 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

Neonate Infestation ............................................................25 Third Instar Infestation ......................................................26

Greenhouse ....................................................................................26

Neonate Infestation ............................................................26 Third Instar Infestation ......................................................27

Field ...............................................................................................27

Discussion ..................................................................................................29 3 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

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

LIST OF FIGURES Figure 1 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

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 = 2nd instar; 132 degree-days = 4th instar). Bars within each graph with different letters are significantly different (Tukey-Kramer test, P < 0.05). Error bars represent SEM.........38

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

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 3rd 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 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 3rd instars were dissected after 567 degree-days for pupal recovery. Overall, recovery was very low; none of the experiments yielded significant results......................................................41

Figure 6 Pupal weight comparison from third instar infested plants in the

field. Average weight of pupae recovered from 3rd 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 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 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 Figure 10 Cucumber induced defenses. 3rd 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

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.

1

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 plant-

insect 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.

2

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 meta-

analysis by Ali and Agrawal (2012), the identification of generalist or specialist is still

unclear because of experimental limitations. Often researchers place insects into self-

determined 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

3

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).

4

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

5

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

6

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

7

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).

8

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

9

mating, the females lay multiple egg masses of 15-30 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 3rd

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 (5th 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

10

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

11

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

12

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 non-

compliance 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 4th instars fed much more on non-

Bt leaf tissue than on Bt leaf tissue, but the 4th 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

13

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

14

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 3rd instar ECB.

4. Determine feeding preferences based on leaf tissue consumption by 3rd instar

ECB when presented with host choices.

15

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

16

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 100

17

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.

18

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).

19

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 20-30°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.

20

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 cm2 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 cm2 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.

21

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.


For 3rd instar experiments, five individuals were placed on different leaves of the

plant to prevent crowding effects. Additional sweet corn plants were infested with 3rd

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

22

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

23

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 mid-

development (4th 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.


For 3rd instar experiments, the center plant of a grouping was infested with five 3rd

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 3rd 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.

24

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 mid-

development 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 2nd instar; 3rd to 4th instar) based on degree-day

accumulation (2nd = 106 degree-days; 4th = 132 degree-days) was analyzed using a one-

way 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.

25

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 2nd 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).

26


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

27

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.


In the 3rd 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

28

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).

29

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 3rd 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 3rd instars. Cucumber was a poor host for neonates

and an intermediate host for 3rd 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 3rd 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

30

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 3rd 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.

31

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.

32

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

33

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

34

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.

35

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.

36

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 16.30 ± 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 32.84 ± 2.26 a

Growth Chamber: Third Instar Infested, Pupal Weight c


Greenhouse: Neonate Infested, Mid-Development Weight d

Near Isoline 9.77 ± 0.63 a Sweet Corn 18.16 ± 1.27 b

Greenhouse: Neonate Infested, Pupal Weight e


Greenhouse: Third Instar Infested, Pupal Weight f


Field: Third Instar Infested, Pupal Weight g

Non-Bt Corn 80.17 ± 8.82 a Tomato 45.71 ± 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).

37

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).

0

0.2

0.4

0.6

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0 200 400 600 800 1000

Surv

ival

Pro

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lity

Degree-Day Accumulation

Near Isoline (a)

Sweet Corn (a)

Cucumber (c)

Cry1F (c)

Green Bean (b)

Tomato (c)

38

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 = 2nd instar; 132 degree-days = 4th instar). Bars within each graph with different letters are significantly different (Tukey-Kramer test, P < 0.05). Error bars represent SEM.

0

20

40

60

80

100

N SC C F GB T

Aver

age

% S

urvi

val

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Aver

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% S

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Host Plant

A.

B.

ab a

bc c

abc

bc

a ab

bc

c

c c

39

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).

0

0.2

0.4

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0 100 200 300 400 500 600 700 800

Surv

ival

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Degree-Day Accumulation

Near Isoline (a)

Sweet Corn (a)

Cucumber (b)

Cry1F (c)

Green Bean (c)

Tomato (c)

40

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 3rd 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).

.

0

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Host Plant

A.

C.

B.

a a

b b b b b

a

b

c c c c c

a a

b b b

b b

41

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 (two- way 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 3rd instars were dissected after 567 degree-days for pupal recovery. Numbers did not differ for any trial (P > 0.05).

0

0.05

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42

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).

Figure 6 Pupal weight comparison from third instar infested plants in the field.Average weight of pupae recovered from 3rd 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.

0

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Non Bt Corn Tomato

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Host Plant

43

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 3rd instar, potentially obtaining sub-lethal doses of Bt toxin as

44

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 3rd instar

ECB, and (2) determine feeding preferences based on leaf tissue consumption by 3rd

instar ECB when presented with a choice.

45

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

46

(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

47

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 20-30°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 (cm2) 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

48

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 3rd 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 3rd 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

49

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 cm2. Multiplying the dry biomass per cm2

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:

1.

2.

3.

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

50

host against the other host offered. The dry biomasses consumed were analyzed using a

one-sample t-test in Excel 2011.

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

51

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

52

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

53

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 3rd to 4th 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

54

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.

55

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 (two-

way 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

0 2 4 6 8

10 12 14 16 18

N SC C F GB SQ T

Dry

Bio

mas

s Con

sum

ed (m

g)

Host Plant

a

cd c

b

d d d

56

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 (two- way 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.

-4

-2

0

2

4

6

8

N SC C F GB SQ T

Aver

age

Wei

ght C

hang

e (m

g)

Host Plant

a

a

b bc

cd d d

57

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.

B.

0

1

2

3

4

5

6

7

8

9

F T

Dry

Bio

mas

s Con

sum

ed (m

g)

Host Plant

0

1

2

3

4

5

6

7

8

9

SC F T GB C

Dry

Bio

mas

s Con

sum

ed (m

g)

Host Plant Compared to Near Isoline

N

Other

* *

* * A.

58

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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, 3rd 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.

67

In the greenhouse, twenty cucumber plants were raised until they accumulated

1384.5 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 3rd 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 3rd 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

68

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 < 0.1354), 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 75-100% 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.

69

Figure 10 Cucumber induced defenses. 3rd 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).

Host Plant Criteria

0

0.02

0.04

0.06

0.08

0.1

0.12

infested non-infested

Dry

Bio

mas

s Con

sum

ed

(mg)

0 0.2 0.4 0.6 0.8

1 1.2 1.4 1.6 1.8


Dry

Bio

mas

s Con

sum

ed

(mg)

0

1

2

3

4

5

6

7


Dry

Bio

mas

s Con

sum

ed

(mg)

A.

B.

C.

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