Novel Techniques for Evaluating the Potential Host

Home , Kairomone, Telenomus podisi, The Exposed (novel), Trissolcus basalis

NOVEL TECHNIQUES FOR EVALUATING THE POTENTIAL HOST

RANGE OF CANDIDATE BIOLOGICAL CONTROL AGENT

TRISSOLCUS JAPONICUS (HYMENOPTERA: PLATYGASTRIDAE)

Sean M. Boyle

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 2017

NOVEL TECHNIQUES FOR EVALUATING THE POTENTIAL HOST

RANGE OF CANDIDATE BIOLOGICAL CONTROL AGENT

TRISSOLCUS JAPONICUS (HYMENOPTERA: PLATYGASTRIDAE)

Sean M. Boyle

Approved: ______Judith Hough-Goldstein, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee

Approved: ______Jacob Bowman, Ph.D. Chair of the Department of Entomology and Wildlife Ecology

Approved: ______Mark Rieger, Ph.D. Dean of the College of Agriculture and Natural Resources

Approved: ______Ann L. Ardis, Ph.D. Senior Vice Provost for Graduate and Professional Education ACKNOWLEDGMENTS

I would like to thank my two co-advisors, Dr. Judy Hough-Goldstein and Dr. Kim Hoelmer, for their unwavering support and guidance, as well as for giving me the opportunity to pursue my graduate degree. I would also like to acknowledge my committee members, Dr. Don Weber and Dr. Doug Tallamy, for providing their abundant knowledge and unique perspectives to this project. I would like to express my sincere gratitude to the people at the USDA-ARS Beneficial Insects Introductory Research Unit, especially Kathy Tatman, Patty Stout, and all the BMSB RSAs, whose efforts facilitated the success of my laboratory experiments and field research. To all the professors, staff, and fellow graduate students in the ENWC department, thank you for your ever-present enthusiasm and encouragement. You have made the past two years truly enjoyable. Finally, I am forever grateful for the endless love and support from my family and friends. I cannot thank you enough for being such an incredible source of motivation and strength throughout this experience.

iii TABLE OF CONTENTS

LIST OF TABLES ...... vi LIST OF FIGURES ...... vii ABSTRACT ...... ix

Chapter

1 USING KAIROMONES AS A HOST RANGE EVALUATION TOOL ...... 1

1.1 Introduction ...... 1 1.2 Materials and Methods ...... 5

1.2.1 Insects ...... 5 1.2.2 Plants ...... 6 1.2.3 Leaf Surface Contamination by Stink Bug Footprints ...... 7 1.2.4 Bioassay Procedure ...... 7 1.2.5 Statistical Analysis ...... 9

1.3 Results ...... 10

1.3.1 Residence Time ...... 10 1.3.2 Linear Walking Velocity ...... 11 1.3.3 Angular Walking Velocity ...... 13

1.4 Discussion ...... 15

2 PARENTAL HOST SPECIES INFLUENCES ON THE HOST- FORAGING PREFERENCES OF TRISSOLCUS JAPONICUS (HYMENOPTERA: PLATYGASTRIDAE) ...... 21

2.1 Introduction ...... 21 2.2 Materials and Methods ...... 27

2.2.1 Insects ...... 27 2.2.2 Plants ...... 29 2.2.3 Kairomone Contamination of Leaf Surfaces ...... 29 2.2.4 Behavioral Bioassay Procedure ...... 30 2.2.5 No-Choice Tests ...... 32 2.2.6 Statistical Analysis ...... 33

iv 2.3 Results ...... 34

2.3.1 Residence Time ...... 34 2.3.2 Linear Walking Velocity ...... 36 2.3.3 Angular Walking Velocity ...... 38 2.3.4 No-Choice Parasitism Rates ...... 39 2.3.5 Exposed Egg Masses ...... 40 2.3.6 Parasitoid Size ...... 41 2.3.7 Emergence from Parasitized Egg Masses ...... 42 2.3.8 Parasitoid Development Time ...... 43

2.4 Discussion ...... 44

3 DETERMINING HABITAT OVERLAP BETWEEN HALYOMORPHA HALYS AND PODISUS MACULIVENTRIS IN NORTHERN DELAWARE . 53

3.1 Introduction ...... 53 3.2 Materials and Methods ...... 56

3.2.1 Trap Design and Deployment ...... 56 3.2.2 Study Sites ...... 57 3.2.3 Statistical Analysis ...... 59

3.3 Results ...... 61

3.3.1 2016...... 62

3.3.1.1 Habitat ...... 62 3.3.1.2 Sampling Period ...... 63 3.3.1.3 Habitat, Sampling Period, and Species ...... 64

3.3.2 2017...... 65

3.3.2.1 Habitat ...... 66 3.3.2.2 Sampling Period ...... 66 3.3.2.3 Habitat, Sampling Period, and Species ...... 68

3.4 Discussion ...... 68

REFERENCES ...... 73

v LIST OF TABLES

Table 2.1: Total number of egg masses parasitized, percent eggs parasitized and mean percentages (± standard deviation) of all exposed egg mass outcomes for each treatment (n = 25) in no-choice tests. Asterisks indicate significant differences between exposed egg mass species treatments that share the same parasitoid strain (Fisher’s Exact test/ Bonferroni correction, *P < 0.0125; Tukey’s HSD **P < 0.05) ...... 40

Table 2.2: Mean right hind tibia (RHT) length and weight (± standard deviation) of H and P strain T. japonicus used in no-choice tests...... 41

Table 2.3: Mean percentage of emerged T. japonicus (± standard deviation), percentage suitability, and mean development time for T. japonicus per parasitized egg mass in no-choice tests. Asterisks indicate significant differences between P-strain treatments (Fisher’s Exact test/ Bonferroni correction, *P < 0.0125; Tukey’s HSD **P < 0.05) ..... 44

Table 3.1: 2016 and 2017 H. halys and P. maculiventris captures in the traps of each habitat per sampling period. Letters indicate significantly different spring capture totals between habitat treatments for each species in the same year (Tukey’s HSD, P < 0.05)...... 61

vi LIST OF FIGURES

Figure 1.1: Experimental set up of camera recording T. japonicus within the arena. Monitor displays EthoVision XT tracking and measurement variables...... 9

Figure 1.2: Mean residence of T. japonicus on leaf substrates. Bars indicate ± SE. Shared number of asterisks indicates no significant difference between leaf substrate treatment means (Tukey’s HSD, α = 0.05). Letters show significantly different kairomone treatment means within each leaf substrate treatment (Tukey’s HSD, α = 0.05)...... 11

Figure 1.3: Mean linear walking velocity of T. japonicus on leaf substrates. Bars indicate ± SE. Shared number of asterisks indicates no significant difference between leaf substrate treatment means (Tukey’s HSD, α = 0.05). Letters show significantly different kairomone treatment means within each leaf substrate treatment (Tukey’s HSD, α = 0.05). ... 13

Figure 1.4: Mean angular walking velocity of T. japonicus on leaf substrates. Bars indicate ± SE. Letters show significantly different kairomone treatment means within each leaf substrate treatment (Tukey’s HSD, α = 0.05)...... 15

Figure 2.1: Mean residence time of H and P strain T. japonicus on G. max leaf substrates. Bars indicate ± SE. Letters show significantly different means within T. japonicus strain treatments (Tukey’s HSD, α = 0.05) ... 36

Figure 2.2: Mean linear walking velocity of H and P strain T. japonicus on G. max leaf substrates. Bars indicate ± SE. Letters show significantly different means within T. japonicus strain treatments (Tukey HSD, α = 0.05) ...... 37

Figure 2.3: Mean angular walking velocity of H and P strain T. japonicus on G. max leaf substrates. Bars indicate ± SE. n.s. indicates no significant differences within T. japonicus strain treatment means (ANOVA, α = 0.05)...... 38

vii Figure 2.4: Linear regression of right hind tibia (RHT) length and weight for all T. japonicus females used in no choice tests. Different parasitoid parental host species displayed with circular points (H. halys) and square point (P. maculiventris). Trendline fitted to the data (P < 0.0001; R2 = 0. 957, y = 0.000685x - 0.165). Area within dashes indicates 95% confidence interval...... 42

Figure 3.1: Total 2016 H. halys and P. maculiventris adults collected during the early spring and late spring sampling periods in woodland (black), fragmented (orange), and agriculture (blue) habitats...... 63

Figure 3.2: 2016 mean captures of H. halys (grey) and P. maculiventris (striped) at each habitat during early and late spring. Bars indicate ± SE. Letters denote significantly different capture means of each species during both sampling periods in woodlands. (Tukey’s HSD, P < 0.05). ns indicates no significant differences between the capture means within habitat treatments...... 64

Figure 3.3: Total 2017 H. halys and P. maculiventris adults collected during the early spring and late spring sampling periods in woodland (black), fragmented (orange), and agriculture (blue) habitats...... 66

Figure 3.4: 2017 mean captures of H. halys (grey) and P. maculiventris (striped) at each habitat during early and late spring. Bars indicate ± SE. ns indicates no significant differences between the capture means within habitat treatments...... 67

viii ABSTRACT

Native to eastern Asia, the brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), is a polyphagous invasive pest in North America and Europe. Since its initial discovery in Allentown, Pennsylvania in 1996, H. halys has spread to 44 states in the continental US, many of which have reported significant economic damages to agriculture. One promising management strategy for H. halys currently being investigated is classical biological control, or the introduction of natural enemies. Specifically, egg parasitoid Trissolcus japonicus (Ashmead) (Hymenoptera: Platygastridae) is considered the primary candidate biocontrol agent for field release, making it the subject of rigorous laboratory host range testing. However, these laboratory host range evaluations are ecologically-constricted and only provide the physiological host range of T. japonicus. Additional factors influencing an egg parasitoid’s true host range, such as its reception of host-related semiochemicals, effects of its parental host species on host location, and habitat overlap between suitable host species have yet to be adequately studied. Therefore, experiments focusing on these vital aspects of T. japonicus host preference were conducted to formulate a more accurate prediction of the parasitoid’s host specificity and ecological host range. To investigate if T. japonicus shows preference for H. halys adult kairomones over those of a physiologically-suitable, native species’, we conducted behavioral assays exposing female parasitoids to various leaf surface substrates contaminated with the chemical footprints of gravid female H. halys or Podisus maculiventris. In all

ix three leaf substrate treatments, Trissolcus japonicus displayed clear preferences for leaf surfaces contaminated by its coevolved host H. halys. Female parasitoids resided significantly longer on H. halys contaminated leaf surfaces than on leaf surfaces contaminated by P. maculiventris. These results indicate that T. japonicus may be able to determine the suitability of a potential host using host-related chemical cues it perceives in the environment. To address possible influences of parental host species on T. japonicus host specificity, separate parasitoid strains were established using H. halys and P. maculiventris eggs as hosts. Female T. japonicus from both strains were exposed to soybean (Glycine max) leaf surfaces contaminated by adult kairomones of either stink bug species. Also, females from each strain were subject to no-choice tests where they were exposed to either H. halys or P. maculiventris egg masses in large mesh cages. Lastly, to determine possible phenotypic plasticity of T. japonicus, each female used in the no-choice tests were weighed and their right hind tibiae were measured. Results from the behavioral assays suggest that T. japonicus exhibits some degree of host fidelity while searching for hosts. Females which emerged from P. maculiventris eggs resided for equal amounts of time on leaves contaminated with kairomones from either stink bug species. Differences in no-choice test performance were not found between the two strains, as parasitism rates and host suitability were determined to be dependent on exposed host species and not the parasitoid strain. T. japonicus which emerged from H. halys possessed longer right hind tibiae and weighed roughly twice as much as wasps which emerged from P. maculiventris. To determine potential spatiotemporal overlap between H. halys and P. maculiventris, pheromone traps for both species were placed in woodland, fragmented,

x and agricultural habitats in early and late spring in 2016 and 2017. For both years, woodland traps yielded significantly greater captures than the other two habitats. Significant differences in woodland trap collections between the two species were observed in 2016, yet these differences were not found in 2017. Our trapping data suggest that these two species inhabit similar habitats during their spring activity. All together, our three studies expand on current host range testing protocols by providing novel methodologies for evaluating a biological control agent’s host specificity and its potential risks to non-target species.

xi Chapter 1

USING KAIROMONES AS A HOST RANGE EVALUATION TOOL

1.1 Introduction

Native to eastern Asia, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), the brown marmorated stink bug, has become a serious invasive pest in the United States and Europe (Hoebeke & Carter, 2003; Rice et al. 2014; Wermelinger et al., 2008). Since its initial discovery in 1996 in Allentown, Pennsylvania, H. halys has spread to 44 states, and is considered an agricultural pest in 20 of them (NEIPM 2017). The mid-Atlantic states can be considered the epicenter of the H. halys invasion, as major economic losses have been reported there for nearly a decade (Leskey et al., 2012a, b). Arguably the most alarming attribute facilitating H. halys’s adverse effects on agriculture is its highly polyphagous herbivory on the fruiting bodies of a vast variety of plants (Lee et al., 2013). Over 100 plant species have been documented as H. halys hosts, including many economically important tree fruit, vegetable, and field crop species (Bergmann et al., 2016). Most notably, peach, apple, pear, cherry, grape, blueberry, green bean, peppers, tomato, eggplant, sweet corn, and soybean crops have all suffered significant damage from H. halys feeding (Nielsen & Hamilton, 2009; Leskey et al., 2012a, b; Kuhar et al., 2012; Cissel et al., 2015; Nielsen et al., 2011). In addition to crop fruits, the ability of H. halys to feed on the fruits of native and ornamental plant species allows it to successfully develop from nymph to adult stages in urban and natural landscapes (Acebes-Doria et al., 2016). Current management

1 regimes for H. halys primarily involve broad-spectrum insecticide application to at- risk crops within agricultural systems (Nielsen et al. 2008). Unfortunately, this insecticide use is largely unsustainable, as it is effective in specific areas (e.g. within agricultural systems) for only short periods of time, forcing growers to perform multiple applications per growing season (Blaauw et al., 2015). Repeated use of broad-spectrum insecticides to control H. halys also disrupts integrated pest management (IPM) programs, causing deleterious effects on natural enemy populations and consequently promoting secondary pest outbreaks (Penca & Hodges, 2017; Leskey & Hamilton, 2013). One promising alternative management strategy for H. halys currently being investigated is classical biological control. Foreign exploration for natural enemies of H. halys in its native range of China yielded the discovery of Trissolcus japonicus (Ashmead) (Hymenoptera: Platygastridae), an egg parasitoid which is now considered the primary candidate biological control agent (Talamas et al., 2013; Yang et al. 2009). Due to its high levels of H. halys parasitism, T. japonicus is recognized as the predominant egg parasitoid of H. halys in China, and for this reason, is currently the subject of rigorous quarantine experimentation in several USDA-ARS laboratories (Qui et al., 2007). Over the last seven years, quarantine studies have focused on assessing T. japonicus potential host range in the US by exposing the wasp to eggs from as many native pentatomid species as possible. Although these host range studies have provided a substantial baseline knowledge of the potential host range of T. japonicus, they only measure the wasp’s behavior after it has encountered a pentatomid egg mass. The host location and host selection processes of an egg parasitoid involve several complex steps mediated by chemical and physical stimuli

2 from various sources within the parasitoid’s habitat (Godfray, 1994). Host specificity tests incorporating other aspects of the egg parasitoid host location and host selection processes are needed to bolster our efforts in establishing a reliable, more predictive host range for T. japonicus. In a stepwise sequence of events, egg parasitoids depend largely on their reception of host-related semiochemical cues to successfully locate a suitable host (Vet & Dicke, 1992). First, egg parasitoids exploit long-range, volatile semiochemicals such as plant volatiles and host species’ pheromones to locate the host’s general habitat (Vinson, 1976; Dicke & Vet, 1999). Then, short-range contact semiochemicals, like chemical traces from adult and immature host stages and host egg adhesive chemicals, are used to find the host within a given habitat and determine the host’s overall suitability (Meiners & Peri, 2013; Bin et al. 1993). The responses of platygastrid parasitoids to various semiochemicals are well documented, as many studies have described the parasitoids’ characteristic host acceptance behaviors after exposing them to plant volatiles (Colazza et al. 2004; Moraes et al. 2005; Michereff et al. 2013), adult and immature contact kairomones (Colazza et al., 1999; Conti et al. 2003; Salerno et al. 2006; Peri et al. 2006), and host egg kairomones (Sales et al. 1980; Conti et al. 2003). Specifically, Trissolcus species displayed distinct periods of arrestment behavior, remaining motionless while their antennae maintained contact with a kairomone-contaminated surface, as well as decreased flight responses, decreased walking speeds, and increased turning rates when exposed to substrates that had previously been walked on by adult stink bugs (Colazza et al., 1999; Conti et al., 2003). Trissolcus wasps have also been shown to augment their behavioral responses when exposed to the adult kairomones of a

3 preferred, coevolved host versus a less preferred, non-coevolved host, suggesting these parasitoids can evaluate the host suitability of a particular species based on the contact kairomones perceived during the host location process (Conti et. al., 2004; Salerno et al. 2006). In addition, previous studies have provided insight into the role played by leaf surface epicuticular waxes in parasitoid host foraging, recognizing that this layer facilitates kairomone attachment and persistence on leaf surfaces (Colazza et al. 2009). To date, there has been minimal research on the response of T. japonicus to adult contact kairomones from H. halys or native, physiologically-suitable pentatomids. Categorizing the potential biological control agent’s behaviors when exposed to adult kairomones from native pentatomid species would help secure a more accurate prediction of its ecological host range in the US. Here, we investigate the behavioral responses of T. japonicus when exposed to adult contact kairomones from gravid (i.e. carrying eggs) female H. halys and native, beneficial Podisus maculiventris (Say) (Pentatomidae: Asopinae). P. maculiventris is considered an important generalist predator of many agricultural pests, and is thought to be distributed among habitats that overlap with those of H. halys (McPherson, 1980; Culliney, 1986; Wiedenmann et al. 1994). Quarantine host range evaluations indicate P. maculiventris is a physiologically suitable host for T. japonicus and is therefore viewed as an at-risk native species for T. japonicus non-target effects. Yet, no research has determined if T. japonicus can actively search for the predatory stink bug using the aforementioned kairomone cues. Our objective was to determine whether or not T. japonicus would respond to P. maculiventris adult kairomones, and compare the responses to those exhibited by T. japonicus when exposed to its coevolved, preferred host H. halys. In addition, we tested three different leaf surfaces as substrates for

4 kairomone contamination not only to simulate the diverse habitats of H. halys and P. maculiventris, but also to display any variation in T. japonicus host searching behavior while present on leaves with differing epicuticular waxes. Our experiments provide novel methods that further elucidate the host specificity of T. japonicus through the study of ecological factors associated with the egg parasitoid’s host location and host acceptance behaviors.

1.2 Materials and Methods

1.2.1 Insects

Laboratory colonies of both H. halys and P. maculiventris were established from locally collected individuals (New Castle County, DE) between March and

October 2016. Both species were reared in growth chambers (25±2°C, 70±10% relative humidity, 16L: 8D photoperiod). H. halys adults were held in 30.5 cm3 mesh cages (BioQuip Products, Inc.), while P. maculiventris were held in ≈ 1 L clear plastic food containers with ventilated lids. Separate cages and containers were used for nymphs and adults. All stages of H. halys were fed common beans (Phaseolus vulgaris Linnaeus), carrots (Daucus carota sativus Hoffman), apples (Malus pumila Miller), and sunflower seeds (Helianthus annuus Linnaeus) 2-3 times per week. All

P. maculiventris stages were fed greater waxworm larvae (Galleria mellonella) (Linnaeus) three times per weeks, as well as provided with common bean pods twice per week. Both species were given water via saturated pieces of cotton placed in plastic bottle lids. Common bean plants and pieces of foam mesh were used as oviposition substrates for H. halys and P. maculiventris, respectively, and egg masses were collected daily. Pairs of adult stink bugs (1 male and 1 female) were isolated in

5 separate ≈ 1 L plastic containers to ensure mating. Once females displayed signs of gravidity (enlarged distended abdomen), they were then used to apply kairomones to leaf substrates for the assays. A colony of T. japonicus was started from parasitized H. halys egg masses from Dr. Christopher Bergh at the Alson H. Smith Jr. Agricultural Research and Extension Center in Winchester, Virginia in August 2015. These egg masses were placed in the field at the Winchester site as sentinel egg masses and subsequently parasitized by the adventive T. japonicus population. When rearing subsequent generations, 1-2 mated female wasps were exposed to one H. halys egg mass (< 24 h old) in plastic test tubes for 2-3 days, and then removed. Parasitized egg masses were maintained in growth chambers (25±2°C, 70±10% relative humidity, 16L: 8D photoperiod) within the USDA-ARS Beneficial Insect Introduction Research Unit (BIIRU) (Newark, Delaware) quarantine facility and checked daily for emergence. Honey was provided in the test tubes and replaced as needed. Within 24 h of emergence, female wasps were isolated in separate plastic test tubes with a single male to ensure mating. After 24 h of isolation with a male, a female was considered mated and ready for the assays. All females used for the behavioral assays were separated by approximately 15 generations from the field collected individuals.

1.2.2 Plants Soybean (Glycine max Merrill), apple (M. pumila), and red maple (Acer rubrum Linnaeus) were used as plant substrates for the bioassays. All three species were chosen based on their H. halys host suitability and prevalence within agricultural, forested, and fragmented landscapes. Soybean plants were grown under greenhouse conditions from seed (Viking 2265; Johnny’s Selected Seeds) in plastic pots using a

6 pro-mix potting soil and watered as needed. Apple leaves were acquired from Milburn Orchards (Elkton, MD), and red maple leaves were collected from trees in an unmanaged woodland adjacent to BIIRU (Newark, DE). Before being used in the assays, all leaves were hand-washed in dilute soapy water (common dish detergent) followed by a water rinse and left to dry on paper towels. Once both the adaxial and abaxial leaf surfaces were dry, leaves were then prepared for stink bug footprint contamination.

1.2.3 Leaf Surface Contamination by Stink Bug Footprints Individual red maple and apple leaves were cut into 16 cm2 squares (with the leaf’s midvein bisecting the square) before being exposed to gravid female stink bugs. Due to rapid wilting when cut, soybean leaves (≈ 16 cm2) were left intact for the assays. Leaf substrates were placed in a glass petri dish (8 cm diameter, 1.5 cm height) and a single gravid female stink bug was confined to the adaxial leaf surface under a ventilated plastic cover (≈ 2 cm diameter, ≈ 0.5 cm height) for 30 minutes. The stink bug was manually forced to walk in a uniform path by moving the plastic cover down the midvein of the leaf, stopping at 3 separate locations for 10 minutes each. After the 30 minutes, the stink bug was removed. Leaf substrates contaminated by feces were not used for the experiments. Leaf substrates that were not exposed to stink bug footprints were used as controls.

1.2.4 Bioassay Procedure

All experiments were conducted between 900 and 1600 hours in a climate- controlled room at 23±1 ºC and 35±10 % relative humidity within a quarantine facility. Only 2-3 day-old, mated, naïve female T. japonicus were used for the

7 experiments. All leaf substrates were used in bioassays within one hour of being contaminated by the stink bug footprints. To begin each bioassay, a single female wasp was placed on the bottom surface of the petri dish arena adjacent to the leaf substrate. This initial wasp placement allowed for a more accurate start and end time to each trial, as the wasp was able to move freely on and off the leaf substrate. Continuous behavioral observation began once the wasp walked onto the adaxial leaf substrate surface and displayed arrestment behavior. Since arrestment behavior was never observed on control leaf substrates, behavioral observation began once the wasp walked onto the adaxial leaf surface and showed antennal contact with the substrate. Trials in which the wasp immediately flew away from or walked off the leaf substrate without performing arrestment behavior (excluding the control treatment) or allowing their antenna to make contact with the leaf surface were excluded from our analyses. All trials ended when the wasp flew away from or walked off the substrate. The arena was illuminated from below to optimize the contrast between the wasp and the substrate surface, making it visible to the camera (ICD-49, Ikegami Tsushinki Co., Ltd., Tokyo, Japan) mounted directly above the center of the arena (Fig. 1.1). The camera was connected to a video monitor and desktop PC which simultaneously recorded, tracked, and processed the wasp’s movement and behavior using EthoVision XT 8.0 (Noldus Information Technology, Wageningen, The Netherlands) motion tracking software. EthoVision XT 8.0 software recorded all observational data for each bioassay. This included the wasp’s total residence time (i.e. time from when the wasp first walked onto the substrate surface until it flew away from or walked off the substrate surface), mean linear walking velocity (mm/s), and angular walking velocity (°/s) (i.e. turning rate) while on the leaf substrate surface.

8 Each wasp was used for only one replicate, and twenty-five replicates were conducted for each treatment.

Figure 1.1: Experimental set up of camera recording T. japonicus within the arena. Monitor displays EthoVision XT tracking and measurement variables.

1.2.5 Statistical Analysis

The distributions of all variable values for each treatment were first evaluated for normality and homoscedasticity using a Shapiro-Wilk test (α = 0.05). The angular walking velocity data was determined to be normally distributed, while the data for residence time and linear walking velocity were heteroscedastic or non-normally distributed and therefore were normalized by logarithmic transformations. All behavioral responses to the three kairomone and leaf substrate treatments were tested using one-way analyses of variance (ANOVA) followed by a Tukey’s Honest Significant Difference (HSD) test (α = 0.05) for multiple comparisons among means. A two-way ANOVA was conducted to determine the presence of a significant

9 interaction between the independent categorical variables of leaf substrate and kairomone species. All statistical analyses were completed using JMP Pro 13.0.0 Statistical Software for Mac (SAS Institute Inc., Cary, NC).

1.3 Results

1.3.1 Residence Time

Significant differences were observed among kairomone treatment means (df = 2, 224, F = 247.47, P < 0.0001). Differences were also found among plant substrate treatments (df = 2, 224, F = 4.0503, P = 0.0188) (Fig. 1.2). Naïve T. japonicus females exhibited strong preferences for leaf substrates contaminated by H. halys kairomones. On average, the wasps spent approximately twice as much time on red maple (t = 169.4 ± 14.5 s, P < 0.0001), apple (t = 96.30 ± 9.84 s, P = 0.0003), and soybean (t = 109.8 ± 13.5 s, P = 0.0009) leaf substrates contaminated by H. halys than on leaf substrates contaminated by P. maculiventris (t = 69.41 ± 10.2 s, 50.43 ± 7.06 s, & 54.95 ± 8.03 s, respectively). T. japonicus spent significantly less time on the red maple (t = 12.19 ± 3.23 s, P < 0.0001), apple (t = 8.809 ± 1.50 s, P < 0.0001), and soybean (t = 13.41 ± 2.02 s, P < 0.0001) leaf substrates of the control group than on the leaf substrates contaminated with kairomones from either stink bug species (Fig.

1.2). Specifically, the parasitoid’s mean residence times on leaf substrates contaminated by H. halys and P. maculiventris kairomones were 11 and 5 times greater versus control substrates, respectively. There was no significant interaction found between kairomone and leaf substrate treatments (df = 4, F = 2.349, P = 0.0554), as the time spent by T. japonicus on leaf substrates contaminated by a given

10 stink bug kairomone was not dependent on the specific leaf substrate possessing the the kairomone.

Mean Residence Time Red maple Soybean Apple 200 Plant Substrate * ** */** Apple A Red maple Soybean 175

150

125 A

A 100

Time (s) B 75 B B 50

25 C C C

0 H. halys Podisus Control H. halys Podisus Control H. halys Podisus Control Kairomone Figure 1.2: Mean residence of T. japonicus on leaf substrates. Bars indicate ± SE. Shared number of asterisks indicates no significant difference between leaf substrate treatment means (Tukey’s HSD, α = 0.05). Letters show significantly different kairomone treatment means within each leaf substrate treatment (Tukey’s HSD, α = 0.05).

1.3.2 Linear Walking Velocity

Significant differences were observed in the linear walking velocities of T. japonicus among kairomone (df = 2, 224, F = 87.243, P < 0.0001) and plant substrate treatments (df = 2, 224, F = 40.058, P < 0.0001) (Fig. 1.3). Within the red maple leaf substrate treatment, wasps walked 35% slower on leaf substrates contaminated by H.

11 halys kairomones (ν = 2.119 ± 0.149 mm/s, P = 0.0229) than on leaf substrates contaminated P. maculiventris kairomones (ν = 3.257 ± 0.249 mm/s). However, no significant differences in the linear walking speed of T. japonicus were observed between H. halys and P. maculiventris kairomone groups in the apple (P = 0.2059) or soybean (P = 0.4489) leaf substrate treatments (Fig. 1.3). T. japonicus walked roughly 2-3 times faster in the red maple (P < 0.0001), apple (P < 0.0001), and soybean (P < 0.0001) control treatments than in either the H. halys or P. maculiventris kairomone treatments. The linear walking velocity of T. japonicus on leaf substrates contaminated by a given stink bug kairomone was not dependent on the specific leaf substrate on which the wasp walked, as no significant interaction (df = 4, F = 1.805, P = 0.1290) was found between the two variables.

12 Mean Linear Walking Velocity Red maple Apple Soybean 12 Plant Substrate * * ** Apple Red maple Soybean

10 C

6 Velocity (mm/s) Velocity B 4 B A

A A A 2 A

0 H. halys Podisus Control H. halys Podisus Control H. halys Podisus Control Kairomone Figure 1.3: Mean linear walking velocity of T. japonicus on leaf substrates. Bars indicate ± SE. Shared number of asterisks indicates no significant difference between leaf substrate treatment means (Tukey’s HSD, α = 0.05). Letters show significantly different kairomone treatment means within each leaf substrate treatment (Tukey’s HSD, α = 0.05).

1.3.3 Angular Walking Velocity

Significant differences were observed in the angular velocities (�) of T. japonicus among kairomone treatments (df = 2, 224, F = 10.346, P < 0.0001), but were not found among plant substrate treatments (df = 2, 224, F = 0.9109, P = 0.4037). Within the red maple treatment, wasps displayed greater angular velocities on leaves contaminated by H. halys (ω = 127.72 ± 3.06 º/s, P = 0.0001) and P. maculiventris (ω = 113.19 ± 4.23 º/s, P = 0.029) kairomones than on the control leaves (ω = 91.21 ± 8.88 º/s) (Fig. 1.4). Similarly, within the apple treatment wasp angular velocities were

13 greater on leaves contaminated by H. halys (ω = 123.23 ± 4.07 º/s, P = 0.0041) and P. maculiventris (ω = 118.83 ± 5.43 º/s, P = 0.0183) kairomones than on the control leaves (ω = 95.26 ± 7.81 º/s). No significant differences were found between the wasps’ angular velocities in leaf substrate treatments contaminated by H. halys and P. maculiventris kairomones, nor among any of the soybean leaf substrate treatments (Fig. 1.4). Consistent with these trends, there was a significant interaction observed between the leaf substrate and kairomone variables, meaning the angular velocity of T. japonicus on leaf substrates contaminated by a given stink bug kairomone was dependent on the specific leaf substrate on which the wasp walked (df = 4, F = 3.366, P = 0.0107).

14 Mean Angular Velocity Apple Red maple Soybean 140 Plant Substrate A Apple A A Red maple Soybean A 120 A A A B B 100

Angular Velocity (deg/s) AngularVelocity 40

0 H. halys Podisus Control H. halys Podisus Control H. halys Podisus Control Kairomone Figure 1.4: Mean angular walking velocity of T. japonicus on leaf substrates. Bars indicate ± SE. Letters show significantly different kairomone treatment means within each leaf substrate treatment (Tukey’s HSD, α = 0.05).

1.4 Discussion

Paramount in any successful biological control program is acquiring a comprehensive understanding of the potential agent’s host range in the geographic location where the targeted pest currently resides. When considering the infinite number of interactions an agent can experience in the field, accurately assessing an agent’s host range is a difficult task to accomplish, one that demands a multipronged approach addressing as many of these interactions as possible. Our examination of the behavioral responses of T. japonicus to leaf surfaces contaminated by adult H. halys

15 and P. maculiventris contact kairomones offers valuable insight on a critical step in the egg parasitoid’s host location process. Here, T. japonicus demonstrated clear preferences for the host-related cues of its preferred, coevolved host, H. halys. This preference is best exhibited by the wasp’s significantly longer residence times on H. halys kairomone-contaminated leaf substrates than on P. maculiventris kairomone-contaminated leaf substrates (Fig. 1.2). Our residence time data suggests that T. japonicus may be able to determine the suitability of a potential host using indirect host-related cues it perceives, ultimately resulting in an increase or decrease in motivation while engaged in host foraging behaviors. The ability of other Trissolcus species to correctly evaluate a host’s suitability using chemical cues produced by non-host life stages has been previously confirmed in the literature. For example, T. brochymenae and T. basalis were both found to reside longer on substrates contaminated by adult kairomones from their coevolved pentatomid hosts, Murgantia histrionica and Nezara viridula, respectively, versus kairomones from non-coevolved pentatomid hosts (Conti et al., 2004; Salerno et al., 2006). However, a recent study performed by Hedstrom et al. (2017) showed no difference between the residence times of T. japonicus on filter paper substrates contaminated by H. halys and three native herbivorous stink bug species. These statistically indistinguishable behavioral responses by T. japonicus may be a result of similar chemical compositions of the adult H. halys and native pentatomid species’ kairomone residues since phytophagous pentatomid species are known to share several molecular components that comprise their wide variety of semiochemical secretions (Weber et al., 2018).

16 Thus far, almost all studies on egg parasitoid responses to indirect host-related contact kairomones have used filter paper as a substrate for kairomone contamination (Colazza et al., 2007). This approach does not adequately mimic the role of an intact plant cuticle during parasitoid-host interactions in nature, because it excludes the capacity of plant surfaces to promote or suppress contact kairomone attachment and persistence on a plant tissue substrate (Rostás et al., 2008; Colazza et al., 2009; Noldus et al., 1991). By mechanically removing the epicuticular wax layer from the leaf surfaces of broad bean plants (Vicia faba), Colazza et al. (2009) verified that epicuticular wax crystals were responsible for the adsorption of contact kairomones from mated female N. viridula. More important, they discovered that the egg parasitoid T. basalis displayed its characteristic host foraging behaviors only when exposed to kairomone-contaminated leaves with intact epicuticular wax layers or to polymer films that had been used to remove the epicuticular wax layers from contaminated leaves. Our study strengthens the idea that epicuticular waxes of leaf surfaces can influence an egg parasitoid’s ability to perceive host-related contact kairomones, as significant differences in the residence times and linear walking velocities of T. japonicus were observed among the three plant substrate treatments (Figs. 1.2, 1.3). Roughly twenty-three plant cuticular wax types have been described for all major groups of angiosperms, with each type possessing a diverse blend of molecular features (Barthlott et al., 1998). A. rubrum (Sapindales), M. pumila (Rosales), and G. max (Fabales) differ phylogenetically at the order level so it can be hypothesized that their epicuticular wax structures differ considerably, which may differentially impact the tritrophic interactions involved in T. japonicus host location (Müller & Riederer, 2005). Future research is needed to build a broader understanding

17 of how the epicuticular waxes of other H. halys host plants, especially those used for oviposition, influence the host foraging behaviors of T. japonicus. The results from our behavioral assays of T. japonicus suggest that the egg parasitoid can recognize and respond to P. maculiventris adult contact kairomones on leaf surfaces. T. japonicus mean residence time, linear walking velocity, and angular velocity (in red maple and apple treatments) on leaf substrates contaminated by P. maculiventris kairomones were all significantly different than the control (Fig. 1.2, 1.3, 1.4). These results are not surprising, since Conti et al. (2004), Salerno et al. (2006), and Hedstrom et al. (2017) demonstrated that Trissolcus spp. positively responded to adult kairomones of non-coevolved pentatomid species. As mentioned earlier, egg parasitoids must be able to exploit several host-related semiochemicals in order to successfully locate a suitable host (Vet & Dicke, 1992). Although T. japonicus may respond to the adult contact kairomones of P. maculiventris, the predaceous stink bug’s biology suggests such exposure may be unlikely to occur. While P. maculiventris will occasionally feed on plant tissue when prey species are scarce, its diet predominantly consists of Lepidoptera and Coleoptera larvae (Warren & Wallis, 1971; McPherson, 1980). Therefore, the majority of its feeding does not stimulate the release of herbivory-induced plant volatiles (HIPVs). Platygastrid parasitoid species are attracted to HIPVs and may utilize them to locate a host’s general habitat (Dicke & Hilker, 2003; Colazza et al. 2004; Moraes et al. 2005; Conti et al., 2008; Michereff et al., 2013). Without a consistent environmental presence of HIPVs associated with P. maculiventris, it would be difficult for T. japonicus to locate habitats containing P. maculiventris egg masses, thus minimizing the risk of non- target effects on P. maculiventris and other predatory pentatomid species.

18 Habitat overlap between H. halys and P. maculiventris is a plausible occurrence, as P. maculiventris is known to occur in many agroecosystems, including soybean, corn, and apple, as well as in natural shrubland and woodland landscapes (Culliney, 1986; Wiedenmann et al. 1994). Potential exists then for incidental exposure of T. japonicus to host-related contact kairomones from P. maculiventris adults, and possibly P. maculiventris eggs, while foraging for the targeted H. halys eggs. Given the significantly longer mean residence times of T. japonicus on H. halys contaminated leaf substrates, we can predict that in the presence of both species’ adult contact kairomones, T. japonicus is likely to have increased motivation to search for H. halys rather than P. maculiventris. Moreover, shorter mean residence times of T. japonicus when exposed to adult P. maculiventris kairomones can be viewed as partial or incomplete host acceptance behavior. These results mirror those of the no-choice host range tests previously conducted with T. japonicus that report only partial host suitability of P. maculiventris, as fully developed T. japonicus adults emerged from less than twenty percent of exposed eggs (Hoelmer et el., unpublished). So, even if an exposure between T. japonicus and either P. maculiventris contact kairomones or eggs occurs, we can expect non-target effects to be reduced. The discovery and establishment of adventive populations of T. japonicus in six mid-Atlantic states (MD, VA, DE, NJ, NY, and PA), Oregon, and Washington presents a unique opportunity for the classical biological control program’s ongoing host range analyses (Talamas et al., 2015; Herlihy et al. 2016; NEIPM, 2017; Morin et al., unpublished; Milnes et al. 2016). This integration of adventive T. japonicus into host specificity testing allows us to compare the thoroughly tested host range of quarantine laboratory T. japonicus populations to the untested host range of the

19 adventive populations. Confirmed genetic differences between adventive and quarantine T. japonicus emphasize the importance of and demand for future studies comparing the two distinct populations (Bon et al., unpublished). In addition, results from future laboratory host range tests of the adventive population can also be compared to sentinel and naturally-laid egg survey data to identify potential differences in parasitism rates, further aiding our goals to better understand the parasitoid’s ecological host range and potential non-target effects. This study’s utilization of adventive T. japonicus has provided a glimpse into how this parasitoid is currently searching for suitable hosts in the field. By using different leaf surfaces as substrates for host-related kairomone contamination, we were able to apply a key ecological component in the egg parasitoid host foraging process to T. japonicus host range evaluations. Future host specificity examinations of T. japonicus, and of other prospective biological control agents, should consider employing ecologically integrative techniques to build upon existing host range understandings and ultimately to improve the efficacy of biological control programs as a whole.

20 Chapter 2

PARENTAL HOST SPECIES INFLUENCES ON THE HOST-FORAGING PREFERENCES OF TRISSOLCUS JAPONICUS (HYMENOPTERA: PLATYGASTRIDAE)

2.1 Introduction

The brown marmorated stink bug (BMSB), Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), is a highly polyphagous insect native to eastern Asia and is currently considered a serious nuisance and agricultural pest across North America and Europe (Hoebeke & Carter, 2003; Rice et al. 2014; Wermelinger et al., 2008). H. halys was initially discovered in the United States in 1996 and has since spread to 44 states, many of which have reported significant economic damages to agriculture (NEIPM 2017). The stink bug can feed and develop on over 100 plant species, including important tree fruit, vegetable, and field crops (Bergmann et al., 2016). Crops such as apples, peaches, cherries, grapes, blueberries, peppers, tomatoes, sweet corn, and soybean are all susceptible to high levels of H. halys feeding (Nielsen & Hamilton, 2009; Leskey et al., 2012a, b; Kuhar et al., 2012; Cissel et al., 2015; Nielsen et al., 2011). Previous H. halys outbreaks in the mid-Atlantic states have displayed the pest’s propensity to inflict major damages to crop yields with estimates upwards of 90 % losses to stone fruit yields as well as $37 million in apple orchard losses (Leskey & Hamilton, 2010; American/Western Fruit Grower, 2011). H. halys generalist feeding ability has also allowed it to utilize many native and ornamental plant species to

21 successfully complete its development through each life stage (Acebes-Doria et al., 2016). In addition, H. halys adults and nymphs are highly capable dispersers, moving considerable distances between host plant habitats as well as from one landscape to another (Lee et al., 2014; Lee & Leskey 2015; Wiman et al. 2014). These apparent behavioral characteristics of polyphagy and mobility classify H. halys as a landscape- level pest, enabling it to colonize urban, natural, and agricultural habitats (Lee et al., 2013b; Joseph et al., 2014). Such traits have accelerated its geographic invasion and have stymied the effectiveness of current management strategies to suppress local populations (Morrison et al., 2015). Management strategies for H. halys largely consist of broad-spectrum insecticide (e.g. neonicotinoid and pyrethroid) applications within agricultural systems (Nielsen et al., 2008). In order to consistently prevent H. halys damage to at-risk crops, growers are forced to apply these insecticides multiple times throughout the growing season, ultimately resulting in temporary, localized control of BMSB populations (Leskey et al., 2012b; Blaauw et al., 2015). Repeated broad-spectrum insecticide applications are notorious for causing a variety of negative consequences to integrated pest management (IPM) programs (Debach & Rosen, 1991). Particularly, natural enemy populations are suppressed or even removed altogether, which subsequently can lead to secondary pest resurgences (Penca & Hodges, 2017; Leskey & Hamilton, 2013). The introduction of natural enemies (i.e. classical biological control) of H. halys is considered a more promising, long-term strategy for managing the pest’s populations at a landscape-level scale (Rice et al., 2014). The Asian egg parasitoid, Trissolcus japonicus (Ashmead) (Hymenoptera: Platygastridae) is the primary natural enemy of H. halys in its native range, where it has been documented to

22 parasitize 50 to 80 % of H. halys egg masses found in the field (Qui et al., 2007; Talamas et al., 2013; Yang et al. 2009; Zhang et al., 2017). For this reason, T. japonicus is recognized as the key candidate biological control agent for H. halys and has been the subject of intensive USDA-ARS quarantine studies over the last seven years. These quarantine studies have concentrated their efforts on testing the host specificity of T. japonicus by exposing the parasitoid to eggs from a broad range of native pentatomid species. To date, results from host range experimentation have provided us with a baseline understanding of T. japonicus fundamental host range. Although T. japonicus shows overt preference for H. halys in no-choice and choice host range tests, the wasp is physiologically capable of successful development within several native stink bug species (Hoelmer et al., unpublished; Hedstrom et al., 2017). Still, the efficacy of biological control programs’ host specificity evaluations has been questioned by a number of scientists (Hawkins & Marino, 1997; Louda et al., 2003a; Howarth, 1983; Strong & Pemberton, 2001). They claim that an exotic agent’s host range is exceedingly difficult to accurately assess using simplistic laboratory procedures, as the stochastic nature of ecological systems and the vast diversity of interactions within them are too complex. The majority of host range tests conducted for T. japonicus have, in fact, been simplified to only measure the direct interaction between the parasitoid and potential host, and therefore exclude the ecologically vital steps of the egg parasitoid host foraging process (Godfray, 1994). This host foraging process is mediated by a parasitoid’s perception of various host-related chemical cues (i.e. semiochemicals) which not only relay crucial information regarding the general habitat and location of a prospective host, but may also offer the parasitoid detailed

23 information concerning a host’s suitability (Vet & Dicke, 1992; Fatouros et al., 2008). By integrating semiochemicals into current T. japonicus host specificity testing regimes, a more confident prediction of the wasp’s ecological host range may be obtained. Egg parasitoids rely heavily on the presence of semiochemicals in their environment to bridge the immense spatial gap between them and their often inconspicuous hosts (Conti & Colazza, 2012). Also known as the infochemical detour strategy, the ability of egg parasitoids to exploit a variety of chemical compounds associated with host species’ life stages other than the ones they parasitize is crucial for successful host location (Vet & Dicke, 1992; Vinson 1998). Volatile compounds like herbivory-induced plant volatiles (HIPVs) and host species’ pheromones lead female parasitoids towards the general habitat (e.g. canopy of a tree) of a potential host, while chemicals deposited on plant substrates (e.g. contact kairomones), such as hydrocarbon residues left by adults and immatures host stages, further motivate parasitoids to search intensively for a host in their local vicinity (Conti et al., 2008; Aldrich et al., 1995; Meiners & Peri, 2013). Laboratory studies which exposed platygastrid parasitoids to HIPVs (Moraes et al. 2005; Michereff et al. 2013), pheromones (Bruni et al., 2000; Conti et al., 2003), and host species adult contact kairomones (Colazza et al., 1999; Salerno et al. 2006; Peri et al. 2006) have observed characteristic egg parasitoid host recognition behaviors. These behaviors included parasitoid arrestment or periods of motionlessness, decreased flight responses, slower walking velocities, and increased turning rates (Colazza et al., 1999). Quantifying the magnitude of semiochemical- induced behaviors is an effective technique for determining an egg parasitoid’s

24 preference to search for one suitable host over another (Conti et. al., 2004). For instance, Trissolcus spp. have displayed dissimilar behavioral responses when exposed to semiochemicals from coevolved hosts versus non-coevolved hosts (Salerno et al. 2006; This thesis, Chapter 1). Comparative analyses of T. japonicus behavioral responses to semiochemicals from H. halys and from selected physiologically suitable native pentatomids would provide valuable knowledge on the wasp’s host foraging preferences, and ultimately expand our current understanding of its host specificity. The manipulation of a parasitoid’s informational and physiological state is another approach that would enhance the ecological complexity of current T. japonicus host range evaluations and strengthen the parasitoid’s non-target risk assessment (Withers & Browne, 2004). Particularly, a parasitoid’s parental host species has been shown to influence its phenotypic traits, associative learning, lifetime fecundity, and host preferences (Abram et al., 2015; Hastings & Godfray, 1999; Turlings et al., 1993; Orr et al. 2000). Many studies of host learning in hymenopterous parasitoids demonstrate distinct, experience-induced changes in the behavioral responses of females to previously accepted (i.e. familiar) hosts and to biotic elements (e.g. host odors and remains, plant volatiles, rearing substrate, etc.) comprising the substrate-host complex (SHC) (Cortesero & Monge, 1994; Vet et al., 1995; Dauphin et al., 2009; Aquino et al., 2012). Yet, parental host species effects have not been adequately identified for T. japonicus. Only a single study has documented rudimentary phenotypic differences in the wasp’s size when emerging from different hosts (Medal & Smith, 2015). Previous studies suggest positive intraspecific correlations between egg parasitoid size and reproductive traits such as female egg loads and total offspring produced (Boivin, 2010; Boivin & Martel, 2012).

25 Interestingly, an egg parasitoid’s vagility, or propensity to move throughout its environment in search of hosts, was also positively correlated with the body size of certain egg parasitoid species (Bennet & Hoffman, 1998; Abram et al., 2015). Allahyari et al. (2004) examined parental host effects on egg parasitoid fitness parameters for Trissolcus grandis when reared on its coevolved host versus a non- coevolved host, concluding that the observed increases in host handling time for smaller T. grandis females reared on the non-coevolved host could indicate reductions in the parasitoid’s lifetime fecundity. Since T. japonicus parasitizes pentatomid species other than H. halys in its native Asian range, and can also successfully develop in several North American species, understanding the influences that the parental host species may have on T. japonicus host preferences is necessary for a comprehensive non-target risk assessment (Zhang et al., 2017; Hoelmer et al., unpublished; Hedstrom et al., 2017). Here, we have taken a “devil’s advocate” approach to the non-target risk assessment of T. japonicus by simulating a non-target parasitism event and examining its influence on the candidate agent’s host specificity. Our present study tested how the parental host species of T. japonicus affects its host foraging behaviors and its capacity to successfully locate and parasitize a suitable host. To do so, we established two separate T. japonicus laboratory strains by rearing the parasitoid on its coevolved host, H. halys, and on a native, physiologically suitable host, Podisus maculiventris (Say) (Pentatomidae: Asopinae). P. maculiventris is an important generalist predator of many agricultural pests, and is thought to be distributed among habitats which overlap with those of H. halys (McPherson, 1980; Culliney, 1986; Wiedenmann et al. 1994). Our previous research revealed stark differences in the behavioral responses of

26 T. japonicus (reared on H. halys) when exposed to H. halys and P. maculiventris adult kairomones on plant substrates (This thesis, Chapter 1). With these results in mind, our current study exposed the two T. japonicus strains to plant substrates contaminated by H. halys and P. maculiventris adult kairomones in order to identify if the host species from which T. japonicus emerges affects the wasp’s kairomone-induced behavioral responses. To expand our investigation of parental host influence, we then conducted no-choice host range tests for each T. japonicus strain in large, complex arenas so their abilities to successfully locate and parasitize H. halys and P. maculiventris could be more holistically evaluated. By comparing the performances of the two T. japonicus strains, we expect to acquire novel insight into how non-target parasitism events and parental host species may impact the host specificity and ecological host range of T. japonicus.

2.2 Materials and Methods

2.2.1 Insects

Laboratory colonies of both H. halys and P. maculiventris were established from locally collected individuals (New Castle County, DE) between March and May

2017. Both species were reared in growth chambers (25±2°C, 70±10% relative humidity, 16L: 8D photoperiod). H. halys adults were held in 30.5 cm3 mesh cages (BioQuip Products, Inc.), while P. maculiventris were held in ≈ 1 L clear plastic food containers with ventilated lids. Separate cages and containers were used for nymphs and adults. All stages of H. halys were fed green pods of common beans (Phaseolus vulgaris Linnaeus), carrot roots (Daucus carota sativus Hoffman), whole apple fruits (Malus pumila Miller), and sunflower seeds (Helianthus annuus Linnaeus) 2-3 times

27 per week. All P. maculiventris stages were fed greater waxworm larvae (Galleria mellonella) (Linnaeus) three times per weeks, as well as provided with P. vulgaris green pods twice per week. Both species were given water via saturated pieces of cotton placed in plastic bottle lids. Common bean plants and pieces of foam mesh were used as oviposition substrates for H. halys and P. maculiventris, respectively, and egg masses were collected daily. Pairs of adult stink bugs (1 male and 1 female) were isolated in separate ≈ 1 L plastic containers to ensure mating. Once females displayed signs of gravidity (enlarged distended abdomen), they were then used to apply kairomones to leaf surfaces for the assays. A colony of T. japonicus was started from parasitized H. halys egg masses from Dr. Christopher Bergh at the Alson H. Smith Jr. Agricultural Research and Extension Center in Winchester, Virginia in August 2015. These egg masses were placed in the field at the Winchester site as sentinel egg masses and subsequently parasitized by the adventive T. japonicus population. To establish the two different strains of T. japonicus, 1-2 mated female wasps, which had emerged from H. halys eggs, were exposed to a single egg mass (< 24 h old) from either H. halys or P. maculiventris in plastic test tubes for 2-3 days, and then removed. Parasitized egg masses were maintained in growth chambers (25±2°C, 70±10% relative humidity, 16L: 8D photoperiod) within the USDA-ARS Beneficial Insect Introduction Research Unit (BIIRU) (Newark, Delaware) quarantine facility and checked daily for emergence. Honey was provided in the test tubes and replaced as needed. Within 24 h of emergence, female wasps were isolated in separate plastic test tubes with a single male (emerged from the same host species) to ensure mating. After 24 h with a female, male wasps were removed. T. japonicus females were 2-3 days old when used

28 for the behavioral observation assays, while 3-5 day old females were used for the no- choice tests. All females used for the behavioral assays and no-choice tests were reared between 15-25 generations from the field collected individuals.

2.2.2 Plants Soybean (Glycine max Merrill) (Viking 2265; Johnny’s Selected Seeds) and common bean (Phaseolus vulgaris) (Johnny’s Selected Seeds) were grown from seed under greenhouse conditions in plastic pots using a pro-mix potting soil and watered as needed. To optimize total plant surface area, clusters of 3-5 P. vulgaris plants were grown to approximately 25-30 cm in height. Before being contaminated by stink bug kairomones, G. max leaves and P. vulgaris plant clusters were hand-washed in dilute soapy water (common dish detergent) followed by a water rinse and left to dry. Once leaf surfaces appeared dry, plant materials were then prepared for stink bug kairomone contamination.

2.2.3 Kairomone Contamination of Leaf Surfaces

For the behavioral bioassay, whole G. max leaves (≈ 16 cm2) were placed in glass petri dishes (8 cm diameter, 1.5 cm height) and a single gravid female stink bug was confined to the adaxial leaf surface under a ventilated plastic cover (≈ 2 cm diameter, ≈ 0.5 cm height) for 30 minutes. The stink bug was manually forced to walk in a uniform path by moving the plastic cover down the midvein of the leaf, stopping at 3 separate locations for 10 minutes each. After the 30 minutes, the stink bug was removed. Leaf substrates on which bugs had deposited frass were not used for the experiments. Leaf substrates that were not exposed to stink bug footprints were used as controls.

29 For the no-choice tests, two gravid stink bug females were allowed to walk freely on P. vulgaris plant clusters for one hour. Fine plastic mesh covers were used to restrict the stink bugs to the plant surfaces. Stink bugs were monitored continuously in order to ensure they maintained contact with plant surfaces during the entire contamination period. After one hour, the stink bugs were removed. Plant clusters with frass or newly deposited eggs were discarded from the no-choice tests. A single H. halys or P. maculiventris egg mass (< 24 h old) was affixed to the underside of a leaf in each P. vulgaris cluster using small plastic clips. A particular location to affix an egg mass was chosen based on observations made during the kairomone- contamination period, as leaves that were walked on most often during the contamination period were selected for egg mass attachment. Egg masses for H. halys and P. maculiventris were kept on the substrate on which they were oviposited (P. vulgaris leaves and foam mesh, respectively). Small pieces of trimmed substrates possessing egg masses were removed from the stink bug colonies, leaving only enough substrate material as needed to attach the egg masses to the leaf surface. For each plant cluster, the species of egg mass corresponded with the stink bug species used for plant surface kairomone contamination.

2.2.4 Behavioral Bioassay Procedure

All experiments were conducted between 900 and 1600 hours in a climate- controlled room at 21±2 ºC and 35±10 % relative humidity within a quarantine facility. Only 2-3 day-old, mated, naïve female T. japonicus were used for the experiments. All G. max leaf substrates were used in bioassays within one hour of being contaminated by the stink bug footprints. To begin each bioassay, a single female wasp from either the H. halys strain or the P. maculiventris strain (referred to

30 hereafter as the H strain and P strain) was gently placed on the bottom surface of the petri dish arena adjacent to the leaf substrate. This initial wasp placement allowed for a more accurate start and end time to each trial, as the wasp was able to move freely on and off the leaf substrate. Continuous behavioral observation began once the wasp walked onto the adaxial leaf substrate surface and displayed arrestment behavior. Since arrestment behavior was never observed on control leaf substrates, behavioral observation began once the wasp walked onto the adaxial leaf surface and showed antennal contact with the substrate. Trials in which the wasp immediately flew away from or walked off the leaf substrate without performing arrestment behavior (excluding the control treatment) or allowing their antenna to make contact with the leaf surface were excluded from our analyses. All trials ended when the wasp flew away from or walked off the substrate. The arena was illuminated from below in order to optimize the contrast between the wasp and the substrate surface, making it visible to the camera (ICD-49, Ikegami Tsushinki Co., Ltd., Tokyo, Japan) mounted directly above the center of the arena. The camera was connected to a video monitor and desktop PC which simultaneously recorded, tracked, and processed the wasp’s movement and behavior using EthoVision XT 8.0 (Noldus Information Technology, Wageningen, The Netherlands) motion tracking software. EthoVision XT 8.0 software recorded all observational data for each bioassay. This included the wasp’s total residence time (i.e. time from when the wasp first walked onto the substrate surface until it flew away from or walked off the substrate surface), mean linear walking velocity (mm/s), and angular walking velocity (°/s) (i.e. turning rate) while on the leaf substrate surface. Each wasp was used for only one replicate, and twenty-five replicates were conducted

31 for each treatment. The combinations of T. japonicus strain and stink bug species kairomone created a total of four different treatments.

2.2.5 No-Choice Tests An individual kairomone-contaminated P. vulgaris plant cluster possessing either a matching H. halys or P. maculiventris egg mass was placed in mesh cages

(30.5 cm3; BioQuip Products, Inc.) along with one mated, naïve T. japonicus female from the H strain or the P strain. The mesh cages were then placed in growth chambers (25±2°C, 70±10% RH, 16L: 8D) for 24 (±1) hours. After this 24 h period, the wasp was removed and frozen. The egg mass was collected, placed in a plastic petri dish and incubated in growth chambers (25±2°C, 70±10% RH, 16L: 8D). We conducted four separate treatments, each consisting of an H strain or a P strain wasp exposed to either an H. halys or a P. maculiventris egg mass. Twenty-five replicates were performed for each treatment. All wasps used were allowed one week to dry in ventilated test tubes and were subsequently weighed individually with a high-resolution microbalance (Mettler Toledo MX5, Mettler-Toledo LLC, Columbus, Ohio). Also, each wasp’s right hind tibia (RHT) was measured with micro-imaging software (Olympus cellSens Standard 1.11, Olympus Corporation, Tokyo, Japan) using a digital camera (Olympus DP72) mounted to a stereo microscope (Olympus SZX16). For each egg mass used we recorded the number of emerged wasps and hatched stink bug nymphs, and dissected all remaining eggs to determine egg mortality (total dead or undeveloped parasitoids and nymphs). Development time (the number of days from oviposition to wasp emergence) of T. japonicus was recorded for all egg masses that were successfully parasitized during the no-choice tests.

32 2.2.6 Statistical Analysis For the behavioral bioassay data, the distributions of all variable values for each treatment were first evaluated for normality and heteroscedasticity using a Shapiro-Wilk test (α = 0.05) and Bartlett’s test (α = 0.05), respectively. Residence time, linear walking velocity, and angular velocity data displayed heteroscedastic or non-normal distributions, so they were all normalized by logarithmic transformations. A two-way analysis of variance (ANOVA) was performed to determine the presence of a significant interaction between the independent nominal variables of kairomone type and T. japonicus parental host species. We also used a Tukey’s Honest Significant Difference (HSD) test (α = 0.05) to compare the response means within each treatment. Differences in parasitism rates (proportion of egg masses successfully parasitized) among treatments in the no-choice tests were analyzed using Fisher’s

Exact Test of Independence (α = 0.05). Also, a post hoc analysis of the parasitism rates between treatments was performed using Fisher’s Exact Test of Independence with a Bonferroni correction (comparison-wise α = 0.0125) to account for each pairwise comparison (McDonald, 2014). The counted outcomes of each exposed egg mass (number of emerged parasitoids, hatched nymphs, and egg mortality) were converted into percentages (counted outcome/number of eggs in exposed egg mass) and their distributions were analyzed using the Shapiro-Wilk test (α = 0.05) and Bartlett’s test (α = 0.05). A two-way ANOVA and Tukey’s HSD test were used to identify a potential interaction between parasitoid strain and exposed egg mass species as well as compare the mean outcome percentages between treatments. H and P strain T. japonicus RHT length and weight measurement values were identified as homoscedastic (normal), and therefore were compared using a one-way ANOVA (α =

33 0.05). In addition, we used a linear regression to distinguish a possible relationship between T. japonicus RHT length and weight. Data from no-choice tests that resulted in successful parasitism of exposed egg masses were partitioned from the entire no-choice data set in order to compare the number of emerged parasitoids between treatments. The difference between the two species host suitability levels (proportion of egg masses with > 50% parasitoid emergence) were compared using a Fisher’s Exact Test of Independence (α = 0.05) along with a post hoc analysis of host suitability between treatments with a Bonferroni correction (comparison-wise α = 0.0125) to account for each pairwise comparison (McDonald, 2014). The number of emerged T. japonicus from successfully parasitized egg masses was normalized with an arcsine transformation, and then analyzed using a two-way ANOVA and Tukey HSD (α = 0.05). Here, the two-way ANOVA allowed us to not only characterize the effects of parental host species (wasp strain) and exposed egg mass species on T. japonicus emergence, but to also demonstrate if these two nominal variables had an interaction that significantly affected the number of emerged wasps. Lastly, T. japonicus development time in successfully parasitized egg masses was analyzed using a one-way ANOVA. Statistical analyses were completed using JMP Pro 13.0.0 Statistical Software for Mac (SAS Institute Inc., Cary, NC).

2.3 Results

2.3.1 Residence Time

There was a significant interaction between parasitoid strain and kairomone treatments (df = 2, F = 6.991, P = 0.0013), meaning the time T. japonicus spent on

34 kairomone-contaminated leaf substrates was dependent on the parental host species of the parasitoid. When exposed to leaf substrates contaminated by P. maculiventris kairomones, the mean residence time of P strain wasps (t = 132.9 ± 8.029 s) was 2.4 times greater than the residence time of H strain wasps (t = 54.95 ± 8.03 s) (P < 0.0001) (Fig. 2.1). However, the mean residence times of H strain (t = 109.8 ± 13.5 s) and P strain (t = 89.71 ± 8.707 s) T. japonicus were not significantly different when exposed to leaf surfaces with H. halys kairomones (P = 0.9941). Within the H strain wasp treatment, T. japonicus mean residence time on H. halys-contaminated leaf substrates was about twice as long as the wasp’s residence time on P. maculiventris-contaminated leaf substrates (P = 0.0025) (Fig. 2.1). Yet within the P strain treatment, T. japonicus females did not resided any longer on H. halys-contaminated leaf substrates than on P. maculiventris-contaminated leaf substrates (P = 0.729). Within both T. japonicus strain treatments, residence times on leaf substrates contaminated by either stink bug species kairomone were significantly greater than on the control leaf substrates (P < 0.0001).

35 H-strain P-strain 160 Kairomone A Control H. halys Podisus 140 A 120

A 100

Time (s) Time B 60

B 20 C

0 H. halys Podisus Control H. halys Podisus Control

2.3.2 Linear Walking Velocity

There was a significant interaction between parasitoid strain and kairomone treatments (df = 2, F = 6.372, P = 0.0022), as the linear walking velocity of T. japonicus on kairomone-contaminated leaf substrates was dependent on the parental host species of the parasitoid. When exposed to the control leaf substrates, P strain wasps walked over 2.5 times slower (ν = 1.313 ± 0.142 mm/s) than H strain wasps (ν = 3.537 ± 0.444 mm/s) (P < 0.0001) (Fig. 2.2). P strain wasps also walked significantly slower (ν = 0.988 ± 0.0703 mm/s) than H strain wasps (ν = 1.777 ± 0.164 mm/s) while exposed to leaf substrates with P. maculiventris kairomones (P = 0.0002). H strain

36 wasps on control leaf substrates walked significantly faster than on either H. halys (ν = 1.462 ± 0.104 mm/s) or P. maculiventris contaminated leaf substrates (P < 0.0001) (Fig. 2.2). No significant differences were found between the linear walking velocities of H strain wasps on either stink bug kairomone leaf substrate treatment (P = 0.4489). Within the P strain T. japonicus treatments, no differences were observed between the wasp’s linear walking velocities on H. halys (ν = 1.146 ± 0.116 mm/s), P. maculiventris, or control kairomone leaf substrate treatments (P = 0.996).

H-strain P-strain 4.5 Kairomone Control H. halys A Podisus 4.0

3.5

3.0

2.5

B 2.0

B A Linear velocity (mm/s) velocity Linear 1.5 A A 1.0

0.5

0.0 Control Podisus H. halys Control Podisus H. halys

37 2.3.3 Angular Walking Velocity Contrasting with both residence time and linear walking velocity data, no significant interaction between parasitoid strain and kairomone treatments was found in the T. japonicus angular walking velocity means (df = 2, 149, F = 0.126, P = 0.882). Further, no differences were observed in T. japonicus mean angular walking velocities between parasitoid strain (df = 1, 149, F = 0.602, P = 0.439) or among stink bug kairomone (df = 2, 149, F = 0.256, P = 0.774) (Fig. 2.3).

H-strain P-strain 140 Kairomone Control H. halys ns ns Podisus 120

100

60 Angular velocity (deg/s) velocity Angular

0 Control H. halys Podisus Control H. halys Podisus

38 2.3.4 No-Choice Parasitism Rates T. japonicus females of both H. halys and P. maculiventris strains displayed varied abilities to successfully parasitize host eggs during the no choice tests (Table 1). Despite emerging from different host species, each parasitoid strain parasitized H. halys and P. maculiventris egg masses at similar rates. Thus, H and P strain T. japonicus females successfully parasitized 72% (n = 18) and 88% (n = 22) of the H. halys egg masses, respectively. These proportions of successfully parasitized H. halys egg masses did not differ by strain (P = 0.289) (Table 2.1). Moreover, the proportions of successfully parasitized P. maculiventris egg masses also did not differ by wasp strain (P = 0.754). As hypothesized, H-strain female T. japonicus successfully parasitized significantly more H. halys egg masses than P. maculiventris egg masses (n = 6) (P = 0.0016) (Table 2.1). P strain female wasps also parasitized significantly more H. halys egg masses than P. maculiventris egg masses (n = 8) (P = 0.0001).

39 Table 2.1: Total number of egg masses parasitized, percent eggs parasitized and mean percentages (± standard deviation) of all exposed egg mass outcomes for each treatment (n = 25) in no-choice tests. Asterisks indicate significant differences between exposed egg mass species treatments that share the same parasitoid strain (Fisher’s Exact test/ Bonferroni correction, *P < 0.0125; Tukey’s HSD **P < 0.05)

Parental host Exposed egg mass n % eggs % Emerged % % species species parasitized parasitized parasitoids Hatched Egg Mortality (Mean # of eggs) egg masses nymphs

H. halys H. halys 18 72.0 61.1 ± 41.3 16.0 ± 27.8 23.0 ± 22.5 (27.3) P. maculiventris H. halys 22 88.0 64.9 ± 30.0 7.56 ± 14.5 27.6 ± 20.4 (27.7) H. halys P. maculiventris 6 * 24.0 * 16.6 ± 31.5** 29.5 ± 33.4 53.9 ± 33.3** (26.7) P. maculiventris P. maculiventris 8 * 32.0 * 14.1 ± 25.4** 23.4 ± 23.3 62.6 ± 25.2** (27.8)

2.3.5 Exposed Egg Masses No interaction between T. japonicus strain and exposed egg mass species was identified in the mean percentage of emerged parasitoids (df = 1, 99, F = 0.233, P = 0.631), hatched nymphs (F = 0.048, P = 0.827), and egg mortality (F = 0.152, P = 0.698). However, the exposed egg mass species had a significant effect on the percentage of emerged parasitoids (df = 1, 99, F = 53.41, P < 0.0001), hatched nymphs (F = 8.163, P = 0.0052), and egg mortality (F = 40.70, P < 0.0001). When exposed to H. halys eggs, H and P strain females produced significantly more offspring than they did when exposed to P. maculiventris eggs (P < 0.0001) (Table 2.1). Also, exposed P. maculiventris egg masses had greater mean percentages of dead eggs (egg mortality) than H. halys eggs after exposure to both H strain (P = 0.0003) and P strain (P < 0.0001) parasitoid treatments.

40 2.3.6 Parasitoid Size H strain T. japonicus females possessed significantly longer right hind tibiae (df = 1, 99, F = 10006.7, P < 0.0001) as well as being significantly heavier (df = 1, 99, F = 699.29, P < 0.0001) than P strain parasitoids (Table 2.2). H strain RHT length and weight were positively correlated (P < 0.0001, R2 = 0.647, y = 0.000683x - 0.164). P strain females displayed a similar correlation (P < 0.0001, R2 = 0.654, y = 0.000645x - 0.152). Pooling both strains together, T. japonicus RHT length and weight were shown to have a highly significant, positive correlation (P < 0.0001, R2 = 0.957, y = 0.000685x - 0.165) (Fig. 2.4).

Table 2.2: Mean right hind tibia (RHT) length and weight (± standard deviation) of H and P strain T. japonicus used in no-choice tests.

Parental host species Mean RHT length (��) Mean Weight (mg)

H. halys 447 ± 16.3 0. 141 ± 0.0138

P. maculiventris 345 ± 16.0 0.071 ± 0.0127

41 0.20

0.15

0.10 Weight (mg) Weight

0.05

0.00 300 350 400 450 500 RHT Length (micrometers) Figure 2.4: Linear regression of right hind tibia (RHT) length and weight for all T. japonicus females used in no choice tests. Different parasitoid parental host species displayed with circular points (H. halys) and square point (P. maculiventris). Trendline fitted to the data (P < 0.0001; R2 = 0. 957, y = 0.000685x - 0.165). Area within dashes indicates 95% confidence interval.

2.3.7 Emergence from Parasitized Egg Masses

Similar to the exposed egg mass outcomes, no significant interaction was displayed between T. japonicus strain and exposed host species (df = 1, 53, F = 0.2661, P = 0.6082), indicating that parasitoid emergence from either host was not dependent on the parental host species of T. japonicus females used in the no-choice tests. The mean percentages of emerged T. japonicus differed significantly between exposed host species treatments (df = 1, 53, F = 12.568, P = 0.0009) (Table 2.3). As a whole, H. halys egg masses were found to be significantly more suitable than P.

42 maculiventris egg masses (P = 0.0015), as approximately 93% of parasitized H. halys egg masses, versus 50% of parasitized P. maculiventris egg masses, were considered suitable. Parasitized H. halys egg masses resulted in greater parasitoid emergence than parasitized P. maculiventris egg masses (P = 0.0171) for the P strain treatment. Also, H. halys egg masses were found to be significantly more suitable than P. maculiventris egg masses for P strain wasps (P = 0.0067). The percentages of emerged parasitoids for H strain T. japonicus in successfully parasitized egg masses were not significantly different between the two host species (P = 0.1968) (Table 2.3). Similarly, no significant difference in either host’s suitability was found for the H strain wasps (P = 0.143). These statistically insignificant findings for emerged parasitoid percentage and host suitability between H strain wasp treatments are an obvious result of widely differing sample size of parasitized egg masses.

2.3.8 Parasitoid Development Time No significant differences in the development time (number of days from T. japonicus oviposition to parasitoid emergence) were observed among the different treatments (df = 1, 53, F = 0.544, P = 0.6544) (Table 2.3).

43 Table 2.3: Mean percentage of emerged T. japonicus (± standard deviation), percentage suitability, and mean development time for T. japonicus per parasitized egg mass in no-choice tests. Asterisks indicate significant differences between P strain treatments (Fisher’s Exact test/ Bonferroni correction, *P < 0.0125; Tukey’s HSD **P < 0.05)

Parental host Exposed egg n parasitized % suitable % Emerged Development species mass species egg masses egg masses parasitoids time (days) (> 50% parasitism)

H. halys H. halys 18 (17) 94.4 84.8 ± 16.4 17.6 ± 2.83

P. maculiventris H. halys 22 (20)* 90.9 73.7 ± 18.6** 17.5 ± 1.53

H. halys P. maculiventris 6 (4) 66.7 69.2 ± 20.2 18.5 ± 1.97

P. maculiventris P. maculiventris 8 (3)* 37.5 44.1 ± 26.5** 17.1 ± 1.25

2.4 Discussion The results of our T. japonicus foraging behavior assays offer unique insight into how the egg parasitoid’s parental host species may influence its capacity to locate H. halys as well as at-risk, non-target pentatomid species. Differences observed between H and P strain residence times and linear walking velocities on kairomone- contaminated leaf substrates suggests some degree of host fidelity is present during the host foraging process of T. japonicus (Fig. 2.1, 2.2). H strain T. japonicus displayed clear preferences for the kairomones of its parental host, residing on H. halys- contaminated leaf surfaces for nearly twice as long as it did on P. maculiventris- contaminated leaf surfaces. However, these distinct preferences for the adult kairomones of its coevolved host seem to disappear when the parasitoid emerges from a suitable non-coevolved host, as P strain T. japonicus spent equal amounts of time on

44 leaf surfaces possessing either stink bug species’ chemical cues. The amplified response of P strain T. japonicus females to their parental host’s kairomones might be the consequence of the wasps’ exposure to parental host remains during and/or after eclosion (Corbet, 1985; Vet & Groenewold, 1990). Previous studies have proposed that early adult experiences, such as antennation with the natal host chorion, can play a key role in shaping the initial host-seeking behavior of parasitoids (Giunti et al., 2015). Since only naïve T. japonicus females were used, their parental egg masses would have been the only source of host-related chemical cues they came in contact with prior to encountering adult kairomones in the behavioral assays. If similar chemical compounds are present in both the host egg and adult footprint residues, it would reinforce the idea that early adult associative learning can influence the responsiveness of T. japonicus to adult contact kairomones while foraging for a suitable host. For this reason, chemical composition analyses of host egg and adult derived kairomone compounds are needed to distinguish any chemical similarities. From a host specificity perspective, the elevated behavioral responses of P strain females to adult P. maculiventris kairomones could indicate increased preference and/or motivation to search for its parental host, potentially leading to future non-target attacks on the native species. But a close inspection of the significant differences in linear walking velocity and in size between the two T. japonicus strains suggests the reverse (Fig. 2.2, 2.4). P strain females walked considerably more slowly than H strain wasps and showed no differences in linear walking velocity between the different kairomone treatments (including the control). In terms of size, H strain wasps possessed 30% longer right hind tibiae (RHT) and weighed approximately twice as much as P strain wasps (Table 2.2; Fig. 2.4).

45 Together these findings plausibly describe a positive relationship between T. japonicus size and walking speed, a common association observed in many parasitoid species (Bennet & Hoffman, 1998; Romeis et al., 2005; Abram et al., 2015). The relationship is noteworthy because walking speed has previously been used as a proxy for parasitoid vagility, namely the ability of a parasitoid to move between host patches within the same habitat (Visser, 1994). The slower walking velocities for smaller P strain T. japonicus females may then suggest a reduced ability to actively search for a host, which could negatively impact the rate of encountering their non-target parental host. The disadvantageous size-induced effects to P strain T. japonicus vagility were likely magnified by the pubescence of the soybean leaf surfaces used as kairomone substrates. The smaller P strain females clearly displayed difficulty while walking on the pubescent leaf surfaces. This observation parallels those of other egg parasitoid studies; for example, Trichogramma spp. were shown to walk more slowly and to parasitize fewer hosts on pubescent varieties of cotton than on glabrous varieties (Treacy et el., 1986; Keller, 1987). Overall, smaller T. japonicus females emerging from non-target hosts like P. maculiventris may have reduced host foraging abilities, especially within habitats containing plants with complex leaf surface morphologies, resulting in a potential decreased likelihood of future non-target parasitism. The absence of significant differences between H and P strain T. japonicus that were exposed to the same host is noteworthy when attempting to unveil possible effects parental host species may have on the parasitoid’s ability to successfully locate and parasitize H. halys or other physiologically suitable pentatomid host species. Both parasitoid strains displayed high levels of parasitism when exposed to H. halys egg masses and low levels of parasitism when exposed to P. maculiventris egg masses

46 (Table 2.1). Similar rates of parasitism between the two strains indicate that even when T. japonicus emerges from a non-target species, it still holds strong preference for its coevolved host H. halys. Also, low parasitism rates of P. maculiventris egg masses for P strain wasps imply that T. japonicus females emerging from a given non- target host species do not necessarily increase their preference for or parasitism success of that particular species. When attempting to accurately predict the possible negative effects T. japonicus non-target attacks may have on native stink bug populations, the fate of every egg within each non-target egg mass encounter should be considered. In our no choice tests, about 15% of all exposed P. maculiventris eggs resulted in the emergence of T. japonicus. Laboratory no-choice tests in general tend to overestimate the non-target effects of a biological control agent (Haye et al., 2005), so theoretically this low detected percentage of emerged T. japonicus could be further diminished in the field. After conducting no-choice tests and analyses of field- collected egg masses, Zhang et al. (2017) found that in its native range T. japonicus parasitized only 10% of non-target Plautia crossota (Stål) eggs in the field versus roughly 80% parasitism of the non-target’s eggs in no-choice tests. The sustained preference for H. halys in conjunction with low overall fecundity in P. maculiventris eggs for both parasitoid strains suggests the probability of an established P strain T. japonicus population in the field to be low. Studies examining the parasitism of field- collected P. maculiventris egg masses within the known US geographic regions of adventive T. japonicus populations would offer valuable additional insight into how reliable our no-choice test data is for predicting non-target effects. The significant differences in the levels of host suitability (= proportion of parasitized egg masses with > 50% parasitoid emergence) between H. halys and P.

47 maculiventris support the suggestion that P. maculiventris is a low quality host for T. japonicus (Table 2.3). High egg mortality percentages in P. maculiventris egg masses found in both parasitoid strains could be considered another indicator that the predatory stink bug is an unfavorable host (Table 2.1). Moreover, the significantly lower level of host suitability observed in the P strain wasps exposed to P. maculiventris eggs demonstrates that non-target host quality remains constant for T. japonicus regardless of the parasitoid’s parental host species. Similar findings in future no-choice tests exposing T. japonicus females reared on other at-risk non-target pentatomids to the egg masses of their respective parental hosts would validate the continuity of T. japonicus host preference and suitability displayed in our study. In addition, we only reared T. japonicus on P. maculiventris for one generation. Recognizable increases in host suitability and host preferences for a newly associated host may not be developed until a parasitoid completes successive generations within that host. For instance, two laboratory lines of braconid parasitoid Aphidius ervi (Haliday) reared continuously on either fox glove aphid, Aulacorthum solani (Kaltenbach), or pea aphid, Myzus persicae (Sulver), varied considerably in the parasitism rates of the two host species, with the fox glove A. ervi strain parasitizing significantly more fox glove aphids than the pea aphid strain (Henry et al., 2010). Repeating our no-choice tests using an older line of P strain wasps would determine the plasticity of T. japonicus host preferences and P. maculiventris host suitability. Low host suitability does not always translate into an egg parasitoid’s rejection of its encountered host. The generalist platygastrid parasitoid Telenomus podisi was shown to readily accept (i.e. oviposit within) H. halys eggs even though it was completely incapable of successfully developing within the invasive stink bug eggs

48 (Abram et al., 2014). Based on exhibited responses to adult contact kairomones in the behavioral assays and minor parasitism in the no-choice tests, we can conclude that T. japonicus has some ability to locate, accept, and parasitize P. maculiventris egg masses. However, our study did not integrate behavioral observations of T. japonicus when exposed to plant volatiles (e.g. HIPVs) or adult host species volatiles (e.g. pheromones and metathoracic secretions). These volatiles have been shown to incite pronounced effects on egg parasitoid host location by providing useful information concerning the general habitat where a host can be found (Michereff et al. 2013; Colazza et al., 1999; Conti & Colazza, 2012). In some cases, certain platygastrid species have confirmed their preferences for a coevolved host versus a non-coevolved host by responding more strongly to volatile kairomones produced by adults of the coevolved host (Conti et al., 2004; Salerno et al., 2006). Also, aggregation pheromones produced by adult male P. maculiventris have been shown to stimulate host-seeking behavior of its egg parasitoid Telenomus calvus (Aldrich et al., 1984; Bruni et al., 2000). Whether or not T. japonicus is stimulated by P. maculiventris pheromones or other adult volatiles has yet to be understood. Many pentatomid species share similar volatile semiochemical compounds, so it is plausible that adult H. halys and P. maculiventris volatiles may share chemical constituents, even though their aggregation pheromones differ (Weber et al., 2018). If so, T. japonicus may then be predicted to have some level of response to P. maculiventris adult volatiles, which could indicate its ability to accurately locate the non-target’s habitat. On the other hand, T. japonicus has already shown strong behavioral responses to the adult H. halys volatile n-tridecane (Zhong et al., 2017). Intriguingly, n-tridecane is also emitted as HIPV by common bean (P. vulgaris) plants in response to H. halys feeding (Fraga et

49 al., 2017). With these studies in mind, we can confidently accept that T. japonicus actively utilizes both adult volatiles and HIPVs to locate H. halys in the field. This cannot be completely said for T. japonicus searching for P. maculiventris since the non-target, although may facultatively feed on plants when prey is scarce, is predominantly a predaceous stink bug whose feeding does not promote the release of HIPVs. Thus, it can be predicted that T. japonicus will have more opportunities to successfully locate habitats containing H. halys due to the greater abundance of semiochemicals conveying the presence of its coevolved host. H. halys population dynamics and seasonality may also lead to the invasive pest’s higher probability of being located by T. japonicus over non-target species. H. halys polyphagy and omnipresence in woodlands, urban and fragmented landscapes, and agroecosystems facilitates its dominance as the primary pentatomid species in regions where it has definitively established (Nielsen & Hamiliton, 2009). Furthermore, H. halys spring emergence from overwintering sites has been documented between March and April in temperate regions of the US, but they do not begin laying eggs until June (Nielsen et al., 2008). Although data on the spring emergence and oviposition time periods has not been completely studied for P. maculiventris, simple population sampling studies have displayed early spring emergence and mid to late spring egg-laying (Aldrich et al., 1984; De Clercq, 2000). This hypothesized early first oviposition time period for P. maculiventris may act as temporal barrier to T. japonicus parasitism, as the egg parasitoid spring emergence would more likely be phenologically synchronized with its coevolved host. In its native range of northern China, T. japonicus rarely parasitized suitable hosts other than H. halys in May or June, and was found to be the predominant egg parasitoid of

50 H. halys, P. crossota, and D. baccarum in July, August, and September (Zhang et al., 2017). Mid to late season prominence of T. japonicus could signify synchronization with H. halys and other herbivorous pentatomids with similar life histories. The relative absence of T. japonicus during late spring and early summer months may also have considerable effects on its host foraging preferences. As mentioned earlier, associative learning is a major factor influencing parasitoid host foraging success and can determine the magnitude at which parasitoids search for a particular host (Vet et al., 1995; Vinson, 1998). Innate responses to the various aforementioned chemical cues during the host foraging process are reinforced and learned if the parasitoid is successful in locating a suitable host and oviposition occurs (Colazza et al., 2010). Behavioral analyses conducted by Peri et al. (2006) demonstrated that Trissolcus basalis females adjusted their responses to adult kairomone residues according to prior oviposition experience; stronger responses were observed after successful host location and parasitism, while decreased or weaker responses were a result after no host egg mass was encountered. If T. japonicus learns in a similar manner as its congener, we would expect host-seeking females to respond more strongly to semiochemicals associated with previously encountered hosts. The probability of H. halys being the initial host encountered by T. japonicus appears to be high, considering its likely phenological synchrony with H. halys. As a result, H. halys-related semiochemicals would provoke stronger behavioral responses during T. japonicus host foraging, and ultimately lead to future H. halys parasitism. Since P. maculiventris is able to lay eggs during the entirety of its life span, chances of the native being the initial host encountered by a T. japonicus female do exist (De Clercq, 2000). But due to the lower diversity of P. maculiventris semiochemicals compared to

51 those of H. halys, fewer opportunities to perceive the non-target chemical cues could dampen their reinforcement as reliable indicators of a suitable host and may consequentially be “forgotten” by individual T. japonicus females over time (Dauphin et al., 2009). Our behavioral observations and no-choice tests comparing two separate strains of T. japonicus provide novel techniques for assessing egg parasitoid host specificity. As seen in the past, biological control agents released to control invasive pests can contribute to unforeseen deleterious impacts on native fauna (Simberloff & Stiling, 1996). With a “devil’s advocate” approach to classical biological control host range testing, we were able to evaluate non-target risks of T. japonicus by viewing the candidate agent through a unique lens, a lens that can be described as proactive rather than reactive. By employing a creative method that simulates a relevant ecological scenario, we have gotten the proverbial ball rolling towards a more accurate host range prediction for T. japonicus. Future research exploring how parental host species influence T. japonicus host foraging behaviors and host preferences is needed to attain a complete understanding of the possible threats the parasitoid poses to native species and its overall potential to successfully control H. halys.

52 Chapter 3

DETERMINING HABITAT OVERLAP BETWEEN HALYOMORPHA HALYS AND PODISUS MACULIVENTRIS IN NORTHERN DELAWARE

3.1 Introduction The spatial and temporal coexistence of an invasive pest targeted for suppression, and a native species worthy of protection, is a serious concern for any biological control program’s non-target risk assessment. Certain native species may have increased risks of non-target attacks from a potential agent if they share similar habitats with the target species (Barratt et al., 2000). More often than not, biological control programs focus much of their efforts on laboratory host range testing to evaluate native species’ host suitability for a given agent (Louda et al. 2003). Although these host range evaluations garner crucial information for establishing an agent’s physiological host range, they alone cannot provide a comprehensive understanding of how an agent will behave in a new environment (Simberloff & Stiling, 1996). Many ecologists suggest incorporating various ecological factors that may influence a potential agent’s effectiveness in controlling the targeted pest when attempting to predict the risks of non-target effects (Louda, 1999; Strong & Pemberton, 2001). Seasonal monitoring of invasive species’ populations using pheromone traps has been shown to be an effective technique for identifying preferred habitats, landscape-level movement, and overall phenologies of exotic pests in their new geographic region (Bogich et al., 2008). Pheromone trapping has been especially

53 important for the brown marmorated stink bug (BMSB), Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), as this serious nuisance and agricultural pest continues to invade new regions across the United States, Canada, and Europe (Rice et al. 2014; Gariepy et al., 2014; Haye et al., 2014). Since its initial discovery in Allentown, Pennsylvania in 1996, H. halys has spread to 44 states causing major economic losses in a variety of crops systems (Hoebeke & Carter, 2003; NEIPM, 2017; Leskey et al., 2012a). Its extreme polyphagy and propensity to disperse considerable distances in short time frames has facilitated its invasion (Bergmann et al., 2016; Lee & Leskey, 2015; Wiman et al., 2014). Specifically, H. halys ability to develop on fruits of a wide spectrum of cultivated crops (e.g. apples, peaches, blueberries, tomatoes, peppers, sweet corn, and soybean), ornamentals (e.g. Paulownia tomentosa (Thunberg) (Paulowniaceae), Catalpa spp. (Bignoniaceae), and Ailanthus altissima (Mill.) Swingle (Simaroubaceae)), and natives (e.g. Acer spp. (Sapindaceae), Cercis canadensis (Linnaeus) (Fabaceae), Prunus serotina (Ehrhart) (Rosaceae), Juglans nigra (Linnaeus) (Juglandaceae), and Cornus florida (Linnaeus) (Cornaceae)) has promoted its ubiquity in agricultural systems, fragmented human landscapes, and unmanaged woodlands habitats (Rice et al., 2014; Lee et al., 2014; Joseph et al., 2014). The omnipresence of H. halys in a diversity of habitats provokes concern in connection with a variety of suppression tactics (Morrison et al., 2015). Ecologically deleterious pesticide application in agricultural systems is only sporadically effective at reducing local H. halys populations, because H. halys can avoid treated areas by utilizing unmanaged woodland habitats adjacent to crop systems (Kuhar et al., 2012; Blaauw et al. 2015). The invasive pest’s presence in natural forested habitats also

54 causes concern for potential biological control efforts, since many native stink bug species occur in woodland areas (McPherson, 1982). The egg parasitoid Trissolcus japonicus (Ashmead) (Hymenoptera: Scelionidae), is the primary natural enemy in the native range of H. halys, where it has been documented to parasitize 50 to 80% of H. halys egg masses found in the field (Qui et al., 2007; Talamas et al., 2013; Yang et al. 2009). For the last seven years, this parasitoid has been the subject of rigorous host range examinations in USDA-ARS quarantine facilities. Results from these host range tests have shown the parasitoid’s clear preferences for its coevolved host, but have also indicated the physiological suitability of several native pentatomid species (Hoelmer et al., unpublished; Hedstrom et al., 2017). Field studies in northern China have shown that T. japonicus several other pentatomid species occupying similar habitats with H. halys (Zhang et al., 2017). For this reason, a more detailed understanding of possible habitat overlap between H. halys and native stink bug species in invaded regions is needed to comprehensively assess the non-target risks associated with T. japonicus. The primary objective of the present study was to simultaneously monitor H. halys and native, beneficial Podisus maculiventris (Say) (Pentatomidae: Asopinae) activity over the growing season within different habitats. P. maculiventris is considered an important generalist predator of many agricultural pests, and is thought to be distributed among habitats which overlap with those of H. halys (McPherson, 1980; Culliney, 1986; Wiedenmann et al. 1994). This predatory stink bug is also a physiologically suitable host of T. japonicus, making it an at-risk species of non-target attacks. Spring activity for H. halys and P. maculiventris consists mainly of locating initial food sources, mating, and ovipositing the F1 generation (Saulich and Musolin,

55 2012). Here, we baited separate traps with synthetic aggregation pheromone lures known to attract the two species. Identifying any temporal or spatial habitat overlap between H. halys and P. maculiventris will provide important assistance with developing a reliable prediction of the non-target risks T. japonicus poses to native pentatomids.

3.2 Materials and Methods

3.2.1 Trap Design and Deployment

Pheromone traps used for each species consisted of 3.785 L transparent cylindrical plastic containers (Uline, Pleasant Prairie, Wisconsin) with two holes cut on opposite sides where a conical piece of plastic screening was inserted projecting inward. For P. maculiventris, traps were baited with rubber lures that had been infused with a blend of (E)-2-hexenal and racemic α-terpineol, a synthetic mimic of the male sex pheromone (provided by Ashot Khrimian, Beltsville Agricultural Research Center, Beltsville, Maryland) (Aldrich et al., 1978). Baits were replaced once per week. In 2016, H. halys traps were baited with methyl (E,E,Z)-2,4,6- decartienoate (MDT) (Phercon®, Trécé Incorporated, Adair, Oklahoma), a synthetic aggregation pheromone for Plautia stali Scott (Hemiptera: Pentatomidae), which is cross attractive to H. halys (Khrimian et al., 2008). The MDT lures were replaced every four weeks. The pheromone lure type was changed between 2016 and 2017 in order to maintain consistency with other H. halys trapping studies being conducted throughout the US. In 2017, H. halys traps were baited using a combination of MDT and two components of the H. halys aggregation pheromone, (3S,6S,7R,10S)-10,11- epoxy-1-bisabolen-3-ol and (3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol (AgBio,

56 Inc., Westminster, Colorado). Due to its controlled release formula, the combination lure was replaced every 12 weeks as recommended by the manufacturer. At each location, traps for both species were hung from trees approximately 2 m above the ground and separated by about 50 m. In 2016, traps were deployed from 28 March to 19 October. In 2017, traps were deployed from 15 March to 20 October.

3.2.2 Study Sites Trap locations were chosen based on specific criteria. A preliminary pheromone trapping study for P. maculiventris conducted throughout northern Delaware (New Castle County, Delaware) in 2015 provided initial information on the local P. maculiventris populations. We partitioned our study sites into three distinct habitat types: (1) dense woodlands, (2) fragmented or urban landscapes, and (3) agricultural systems. Our 2015 data reflected findings from previous studies, suggesting deciduous forests and the areas bordering them were the predominant sites for P. maculiventris overwintering (McPherson, 1982). Also, deciduous woodlands and natural areas in northern Delaware contain many native woody host plants of H. halys, notably Acer spp., Cercis canadensis, Cornus florida, Juglans nigra, and Prunus serotina (NEIPM, 2017). As mentioned earlier, agroecosystems are broadly used habitats for both P. maculiventris and H. halys, so agricultural systems possessing at-risk crops were chosen. Wooded borders near our selected agricultural study sites commonly contained non-native plants known to be important H. halys hosts such as Ailanthus altissima, Morus alba, Paulownia tomentosa, and Catalpa spp. To maximize the ecological consistency among sites within the same habitat type, we maintained several spatial constants. All woodland sites were at least 100 m from the forest edge in all directions. Fragmented and managed landscape locations

57 were all located in state natural areas and county parks where common landscaping practices (e.g. weekly mowing) were performed and were in close proximity to large residential areas. Traps in agricultural systems were hung from trees in unmanaged edge habitats adjacent to cultivated crop cultures. In 2016, three separate sites were chosen for each habitat. Dense woodland patches separated by at least 2 km within White Clay Creek State Park (Newark, DE) were chosen for the interior forest sites. For the fragmented landscape sites, traps were placed in two separate areas of Fair Hill Natural Resource Management Area (Elkton, MD) and one location at Middle Run Natural Area (Newark, DE). For our agricultural sites, we selected two distinct locations at Milburn Orchards (Elkton, MD), one adjacent to a perimeter row of apple and the other bordering an edge row of peach. The third agricultural trap site was located on the edge of a corn field on the University of Delaware farm (Newark, DE). In 2017, four separate locations were used for each habitat type. In addition to the three interior forest sites at White Clay Creek SP, a fourth site was selected in Iron Hill Park (Newark, DE). Previously selected sites within Fair Hill Natural Resource Management Area and Middle Run Natural Area were used again for fragmented landscape habitats. Two new fragmented landscape locations, Carousel Park (Wilmington, DE) and Judge Morris Estate (Newark, DE), were added. Along with the three trap sites at Milburn Orchards and the University of Delaware farm, Orzada Farms (Newark, DE) ornamental tree nursery was selected as a fourth agricultural site. This nursery possessed many genera of trees (e.g. Acer, Prunus, Ilex, Ulmus, and Zelkova) that are known to be used by H. halys for feeding and oviposition. In both

58 years, traps were checked twice a week through May, followed by once per week until the last sampling date in October. The number of P. maculiventris and H. halys adults caught in traps at each site was recorded. The total number of stink bugs caught was then divided into two sampling periods corresponding to when they were collected: (1) early spring, the last week of March through the third week in April (24 March to 23 April) and (2) late spring, the last week of April through the third week of May (24 April to 23 May). The specific sampling periods were selected on the basis of consistent collections of adults and according to the hypothesized temporal overlap in overwintering emergences of both species. For both years, the number of stink bugs collected from 1 June to the end of the sampling period were not used in our statistical analyses as very few P. maculiventris adults (< 30) and large numbers of H. halys (> 1500) adults were collected. These striking collection disparities were expected since P. maculiventris adults of the F1 generation do not respond to the pheromone used (Aldrich et al., 1984), and while late summer is considered the peak for the H. halys populations when majority of the F1 generation have reached the adult life stage (Nielsen and Hamilton, 2009). Habitat and sampling time frame were defined as treatments with individual traps per habitat and number of collection dates per sampling time frame as replicates.

3.2.3 Statistical Analysis The number of stink bugs collected varied widely among each trap location and collections dates within each sampling period, including many zero counts. The prevalence of zero values for counts per collection date created heteroscedasticity in the raw data sets for both years. To minimize zero data counts, the number of adult

59 bugs collected from all traps were pooled as total counts per habitat on a given collection date. This data summation method did not eliminate all zero data counts, and differences between treatment variances still produced non-normal distributions

(Shapiro-Wilk test, � = 0.05). A constant (1.5) was added to each summed count to achieve positive, non-zero values so a logarithmic transformation could be performed (McDonald, 2014). Once the data was normalized, it was then subject to a three-way analysis of variance (ANOVA) in order to compare effects of species, habitat type, and sampling period on our observed stink bug counts, as well as to identify significant interactions between the three nominal variables. Multiple comparisons were also performed using a Tukey’s Honest Significant Difference (HSD) test (� = 0.05) to determine differences between variable means. Comparisons between 2016 and 2017 collection data were not performed since they differed in the exact locations and number of study sites, and the lures used for H. halys. Statistical analyses were completed using JMP Pro 13.0.0 Statistical Software for Mac (SAS Institute Inc., Cary, NC).

60 3.3 Results

2016 2017 Woodland Fragmented Agriculture Woodland Fragmented Agriculture Podisus

Early Spring 169 26 21 163 53 10

Late Spring 11 12 2 49 23 14

June & July 10 4 0 0 1 0

August to 3 4 1 1 2 1 October

A Spring Total 180 38 B 23 B 212 A 76 B 24 B

Season Total 193 46 24 213 79 25 H. halys

Early Spring 49 0 0 126 0 5

Late Spring 134 16 3 182 16 62

June & July 2 3 0 1 3 7

August to 616 634 446 118 853 942 October

Spring Total 183 A 16 B 3 B 308 A 16 B 67 B

Season Total 801 653 449 427 872 1016

61 3.3.1 2016 A total of 241 P. maculiventris adults were caught in nine pheromone traps from 24 March to 31 May. Of the 241, 216 adults were caught in early spring (24 March to 23 April), while only 25 adults were caught in late spring (24 April to 23 May). For H. halys, 202 adults were collected overall with 49 and 153 adults captured in early spring and late spring, respectively. The first P. maculiventris individuals of the spring were collected on 28 March at two of the three woodland sites. Eighteen days later, the first H. halys adults were trapped on 12 April in the same woodland locations as the initial P. maculiventris individuals.

3.3.1.1 Habitat

The number of P. maculiventris caught in traps differed significantly among the three habitats (df = 2,47, F = 6.734, P = 0.0028) (Table 3.1). There were also significant differences between the capture totals of H. halys among the three habitats (df = 2,47, F = 15.51, P < 0.0001). Trap captures of both H. halys and P. maculiventris were significant greater in the woodland sites than in the fragmented or agricultural sites (P < 0.05). No differences in the number of trapped stink bug adults of either species were found between fragmented and agricultural locations (Table 3.1).

62 50

40 H. halys

40 Podisus Number of adults captured of adults Number

0 5/11 3/24 3/28 4/01 4/06 4/09 4/12 4/15 4/19 4/23 4/26 4/29 5/03 5/07 5/16 5/20 5/23 Early Spring Late Spring Date Figure 3.1: Total 2016 H. halys and P. maculiventris adults collected during the early spring and late spring sampling periods in woodland (black), fragmented (orange), and agriculture (blue) habitats.

3.3.1.2 Sampling Period Among all three habitats, a highly significant interaction was observed between the number of P. maculiventris and H. halys collected during the early spring and late spring sampling periods (df = 1, F = 38.62, P < 0.0001). In other words, the number of P. maculiventris and H. halys adults collected was dependent on the sampling period when they were caught. Traps caught more P. maculiventris adults in early spring than in late spring (P < 0.05) (Fig. 3.2). Also, more P. maculiventris adults were caught in early spring than H. halys adults. In late spring this relationship was reversed, as greater numbers of H. halys were trapped than in early spring.

63 Similarly, significantly more H. halys were caught in traps than P. maculiventris in late spring.

Woodland Fragmented Agriculture 30

A 25

20 ns ns

15 Number of adults captured of adults Number 10 B

0 Early Spring Late Spring Early Spring Late Spring Early Spring Late Spring Sampling Period Figure 3.2: 2016 mean captures of H. halys (grey) and P. maculiventris (striped) at each habitat during early and late spring. Bars indicate ± SE. Letters denote significantly different capture means of each species during both sampling periods in woodlands. (Tukey’s HSD, P < 0.05). ns indicates no significant differences between the capture means within habitat treatments.

3.3.1.3 Habitat, Sampling Period, and Species

Notably, a three-way ANOVA found all three independent variables to have a significant interaction on the number of stink bug adults collected (df = 2, F = 13.82, P < 0.0001). This significant interaction suggests that the presence of H. halys and P.

64 maculiventris in woodland, fragmented, and agricultural habitats is dependent on when the two species are surveyed in the spring. In early spring, significantly more P. maculiventris adults were caught in woodlands than H. halys adults (P < 0.05). On the other hand, significantly more H. halys adults were collected in woodlands in late spring than P. maculiventris adults. H. halys captures increased significantly in woodlands between early and late spring, while P. maculiventris captures in woodlands decreased significantly from early spring to late spring. No significant differences in the catches of either species during the two sampling periods were found in fragmented or agricultural habitats.

3.3.2 2017 From 23 March to 31 May, 312 P. maculiventris adults were collected in twelve pheromone traps. In early spring (24 March to 23 April) 226 adults were collected, while 86 adults were collected in late spring (24 April to 23 May). A total of 391 H. halys adults were trapped, with 260 and 131 adults trapped in early spring and late spring, respectively. Larger overall collections in 2017 compared to 2016 could be due to the addition of 3 new trap locations and the change in the H. halys pheromone used. The earliest P. maculiventris individuals were collected on 3 April in traps within all three habitats. On 6 April the first H. halys individuals were caught in only one woodland trap site. Surprisingly, this location was the only woodland site where H. halys was found until the last collection date of the early spring sampling period.

65 3.3.2.1 Habitat Similar to the 2016 results, highly significant differences among habitats was observed in the number of collected H. halys (df = 2,47, F = 6.499, P = 0.0033) and P. maculiventris (df = 2,47, F = 6.236, P = 0.0041) adults (Table 3.1). Again, traps in the woodland habitats collected greater numbers of both stink bug species than traps in the fragmented and agricultural habitats (P < 0.05).

140

120

100 H. halys

140

120 P. maculiventris

100 Number of adults captured 80

0 3/24 3/28 4/03 4/06 4/09 4/12 4/15 4/19 4/23 4/26 4/29 5/03 5/07 5/11 5/16 5/20 5/23 Early Spring Late Spring Date Figure 3.3: Total 2017 H. halys and P. maculiventris adults collected during the early spring and late spring sampling periods in woodland (black), fragmented (orange), and agriculture (blue) habitats.

3.3.2.2 Sampling Period

A significant interaction was found between the number of H. halys and P. maculiventris adults trapped across the three habitats and the sampling period when

66 they were caught (df = 1, F=5.577, P = 0.0205). Although this interaction implies that the number of adults collected of either species is dependent on the time frame when they were collected, this relationship was not perceived in multiple comparison analyses as no differences in the number of H. halys and P. maculiventris adults was seen for early or late spring (Fig. 3.4).

Woodland Fragmented Agriculture 40

35 ns ns ns 30

15 Number of adults captured of adults Number

0 Early Spring Late Spring Early Spring Late Spring Early Spring Late Spring Sampling Period Figure 3.4: 2017 mean captures of H. halys (grey) and P. maculiventris (striped) at each habitat during early and late spring. Bars indicate ± SE. ns indicates no significant differences between the capture means within habitat treatments.

67 3.3.2.3 Habitat, Sampling Period, and Species Unlike our 2016 trap collection data, no three-way interaction was observed between the species of stink bug, habitat, and sampling time (df = 2, F = 0.0591, P = 0.943). This is clearly evident considering there were no significant differences in the number of either species collected across the three habitats during the entire spring survey (df = 1, F = 1.071, P = 0.3037), as well as between the early and late spring sampling periods (df = 1, F = 0.121, P = 0.729).

3.4 Discussion

Our two-year pheromone trap study illustrates how a biological control program can integrate pheromone trapping of both the targeted pest and a native species into the non-target risk assessment of a potential biological control agent. Overall, we observed varying degrees of spatial and temporal overlap between the spring phenologies of H. halys and P. maculiventris. In both years, traps located in unmanaged woodland habitats accounted for an overwhelming majority of the total number of stink bug adults collected (Table 3.1). These results are not surprising, as previous research has suggested H. halys and P. maculiventris overwinter in forested landscapes under bark, within dead, standing trees, and in leaf litter (Lee et al., 2014; Aldrich et al., 1984; Herrick & Reitz, 2004). P. maculiventris was trapped more often than H. halys in forested habitats during early spring (24 March to 23 April) in 2016 (Fig. 3.2); however, H. halys was the species trapped most often in forest sites during late spring (24 April to 23 May). These differences in early and late spring captures between the two species were not observed the following year (Fig. 3.4). Such a dichotomy between years makes it difficult to interpret temporal separation between the two species during their spring activity.

68 A change in one of our woodland trap locations in 2017, due to inaccessibility to a large portion of White Clay Creek State Park (WCCSP), seemed to have a sizable effect on the number of H. halys adults collected in 2017. The new 2017 trap location, although less than 2 km from the original site used in 2016, captured approximately 86% of all H. halys adults in woodlands, including nearly all (≈ 98%) H. halys caught in early spring (Table 3.1; Fig. 3.4). While still meeting our required 100 m from a distinct forest edge, the new location was by far the closest trap site in WCCSP to residential neighborhoods. The site’s proximity to human-made structures may explain the skewed abundance of H. halys, as the invasive bug is notorious for overwintering in structures such as houses, commercial buildings, and barns (Watanabe et al., 1994; Hamilton, 2009; Inkley, 2012). Furthermore, this trap site was the only location in our entire study where H. halys was collected before P. maculiventris. This finding may further emphasize the effects residential areas adjacent to woodlands have on the spring habitat selection of H. halys. Following their emergence from overwintering sites, H. halys adults begin to disperse in search of host plants for initial feeding and mating (Wang & Wang, 1988; Hoebeke & Carter, 2003). Most agricultural crops are not available during early spring, so H. halys’s ability to utilize wild hosts throughout their invaded US range is critical for their early season reproductive success. Many well-known wild hosts of H. halys such as J. nigra, C. canadensis, and P. serotina were common species found in our WCCSP woodland habitat locations (NEIPM, 2017). Interestingly, the only woodland site not in WCCSP, located in Iron Hill Park (Newark, DE), captured just one individual during the spring sampling periods. This park is an isolated forest patch dominated by non-host hardwood species (e.g. beech, sycamore, oak), and therefore

69 may not offer adequate forest habitat for H. halys. On the other hand, P. maculiventris was consistently collected in Iron Hill Park throughout April 2017. The predatory stink bug was not captured as often at this particular site as it was in WCCSP, but its presence within this forest patch with few H. halys hosts may signify spatial separation between the two species. This inference cannot be confidently supported by our limited sample size, so future P. maculiventris spring pheromone trapping in forest habitats lacking ample H. halys host plants are needed to verify this hypothesis. Our study echoes the conclusions made from previous H. halys phenology fieldwork, recognizing unmanaged woodland areas in the mid-Atlantic as important refuge habitat for H. halys populations (Lee et al., 2014; Bakken et al., 2015). Spring habitat partitioning in forested areas allows H. halys to escape current control methods, such as pesticide applications, allowing females to mature, mate, and lay eggs without human suppression. The introduction of the candidate biological control agent Trissolcus japonicus is one of few promising control methods that may prove effective in these unmanaged woodland areas. Before T. japonicus can be released to control H. halys, the egg parasitoid’s behavioral ecology and population dynamics in its native range should be adequately studied so its potential efficacy as a biological control agent, and its risk to native pentatomid species, can be confidently predicted (Duan & Messing, 1997). Aiming to distinguish the ecological host range of T. japonicus in its native range, Zhang et al. (2017) examined naturally laid H. halys and other pentatomid egg masses collected in forested habitats and confirmed T. japonicus as the predominant egg parasitoid of H. halys egg masses. Moreover, their sentinel egg mass surveys conducted from May to September displayed temporal patterns of parasitism, as the

70 majority of T. japonicus parasitism events occurred in July and August. Knowing that T. japonicus successfully parasitizes H. halys in woodland habitats in China, we can make an informed prediction that it will behave in a similar fashion in forests such as the ones surveyed in the present study. Yet, without a complete grasp of native pentatomid oviposition preferences, its potential non-target effects on native species co-occurring in these wooded habitats remains unknown. In the eastern US, it has been suggested that, as a function of degree-day accumulation, mature H. halys females begin to lay eggs in late May or early June on a wide spectrum of host plant species and continue egg-laying until roughly mid- August (Nielsen et al., 2008). Unfortunately, P. maculiventris oviposition in natural landscapes has been poorly studied, with anecdotal evidence describing female egg- laying beginning 1-2 weeks after early spring emergence and continuing throughout the female’s lifetime (De Clercq, 2008). Considering that P. maculiventris is a generalist predator that feeds primarily on Coleoptera and Lepidoptera larvae and can be found across a variety of habitats, it may not have specific locations or host plant preferences for its eggs (Culliney, 1986: Wiedenmann et al., 1994). Our trapping data from both years confirms that P. maculiventris occurs in a variety of habitats during its spring activity, but resides mainly in forested landscapes until the middle to end of May. Another pattern demonstrated in both years of our study is the earlier initial trapping dates for P. maculiventris compared to H. halys (Fig. 3.1, 3.3). If P. maculiventris begins laying eggs not long after emerging from overwintering sites, while H. halys females require longer spring maturation before they can lay eggs, it is possible that their initial oviposition may be temporally separated.

71 The present study demonstrates an effective technique for monitoring the spring emergence of H. halys and P. maculiventris. Even though our results are not consistent across years, clear patterns of habitat overlap in unmanaged woodlands are evident between the two species. To date, no other studies have attempted to compare the spring emergence patterns of H. halys and native stink bug species within different habitats simultaneously. Future research is needed to better understand seasonal co- occurrences of P. maculiventris in wild habitats known to be H. halys reservoirs.

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