University of Florida Thesis Or Dissertation

ABUNDANCE OF HELICOVERPA ZEA (LEPIDOPTERA: NOCTUIDAE) AND OTHER ECONOMIC LEPIDOPTERAN PESTS IN COTTON, SOYBEAN, AND PEANUT IN THE SOUTHEASTERN UNITED STATES

TYLER J. SHAW

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2021

To my parents for their unwavering support of my interest in entomology from a young age

ACKNOWLEDGMENTS

I would like to thank the chair, Dr. Silvana V. Paula-Moraes and members of my supervisory committee, Drs. Francis Reay-Jones, Dominic Reisig, and Philip G. Hahn, for their helpful guidance and support throughout this project. I also want to give my appreciation to the staff at the West Florida Research and Education Center for their hard work in preparing experimental plots, as well as my fellow lab members for their help in population sampling.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 6

LIST OF FIGURES ...... 7

ABSTRACT ...... 8

CHAPTER

1 INTRODUCTION ...... 10

Insect Resistance Management ...... 12 Pyramided Bt traits ...... 14 High Dose/Refuge ...... 15 Host Suitability in Mixed Agroecosystems ...... 17 Fitness Costs of Insect Resistance to Bt crops ...... 19 Overwintering Populations ...... 20 Contribution of this Research ...... 22

2 MATERIALS AND METHODS ...... 23

Comparison of Adult Emergence Trap Designs ...... 23 Abundance of H. zea, A. gemmatalis, and C. includens in Cotton, Soybean, and Peanut in the Southeastern United States ...... 25

3 RESULTS ...... 37

Comparison of Adult Emergence Traps ...... 37 Abundance of Helicoverpa zea (Lepidoptera: Noctuidae) and Other Economic Lepidopteran Pests in Cotton, Soybean, and Peanut in the Southeastern United States ...... 37

4 DISCUSSION ...... 50

LIST OF REFERENCES ...... 62

BIOGRAPHICAL SKETCH ...... 73

LIST OF TABLES

Table page

3-1 Total larval, pupal, and adult abundance of H. zea in focal fields during 2019 and 2020...... 44

3-2 Two-way ANOVA for H. zea populations in field crops cultivated in southeastern U.S...... 45

3-3 Two-way ANOVA for damaged cotton squares and damaged cotton bolls in different crops cultivated in southeastern U.S...... 45

LIST OF FIGURES

Figure page

2-1 Pyramid cage with capture jar and 3D-printed funnel at top. Jay, Florida ...... 32

2-2 Emergence tent and pyramid cage set in fallow field. Jay, Florida...... 32

2-3 Capture jar with 3D-printed funnel connected to the emergence tent. Jay, Florida...... 33

2-4 3D-printed funnel in capture jar with specimens of Helicoverpa zea trapped in the container. Jay, Florida ...... 33

2-5 Pupal digging method in fallow field. Jay, Florida...... 34

2-6 Opening of H. zea pupal chamber in soil. Jay, Florida ...... 34

2-7 H. zea pupa within pupal chamber in soil. Jay, Florida...... 35

2-8 H. zea pupa in diapause with presence of three eye spots (circled)...... 35

2-9 H. zea pupa without presence of three eye spots...... 36

2-10 Globules of fat body as indicated by arrows in H. zea pupa...... 36

3-1 Average number of H. zea adults caught in five pyramid cages compared to the average number of H. zea adults caught in one emergence tent ...... 43

3-2 Relative larval abundance of Helicoverpa zea, Anticarsia gemmatalis, and Chrysodeixis includens from July through October...... 46

3-3 Average number of Helicoverpa zea larvae recorded per every 9.1 meters of crop rows per plot during field sampling in Florida...... 47

3-4 Average number of Helicoverpa zea larvae recorded per every 9.1 meters of crop row per plot during field sampling in North Carolina...... 48

3-5 Average number of Helicoverpa zea larvae recorded per every 9.1 meters of crop rows per plot during field sampling in South Carolina...... 49

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

ABUNDANCE OF HELICOVERPA ZEA (LEPIDOPTERA: NOCTUIDAE) AND OTHER ECONOMIC LEPIDOPTERAN PESTS IN COTTON, SOYBEAN, AND PEANUT IN THE SOUTHEASTERN UNITED STATES

Tyler J. Shaw

May 2021

Chair: Silvana V. Paula-Moraes Major: Entomology and Nematology

Transgenic Bt crops expressing insecticidal toxins from Bacillus thuringiensis (Bt) are valuable tools for the management of economic pests, including the bollworm,

Helicoverpa zea (Lepidoptera: Noctuidae). However, resistance to Bt cotton has been documented in this species since their introduction to U.S. agriculture in 1996.

Understanding the occurrence and relative contribution to populations of this species by different crops of the southeastern United States across growing seasons is critical when designing insect resistance management (IRM) programs. The objective of this study was to measure the larval, pupal, and adult abundance of H. zea in soybean, non-

Bt cotton, Bt cotton, and peanut across the 2019 and 2020 growing seasons in Florida,

South Carolina, and North Carolina. Overwintering populations of H. zea were estimated using two emergence trap designs: the emergence tent and the pyramid cage. The capture rates of adults between these two trap types were compared, resulting in higher capture rates by the pyramid cage. The estimation of the larval populations in different crops indicated that there was no significant difference in H. zea early and late instar abundance between non-Bt cotton and Bt cotton. However, there

were more middle instar larvae in non-Bt cotton compared to Bt cotton. There were low to non-existent pupal and adult populations after the 2019 and 2020 growing seasons in all states. In addition to H. zea, Anticarsia gemmatalis (Lepidoptera: Erebidae) and

Chrysodeixis includens (Lepidoptera: Noctuidae) larvae were detected on soybean in

2019 and 2020 in all three states.

CHAPTER 1 INTRODUCTION

Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), commonly referred to as the corn earworm, tomato fruitworm, or bollworm (New World), is a polyphagous pest with a wide array of host plants, including corn (Zea mays) and sorghum (Sorghum spp.), which are the preferred hosts of this species (Fitt 1989, Gould et al. 2002, Jackson et al.

2008, Head et al. 2010, Cunningham and Zalucki 2014). This pest species impacts agroecosystems across North America, but only overwinters south of the 40th parallel north (Hardwick 1965). The number of generations per year varies with degree days, with four generations in North Carolina (Neunzig 1969) and up to seven generations in southern Florida (Capinera 2001, Quaintance and Brues 1905). The development time for each generation is approximately 30 days (Bishopp and Jones 1907). Helicoverpa zea also has a high capacity to disperse, with bidirectional migrations from south to north and vice versa in the United States, creating mixed populations of immigrant and overwintering populations across the country (Farrow and Daly 1987, Hendrix et al.

1987, Westbrook and López 2010, EPA 2018).

This species was traditionally managed in cotton using a variety of insecticides, in addition to the entomopathogenic bacterium, Bacillus thuringiensis (Bt) as a biopesticide (Kumar et al. 2008). Bacillus thuringiensis produces several insecticidal proteins, including cytolytic crystal proteins (Cry) and vegetative insecticidal proteins

(Vip) (Kumar et al. 2008) each with different target sites (Jurat-Fuentes et al. 2021). Cry and Vip toxins also have the advantage of being highly targeted against lepidopteran pests, while having little to no effect on beneficial insects and no effect on vertebrate physiology (Mendelsohn et al. 2003, Comas et al, 2013, Tabashnik et al. 2017). Despite

these advantages, the efficacy of foliar-applied Bt insecticides is limited due to their inability to contact or be consumed by H. zea, which feeds inside crop tissues, before they degrade (Kumar et al. 2008). However, in 1996, transgenic technology made it possible for corn and cotton plants to express Bt toxins as plant incorporated protectants (PIPs), allowing for more targeted control of insect pests while also decreasing insecticide applications (EPA 2010). The highly targeted nature of Bt technology consequently poses decreased off-target effects such as the suppression of beneficial arthropods typically imposed by insecticides (Hellmich et al. 2008, Hutchison et al. 2010, Romeis et al. 2019). These qualities of Bt technology have contributed to the widescale adoption of Bt crops, with their use increasing from 8% of total corn grown in the U.S. in 1997 to 82% in 2020 and from 15% to 88% of cotton (Gossypium hirsutum) grown in the U.S. within that same time (USDA ERS 2020). However, the risks of resistance in species targeted by Bt technology due to selection pressure have been increasingly documented in the literature (Caprio et al. 1992, Yang et al. 2014,

2021; Brévault et al. 2015, Dively et al. 2016, Tabashnik et al. 2017, Niu et al. 2021).

Since the introduction of Bt crops in the U.S., field-evolved practical resistance against Bt toxins has occurred for some target pests, including H. zea (Gould 1998,

Huang et al. 2011. Tabashnik et al. 2017), with a decrease in susceptibility to Cry1 and

Cry2 Bt toxins (Burd et al. 2003, Luttrell and Ali 2007, Welch et al. 2015. Reisig et al.

2018). There is also an increasing concern for the development of resistance to the more recently introduced Vip toxins, with Vip-resistant alleles reported in field populations of H. zea in Texas (Yang et al. 2020). Field-evolved resistance has been defined as the reduction in the susceptibility of a pest population to a pesticide due to

the repeated exposure of a pest population to that pesticide (Tabashnik et al. 2014).

Practical resistance has been defined as field-evolved resistance which significantly reduces the efficacy of a pesticide in a manner which impacts pest control (Tabashnik et al. 2014). A pest population is considered to have developed practical resistance if over fifty percent of the population consists of resistant individuals, resulting in reduced efficacy of the pesticide (Tabashnik et al. 2014). The development of Bt resistance can lead to control failures and an increase in the use of chemical insecticides (Tabashnik et al. 2017, EPA 2018). Thus, the development of practical resistance to Bt toxins by H. zea is a growing economic concern in the U.S., particularly in cotton where the insect is a major economic pest (Tabashnik et al. 2017, EPA 2018, Reisig and Kurtz 2018).

However, resistance levels in insect pests vary widely across different species and across different landscapes. Some of the factors which contribute to the differing rates of Bt resistance evolution include the differences in host suitability across crops and the predominate native alternative host plants in each region (Gore et al. 2003, Gustafson et al. 2006, Rabelo et al. 2020). The spatial and temporal relationships among crops and native plants in the landscape may also play a role in differential rates of Bt resistance (Storer et al. 2003, Tabashnik et al. 2017). Thus, understanding the contribution of different crops to Bt-targeted pest populations can help to refine predictions of resistance evolution as well as how and why these predictions of resistance evolution vary from region to region and across species (Storer et al. 2003,

Tabashnik et al. 2017).

Insect Resistance Management

In response to the risks imposed on agriculture by field-evolved practical resistance of target pests to Bt crops, several insect resistance management (IRM)

strategies, which aim to reduce the selection for resistance, have been enforced by the

U.S. Environmental Protection Agency (EPA) since the early years of Bt technology adoption (EPA 1998, EPA 2018, Tabashnik et al. 2017). The proposition of IRM programs is based on the assumptions of what is considered a high dose of Bt toxins, the recessive inheritance of resistance, and the occurrence of random mating by targeted pests (Gould 1998, Tabashnik et al. 2013, Brévault et al. 2015, EPA 2018). If mating is random and resistance is recessive, reproduction between Bt-resistant individuals and susceptible individuals will result in the production of heterozygotes which exhibit reduced resistance compared to homozygous resistant individuals, thus stemming the evolution of resistance and avoiding or delaying the risk of practical resistance to Bt crops (Gould 1998, Tabashnik et al. 2013, Brévault et al. 2015). The high dose concept is based on the assumption that Bt crops should provide a high enough dose to kill 95% of individuals heterozygous for a recessive resistance allele and 99.99% of susceptible individuals to reduce the risk of field-evolved practical resistance (Gould 1998). One such IRM strategy built upon these assumptions is termed the high dose/refuge strategy which involves the implementation of high dose Bt crops and the cultivation of non-Bt refuges in the landscape (Gould 1998, EPA 1998,

Bates et al. 2005). However, regulations for these strategies vary across regions for several reasons, including the availability of natural refuges in the area as well as the temporal and spatial co-occurrence of various host crops of the target species (Storer et al. 2003, Head et al. 2010, Carrière et al., 2017, Li et al. 2017, Tabashnik et al. 2017,

EPA, 2018). Thus, these factors and how they relate to the population dynamics of

pests targeted by Bt technology, such as H. zea within heterogenous landscapes should be considered when developing IRM programs.

Pyramided Bt traits

Pyramiding involves the expression of multiple Bt toxins by transgenic crops

(EPA 2018). When Bt crops were first introduced to commercial agriculture in the U.S., there was only one toxin expressed in Bt corn and Bt cotton (EPA 2010). These early Bt crops expressed Cry1Ab in corn and Cry1Ac in cotton, both of which target certain lepidopteran pests (Tabashnik et al. 2013, EPA 2018). However, target pests can more readily overcome the action of a single toxin, compared to multiple Bt toxins, contributing to a higher rate of resistance development in target pest populations relative to pyramided Bt crops (Burd et al. 2003, Luttrell and Ali 2007, Welch et al. 2015,

EPA 2018). Currently, Bt corn expresses combinations of Cry1A.105, Cry2Ab2, Cry1F,

Cry1Ab, and/or Vip3Aa20 while Bt cotton expresses combinations of Cry1Ac, Cry2Ab2,

Cry1Ab, Cry2Ae, Cry1F, or Vip3Aa19 (Dively et al. 2016, EPA 2018). This diversification of toxins with dissimilar domains makes it more difficult for resistance to evolve against any one mode of action (Carrière et al. 2016, Jurat-Fuentes et al. 2021).

However, resistance against certain toxins remains an issue due to the cross-resistance between groups of toxins in some target pests (Anilkumar et al. 2008b, Caccia et al.

2012, Welch et al. 2015, Carrière et al. 2016), which are similar across cotton and corn

(Dively et al. 2016, EPA 2018). Furthermore, the development of resistance in target pests to any one toxin diminishes the redundancy of pyramiding, increasing selection pressure on remaining pyramided toxins (Carrière et al. 2016, Tabashnik et al. 2018).

This is especially true since corn is planted earlier in the season compared to cotton,

posing continuous selective pressure on H. zea populations throughout the year (Gould et al. 2002, Head et al. 2010, EPA 2018).

High Dose/Refuge

To reduce the risk of resistance development, the high dose/refuge strategy is implemented in IRM programs (EPA 1998). This strategy involves the employment of non-Bt refuges co-occurring with crops which produce a high dose of Bt PIPs

(Tabashnik et al. 2017, EPA 1998). While Cry1 and Cry2 remain moderately effective against H. zea, the more recently introduced traits expressing Vip3A, are more effective but still fall below the definition of a high dose (Burd et al. 2003; Luttrell and Ali 2007,

Welch et al. 2015, EPA 1998). In addition to the use of high dose PIPs, non-Bt refuges must be simultaneously available in the landscape (EPA 1998). The objective of non-Bt refuges is to produce unselected pest populations in the landscape which are expected to mate with resistant populations, reducing the selection pressure imposed on target pest populations by the Bt crop by promoting the prevalence of susceptible alleles in the pest population targeted by Bt (EPA 1998, Tabashnik et al. 2008, EPA 2018).

Different approaches of refuges have been proposed and utilized since the introduction of Bt crops, including natural, structured, and blended refuge (EPA 1998,

Tabashnik et al. 2007, EPA 2018). Natural refuge is based on the existence of alternative plant hosts, such as weedy plants or non-Bt crops (Stadelbacher 1981,

Gustafson et al. 2006, Fitt 1989, Li et al. 2017, EPA 1998). Structured refuges of non-Bt crops are planted in agricultural fields as separate plots near stands of Bt crops, as field borders, or as within-field strips or blocks (EPA 1998, EPA 2018). An alternative refuge approach is the random dissemination of non-Bt crops within Bt crop stands, by mixing

Bt and non-Bt crop seed in a determined ratio before planting (Crespo et al. 2015,

Onstad et al. 2017, EPA 2018). This predetermined mixture of Bt/non-Bt seeds is often referred to as “seed blends” or “refuge in the bag” (RIB) (EPA 2018) and has been considered as a method for ensuring compliance of refuge-planting among farmers

(Reisig 2017). Currently, to reduce selection pressure on lepidopteran pests, there is a requirement of 20% refuge for pyramided corn, and 50% refuge for single-toxin corn in cotton-growing regions (EPA 2018). In the southeastern U.S., no refuge is required for

Bt cotton due to the availability of natural refuge (Gould et al. 2002, Head et al. 2010,

EPA 2018). Each refuge approach and the ratios used in relation to Bt crop stands have implications on the rate of evolution of field-evolved resistance (Yang et al. 2014,

EPA 2018, Onstad et al., 2017).

One such implication is the increased cross-pollination in RIB plots (Onstad et al.

2017), which creates a mosaic of varying expression doses of Bt toxins throughout cross-pollinated kernels, providing a low dose in some kernels and, thereby, contributing to the selection of resistant populations of target pests (Yang et al., 2014,

Brévault et al. 2015, Crespo et al. 2015, Caprio et al. 2015). The dispersal capacity of

H. zea larvae, as well as their ability to detect and avoid areas of high Bt concentrations, also contributes to reduced toxin exposure (Gore et al. 2005; Braswell et al. 2019b).

These factors may result in larvae moving to assumed areas of lower Bt concentration in the lower canopy (Gore et al. 2002), although largely unexplored factors, such as nutrients, likely interact with Bt concentration in plant tissues to influence larval movement (Braswell et al. 2019a, Braswell et al. 2019c). Modeling has shown a similar rate of Bt resistance evolution for H. zea between blended and structured refuges of corn (Pan et al. 2016) or when model parameters are altered, more rapid Bt resistance

evolution for H. zea in blended compared to structured refuges of corn (Caprio et al.

2019). These models consider varying expression levels of PIPs across non-Bt corn kernels that have been cross-pollinated by Bt crops (Yang et al. 2014, Brévault et al.

2015, Crespo et al. 2015). However, there are many factors that may contribute to differences in the effects of cross-pollination across regions such as weather and synchrony of pest flight with host development (Onstad et al. 2017). These models remain largely untested in the field for H. zea.

Cross-resistance, as well as differences in toxin expression levels throughout the growing season, can also influence efficacy (Welch et al. 2015, Carrière et al. 2016,

2017). Due to similarities between Cry1 toxins expressed in corn and cotton (Welch et al. 2015, Carrière et al. 2016), larvae feeding on Bt corn available earlier in the season may produce resistant offspring that may then feed on late season crops such as cotton

(Gould et al. 2002, Jackson et al. 2008, Head et al. 2010, Carriere et al, 2017).

Furthermore, Cry1Ac and Cry1F levels were each shown to diminish throughout the year in Bt cotton (Carrier et al. 2017). A reduction of dose in Bt cotton throughout the growing season could, therefore, allow Bt cotton to act as a selective filter for overwintering populations which feed on early season plants such as corn and sorghum

(Gould et al. 2002, Head et al. 2010, Carriere et al. 2017). This hypothesis is supported by findings which show that H. zea survival from Cry1Ac exposure is influenced by the relative abundance and interaction of corn, cotton, and soybean in the landscape

(Arends et al. submitted 2021a).

Host Suitability in Mixed Agroecosystems

One key aspect to be considered in natural refuge recommendations is how suitable an alternative host is to H. zea. Different crops contribute differing population

densities of H. zea to the landscape, which influences the occurrence of susceptible alleles in H. zea populations and the efficacy of alternative hosts to serve as refugia

(Gore et al. 2003, Liu et al, 2004, Jackson et al. 2008). The landscape of field crops in the southeastern U.S. is heterogeneous and dominated by Bt cotton, Bt corn, soybean, and peanut, with each crop contributing to pest densities in differing ways (Storer et al.

2003, Jackson et al. 2008). While H. zea is a polyphagous pest, they predominately feed on host plants that have a C4 carbon cycle, such as corn (DeNiro and Epstein,

1978), which contributes more individuals to the landscape early in the crop season

(Gould et al. 2002, Head et al. 2010). Second generation adults, after developing on corn and sorghum, are the first to oviposit on C3 plants such as cotton, soybean, and peanut in the southern United States (Gould et al. 2002, Jackson et al. 2008, Head et al. 2010). Due to the temporal separation of corn and cotton created by differences in planting date, the population dynamics of H. zea across these crops throughout the growing season may contribute to the evolution of resistance. For instance, Bt corn may serve as a selective filter for a population of resistant individuals early in the season, especially when compliance by farmers to plant the required refuge is low (Reisig 2017).

This filtering effect may be exacerbated due to the expression of similar Cry1A toxins by both corn and cotton (Welch et al. 2015, Carrière et al. 2016). Bt corn damage has also been shown to be dependent on both Bt resistance in H. zea and the abundance of corn in the landscape (Arends et al. submitted 2021b). Selective filtering by corn, which is influenced by these factors, would lead to more resistant individuals which are able to feed on Bt cotton later in the season (Head et al. 2010), although the influence of the cropping system configuration is complex and highly dependent on the abundance of

non-Bt hosts in the environment such as soybean (Arends et al. submitted 2021a).

Therefore, the implementation of refuges in preferred early season hosts such as corn may heavily influence the contribution of resistant H. zea populations by cotton later in the growing season. Similarly, natural refuge, primarily assumed to be soybean, may influence the contribution of resistant individuals that overwinter and infest corn early in the season the following year (Gould et al. 2002, Head et al. 2010). Cotton, however, has been demonstrated to be a less suitable host for H. zea when compared to other non-Bt crops, such as soybean (Gore et al. 2003, Jackson et al., 2008) and is generally not a significant contributor of H. zea populations in the landscape (Jackson et al. 2008,

Gustafson et al. 2006). However, non-Bt crops such as soybean, which overlap temporally with cotton, have been documented as an effective refuge, by contributing to unselected H. zea populations at the same time as Bt cotton, promoting mixed mating of populations from both crop sources (Gould et al. 2002, Gore et al. 2003, Gustafson et al. 2006, Head et al. 2010, Arends et al. submitted 2021a). Furthermore, non-Bt crops, such as peanut and soybean, may also contribute to differences in life history traits across resistant and susceptible populations of H. zea (Gustafson et al. 2006, Jackson et al. 2008, Rabelo et al. 2020), which may also influence rates of resistance evolution.

Thus, documentation of the contribution to H. zea populations from each of these crops will influence our understanding of the population dynamics of H. zea in landscapes of the southeastern U.S., which has implications for the selection of Bt resistance in H. zea populations across the region.

Fitness Costs of Insect Resistance to Bt crops

The resistance to Bt toxins is also influenced by fitness costs across various host plants (Storer et al. 2001, Gore et al. 2003, Gustafson et al. 2006, Wu et al. 2006,

Anilkumar et al. 2008a, Jackson et al 2008, Ives et al., 2011, Reisig and Reay-Jones

2015, Bilbo et al. 2018). Fitness costs in Bt resistant populations of H. zea have been shown to decrease pest survival, development time, and mass (Gassmann et al. 2009).

In addition, pupal weight has been shown to be correlated with Bt resistance (Reay-

Jones et al. 2020), which may also decrease flight capacity as illustrated with the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) (Wu et al.

2006), consequently reducing the ability of adults to disperse and result in low gene flow

(Ives et al. 2011). This reduction in gene flow could increase the occurrence of resistance (Caprio et al. 1992, Ives et al. 2011, Rabelo et al. 2020). Furthermore, previous modeling using Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) has illustrated that sublethal effects by a high concentration of Bt would result in decreased fitness, and thus, decrease the rate of resistance selection (Tabashnik et al. 2004).

Thus, better understanding the occurrence of sublethal effects on targeted pests to Bt crops and their influence on fitness costs refine our understanding of H. zea fitness and population dynamics in the landscape.

Overwintering Populations

While understanding H. zea population ecology during the crop season is important in documenting the population dynamics within a year, it is also important to understand how each crop contributes to the population which emerges the following year after overwintering. This is especially true in late season crops such as cotton, which may act as a selective filter for resistant populations of H. zea to feed on early season plants, such as corn after overwintering (Gould et al. 2002, Jackson et al. 2008,

Head et al. 2010). Induction of diapause in H. zea is correlated with declining temperatures and food availability (Fitt 1989) and has been documented in the southern

United States in areas below the 40th parallel north (Hardwick 1965). These overwintering populations can then migrate to northerly latitudes (Farrow and Daly

1987, Westbrook and López 2010). Thus, the landscape of the southeastern United

States is important in contributing to resistance alleles in H. zea populations in the northern U.S. and Canada.

However, documenting the overwintering survival and emergence of ground- pupating lepidopteran species presents the challenge of recovering emerging adults

(Murray and Wilson 1991, Yang et al. 2014). As such, studies which involve overwintering populations of lepidopterans, such as H. zea, often rely on emergence traps for adult collection (Shiller 1946, Caron et al. 1978, Murray and Wilson 1991).

These traps are deployed in crop fields where H. zea populations are likely to occur.

They are placed over the soil where larvae are expected to have burrowed to pupate and adults will emerge from their pupal chambers (Barber and Dicke 1937). A traditional trap design in the U.S. is known as the pyramid cage (Shiller 1946). However, an alternative approach is adopted in Australia, and is referred to as the “emergence tent.”

Both emergence traps are similar in their overall design and use of sloping walls, which channel the emerging adults through a funnel and into a collection jar, with the funnel serving as a barrier that prevents the adult from moving back out of the jar. The adults are then able to be easily collected for analysis. However, these two traps differ in their construction, coverage area, and overall shape, possibly resulting in differences in capture rates as well as differences in the microclimate they create (Murray and Wilson

1991). Furthermore, individual needs should be considered when choosing the appropriate trap for overwintering survival studies. For example, the pyramid cages are

made of supplies which are cheaper and are easier to build. However, the emergence tent is practical since it can be folded for efficient storage. Also, while the pyramid cage can be easily deployed, the emergence tents require approximately 5-10 minutes to set up. Furthermore, the structural integrity of both trap types should be considered in areas such as the southeastern U.S. where inclement weather, such as hurricanes, pose a potential hazard for trap integrity.

Contribution of this Research

The reports of field-evolved resistance of H. zea to Bt cry toxins expressed in both cotton and corn is a growing threat to the long-term efficacy of Bt technology.

Natural refuge has been adopted in the southern U.S. since 2007 for Bt cotton (EPA

2007), based on studies documenting a large number of cultivated and weed species exploited by the polyphagous behavior of H. zea. Improving the knowledge of H. zea ecology represents an important contribution to IRM recommendations, including overwintering survival and alternative host plant use in the heterogeneous landscape of the southeastern U.S.

The objectives of this study were to:

1. Assess the capture rates between the emergence tent and pyramid cage designs used to document noctuid adult emergence.

2. Describe the abundance of H. zea in Bt and non-Bt cotton, soybean, and peanut in the landscape of the southeastern United States.

CHAPTER 2 MATERIALS AND METHODS

Comparison of Adult Emergence Trap Designs

Comparative rates of capture of H. zea adults between the pyramid cage (Shiller

1946) (Figure 2-1) and emergence tent (Marshall et al. 1996, Duffield and Dillon 2005)

(Figure 2-2) were performed during the 2019 growing season at the University of

Florida, West Florida Research and Education Center, Jay, Florida.

Pyramid cages (Figure 2-1) were built using 2x2 lumber and 1/8” hardware cloth

(YardGard, North Plains, OR) with dimensions of 0.91 m x 1.22 m, covering a total area of 1.11 square meters/cage. Emergence tents (Figure 2-2) were built using 50% white shade cloth (Duffield and Dillon, 2005) (ShadeCloth Store, Mundelein, IL) with dimensions of 3.05 m x 1.83 m to cover a total area of 5.58 sq. meters/tent. Yellow plastic funnels were 3D-printed (FDACS, Gainesville, Florida) and attached to both the emergence tents and the pyramid cages to catch adults emerging from the covered area (Figures 2-1, 2-2, 2-3, 2-4). The funnel had 0.3 cm holes to allow light penetration and a single 1.5 cm hole to allow the adults to move into the capture jar, while reducing the likelihood of the adult moving back out of the capture jar and into the traps (Figure

2-4).

Three experiments comparing H. zea adult capture in emergence tents and pyramid cages were performed in corn plots during the 2019 crop season. The experimental areas were treated with Amdro insecticidal fire ant bait (Ambrands ©,

Atlanta, GA) to prevent predation by Solenopsis invicta (Buren) (Hymenoptera:

Formicidae) (Baldwin et al. 2020).

The first experiment was arranged in a CRD (Complete Randomized Design) with four replicates of non-Bt corn plots with eight rows of 4.5 m x 15.24 m long. A non-

Bt corn hybrid (P1319R) (Corteva©) was cultivated following the agronomic recommendations of the region (Wright et al. 2018b). Artificial infestation was performed with neonates from a laboratory-reared colony (Benzon Company©, Carlisle, PA) during the corn silking stage. Neonates were placed on the corn silk to allow larvae to then infest the corn ear. Visual inspection of each of the corn ears in the plot took place one week after late instars were observed to ensure that larvae began pupation. The corn stalks were manually cut down and removed from the plot. After cutting and removing the corn stalks, one emergence tent per plot was randomly placed facing different orientations (N, S, E, W), either in parallel or perpendicular to crop rows, in each of the four plots. Five pyramid cages were also randomly placed in the middle four rows of each plot. There was a ratio of five pyramid cages to each emergence tent, due to the coverage of one emergence tent (5.58 m2) equaling the coverage area of approximately five pyramid cages (1.11 m2/cage). This ratio of one emergence tent to five pyramid cages was implemented in all three of the adult trapping comparison experiments. The total number of emerged adults captured in the five pyramid cages and one emergence tent were recorded for each sampling date which occurred daily for approximately three weeks.

The second experiment in Florida was also arranged in a CRD with four replications of the same Bt corn hybrid. The plots were 4.5 m x 15.24 m long and were planted in the middle of April and had a natural infestation of H. zea. The infestation of four plots of Bt corn during the dent stage was assessed, to ensure that late instar

larvae observed one week prior in the plot had moved to the ground to pupate. Once no larvae were detected, tents were placed randomly facing either East or West, parallel with the crop rows, with each pyramid cage placed randomly in the plot. The total number of emerged adults captured in the five pyramid cages and one emergence tent were recorded for each sampling date which occurred daily for approximately three weeks.

The third experiment in Florida was performed to document the comparative rates of capture between the two traps using a known number of pupae placed under the tent and cages. One replication was used due to the limited availability of traps. Five pyramid cages and one emergence tent were placed randomly in a fallow field where no crops were previously planted. A total of 50 H. zea pupae from a field population- derived colony were placed in five separate containers (10 pupae/container), filled with vermiculite, and placed randomly under the tent. A total of 10 pupae were placed in a container filled with vermiculite and randomly placed under each pyramid cage. The total number of emerged adults captured in the five pyramid cages and one emergence tent were recorded for each sampling date which occurred daily for approximately two weeks.

Data analysis. Paired t-tests were used to compare the number of adults caught between the two trapping methods for each of the three experiments (RStudio, Version

Abundance of H. zea, A. gemmatalis, and C. includens in Cotton, Soybean, and Peanut in the Southeastern United States

The quantification and population dynamics of H. zea (Boddie) (Lepidoptera:

Noctuidae) in Florida, North Carolina, and South Carolina, was conducted by sampling

the larval, pupal, and adult abundance in in Bt cotton (Cry1Ac + Cry1F) (PHY312WRF)

(Corteva©), non-Bt cotton (DP1822XF) (BAYER©), and soybean (AG52X9) plots over two growing seasons (2019 and 2020). In addition to Bt cotton, non-Bt cotton, and soybean, experimental plots of peanut (Georgia 06G) were also evaluated in Florida.

Larvae, pupae, and adults of H. zea were sampled in four locations across three states.

The study was performed in two locations in North Carolina: Rocky Mount, North

Carolina, and North Carolina State University, Vernon James Research & Extension

Center in Plymouth, North Carolina. In South Carolina the study was performed at the

Pee Dee Research and Education Center, Clemson University, Florence, South

Carolina. In Florida, the study was performed at the West Florida Research and

Education Center, University of Florida, Jay, Florida.

Both locations in North Carolina had one field of each crop, equaling six fields. In

Plymouth, North Carolina, the cotton experimental fields were 65.84 m x 144.78 m, and the soybean fields were 65.84 m x 365.76 m. In Rocky Mount, North Carolina, each field was 45.72 m x 91.44 m. Both cotton and soybean had a row spacing of 0.91 m at each

North Carolina location. In Plymouth and Rocky Mount, North Carolina, cotton and soybean were planted on June 19th, 2019 and June 3rd, 2020. South Carolina had two blocks of each crop in a completely randomized design. Cotton fields had 0.97 m row spacing and soybean fields had 0.76 m row spacing. One Bt cotton field had the dimensions of 28.04 m x 103.63 m, while the other Bt cotton fields were 29.26 m x

96.93 m. Non-Bt cotton fields equaled 28.04 m x 92.35 m and 120.70 m x 97.54 m.

Soybean fields in South Carolina were 27.43 m x 97.54 m and 36.58 m x 92.35 m. For the 2019 season in South Carolina, cotton was planted May 21st and soybean was

planted May 22nd. For the 2020 growing season in South Carolina, cotton was planted

June 9th, and soybean was planted June 4th. Each crop in Florida (Bt cotton, non-Bt cotton, soybean, and peanut) was planted in plots of eight rows with dimensions of 7.32 m x 15.24 m. All sixteen plots were planted 7.32 m apart with 4.57 m borders. In Florida, the experiment was planted May 22nd, for the 2019 season and May 25th for the 2020 season. Each of the experimental plots, excluding planting date in each location was cultivated following the agronomic recommendations from the respective region

(Dunphy and Roberson, 2017; Wright et al., 2017, 2018a, 2018c; Jones et al. 2019;

Edmisten et al., 2020). In addition, no insecticides targeting lepidopterans other than seed treatments were applied to any of the experimental plots in all locations. In addition, Orthene (The Scotts Company LLC, Marysville OH) was applied in North

Carolina plots and Amdro insecticidal fire ant bait (Ambrands ©, Atlanta, GA) applied in

Florida plots to encourage H. zea larval survival and decrease predation by fire ants, respectively.

The quantification of H. zea and other lepidopteran species was performed by larval sampling once the flowering stage began (reproductive stage). Sampling took place approximately every other week from August 1st, 2019 to September 11th, 2019 in

Rocky Mount, and August 2nd, 2019 to September 13th, 2019 in Plymouth for a total of four sampling dates in Rocky Mount and Plymouth, North Carolina for the 2019 growing season. For 2020, in Rocky Mount, North Carolina, sampling took place every two weeks from August 1st, 2020 to September 8th, 2020 for a total of four sampling dates.

For 2020 in Plymouth, North Carolina, sampling took place every two weeks from

August 6th, 2020 to September 16th, 2020 for a total of four sampling dates. For South

Carolina in 2019, sampling took place every two weeks from July 23rd to September

24th, for a total of five sampling dates for both replicates. For South Carolina in 2020, sampling took place every week from August 5th to October 7th, for a total of eight sampling dates for each replicate. In Florida in 2019, sampling took place every two weeks from July 26th to September 20th, for a total of five sampling dates. In Florida in

2020, sampling took place every two weeks from July 27th to October 5th for a total of six sampling dates. The species of concern in this sampling was H. zea, however, the abundance of Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae) and Anticarsia gemmatalis (Hübner) (Lepidoptera: Erebidae) were also recorded. The larval sampling technique differed depending on the crop. In cotton, 100 bolls and 100 squares were randomly selected and visually inspected in each plot. The number of damaged squares was not recorded in South Carolina. The number of live larvae, squares with injury, and bolls with injury were recorded. Damaged squares and bolls were assumed to be H. zea damage due to the feeding patterns by H. zea, which is characterized by a round hole in the cotton boll, as well as the high lethality of pyramided Bt toxins in cotton towards pests such as H. virescens, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) and

Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae), eliminating the possibility of pest damage caused by these pests (Reisig et al. 2018). In peanut and soybean, larval sampling was performed by randomly placing a drop cloth 0.91 square meters in area underneath the plants in between rows within the center of each plot. Plants on both sides of the drop cloth were shaken vigorously with five samples per plot. Each of the five beat cloth samples per plot were pooled prior to data analysis to give a larval density for every 9.1 meters per plot. For each sample, the instars were categorized

into: 1) early instars, which included instars one to two; 2) middle instars, which included instars three to four; and 3) late instars, which included instars five or more. The total number of larvae per species and instar was recorded. The instar categories were not recorded in South Carolina.

The quantification of H. zea pupae in each crop and occurrence of diapause was documented in the experimental plots after harvest, during the fallow season (winter

2020 and winter 2021) in February. In Florida and North Carolina, pupal excavation

(Figure 2-5) was performed by removing the ground vegetation and carefully scraping away the top layer of soil with a garden hoe and shovel to reveal the opening of the diapause chambers. In Florida two sections of soil with an area of 2.78 square meters in a total of 5.56 square meters per plot were excavated. The pupal chambers were easily identified by their round shape, which was typically about 1.5 cm in diameter (Figure 2-

6). In Florida, when pupal chambers were revealed, a square section of soil with dimensions of approximately 15.24 cm x 15.24 cm and 15.24 cm deep was removed to expose the pupae. In North Carolina, an area of approximately 86.86 square meters per plot was excavated. Soil from this area was placed onto a shaker table to sift the soil for pupae or the soil was turned over approximately 30 cm deep to expose pupae. In South

Carolina, a plough was used to overturn soil to expose pupae in an area of 91.44 m x

12.19 m totaling 1,113.74 square meters per plot. Once pupae were recovered, they were placed in plastic cups and taken to the laboratory to record weight, sex, diapause condition and stage. Sex was determined following the description of Hardwick (1965) while diapause stage was determined following Murray and Wilson (1991) and Phillips and Newsom (1966). Pupae were determined to be in diapause by the presence of

three eyes spots (Figure 2-8), which disappear in later stages of development (Figure 2-

9), as well as the presence of fat body (Figure 2-10). Condition of pupae such as parasitism, predation, damage, and disease were also assessed and recorded.

Emergence of H. zea adults in the experimental plots during and after the crop season was recorded by placing pyramid cages and emergence tents in the experimental fields in North Carolina and South Carolina, while the Florida location only used emergence tents. In all locations, emergence traps were placed approximately one week after the final larval sampling date for that state. One emergence tent and four pyramid cages, totaling 18 emergence tents and 72 pyramid cages, were randomly placed in each experimental plot in North Carolina, but at least 10 meters from the field edge. In South Carolina, four emergence tents were randomly placed in each cotton plot and two in each soybean plot, while ten pyramid cages were placed in each soybean and cotton plot, totaling 60 pyramid cages and 20 emergence tents. All traps were placed at least ten meters from the field edge. In Florida, two emergence tents were placed per experimental plot. In Florida, emergence traps were placed randomly in each plot within the center four rows and at least two meters from the plot edge. Traps were checked three times a week in Florida and twice a week in North and South Carolina.

The traps were left in the fields after the growing season through the fallow season until

May for both years. After the 2019 growing season, only H. zea adults were recorded in all locations. After the 2020 growing season, adults of H. zea, A. gemmatalis, and C. includens were recorded at the Florida location while no adults were recorded in North and South Carolina.

Data analysis. The H. zea total larval abundance, early instar abundance, middle instar abundance, late instar abundance, C. includens larval abundance, A. gemmatalis larval abundance, number of damaged squares per 100 squares, and number of damaged bolls per 100 bolls were each separately analyzed with the fixed effects of state (Florida, South Carolina, and North Carolina), crop (Bt cotton, non-Bt cotton, soybean, and peanut), months sampled, and the interaction of state and crop were analyzed using generalized linear models (GLMs) with a Poisson family, and

“observation”” and “plot” as random effects. An observation-level random effect in which each data point is subjected to a different random effect was included in the GLM to account for overdispersion of the data. Due to the absence of H. zea larvae in Bt cotton in Florida and only one larva detected during the sampling in soybean in South

Carolina, the interaction of state and crop was removed from the model prior to analysis. Since plots of peanut was only included in Florida, with no sampling dates occurring in October or July for the North Carolina locations, the factors, peanut, July, and October were excluded in all GLMs. For each GLM, analysis of variance was used to determine numerator degrees of freedom, p-values, and F values. The means were compared using pairwise contrasts to calculate back-transformed means and attain z- scores. All analyses were performed in RStudio, Version 1.1.463 – © 2009-2018

RStudio, Inc.

Figure 2-1. Pyramid cage with capture jar and 3D-printed funnel at top. Jay, Florida. Photo courtesy of author.

Figure 2-2. Emergence tent and pyramid cage set in fallow field. Jay, Florida. Photo courtesy of author.

Figure 2-3. Capture jar with 3D-printed funnel connected to the emergence tent. Jay, Florida. Photo courtesy of author.

Figure 2-4. 3D-printed funnel in capture jar with specimens of Helicoverpa zea trapped in the container. Jay, Florida. Photo courtesy of author.

Figure 2-5. Pupal digging method in fallow field. Jay, Florida. Photo courtesy of author.

Figure 2-6. Opening of H. zea pupal chamber in soil. Jay, Florida. Photo courtesy of author.

Figure 2-7. H. zea pupa within pupal chamber in soil. Jay, Florida. Photo courtesy of author.

Figure 2-8. H. zea pupa in diapause with presence of three eye spots (circled). Photo courtesy of author.

Figure 2-9. H. zea pupa without presence of three eye spots. Photo courtesy of author.

Figure 2-10. Globules of fat body as indicated by arrows in H. zea pupa. Photo courtesy of author.

CHAPTER 3 RESULTS

Comparison of Adult Emergence Traps

Overall, the number of adults captured in pyramid cage was significantly greater than the number of adults captured per emergence tent in the artificial infestation of non-Bt corn study (AI non-Bt) (t = 2.4938, DF = 15, p = 0.0276), the natural infestation of Bt corn study (NI Bt) (t = 2.6112, DF = 15, p = 0.0197), and the pupal placement study (PP) (t = 3.873, DF = 5, p = 0.0112) (Figure 3-1).

Abundance of Helicoverpa zea (Lepidoptera: Noctuidae) and Other Economic Lepidopteran Pests in Cotton, Soybean, and Peanut in the Southeastern United States

Overall, during the larval field sampling performed in Florida, North Carolina, and

South Carolina, H. zea (n = 208) was the third most abundant species, following C. includens (n = 420), and A. gemmatalis (n = 1,765) (Figure 3-2, Table 3-1).

Helicoverpa zea larval abundance was significantly affected by crop (p = 0.0183)

(Table 3-2). The average abundance of this species in non-Bt cotton (CI95% = [0.21,

1.00] mean = 0.4537) was 4.04 times as high as the abundance in Bt cotton (CI95% =

[0.04, 0.30], mean = 0.1124), and 5.45 times as high as soybean (CI95% = [0.03, 0.27], mean = 0.0833), with the average abundance in Bt cotton being 1.35 times higher than soybean. The larval abundance in non-Bt cotton was significantly higher than in soybean (z = -2.232, p = 0.0416), but not significantly different than in Bt cotton (z = -

2.32, p = 0.0660). Helicoverpa zea larval abundance was also affected by state (p ≤

0.0001) (Table 3-2). The average larval abundance of this species in North Carolina

(CI95% = [0.38, 1.85], mean = 0.8372) was 6.21 times as high as the larval abundance in

South Carolina (CI95% = [0.05, 0.37], mean = 0.1348) (z = -2.870, p = 0.0114) and 22.20

times as high as Florida (CI95% = [0.01, 0.12], mean = 0.0377) (z = -4.528, p < 0.0001), with the average larval abundance in South Carolina being 3.58 times as high as the average larval abundance in Florida. No significant difference in the abundance of H. zea was detected between Florida and South Carolina populations (z = -1.706, p =

0.2029) (Table 3-2).

There was no effect of crop on the abundance of early instar larval abundance of

H. zea (p = 0.2975). However, there was an effect of month on early instar larval abundance (p =0.0159). The average early instar abundance in August (CI95% = [0.073,

0.394], mean = 0.1695) was 24.78 times as high as the early instar larval abundance in

September (CI95% = [0.001, 0.10], mean = 0.0116) (z = 2.412, p = 0.0159) (Table 3-2).

The occurrence of H. zea early instar larvae was also different between Florida and

North Carolina (p ≤ 0.0001) (Table 3-2), with the average early instar larval abundance being 25.87 times as high in North Carolina (CI95% = [0.07, 0.70], mean = 0.2251) compared to Florida (CI95% = [0.002, 0.05], mean = 0.0087) (z = -3.933, p = 0.0001). In the case of middle instar larvae, the average larval abundance in North Carolina (CI95%

= [0.12, 0.65], mean = 0.2787) was 25.34 times as high as Florida (CI95% = [0.003, 0.05], mean = 0.011) (z = -4.819, p = <0.001) (Table 3-2). Middle instar abundance decreased over time, with the average larval abundance in August (CI95% = [0.10, 0.38], mean =

0.1857) being 11.05 times as high as the average larval abundance in September

(CI95% = [0.003, 0.09], mean = 0.0168) (z = 2.969, p = 0.0030) (Table 3-2). The abundance of middle instar larvae varied among crops (p = 0.0011) (Table 3-2). The average middle instar abundance was 6.78 times greater in non-Bt cotton (CI95% = [0.07,

0.45], mean = 0.1730) than Bt cotton (CI95% = [0.007, 0.10], mean = 0.0255) (z = -3.205,

p = 0.0039) and 4.40 times greater than the average larval abundance in soybean

(CI95% = [0.01, 0.14], mean = 0.0393) (z = 2.735, p = 0.0171). Average late instar abundance was 10.56 times as high in North Carolina (CI95% = [0.07, 0.47], mean =

0.1859) than in Florida (CI95% = [0.005, 0.07], mean = 0.0176) (z = -2.755, p = 0.0059).

Late instar abundance was also influenced by crop (p = 0.0206) (Table 3-2). The average late instar abundance in non-Bt cotton (CI95% = 0.11, 0.57], mean = 0.2486) was 12.43 times as high as soybean (CI95% = [0.003, 0.13], mean = 0.0200) (z = 2.443, p = 0.0387). The average late instar abundance in non-Bt cotton was 6.59 times as high as Bt cotton (CI95% = [0.11, 0.17], mean = 0.0377) (z = -2.218, p = 0.0682) while the average late instar larval abundance was only 0.53 times greater in Bt cotton than soybean (z = 0.537, p = 0.8532). Late instar abundance was not significantly different through the sampling months (p = 0.9685) (Table 3-2).

The number of damaged cotton squares per 100 square sample was estimated only in North Carolina and Florida, and there was no interaction between state and crop on damaged cotton squares (p = 0.4588) (Table 3-3). The number of damaged cotton squares per 100 square sample was significantly different in Bt cotton and non-Bt-cotton

(p = 0.0454) (Table 3-3). The average number of damaged squares detected in non-Bt cotton (CI95% = [2.22e-16, inf], mean = 0.0073) was 4.38 times as high as Bt cotton

(CI95% = [2.2e-16, inf], mean = 0.0021) (z = -2.003, p = 0.0452). The number of damaged cotton squares was also significantly different among states (p = 0.0008)

(Table 3-3). The average number of damaged squares recorded was 9.17 times higher in North Carolina (CI95% = [2.2e-16, inf], mean = 0.0110) than in Florida (CI95% = [2.22e-

16, inf], mean = 0.0014) (z = -3.230, p = 0.0012).

Similarly, the overall number of damaged cotton bolls was significantly affected by crop (p = 0.0052) and state (p = 0.0015), but not their interaction (p = 0.6516) (Table

3-3). The average number of damaged bolls recorded was 3.99 times higher in non-Bt cotton (CI95% = [0.51, 2.77], mean = 1.186) than Bt cotton (CI95% = 0.11, 0.78], mean =

0.297) (z = -2.617, p = 0.0089). The average number of damaged cotton bolls in South

Carolina (CI95% = [0.49, 2.93], mean = 1.199), was only 1.10 times as high as North

Carolina (CI95% = 0.32, 3.72, mean = 1.088) but 7.44 times as high as Florida (CI95% =

0.06, 0.45], mean = 0.161). The number of damaged cotton bolls per 100 boll sample in

Florida was significantly different from North Carolina (z = -3.251, p = 0.0033) and

South Carolina (z = -2.708, p = 0.0186). North and South Carolina were not significantly different in number of damaged cotton bolls per 100 boll sample (z = 0.146, p = 0.9833).

Pupal digging during the fallow season indicated that populations of H. zea across the states were low, with zero pupae occurring in Florida after the 2019 and

2020 growing seasons and zero pupae recovered from all states after the 2020 growing season (Table 3-1). In North Carolina, there were a total of six pupae recovered after the 2019 growing season, with four pupae from non-Bt cotton and two pupae from Bt cotton (Table 3-1). The average weight of pupae from non-Bt cotton in North Carolina equaled 398.50 mg (SE ± 0.02), while the average weight of pupae from Bt cotton was not able to be attained due to damage to one of the two pupae while the remaining undamaged pupa weighed 436 mg. Pupal populations were highest in South Carolina, with a total of 74 pupae recovered from soybean and three pupae recovered from non-

Bt cotton following the 2019 growing season, and zero pupa was recovered following the 2020 growing season (Table 3-1). The average pupal weight of in South Carolina

was 524.41 mg (SE ± 10.56) from soybean plots, and 449.1 mg (SE ± 66.57) in non-Bt cotton.

Adult populations of H. zea trapped in emergence tents and pyramid cages during the fallow season across all states and years were overall low, with zero adults captured in Florida following the 2019 growing season. Only one adult was captured in non-Bt cotton following the 2020 growing season (Table 3-1). There were no adults captured in South Carolina and North Carolina following the 2019 growing season

(Table 3-1). Because of this low adult trapping in the previous year, and limitations due to the COVID-19 pandemic, no trapping was performed in the 2020 fallow season in

North Carolina and South Carolina.

Anticarsia gemmatalis was the most abundant species detected during the larval sampling in soybean, and the overall abundance of A. gemmatalis was significantly different across states (p ≤ 0.001) (Table 3-2). The average larval abundance of this species in soybean in Florida (CI95% = [2.71, 82.72], mean = 14.977), was 26.74 times as great as soybean in North Carolina (CI95% = [0.09, 2.29], mean = 0.560), and 30.84 times as great as soybean in South Carolina (CI95% = [0.07, 6.15], mean = 0.484), with the average larval abundance in North Carolina being only 1.16 times higher than South

Carolina. The larval abundance was significantly higher in Florida than in South

Carolina (z = 3.314, p = 0.0027) and in North Carolina (z = 3.3459, p = 0.0016).

However, no difference between A. gemmatalis larval abundance was detected between North Carolina and South Carolina (z = -0.121, p = 0.9920). The effect of crop in the abundance of this species was only estimated for Florida, which had experiment plots of peanut and soybean. Anticarsia gemmatalis occurred in soybean (CI95% = [3.05,

26.59], mean = 9.57) 4.18 times as much as peanut (CI95% = [0.67, 6.26], mean = 2.29)

(z = -4.854, p <0.0001) (Tables 3-1 and 3-2).

During the adult trapping and pupal digging in the fall of 2020 in Florida, six adult specimens of A. gemmatalis were captured in peanut, while zero adult specimens of C. includens were recovered (Table 3-1). Pupae of A. gemmatalis were not recovered during the sampling (Table 3-1).

Chrysodeixis includens was the second most abundant species detected during the larval sampling in soybean (Table 3-1). The overall abundance was significantly affected by state (p = 0.0453) (Table 3-2). The average larval abundance in South

Carolina (CI95% = [4.04, 27.22], mean = 10.480), was 2.42 times higher than North

Carolina (CI95% = [2.14, 8.74] mean = 4.33), and 7.33 times higher than Florida (CI95% =

[0.62, 3.31], mean = 1.43), with the average larval abundance in North Carolina being

3.03 times as high as Florida. Larval abundance of C. includens in Florida and South

Carolina differed significantly (z = -0.0351), with North Carolina not differing significantly from Florida (z = -1.244, p = 0.4271) or South Carolina (z = 1.244, p = 0.4273).

Chrysodeixis includens occurred in soybean (CI95% = [2.22e-16, inf], mean = 0.0066)

3.67 times as much as in peanut (CI95% = [2.2e-16, inf] mean = 0.0022) (z = -3.126, p =

0.0018). During pupal digging, this species was not recovered since this species pupates in the plant canopy.

2 1.8 A 1.

1. adults captured adults

1.2 A

.8 B . A B . B .2

A erage number of number erage A P ramid ages Emergence P ramid ages Emergence P ramid ages Emergence ( ) ent (1) ( ) ent (1) ( ) ent (1) AI non Bt NI Bt PP Experiment

Figure 3-1. Average number of H. zea adults caught in five pyramid cages compared to the average number of H. zea adults caught in one emergence tent in three separate emergence traps comparison experiments. “AI - non-Bt” indicates the experiment in Florida in which traps were placed in non-Bt corn and artificially infested with H. zea neonates. “NI – Bt” indicates the experiment in Florida in which traps were placed in plots of Bt corn that were naturally infested with H. zea. “PP” indicates the experiment in Florida in which pupae of H. zea were placed under each trap. Means sharing the same letter are not statistically different at p-value ≤0.05, using pair-wise t-tests comparing traps and tents in each trial. Error bars indicate standard errors

Table 3-1. Total larval, pupal, and adult abundance of H. zea in focal fields during 2019 and 2020. Crop N. of Avg. N. of N. of Avg. N. of (Pupae/m2)/(Larvae/m2) N. of Avg. N. of larvae larvae/m pupae pupae/m2 adults adults/m2 Florida Bt-cotton 1 NA 0 0 NA 0 0 Non-Bt 7 NA 0 0 NA 1 0.022 cotton Soybean 1 0.0025 0 0 0 0 0 Peanut 4 0.0099 0 0 0 0 0

North Carolina Bt-cotton 23 NA 2 0.0058 NA 0 0 Non-Bt 94 NA 4 0.012 NA 0 0 cotton Soybean 56 0.38 0 0 0 0 0

South Carolina Bt-cotton 9 NA 0 0 NA 0 0 Non-Bt 12 NA 3 0.00067 NA 0 0 cotton Soybean 1 0.0068 74 0.017 2.5 0 0 Four plots of each crop type were grown in Florida, while two plots of each crop were grown in North and South Carolina. Number of larvae are total found for 11 sampling trips in Florida, 8 for North Carolina, and 12 for South Carolina. Number of pupae found per 22.20 m2 in Florida, 347.33 m2 in North Carolina, and 4,454.96 m2 in South Carolina. Number of adults captured in emergence cages and tents, covering 10.02 m2 in North Carolina, 22.6 m2 for each soybean plot in South Carolina, 33.42 m2 for each cotton plot in South Carolina, and 11.6 m2 for each Florida plot.

Table 3-2. Two-way ANOVA for H. zea populations in field crops cultivated in southeastern U.S. Fixed effects Chi-square nDF1 dDF2 p-value H. zea total larval abundance State 18.5 2 290 <0.0001 Crop 5.7 2 290 0.0183 H. zea early instar abundance State 14.8 1 223 <0.0001 Crop 1 2 222 0.2975 Month 5.8 1 223 0.0159 H. zea middle instar abundance State 18.9 1 223 <0.0001 Crop 5.8 2 222 0.0011 Month 8.8 1 223 0.003 H. zea late instar abundance State 5.7 1 223 0.0059 Crop 3.9 2 222 0.0206 Month 0.002 1 223 0.9685 A. gemmatalis larval abundance State 8.6 2 290 0.0002 C. includens larval abundance State 3.1 2 290 0.0453 1nDF depicts the numerator degrees of freedom 2dDF depicts the denominator degrees of freedom

Table 3-3. Two-way ANOVA for damaged cotton squares and damaged cotton bolls in different crops cultivated in southeastern U.S. Fixed effects Chi-square nDF dDF p-value Damaged cotton squares State 15.7517 1 223 0.0008 Crop 3.4531 1 223 0.0454 State x crop 0.5489 1 223 0.4588 Damaged cotton bolls State 6.2348 2 222 0.0015 Crop 7.2766 1 223 0.0052 State x crop 0.4238 2 222 0.6516 1nDF depicts the numerator degrees of freedom 2dDF depicts the denominator degrees of freedom

Figure 3-2. Relative larval abundance of Helicoverpa zea, Anticarsia gemmatalis, and Chrysodeixis includens from July through October in Bt cotton (BtC), non-Bt cotton (nBtC), peanut (P), and soybean (SB) fields in Florida (FL), North Carolina (NC), and South Carolina (SC). 2019 to 2020 crop seasons.

0.35

0.3

0.25 larvae per 9.1per larvae

0.2 BtC nBtC 0.15

P meters perplot meters

Helicoverpa zea Helicoverpazea SB 0.1

0.05 Average of Average 0 Jul Aug Sep Oct FL

Figure 3-3. Average number of Helicoverpa zea larvae recorded per every 9.1 meters of crop rows per plot during field sampling in Florida (FL) in each crop. “Bt ” represents Bt cotton, “nBt ” represents non-Bt cotton, “P” represents peanut, and “SB” represents so bean. 2 19 to 2 2 crop seasons. Error bars represent standard error.

8 larvae per 9.1 9.1 per larvae

6 BtC nBtC

meters per plot per meters SB

Helicoverpa zea zea Helicoverpa 4

2 Average of Average

0 Aug Sep NC

Figure 3-4. Average number of Helicoverpa zea larvae recorded per every 9.1 meters of crop row per plot during field sampling in North Carolina (NC) in each crop. “Bt ” represents Bt cotton, “nBt ” represents non-Bt cotton, “P” represents peanut, and “SB” represents so bean. 2 19 to 2 2 crop seasons. Error bars represent standard error.

0.9

0.8

0.7

larvae per 9.1 9.1 per larvae 0.6

0.5 BtC 0.4 nBtC

meters per plot per meters SB

0.3 Helicoverpa zea zea Helicoverpa

0.2

0.1 Average of Average

0 Jul Aug Sep Oct SC

Figure 3-5. Average number of Helicoverpa zea larvae recorded per every 9.1 meters of crop rows per plot during field sampling in South Carolina (SC) in each crop. “Bt ” represents Bt cotton, “nBt ” represents non-Bt cotton, “P” represents peanut, and “SB” represents soybean.2019 to 2020 crop seasons. Error bars represent standard error.

CHAPTER 4 DISCUSSION

Population dynamics of species targeted by Bt technology, within and across growing seasons is an important aspect when designing IRM programs. To do so, the contribution of all life stages by various crops must be determined. This makes the use of emergence traps important because they allow researchers to directly determine the relative contributions to pest populations by different host plants, whereas pheromone traps capture widely dispersing adults originating from a variety of different host sources in the landscape (Gould et al. 2002, Jackson et al. 2008, Head et al. 2010). Capture of emerging ground-pupating lepidopterans with emergence traps is also an important method to document overwintering populations of pests in agroecosystems (Shiller

1946, Caron et al. 1978, Murray and Wilson 1991). In the present study, the pyramid cage captured more H. zea adults in corn plots, where the pest pressure of this species was highest. However, the ability for the pyramid cages to be randomly dispersed throughout the experimental plot may allow the traps to cover more variable areas of pupal densities. Thus, when limited in the number of emergence traps that can be used, the emergence tent may be a better option. However, these traps differ in their designs and materials, which also need to be considered when choosing the most appropriate trap to be adopted for overwintering studies. For example, the shade cloth construction may make the emergence tent more vulnerable to high winds, while the low center of gravity and heavier weight may make the pyramid cage preferable for use in such conditions. In September 2020, hurricane Sally posed a potential hazard to emergence tents in Florida, and the emergence tents were removed, creating a temporary break in data collection. Hurricane Dorian in 2019 also inflicted damage on emergence tents in

North Carolina. However, this likely did not affect statistical analyses due to the low capture of adults across the study. Thus, these requirements need to be considered when choosing the most appropriate trap type for overwintering studies on Lepidoptera.

The results of the quantification of the larval, pupal, and adult abundance of H. zea indicated overall low populations of this species in the study performed. However,

H. zea larval abundances differed significantly among states. The larval abundance of

H. zea was significantly higher in North Carolina than both Florida and South Carolina.

Previous research has illustrated the high occurrence of H. zea larvae and its corresponding damage in North Carolina as well as high resistance ratios of H. zea populations in this state (Reisig et al. 2018). However, the objective of the present study was to provide comparative abundance rates of H. zea populations in all life stages, as well as damage across the southeastern U.S., which has not been previously reported.

Thus, despite the relatively higher H. zea larval abundance reported in North Carolina, subsequent pupal sampling and adult trapping in this state did not result in high pupal and adult populations during the fallow season. Factors such as cold winters, rainfall, soil type, soil moisture, and predation may have played a role as mortality factors for this species during the study (Murray and Zalucki 1990, Morey et al. 2012, Zheng et al.

2013, Baldwin et al. 2020).

When comparing the abundances of H. zea larvae between non-cotton (soybean and peanut) and cotton (Bt and non-Bt) hosts, there were unexpectedly low larval populations in soybean. In soybean across all states, H. zea larval abundance was significantly lower than non-Bt cotton, but not significantly different from Bt cotton. In addition, the abundance of H. zea larvae in peanut plots in Florida did not differ

significantly from Bt or non-Bt cotton. Furthermore, when larval abundance is separated into early, middle, and late instars, larval mortality in non-Bt cotton is likely relatively low compared to soybean. Even though mortality was not recorded in the present study, lower mortality in cotton is evidenced by the lack of a significant difference in early instar counts across soybean, non-Bt and Bt cotton contrasted with significantly more middle and late instar larvae on non-Bt cotton when compared to soybean. These results indicate that sampling of early instar larvae across crops likely occurred before mortality played a role in decreasing larval abundance of H. zea in soybean and peanut (Zalucki et al. 2002, Baldwin et al. 2020). These results seemingly conflict with previous research which has suggested that non-cotton hosts, including soybean, contribute more to the population than cotton when both cotton and non-cotton hosts are concurrent in the landscape (Jackson et al. 2008, Head et al. 2010). According to the price of soybean being $14.10 per bushel (March 10th, 2021), the current economic injury level for H. zea in soybean with a 91.44 cm row spacing is approximately 0.57 larvae per 0.3 meters (North Carolina State University Extension 2021, MacroTrends

2021). The average number of larvae per meter exceeded this threshold in soybean in all states with the exception of South Carolina (Table 1). However, sample sizes were low in all states, with an overall low larval abundance recorded across all crops in all states. In addition, larval abundance cannot be equivalently compared between cotton and non-cotton hosts due to unequal sampling methods between the two crops (beat cloth in soybean and peanut, visual inspection of 100 squares and 100 bolls in cotton).

Thus, the higher larval abundance found in non-Bt cotton in the present study may not accurately reflect its role as a potential refuge of unselected populations of H. zea.

Therefore, more research using high number of replications, and equal sampling methods to estimate larval densities in both cotton and non-cotton hosts would provide more robust data regarding the relative abundance of H. zea populations in these crops.

Results of the present study, which showed low occurrence of H. zea larvae in soybean and peanut, agree with results indicating that corn and weeds may be the most significant contributors of H. zea populations in cotton agroecosystems (Gould et al.

2002, Head et al. 2010, EPA 2018). For example, high numbers of Physallis spp., which have been shown to support the taxonomic related species Heliothis virescens

(Fabricius) (Lepidoptera: Noctuidae) (Sudbrink and Grant 1995) were observed in

Florida plots in 2019 during larval sampling. However, evaluation of this genus as a potential host plant of H. zea was not able to be conducted due COVID-19 restrictions during the 2020 crop season, which prevented comparative life tables. Further research on the suitability of weed plants as host plants of H. zea, and their contribution of unselected populations in the landscape of Bt cotton for this region should be conducted.

The H. zea larval abundance across all instars did not differ significantly between

Bt and non-Bt cotton across all states. However, the differences in early, middle, and late instar abundance indicate that high rates of early instars in Bt cotton may be attributed to sampling occurring before larval mortality factors took place (Zalucki et al.

2002, Braswell et al. 2019b). Furthermore, comparable early instar abundance between

Bt and non-Bt cotton is likely due to similar oviposition rates between non-Bt and Bt cotton. Previous research has illustrated higher rates of H. zea oviposition on Bt cotton compared to non-Bt cotton (Braswell et al. 2019b). However, this is likely related to a

density-dependent behavior in which adults oviposit on Bt-cotton due to a high abundance of larvae on non-Bt cotton, and therefore is likely not due to discrimination between Bt and non-Bt plants by adults (Braswell et al. 2019b). Thus, the relatively low densities of H. zea larvae observed in the present study when compared to the study by

Braswell et al. (2019a) may have contributed to similar oviposition rates between Bt and non-Bt cotton as evidenced by similar early instar abundance between Bt and non-Bt cotton. Furthermore, early instar mortality was likely greater in Bt cotton than non-Bt cotton, as indicated by higher rates of middle instar larvae in non-Bt cotton compared to

Bt cotton. Additionally, there was a significantly higher number of damaged squares and bolls in non-Bt cotton compared to Bt cotton across all three states. However, the lack of a significant difference in the abundance of late instar larvae between non-Bt and Bt cotton may indicate the occurrence of resistance in larvae surviving and reaching later development stages, which agrees with studies documenting high rates of Bt resistance across the southeastern U.S (Bilbo et al. 2019, Kaur et al. 2019, Rabelo et al. 2020, Niu et al. 2021). Such extensive resistance across a large region can have widescale implications when considering the mixed landscapes of each state and their contribution to overwintering populations, which infest early-season crops, such as corn, in the following year. Previously, it has been shown that most H. zea adults originate from C4 plants such as corn early in the season, while there is a short window in which a majority of adults originate from cotton (Gould et al. 2002, Head et al. 2010). However, this research took place in an area of mixed Bt and non-Bt cotton. Thus, the current landscape of the southeastern U.S., which is dominated by Bt cotton, likely contributes to lower abundances of H. zea adults originating from cotton. If resistance is to

increase, the relative contribution of cotton would also increase, enhancing the selective filtering effect between Bt cotton and Bt corn across growing seasons. Thus, understanding the abundance of overwintering H. zea pupae and emerging adults as they relate to larval populations and crop damage better clarifies the population dynamics of this Bt-resistant pest across growing seasons.

In North Carolina, the only contribution of overwintering pupal population by Bt cotton was two pupae (0.006 pupae/m2) following the 2019 growing season, with four pupae recovered from non-Bt cotton plots (0.012 pupae/m2) that same year. No pupae were recovered following the 2020 growing season. In addition, no adults were captured in the plots in North Carolina following the 2019 growing season. Thus, while larval populations were highest in North Carolina across both years, there were low rates of overwintering survival in this region (Caron et al. 1978). Previously, late-planted corn, a preferred host of H. zea, which supports high populations of larvae indicated to support an average of 3.4% of larvae which developed into diapausing pupae, survived through the winter, and emerged as adults across three counties in North Carolina (Caron et al.

1978). Adult emergence after the fallow season was determined to make up 0% of larvae for this region in the present study. However, cropping history has been indicated to influence Cry1Ac bioassay survival, illustrating overwintering occurrence in this region, especially in soybean (Arends et al., submitted). Thus, due to the lower preference of H. zea for soybean and cotton compared to corn (Jackson et al. 2008,

Head et al. 2010, Cunningham and Zalucki 2014), with Bt and non-Bt cotton contributing to lower survival rates (Gore et al. 2003), successful adult overwintering in North

Carolina was likely not high enough to result in spring adult capture in our study. This is

especially true considering the low larval and pupal abundances recorded throughout the study.

South Carolina had the highest abundance of H. zea pupae recovered from soybean with a total 74 pupae recovered in 2019 (0.017 pupae/m2). However, there was relatively less pupae recovered from Bt and non-Bt cotton when compared to North

Carolina, with three pupae recovered from non-Bt cotton plots (0.001 pupae/m2) and zero pupae from non-Bt cotton in 2019. This result alone is consistent with research which shows that soybean is the biggest contributor to H. zea populations (Gore et al.

2003, Jackson et al. 2008), although the larval abundance in soybean was overall low, during the crop season sampling. In addition, no pupae were recovered from any state following the 2020 growing season. Furthermore, no H. zea adults were collected in emergence traps in 2019 in South Carolina. Thus, future studies should increase the experimental area and number of the fields in each state to better determine the contribution of soybean to susceptible populations of H. zea adults in South Carolina.

In Florida, no pupae were recovered during the sampling in both years, with one adult recovered from non-Bt cotton following the 2020 growing season. Little to no capture of adults in all three states is likely related to previously mentioned abiotic mortality factors which impact pupal survival (Williams and Stinner 1987, Murray and

Zalucki 1990, Morey et al. 2012). Even though weather data were not correlated with adult emergence rates for any of the states in the study, this region experienced inclement weather due to hurricane Sally in 2020, which brought 370.8 mm of rain to

Santa Rosa County, Florida (US Department of Commerce 2020). Future studies should focus on a long-term evaluation of the annual impact of weather on the

overwintering survival of H. zea, particularly in the Florida Panhandle, a transition zone between tropical and temperate regions. Furthermore, the abundance of natural enemies such as the red imported fire ant, Solenopsis invicta, is widespread throughout the southeastern U.S. and can significantly reduce H. zea egg numbers in peanut and cotton fields (Ascunce et al. 2011, Baldwin et al. 2020). It is likely that S. invicta is also a significant predator of last instar larvae moving to the ground to pupate (Baldwin et al.

2020).

Overall, the population dynamics of H. zea in mixed landscapes is highly variable across states, with North Carolina exhibiting the highest rates of cotton boll damage and larval populations. South Carolina, while being second in rates of boll damage and larval abundance, simultaneously had the highest overall population of pupae with the most pupae collected from soybean plots. While neither the pyramid cage nor the emergence tent design used in H. zea adult sampling resulted in capture of overwintering adults, this likely reflects low overwintering survival, since emergence trap comparison studies have shown higher capture rates of emerging H. zea adults in corn plots, where pest pressure was higher. Thus, in our study, when considering abundance of pupal populations, soybean is likely an efficacious refuge option in Bt cotton- dominated agroecosystems for in-season populations, at least in South Carolina.

However, a long-term study should be performed on pupal and overwintering adult populations in the region to better determine the contribution of H. zea populations by soybean during the fallow season.

While H. zea was the focal species during field sampling of cotton, soybean, and peanut, A. gemmatalis and C. includens were most abundant species detected during

the larval sampling. Both species are widespread pests across the Southeast (Herzog and Todd 1980), with legumes such as soybean and peanut being the preferred host crops (Waters and Barfield 1989). However, A. gemmatalis is the most economic soybean pest in Florida, in some cases feeding on the crop until the plant is completely defoliated, resulting in heavy yield losses (Herzog and Todd 1980, Guillebeau et al.

2008). While cotton is listed as a host plant of C. includens (Jost and Pitre 2002, Specht et al. 2015), A. gemmatalis is not reported to be a pest of cotton. Furthermore, due to C. includens feeding mostly on cotton leaves, rather than squares and bolls (Smith et al.

1994), this species was not detected in cotton. The results of the present study are consistent with previous findings on host preferences showing high numbers of larvae of both A. gemmatalis and C. includens in peanut and soybean (Martin et al. 1976, Waters and Barfield 1989). Over the two years of this study in Florida, A. gemmatalis had the highest abundance, while C. includens has the lowest. Both species overwinter in southern Florida (Carner et al. 1974, Mitchell et al. 1975, Wilkerson et al. 1986,

Buschman et al. 1977 Smith et al. 1994). Anticarsia gemmatalis migrates to northern

Florida in mid-August (Wilkerson et al. 1986), and C. includens is reported to peak in

August in the Florida Panhandle (Shaw et al., submitted).

South Carolina had the highest average larval abundance of C. includens. In contrast to H. zea populations, North Carolina had the lowest abundance of C. includens and A. gemmatalis among the states under study. Previous research (Beach and Todd 1986) suggests that in areas where cotton and soybean are cultivated, larval populations of C. includens is higher when soybean is the predominant crop. However, larval sampling in this study indicated that abundance across all states was low, and the

limited number of planting dates and number of plots prevent robust conclusions concerning differences in C. includens and A. gemmatalis populations between states.

Because these species have different behaviors and life history traits, when compared to H. zea, pupal and adult emergence during the fallow season was not expected.

These species are not known to overwinter in northern Florida or in more northerly latitudes (Buschman et al 1977, Buschman et al. 1981). This makes studies on pupal and adult populations following the growing season limited to the weeks following harvest. Monitoring of A. gemmatalis and C. includens adults is usually only done by light and pheromone traps (Waddill et al. 1982, Mitchell and Heath 1986), which intercept migrating adults (Lingren et al. 1993). The use of emergence traps would therefore better serve as a method to document the contribution of the mixed landscape to pest populations. Research on overwintering adult populations of C. includens is further limited by its pupation behavior, which occurs on the underside of leaves (Shour and Sparks 1981). Thus, while emergence tents were illustrated to be a viable method of studying ground-pupating lepidopteran species such as H. zea, emergence tents are likely not a viable method for measuring C. includens emergence, especially since they are not reported to overwinter in northern Florida. The use of emergence traps, however, provided information on the contribution of soybean and peanut to the populations of A. gemmatlais. This was possible because this species pupates in loose soil, approximately 2 cm deep (Lee and Johnson 1990). A previous study (Buschman et al. 1981) in Mississippi implemented emergence traps to study A. gemmatalis overwintering survival in the region, using laboratory-reared larvae and pupae placed under emergence traps. In addition, emergence traps were placed in experimental fields

where A. gemmatalis populations naturally occurred in August and September.

However, this study showed no emergence of A. gemmatalis adults, indicating that they did not survive the winter in nearby Mississippi. The authors concluded that A. gemmatalis adults captured early in the year in blacklight traps in Mississippi were likely migrating populations, possibly from south Florida and the Yucatan peninsula

(Buschman et al. 1981). While A. gemmatalis has not been reported to overwinter in northern Florida, it was expected that the capture of emerging adults would be limited to the few weeks after the tents were placed late in the growing season and through the start of the fallow season. However, the capture of A. gemmatalis during the adult trapping in the fallow season indicated latent populations of A. gemmatalis in the region.

Further studies in the Florida Panhandle should investigate overwintering survival of this species in the region. Furthermore, information on the relative contribution of peanut and soybean to pest populations will better refine the knowledge about the population dynamics of this pest.

Overall, the results of the study indicate that H. zea larval, pupal, and adult populations were extremely low within and across the 2019 and 2020 growing seasons in Florida, North Carolina, and South Carolina. Low larval populations in all crops may indicate that these row crops are not significant contributors of H. zea populations.

While absolute comparisons of larval abundance between cotton and non-cotton hosts were not possible due to differences in sampling methods between cotton and non- cotton crops, this study has illustrated that H. zea larval abundance was higher in non-

Bt cotton than Bt cotton across all states. This result indicates efficacy of two-toxin Bt cotton in the southeastern United States. However, an increase in sampling size needs

to be done in these areas to make more definitive conclusions on the contribution of H. zea populations by these crops between growing seasons. Furthermore, the suitability of weedy hosts in the landscape should be assessed. Better understanding the relative contribution of natural refuge could influence decisions in weed spraying and creating boundaries of natural refuge in cotton agroecosystems to incorporate into IRM decisions. Results of this study have also shown that differences in emergence trap designs may also influence rates of capture. Comparisons of these traps in areas of high pest pressure in corn and design aspects should be considered in future studies on overwintering studies. Furthermore, high populations of other lepidopteran pests, and interspecific competition between H. zea, A. gemmatalis and C. includens, should be further investigated (Beach and Todd 1986, Braswell et al. 2019a).

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BIOGRAPHICAL SKETCH

The author, Tyler Shaw was born as the eldest of three children to parents

James and Eva LeAnn Shaw in Daytona Beach, Florida. Growing up in the biodiversity hotspot that is central Florida, Shaw developed a strong passion for finding different insect species in his backyard. Eventually his passion for entomology led him to attend the Uni ersit of Florida to pursue a bachelor’s degree in Entomolog and Nematolog in the fall of 2015. Once there, he started a job at the McGuire Center for Lepidoptera and Biodiversity of The Florida Museum of Natural History. It was at the museum where he participated in curating lepidopteran collections as well as assisting in pollinator ecolog research. During his time as a student, Shaw’s interests pro oked him to get involved in extracurricular activities. He joined the Entomology Club and also became treasurer of the Honey Bee Club. During this time, he developed his skills as a beekeeper as well as an insect photographer and scientific illustrator, sharing these passions with other students along the way. After graduating with his Bachelor of

Science degree in Entomology and Nematology in 2017, Shaw went on to attend graduate school at the Uni ersit of Florida’s West Florida Research and Education

enter (WFRE ) in Ja , Florida in 2 19. During his two ears as a master’s student of entomology at the WFREC, Shaw developed his skills as a researcher by working on two research projects. One of these projects focused on the cross-attraction of plusiine lepidopterans to Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae) sex pheromone lures - research which will help agricultural workers to accurately determine pest populations in their fields. During his time as a master’s student, Shaw also assisted in developing and teaching an online Principles of Entomology course with his advisor, Dr. Silvana V. Paula-Moraes. During this experience, Shaw discovered an

enthusiasm for teaching biological concepts, cementing his desire to pursue a career in academia. Shaw hopes to further develop his understanding of ecology and evolution to contribute to research which aims to conserve natural resources so that future generations may also be inspired by the magnificence of nature.