(Diptera: Tachinidae) of Stink Bugs (Hemiptera: Pentatomidae) in Southwest Ohio

DETERMINANTS OF HOST USE IN TACHINID PARASITOIDS (DIPTERA: TACHINIDAE) OF STINK BUGS (HEMIPTERA: PENTATOMIDAE) IN SOUTHWEST OHIO

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science

MATTHEW W DUNCAN B.S., Wright State University, 2014

2017 Wright State University

WRIGHT STATE UNIVERSITY

GRADUATE SCHOOL

April 7, 2017

I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Matthew W Duncan ENTITLED Determinants of host use in tachinid parasitoids (Diptera: Tachinidae) of stink bugs (Hemiptera: Pentatomidae) in Southwest Ohio BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science.

______John O Stireman III, Ph.D. Thesis Director

______David L. Goldstein, Ph.D., Chair Department of Biological Sciences

Committee on Final Examination

______John O Stireman III, Ph.D.

______Donald F. Cipollini, Ph.D.

______Jeffrey L. Peters, Ph.D.

______Robert E.W. Fyffe, Ph.D. Vice President for Research and Dean of the Graduate School

ABSTRACT

Duncan, Matthew W. M.S., Department of Biological Sciences, Wright State University, 2017. Determinants of host use in tachinid parasitoids (Diptera: Tachinidae) of stink bugs (Hemiptera: Pentatomidae) in Southwest Ohio.

Tachinid parasitoids in the subfamily Phasiinae are important natural enemies of heteropteran bugs. Host location by these flies occurs via antennal reception to the pheromones of their hosts; however little is known regarding the mechanisms which underlie host selection. Halyomorpha halys, the invasive brown marmorated stink bug, represents a potential novel host species in North America. This study was conducted to determine the suitability of H. halys as a host for phasiine species, and to assess cues used in host selection by the species Gymnoclytia occidua. Field attraction to pentatomid pheromones by both phasiines and pentatomids in Southwest Ohio were investigated and preliminary laboratory host-selection experiments were conducted. In 2015, from June 23 to September 16 pyramid-type traps were baited with three pentatomid-pheromone lures and were monitored in agricultural and semi-natural locations. Trap catches included specimens from seven different phasiine species and three different pentatomid species. Host movement is an important factor in parasitoid attraction to host models, this attraction was not affected by pheromone presence, choice and no-choice trials indicate that Gymnoclytia occidua females do not discriminate against H. halys. However, no parasitoids were successfully reared from H. Halys. Field parasitism by a Gymnoclytia occidua female on H. halys was directly observed, and both adults and nymphs of H. halys were found bearing parasitoid eggs in the field. These results suggest that H. halys may be a “sink” for Gymnoclytia occidua and possibly other native phasiine parasitoids in North America.

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TABLE OF CONTENTS

Page

INTRODUCTION………………………………………………………………………..1

The Parasitoid Lifestyle of Tachinidae……………………………….1

Using Tachinids as Biological Control Agents………………………3

Invasive Species………………………………………………………..5

Brown Marmorated Stink Bugs (H. halys), an Emerging Threat….8

Subfamily Phasiinae: Tachinids Parasitoids of Stink Bugs….…...13

Objectives and Hypotheses…………………………………………..15

METHODS……………………………………………………………………………...19

PHEROMONE ATTRACTION IN THE FIELD……………………………...19

Background……………………………………………………………19

Study Area……………………………………………………………...19

Trap Construction……………………………………………………..20

Sampling Periods………………………………………………………20

Data Analysis…………………………………………………………..22

OVIPOSITION ASSAYS……………………………………………………...22

Background……………………………………………………………22

Collecting and Rearing Specimens…………………………………24

Model Host Trials………………………………………………………26

Live Host Trials………………………………………………………...28

Host Suitability (Native vs. Non-native)…………………………..…30

Data Analysis…………………………………………………………..31

RESULTS………………………………………………………………………………32

PHEROMONE ATTRACTION…………………………………………….…32

OVIPOSITION ASSAYS…………..……………………………………….…34

Model Host Trials………………………………………………………34

Live Host Trials……………………………………………………...... 35

Host Suitability (Native vs. Non-native)……………………………..35

DISCUSSION…………………………………………………………………………..37

FIGURES……………………………………………………………………………….44

TABLES…………………………………………………………………………………59

REFERENCES…………………………………………………………………………63

LIST OF FIGURES

Figure Page

1. Spread of the invasive species, Halyomorpha halys, in N. America…...... 44

2. Life cycle of the tachinid parasitoid, Gymnoclytia occidua……....….……..45

3. Map of sampling area…………………………...………….…..…..….………46

4. Pyramid trap assembly…………………………………………...... ………..47

5. Pentatomid pheromone molecule structures…...………………...…………48

6. Collected tachinid species…………………………………………..………...49

7. Insect rearing chambers………………………………………...…..………...50

8. 3d-printed stink bug models and the oviposition arena….….…...………..51

9. Rearing containers of parasitized hosts……………………..…...…………52

10. Total insects attracted to traps (treatments)...….……..………….………..53

11. Total insects attracted to traps (field sites)…….……………………………54

12. Effect of pheromone presence vs. absence on host choice……….……...55

13. Development success of Gymnoclytia occidua larvae……….…………...56

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14. Parasitism rates of Gymnoclytia occidua in the field………………….…..57

15. Oviposition assays results…………………………………………………..58

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LIST OF TABLES

Table Page

1. Sampling locations……..……………………………………………….…….....59

2. Total insects attracted to traps (treatments)………………...………………..60

3. Total insects attracted to traps (field sites)………..………...……….……….61

4. Summary of pheromone attraction results………..…………………………..62

INTRODUCTION

The Parasitoid Lifestyle of Tachinidae

Parasitoid insects undergo their development as parasites of other organisms, killing their host in the process. While a true parasite typically develops on a host without killing it, the development of a parasitoid nearly always results in either sterility or death of the host (Godfray, 1994). It is estimated that approximately 10% of all insects are parasitoids (Eggleton and

Belshaw, 1993). Of this, more than half are parasitoid wasps belonging to the order Hymenoptera (Godfray, 1994). The second most important group of parasitoids are the true flies (Insecta: Diptera) where the lifestyle has evolved in numerous families, most notably, of course, in the family Tachinidae.

Tachinids are a species rich family of true flies (Insecta: Diptera). They are parasitoids that play important roles in shaping the ecological communities of their arthropod hosts (Stireman et al., 2006). Currently there are estimated to be around 8500 described species of Tachinidae; however the total number of species could be twice as high (O’Hara, 2013).

Tachinid larvae are classified as koinobiont parasitoids, which means that their hosts continue feeding and growing while they develop internally (Askew &

Shaw, 1986). They keep their hosts alive by feeding on the haemolymph and non-vital tissues until eventually transitioning to the vital organs. Upon reaching the end of their larval stage they emerge from the host’s body to pupate, killing it in the process (Cerretti et al., 2014; Stireman et al., 2006).

Patterns of host use among Tachinidae can be explained in terms of both ultimate and proximate causes. At the ultimate level the determinants of host use are the evolutionary and historical reasons why a tachinid uses a particular host.

Specifically, tachinids should use hosts that provide their offspring with the best chances of surviving into adulthood and passing on their genes. Adult tachinids, therefore, are expected to have evolved adaptations that make it easier for them to find and discriminate between suitable and unsuitable hosts. At the proximate level, several related processes, driven by behavioral and physiological mechanisms, determine host use in tachinids. These processes include finding the host (host location), choosing to attack the host (host selection), and the ability to develop within the host (larval performance) (Vinson, 1976).

During host location tachinids respond to a hierarchy of sensory stimuli which reveal increasingly specific clues as to the whereabouts of potential hosts

(Godfray, 1994). This typically occurs with the parasitoid first identifying and travelling to the general area where the hosts are located (i.e., host habitat location), and is facilitated by either the recognition of direct host-derived stimuli, indirect stimuli obtained from the food of the host, or a combination of both

(Godfray, 1994; Vet et al., 1991). Parasitoids that have evolved the ability to detect long-range host-derived cues such as intraspecific communication signals

(e.g., pheromone molecules, mating calls, etc.) are essentially able to bypass the host-habitat search altogether and home in on the location of the hosts directly.

As an example, tachinid flies in the tribe Ormiini have evolved tympanal hearing organs which allow them to phonotactically orient towards the mating calls of their orthopteran hosts (Cade, 1975; Robert et al., 1996). Similarly, members of the tachinid subfamily Phasiinae use the male-produced sex pheromones of their hosts as host-finding kairomones (Aldrich et al., 1995; Aldrich et al., 2006).

Kairomones are chemical substances emitted by an organism that get intercepted by another organism, usually at the expense of the emitter.

Since many of their hosts are herbivores, tachinids are valuable both in terms of ecosystem health and as biological control agents to combat invasive pest insects. They can be used as model organisms for studying a number of ecological concepts such as tritrophic interactions, rapid diversification, host- finding behaviors, host range and host specificity. This is primarily because they are such a diverse group of parasitoids, and there is enormous variation in host range among species (Stireman et al., 2006; Eggleton & Gaston, 1992). Many tachinids are reported to have multiple host associations, and some are highly polyphagous. For example, the tachinid species Compsilura concinnata is capable of using nearly 200 species of hosts (Arnaud, 1978).

Using Tachinids as Biological Control Agents

Much of what we know regarding the behavior, ecology, and evolutionary history of tachinids is due to studies investigating their potential use in pest

3 management applications (Grenier, 1988). Many tachinid species have been used successfully as biological control agents to reduce invasive insect populations. The first use of tachinids for applied biological control dates back to the beginning of the 20th century (DeBach, 1974). Since then, over 100 tachinid species have been used for this purpose (Greathead, 1986; Grenier, 1988).

Some of the pests that tachinids have been recruited to control include gypsy moth, brown tailed moth, coconut moth, corn earworm (Grenier, 1988), winter moth (Kimberling et al., 1986), European corn borer (Baker et. al, 1949) and sugar cane borer (DeBach, 1974). Many more tachinid species could potentially be released as bio-control agents against future pest species, or to enhance existing controls. It is vital however, that knowledge regarding the determinants of host use for a potential biological control candidate is established prior to its use in any integrated pest management (IPM) strategy.

The potential negative effects of introducing a parasitoid into a new environment for the purposes of controlling one or more target species must be thoroughly considered. Non-target effects and other undesirable ecological consequences may result if adequate research is not conducted to determine host-specificity and ecological risk (Johnson et al., 2005). The successes of a biological control program will be overshadowed if the parasitoid begins targeting other beneficial insect species as well. For example, the tachinid C. concinnata, mentioned earlier, was one of the species introduced in North America to control gypsy moths. It has since been implicated in the decline of a number of nontarget species, including the giant silk moths (Lepidoptera: Saturniidae; Boettner

4 et al., 2000; Kellogg et al., 2003). Regardless of the past failures and risks associated with using tachinids as biological control agents, they still offer one of the best means of controlling invasive insect pests without requiring increased pesticide use, which can be expensive and environmentally damaging.

Invasive Species

An invasive species is defined as “a species that is non-native to the ecosystem under consideration, and whose introduction causes or is likely to cause economic or environmental harm or harm to human health” (Beck et al.,

2008). In the United States it is estimated that approximately 50,000 species are non-native (Pimentel et al., 2004), and of those approximately 4,300 are considered to be invasive (Corn et al., 1999).

The economic harm caused by these nonindigenous invaders is substantial. In 2004, invasive species were responsible for approximately $120 billion in damages and control costs in the United States alone; invasive insects and other arthropod pest species were responsible for around 16.6% of that cost, or roughly $20 billion (Pimentel et al., 2004). The environmental damage that they cause is equally nontrivial. Biotic invaders can cause or hasten the extinction of native flora and fauna (Mack et al., 2000). In 1998, out of 958 species that were listed as being threatened or endangered, approximately 400 were classified as such because of competition with or predation by invasive species (Wilcove et al., 1998).

Invasive species can also have positive and negative effects on native parasitoids (Chabaane et al., 2015). For instance, native female parasitoids might lay their eggs on a novel invasive species if it bears similar traits to native hosts. If suitable for larval development then the parasitoids would benefit by having access to more host options, and their populations would increase and they may contribute towards controlling the invasive host. If unsuitable, then parasitoids attempting to use it may experience a population decline, as the novel host would be acting as an egg sink (Abram et al., 2014; Davis & Cipollini,

2014). Analogous situations involving plant-insect interactions have been observed in which phytophagous insects show a greater preference for an unsuitable invasive plant over a suitable indigenous plant. For example, Davis and Cipollini (2014) found that Pieris virginiensis butterflies prefer to place more eggs on invasive Alliaria petiolata (garlic mustard) than on their indigenous host plant Cardamine diphylla, despite the fact that A. petiolata is toxic to their developing larvae.

A number of parasitoid species have also been observed placing their eggs on unsuitable hosts. In North America, the wasp Dinocampus coccinellae

(Hymenoptera: Braconidae) does not discriminate between the invasive host species Harmonia axyridis (Coleoptera: Coccinelidae) and its native host

Coleomegilla maculata (Coleoptera: Coccinelidae) (Firlej et al., 2010;

Hoogendoorn and Heimpel, 2002). Larval development is successful on C. maculata but unsuccessful on H. axyridis due to high levels of haemocytic encapsulation (Firlej et al. 2012). Since D. coccinellae wasps do not discriminate

6 against the unsuitable invasive host, the number of eggs it can place on its suitable host, C. maculata, is limited. The presence of H. axyridis therefore directly causes D. coccinellae populations to decline due to mistaken oviposition events, and indirectly causes C. maculata populations to increase by reducing the pressure from parasitoid attacks (Heimpel et al. 2003).

Many of the traits that allow invasive species to outcompete their native counterparts could also represent supernormal stimuli that elicit increased host selection behaviors in some parasitoids. For example, if an invasive host is larger and more active than other neighboring individuals, then it might get chosen more frequently by a host-seeking parasitoid. The preference-performance hypothesis (also referred to as the ‘mother knows best’ hypothesis) states that female preference should reflect offspring performance (Gripenberg et al., 2010).

Although this is typically used in reference to host-plant preferences of phytophagous insects, it can just as easily apply to parasitoid host preferences, in which a female would likely view larger, more active hosts as healthier, and therefore a better investment for her larvae.

Tachinid flies in the subfamily Phasiinae exclusively attack Heteroptera, primarily stink bugs (Pentatomidae) and leaf-footed bugs (Coreidae). Many of their hosts are significant pests of cultivated and uncultivated host plants

(Panizzi, 1997) making phasiines important natural enemies in agricultural and natural habitats. Other hosts of these flies are either less important as pest species or are not pests at all, and parasitism pressure from the flies may contribute to keeping some of these species from reaching pest levels.

Recently, the invasive species Halyomorpha halys, commonly referred to as the brown marmorated stink bug (BMSB), was accidently introduced into

North America. As a brown-colored stink bug it is similar morphologically to many of North America’s indigenous stink bug species, of which many are brown in color. Due to their similarities in appearance to native stink bug hosts and their increasing abundance in many habitats, it makes intuitive sense that some phasiine species might attempt to use BMSB as a novel host. Currently, only one

North American tachinid species, Trichopoda pennipes, has been successfully reared from BMSB (Aldrich et al., 2006). In 2004, Aldrich et al. collected 834

BMSB adults. Upon examination, 17 tachinid eggs were observed on 14 of the

BMSB adults. Of these, two T. pennipes adult flies emerged from two of the

BMSB, while the remaining parasitized BMSB produced no tachinids. More current parasitism rates of BMSB are lacking, but from my own experience in collecting this species they are occasionally found in the wild with tachinid eggs attached to their bodies. Outside of the successful development of T. pennipes on BMSB that was observed by Aldrich et al. (2004), attempts to rear tachinids out of field-parasitized BMSB never produces viable tachinid adults. This suggests the possibility that BMSB could be acting as an egg sink for tachinid species attempting to use it as a novel host.

Brown Marmorated Stink Bugs (H. halys), an Emerging Threat

BMSB is native to Asia where it occurs in the countries China, Korea,

Taiwan and Japan (Hoebeke & Carter 2003). It initially arrived in the United

States as a stowaway in shipping containers destined for Allentown,

Pennsylvania sometime around the year 1996 (Leskey et al., 2012a; Hoebeke &

Carter 2003). Since then it has spread aggressively and can now be detected in approximately 43 states and four Canadian provinces (StopBMSB, 2015) (Figure

1). Its success is largely due to a lack of natural enemies and its ability to develop on more than 300 host plants including vegetables, legumes, small fruits, tree fruits, grapes and ornamentals (Leskey et al., 2012b; Rice et al.,

2014).

As mentioned above, BMSB superficially resembles many of the native brown stink bugs in North America. Adult BMSB, however, are generally larger in size than their native counterparts, and they can be easily recognized by the distinctive alternating light and dark banding patterns on their antennae, legs and abdomen (Leskey et al., 2012b). Females deposit their egg masses on the undersides of leaves with clutch sizes of around 28 pale-green eggs (Leskey et al., 2012b). Upon hatching, BMSB will undergo five nymphal stages before reaching adulthood, and the overall developmental period from egg to adult lasts approximately 50 days (Leskey et al., 2012b). In Asia, BMSB completes a single generation each year in the temperate parts of its range, whereas populations at lower latitudes can complete as many as five to six generations per year (Fujiie,

1985; Hoffmann, 1931). In the United States they typically complete one to two generations per year (Nielsen et al., 2008; Nielsen & Hamilton, 2009; Leskey et al., 2012b).

BMSB is a secondary pest species in Asia where it is largely controlled by a suite of coevolved natural enemies that keep its numbers in check (Funayama,

2002). As with many introduced species, the colonization of a new ecosystem

(i.e., North America) presented an opportunity for BMSB to spread quickly in the absence of natural enemies. When BMSB invades a new area it first becomes a nuisance pest problem, as large numbers of these bugs begin entering man- made dwellings to overwinter en masse (Hamilton, 2009; Inkley, 2012; Leskey et al., 2012b). This is how BMSB affects most Americans. The stink bugs gain access to sheds, garages, and attics towards the beginning of autumn as day lengths shorten and temperatures begin to drop (Inkley, 2012). Many find their way into people’s homes, where the warmer temperatures and artificial lighting cause them to become more active and clumsy, as they often take flight and collide with walls and light fixtures. They do not bite humans and are not known to transmit any human pathogens (Leskey et al., 2012b); however, the foul odor they release when disturbed or crushed, along with the annoyance of having them invade our living spaces, makes BMSB a formidable nuisance pest.

As the invasion of an area progresses further and population sizes increase to higher densities, BMSB becomes an agricultural pest. This highly polyphagous species (~300 known host plants) is one of the primary causes of feeding damage in a number of vegetable crops including pepper, tomato, eggplant, okra, soybean and corn (Kuhar et al., 2012; Leskey et al., 2012b).

Damage also occurs in tree fruit like apple, plum, peach, pear, and cherries, as well as small fruit like grapes and berries (Nielson and Hamilton, 2009; Leskey et al., 2012b). They feed by inserting their stylets into the fruits, pods, stems, leaves and buds of plants, and the damage reveals itself in several ways. For example,

10 tomato and pepper feeding results in the formation of white or yellow scarring on the skin, corn feeding causes kernels to discolor and collapse, damage to apples and stone fruits leaves severe dimpling and internal necrosis, and feeding on beans causes pods to grow into distorted shapes (Kuhar et al., 2012; Leskey et al., 2012b).

Crop losses due to BMSB damage are particularly severe in the Mid-

Atlantic States (Figure 1) where BMSB has become the number one pest species driving management decisions (Leskey & Hamilton, 2011; Leskey et al., 2012a).

During the 2010 growing season, farmers of mid-Atlantic apple and stone fruit crops experienced heavy losses following a mild winter that allowed BMSB populations to explode (Leskey et al., 2012b). That year feeding damage resulted in $37 million dollars in losses for apple farmers (United States Apple

Association, 2010), while some farmers of stone fruit experienced losses of more than 90% of their total crops (Leskey & Hamilton, 2010).

Following the heavy losses experienced in 2010, researchers began investigating ways to control the spread of BMSB in hopes of reducing future damages. A number of management solutions have been attempted including behavioral-based monitoring techniques, attract-and-kill traps and biological control programs (Leskey et al., 2012b). In 2014, the aggregation pheromones of

BMSB were discovered (Khrimian et al., 2014), and since then, pheromone lures have been made available for farmers to use for monitoring BMSB infestation levels in their fields. Traps baited with lures and insecticidal strips can be placed alongside rows of crops giving farmers a rough estimate of their infestation

11 levels. They can then make the decision whether or not levels of infestation are high enough to justify the application of insecticide sprays. The use of insecticides is not always effective, however. BMSB are known to be strong flyers (Lee et al., 2014), so if a farmer decides to spray his/her crops one week, a new wave of BMSB could fly in from adjacent fields the next week.

Currently it appears that classical biological control using the Asian wasp species Trissolcus japonicus (Hymenoptera: Scelionidae) offers the most promise for controlling this invasive pest. It is an egg parasitoid that places its eggs inside of the eggs of BMSB, resulting in high BMSB egg mortality across much of its native range (Rice et al., 2014; Yang et al., 2009). While North

American egg parasitoids are important natural enemies of stink bugs, they rarely develop successfully on BMSB eggs, despite attempts to utilize them (Jones et al., 2014). BMSB eggs therefore act as a sink for native egg parasitoids (Abram et al., 2014). Researchers at the USDA-ARS Beneficial Insect Introduction

Research Unit (ARS/BIIRU) began evaluating T. japonicus in 2007 to determine if it could be released safely into North America as a biological control agent against BMSB (Herlihy et al., 2016). In 2015, despite being under quarantined study in the United States, T. japonicus was reared from BMSB eggs in the wild

(Talamas et al., 2015). Studies have subsequently determined T. japonicus to be a viable candidate for use as a biological control agent, and plans have been announced to mass rear the wasp for trial releases into Oregon and possibly

New York as early as summer 2017 (Ferro, 2017).

While this wasp appears to be a viable solution to the problem of controlling BMSB populations, the question of how this invasive stink bug is affecting native tachinids remains unknown. Does it represent a suitable novel host organism for tachinids, their use of which could strengthen the controls being proposed by the introduction of T. japonicus; or is it an unsuitable host for tachinid development, making it an egg sink that reduces the populations of parasitoids attempting to use it as a host? Answering these questions requires a closer look at tachinid flies from the subfamily Phasiinae.

Subfamily Phasiinae: Tachinids parasitoids of Stink Bugs

Worldwide there are estimated to be around 90 genera of Phasiinae

(Blaschke, 2013), and in the United States there are approximately 81 species divided among 15 genera (O’Hara & Wood, 2004). Despite being the least speciose subfamily of Tachinidae, the phasiines possess considerable morphological diversity. Some are cryptically colored while others possess more vivid coloration and regularly feed at wildflowers, mimicking bees and wasps.

Species in the genus Cylindromyia even have abdomens that superficially resemble those of ichneumonid wasps (see Figure 6C). The genus Trichopoda consists of the ‘feather-legged’ flies that possess uniquely enlarged and flattened setae on their hind tibiae (see Figure 6B). Regardless of morphology, they are all endoparasitoids that feed internally on the bodies of their hosts during larval development.

Phasiines exclusively use true bugs (Heteroptera) as hosts making them the most host-restricted subfamily of Tachinidae. They use the male-produced sex pheromones of their hosts as host-finding kairomones (Aldrich et al., 1995;

Aldrich et al., 2006). Within Heteroptera, the infraorder Pentatomomorpha represent a major host group for the phasiines (Arnaud, 1978). This includes the flat bugs (family Aradidae), the seed bugs and their allies (superfamily

Lygaeoidea), the leaf-footed bugs and their allies (superfamily Coreoidea), the shield bugs or stink bugs (superfamily Pentatomoidea), and the bordered plant bugs (superfamily Pyrrhocoroidea).

Chemical ecologists have recently begun to elucidate the semiochemical library of the true bugs (Millar, 2005) making it possible to investigate the phenomenon of kairomone hijacking observed in phasiine tachinids. Gas chromatographic electroantennogram detector (GC-EAD) experiments have shown that some tachinids are as sensitive to their host’s pheromones as the hosts are themselves (Aldrich & Zhang, 2002), and in some cases the tachinids appear to possess greater sensitivity. For example, Aldrich et al. (2006) observed that Euschistus spp. stink bugs showed little discrimination between their own sex pheromone molecule (methyl [E,Z]-2,4-decadienoate) and a similar organic molecule (ethyl [E,Z]-2,4-decadienoate); however, the tachinid parasitoids of these bugs accurately discriminated between the two.

Objectives and Hypotheses

My research aimed to investigate the possibility that native tachinids could use BMSB as a novel host, and to assess the importance of visual and olfactory cues in host location and selection behavior in the species Gymnoclytia occidua.

To evaluate these questions I conducted three sets of experiments.

I first sought to evaluate the use of host pheromones by native tachinids as kairomones. My primary objective was to determine if any tachinid species are attracted to BMSB pheromones in the field, and to compare this with attraction to pheromones from native stink bugs. For this part of the study I used traps baited with three different pentatomid pheromones, as well as a negative control (MDD,

MDT, BMSB and CONT). Similar studies have been conducted using pentatomid pheromones (Aldrich et al, 2006; Aldrich et al., 2007; Tillman et al., 2010); however, I am not aware of any that have been conducted in Southwest Ohio, and most lacked the inclusion of the recently discovered BMSB pheromone

(Khrimian et al, 2014). Studies that have used BMSB pheromones have mostly focused on the effectiveness of BMSB lures as a tool for monitoring BMSB invasions, and none have reported on tachinid attractions. I predicted that few species of tachinids would be attracted to BMSB lures relative to native stink bugs. Most tachinids in North America have likely not had enough time to evolve the ability to detect BMSB pheromones, and their preferences for native pheromones have likely been reinforced through evolution.

My main prediction for the pentatomids was that they would be more strongly attracted to lures containing their own pheromone molecules than to lures containing pheromone molecules of other pentatomids; i.e., Euschistus spp. bugs should be more attracted to lures containing the Euschistus pheromone

MDD than to any of the other lures, and H. halys should show stronger attraction to BMSB lures than any of the others. Halyomorpha halys might also show a significant level of attraction to MDT lures since they have been reported to be cross attracted to pheromones of the stink bug Plautia stali in their native range

(Sugie et al., 1996; Tada et al., 2001). Plautia stali is an Asian pentatomid that produces the EEZ isomer of MDT pheromone (methyl (E,E,Z)-2,4,6- decatrienoate). The MDT lure that I used contained the EZZ isomer form of the

MDT pheromone (methyl (E,Z,Z)-2,4,6-decatrienoate), which is the pheromone used by the North American stink bug Thyanta custator accerra (Aldrich et al.,

2006). Accordingly, I predicted that T. custator accerra stink bugs would be attracted to these lures.

I then sought to assess the determinants of host use in Gymnoclytia occidua females. This species was chosen as a focal phasiine species to assess the importance of cues in host selection and assess potential impacts of BMSB on native phasiines (see Oviposition Assays: Background in Methods). The first objective here was to investigate whether or not host motion or pheromone presence affects host selection. To accomplish this, I conducted oviposition trials using 3d-printed stink bug models as hosts to allow manipulation of host motion and pheromone presence. I predicted that the tachinid females would show more

16 interest in moving hosts over motionless hosts due to the strong use of visual motion cues observed in other tachinid species (Stireman, 2002; Yamawaki et al., 2002). I also predicted that trials conducted in the presence of BMSB pheromones would elicit the same level of interest in the models as that of trials conducted without pheromones, given that they have no evolutionary history with

BMSB. A second objective was to assess host preference for native versus non- native hosts. This was accomplished by conducting oviposition assays with live

Euschistus spp. stink bugs and BMSB. I predicted that Gymnoclytia occidua females would attack BMSB in greater frequency than their native hosts due to the fact that BMSB are larger and generally more active. As previously mentioned, phasiines utilize specific host-associated pheromone molecules to locate their hosts, so they should be expected to discriminate less at the finer level of host selection. In other words, they will probably be more likely to accept any potential host as long as it resembles a native host.

For the final part of the study I combined laboratory and field data to compare the parasitism rates of BMSB and native Euschistus spp. pentatomids.

Individual parasitized hosts that were collected in the field, as well as ones that were directly observed being parasitized during the oviposition trials in part two were kept alive until they either died naturally or a parasitoid emerged. Here I predicted that BMSB might be a suitable host for Gymnoclytia occidua, if indeed it selects BMSB as a host. Although relatively few tachinids have been reported developing on BMSB successfully, it is not clear if this represents a physiological

17 barrier to using BMSB as a suitable host, or if native North American tachinids have just not recognized BMSB as a suitable host.

METHODS

PHEROMONE ATTRACTION IN THE FIELD

Background

During the period from June – September of 2015 I used baited traps to test the effectiveness of three different pheromone lures in attracting both pentatomids and tachinid parasitoids in agricultural and semi-natural areas around Southwest Ohio. This was also conducted to test if patterns of parasitoid attraction to host kairomones were consistent with previous studies in other areas (Aldrich et al. 2006, 2007; Jang & Park 2010; Weber et al. 2014).

Study Area

The study area where traps were placed included the following locations

(locality codes in parentheses): Carriage Hill MetroPark, Huber Heights, OH

(CAHI); Englewood MetroPark, Englewood, OH (ENGL); Sugarcreek MetroPark,

Bellbrook, OH (SUCR); Patchwork Gardens CSA, Dayton, OH (PTWG); Fulton

Farms site #1, Troy, OH (FULT-1); Fulton Farms site #2, Troy, OH (FULT-2).

More detailed information about each location is provided in Table 1, as well as a map of the study area in Figure 3.

Trap Construction

Pyramid-type traps, designed to attract pentatomids in the field, were constructed following the methods outlined by Morrison et al. (2015). Sheets of black corrugated plastic (Laird Plastics Inc., Dayton, Ohio) were used to create the pyramid bases, and clear half-gallon PET jugs (18x13x13 cm) (Uline,

Pleasant Prairie, Wisconsin) and inverted funnels were used to create the trap tops. The completed traps, when assembled, measured approximately 114 cm tall (Figure 4).

Lures containing known pentatomid aggregation pheromones were purchased from the company AgBio Inc. (Westminster, Colorado). These lures included the aggregation pheromone of Euschistus sp. stink bugs (methyl [E,Z]-

2,4-decadienoate, or MDD), the aggregation pheromone of Thyanta sp. stink bugs (methyl [E,Z,Z]-2,4,6-decatrienoate, or MDT), and a lure containing the two part aggregation pheromone of BMSB ([3S,6S,7R,10S]-10,11-epoxy-1- bisabolen-3-ol and [3R,6S,7R,10S]-10,11-epoxy-1-bisabolen-3-ol, or BMSB)

(Figure 5). The BMSB lure also contained MDT which has been found to work synergistically with BMSB aggregation pheromones (Weber et al. 2014).

A single lure was placed inside each of the trap tops (excluding the traps designated as negative controls) by affixing it to a paper clip suspended from the bottom of the lid. Holes were drilled into the sides of the trap tops (approx. 3 cm dia.) and then covered with mesh screens. This served the dual purpose of allowing adequate dispersal of the pheromones while also providing air

20 circulation to keep trapped specimens alive long enough to collect and bring back to the lab for further experiments.

Sampling Periods

At each of the sampling locations four traps were placed approximately 25 meters apart along edge habitats where wooded areas transitioned directly into either open meadows or agricultural fields. Three of the traps contained the pheromone treatments (MDD, MDT, and BMSB) while the fourth acted as a negative control (CONT) which contained no pheromone lure.

Two sampling periods were conducted between June 23 – July 24 and

August 10 – September 15 during the 2015 field season. Pheromone lures were replaced once on August 10 following a two-week break between sampling periods. Each site was visited an average of two times per week and the numbers of pentatomids and tachinids attracted to each trap were counted.

Individuals were deemed “attracted” to a particular treatment if they entered the trap, were found on the outside of the trap, or if they were observed within a 1m radius of the trap. Live individuals of stink bugs and tachinids were captured and transported back to the lab to establish colonies. Stink bugs were identified to species using a key to the Pentatomidae of Ontario and adjacent areas (Paiero et al., 2013). Tachinids were first identified to genus using a key to North

American Tachinidae (O’Hara and Wood, 2004), and later identified further using keys to species of the various genera (Aldrich, 1926; Brooks, 1946; O’Hara,

2012). Voucher specimens of all species have been deposited in the collection of

J.O. Stireman (JOSC) at Wright State University.

Data Analysis

I used Chi-square goodness-of-fit tests to determine if there was significant variation in the number of individuals attracted to the different pheromone traps and controls for each species of tachinid and pentatomid. Site was also analyzed for goodness of fit.

OVIPOSITION ASSAYS

Background

Gymnoclytia occidua (Walker, 1849) is a species of tachinid fly in the subfamily Phasiinae that is common in the Eastern United States. Males and females of this species can be observed from late May to early October feeding at a number of different wildflower species, including Daucus carota, Erigeron spp., and Solidago spp. These flies exhibit sexual dimorphism, making them easy to distinguish with the naked eye; males are a golden-amber color, whereas females are grey, or silver colored. They range in size from approx. 6 – 9 mm, although females tend to be slightly larger than males. Host records indicate that this species has multiple host associations including the stink bug

(Pentatomidae) species Euschistus servus, E. tristigmus, E. variolarius, Thyanta calceata, and T. custator accerra (Arnaud, 1978; Eger & Ables, 1981; Jones et al., 1996; McPherson et al., 1982; Oetting & Yonke, 1971; Rings & Brooks,

1958;). Superficially these stink bugs are similar in appearance; all possess

22 cryptic brown coloration (Thyanta spp. bugs are green in summer but become brown during fall/winter) and they range from 9-15 mm in size.

Literature regarding the detailed life-histories of most tachinid species is scattered and often non-existent for some species, as is the case for

Gymnoclytia occidua. Aside from the dated host records mentioned above, not much is known about the life-history of this species. Presumably, once male and female adults eclose from their puparia, they seek each other out for the purposes of mating. If both males and females are primed to instinctively home in on the specific pheromones of host organisms, then they will likely encounter mates in the process. This is supported by previous pheromone attraction studies where males and females of related phasiine species were attracted to their host’s pheromones, including Euclytia flava (Townsend), Gymnosoma par

(Walker), Euthera tentatrix Loew, and Hemyda aurata Robineau-Desvoidy

(Aldrich et al., 2006; Aldrich et al., 2007). Once a mated female locates a potential host, she will then begin the host selection process.

It is unknown what cues female Gymnoclytia occidua use to select individual hosts. Since phasiines use highly-specific kairomonal cues during the host-location process, it is possible that Gymnoclytia occidua does not discriminate much between hosts once they are in the vicinity. Other tachinid species use combinations of visual, tactile and chemosensory cues (Burks &

Nettles, 1978; Mondor & Roland, 1997; Monteith, 1956; Roland et al., 1989; Roth et al., 1978; Stireman, 2002; Weseloh, 1980; Yamawaki et al., 2002). A target host might be visually identified by the parasitoid via the host’s characteristic

23 movements, behaviors or fine-detail coloration; tactile recognition of the host might occur via tarsal reception of cell-surface molecules on the host’s cuticle, i.e., “tasting the host” by touching it with their feet (tarsi) (Stireman, 2002). Fine- scale odors produced by the host from frass, salivary secretions, ingested plant compounds, alarm pheromones or other sources can also potentially act as chemosensory elicitors of host-selection in tachinids (Stireman et al., 2006).

Collecting and Rearing Specimens

One of the biggest challenges of this study involved finding the necessary hosts and parasitoids and keeping them alive long enough to perform laboratory assays. Sufficient quantities of Gymnoclytia occidua (males and females) and a variety of both native and non-native hosts needed to be collected in order to conduct behavioral experiments and assess host suitability. I focused my collecting efforts at one of the field sites at Fulton Farms in Troy, Ohio because parasitoids and hosts were most abundant at this location during the pyramid- trap sampling. This site had a particularly abundant Gymnoclytia occidua population, and was also rich in populations of its native hosts, namely

Euschistus spp. stink bugs.

Due to the abundance of food sources at the agricultural sites, and because BMSB is reported to be an increasing threat to agricultural crops, I expected these locations to be where I would collect the most BMSB specimens.

However, they were disproportionately collected in suburban backyards and structures like tool sheds and garages. In my own backyard I would regularly

24 collect an average of around a dozen or more BMSB from a row of mulberry bushes each morning in mid-July. As fall weather returned, BMSB were more regularly found inside dwellings. To capitalize on the overwintering habits of

BMSB, in early October 2016, I sent out a newsletter to the Biology Department at Wright State University requesting that people collect any stink bugs they encounter in their homes and donate them to the lab rather than disposing of them.

Gymnoclytia were typically found on flowers, usually fleabane (Erigeron spp.) or wild carrot (Daucus carota) and were captured using an aerial net. Once inside the net they were quickly transferred to clear plastic vials. Pentatomids were generally spotted resting on leaf surfaces or along plant stems. They were easily captured by holding an empty vial near a bug and then quickly scooping it inside using the lid. In both cases when specimens were caught they were transported back to the lab in the plastic vials which were affixed with lids that had a mesh screen glued over an open hole. This was done to prevent the vials from overheating and to provide air exchange, increasing the chances of specimens surviving the trip back to the lab.

Once back at the lab the specimens were transferred to larger containers.

Each female Gymnoclytia occidua was kept individually in square wide mouth jars approximately 1.9 L in volume purchased from Uline (Pleasant Prairie,

Wisconsin) (Figure 7). These jars were transparent and were placed on their sides underneath a fluorescent light fixture on a 16/8 hours light-dark cycle and a constant average temperature of approximately 21°C. Food was provided in the

25 form of filter paper strips coated with crystalized sugar and suspended inside the containers using paper clips. Petri dishes of fresh water were filled with aquarium gravel to provide the flies with a place to perch while they rehydrated and also to prevent them from drowning. To provide air exchange the lids of each jar were replaced with nylon sleeves that could be twisted and clipped shut. This also functioned as a quick access port for transferring the flies into and out of the container.

Each female parasitoid was paired with a single male and was allowed to mate for a minimum of 24 hours prior to her use in any of the oviposition trials.

Excess males were kept in a bug dorm (299 x 299 x 299 cm) purchased from

BioQuip (Rancho Dominguez, California) (Figure 7). Pentatomids were also kept in a separate multispecies bug dorm and were kept alive by providing them with sugar snap peas (Pisum sativum var. macrocarpon) and dishes of water. The peas were replaced with fresh peas twice weekly, or more frequently in some cases if the peas looked like they were drying out or getting moldy. The water dishes were checked daily and topped off with fresh water approximately every other day to replace what was lost to drinking and evaporation.

Model Host Trials

3D printed stink bug models (Figure 8) were used to experimentally analyze the effects of host movement and pheromone presence on the host- selection process of Gymnoclytia occidua females. An oviposition arena was constructed that consisted of a roughly 31 x 31 cm square platform that sat

26 approximately 15 cm high. A clear sheet of acrylic with the same dimensions was placed on top of the platform. The bottom half of a small plastic terrarium turned upside down and centered on top of the platform completed the arena

(Figure 8). The arena was then placed on a lab bench underneath an array of T5 fluorescent light bulbs.

Stink bug models were printed using a Makerbot Replicator 3D printer at

Proto Build Bar (Dayton, Ohio). A single model was then used to create a silicon mold, from which approximately two dozen more models were cast using liquid synthetic resin. A depression was drilled halfway into the bottom of half of the models and a small neodymium magnet was superglued into place providing a mechanism for manipulating the movements of individual models. The models were then painted brown using enamel-based hobby paints to mimic the appearance of native pentatomids (mainly Euschistus sp.) as well as BMSB

(Figure 8). The models were then distributed into three separate gallon-size zipper-lock bags that contained an MDD lure, a BMSB lure, or no lure. Each bag was sealed and placed inside a second bag to avoid cross-contamination of pheromones between the models.

Finally, a movement apparatus was constructed to facilitate the uniform manipulation of host movements during model trials (Figure 8D). This took the form of a motorized carousel consisting of four metal arms attached to a battery operated gear reduction motor which rotated the arms slowly. When positioned underneath the platform of the arena, the metal arms attracted the magnets on the models and pulled them in a 15 cm diameter circle around the middle of the

27 arena at approximately 1 rpm. The path of movement is illustrated by a red circle in Figure 8C. Early on in the testing phase of the model trials it was determined that this speed was too fast. Rather than adjusting the gear reduction motor, the model trials proceeded by manually rotating the carousel by hand in order to achieve a slower speed that resembled the pace of a walking stink bug.

Model trials began on June 29, 2016, when the first female Gymnoclytia occidua was collected, and ran until July 11, 2016. A total of 28 model trials were conducted during this time period using 11 Gymnoclytia occidua females.

Most of the females were used in multiple trials, regardless of whether or not the female responded to a host during the duration of the trial. On average, females were used approximately two to three times. Each trial consisted of two models being placed in the arena, one moving model and one motionless model. In twenty of the trials, models exposed to the pheromones from the BMSB lures were used, while the remaining eight trials used models that were not exposed to pheromones. Trials began when a female fly was introduced into the arena, and they ended after 10 minutes or upon the fly investigating one of the models.

Landing upon, walking over top of, touching from the side and directed movements toward a specific model were all considered as a choice by that fly.

Live Hosts Trials

Live stink bugs were used in choice and no-choice trials to determine the preferences of Gymnoclytia occidua for native versus novel hosts. These trials began on July 13, 2016 and were conducted intermittently until October 17,

2016, as the availability of both parasitoids and hosts fluctuated throughout the field season. The oviposition arena described above was used for the live host trials; however the movement apparatus was removed as it was no longer necessary.

No-choice trials consisted of a single Gymnoclytia occidua female being presented with between two to eight hosts comprised entirely of either BMSB only or Euschistus spp. In total eight trials were conducted with BMSB only and 7 trials were conducted with Euschistus spp. only. As before, trials lasted for ten minutes or until the first attack was observed. Latency to attack, scored as time in seconds from the beginning of a trial until the first observed attack, was the response variable measured.

Choice trials consisted of a single Gymnoclytia occidua female being presented with an even number of both BMSB hosts and native hosts (i.e., either

Euschistus spp. or Podisus maculiventris). In total, 31 choice trials were conducted using 16 different females. Half of the females (n=8) were only used for single trials, and the remaining females were all used for between two to three trials. One of the females stayed alive in captivity substantially longer than all of the other females and was used for six trials. 20 trials with two of each host type

(4 hosts total) and 11 trials with one of each host type (2 hosts total). The reason that fewer hosts were used in some of the trials was because, at times, the availability of native hosts was much lower than that of BMSB. During the course of these assays, BMSB were always available but native stink bugs had to be continually replenished from the wild. Since the native bugs were the limiting

29 factor, trials that had single individuals of both host were conducted as natives were collected. Trials lasted for a maximum ten minutes or until the first attack was observed. Once again, latency of attack was the response variable measured.

Host Suitability (Native vs. Non-Native)

All of the pentatomids that were attacked during the live host trials were removed from the arena and placed individually in deli-style containers (Figure

9). They were provided with peas and small vials of water and were kept under fluorescent lights on a 16/8 light-dark cycle at approximately 21°C. They were kept alive until either a parasitoid larva emerged or they died naturally.

Parasitized pentatomids that were found in the field or donated from people’s homes were also brought back to the lab and kept alive for parasitoid rearing.

Finally, 30 additional trials were conducted for the sole purpose of parasitizing the hosts, and as such these trials were not timed or closely monitored. During these trials a female fly was placed in the arena with between 2-10 hosts. Hosts were periodically checked to see if they had been oviposited on, and if so those hosts were removed and placed in containers as above. The duration of these extra trials ranged from several minutes up to 60 minutes.

Host bugs upon which Gymnoclytia occidua laid eggs were checked daily for parasitoid emergences. When found, tachinid puparia were often observed underneath one of the peas, or near the openings of the water vials. Puparia were transferred to separate vials to complete eclosion. A small cushion of lightly

30 moistened cotton and small strip of sugar paper placed inside each vial awaited the adult parasitoid. In most cases when puparia were discovered, the hosts were found dead; however, this was not always the case. Hosts that were found alive following parasitoid emergence generally died within 24 hours, but in a few cases the hosts remained alive for at least several days. When hosts were found dead but without any parasitoid emergence, they were left alone for approximately 24 hours. If no parasitoid emerged after this period, the bugs were dissected to investigate for signs of a parasitoid. Records were kept for total numbers of parasitized native and non-native hosts, dates that each of the hosts were attacked and deceased, and the total numbers of parasitoid emergences.

Data Analysis

The model trials were analyzed using chi-square tests to determine the effect of the presence of pheromones on host selection, and T-tests to determine the effect of a moving versus non-moving host. T-tests were used to analyze the differences in mean latency of attack for the choice and no-choice live host trials.

The suitability of native versus non-native hosts for tachinid development was evaluated by comparing the number of successful parasitism events using a Chi- square test. Finally, binomial probability tests were conducted on the oviposition assays to compare differences in the frequencies of host choice between the native and non-native hosts.

RESULTS

PHEROMONE ATTRACTION

The pheromone-baited pyramid traps successfully attracted species of both tachinids and pentatomids (Fig. 10 & 11 and Tab. 2 & 3). The pentatomid taxa that were attracted included Halyomorpha halys (Stål), Chinavia hilaris

(Say), and four different taxa of the genus Euschistus (E. servus (Say), E. tristigmus tristigmus (Say), E. tristigmus luridus Dallas and E. variolarius (Palisot de Beauvois)) (Fig. 10A & 11A). The Euschistus spp. were lumped together because sample sizes were small for most taxa. The species E. tristigmus, however, was by far the most abundant species of Euschistus, comprising nearly 90 percent of individuals attracted of this genus. Seven species of tachinids were attracted to the traps (Fig. 10B & 11B), including Gymnoclytia occidua (Walker), Trichopoda pennipes (Fabricius), Cylindromyia fumipennis

(Bigot), Euthera tentatrix Loew, Euclytia flava (Townsend), Gymnosoma par

Walker and Phasia sp. Latreille. Only one individual of the Phasia sp. was collected at a trap (MDT); however, species identification was not possible because the specimen was damaged during transport back to the laboratory.

All of the pentatomid species exhibited significant variation in attraction to the pheromone lures (Table 4; Table 10A). Euschistus spp. stink bugs were attracted to traps that contained the MDD (n=20) and MDT (n=3) pheromone

32 lures (Fig.10A). BMSB were attracted to traps containing the BMSB lures (n=37) as well as MDT lures (n=12). C. hilaris stink bugs were also attracted to BMSB lures (n=11). No pentatomids were captured at any of the control traps.

Only two of the tachinid species exhibited significant variation in attraction to the pheromone traps, Euthera tentatrix and Gymnoclytia occidua (Figure 10B;

Table 4). All Euthera tentatrix individuals were captured at MDD traps (n=7), whereas Gymnoclytia occidua were captured at MDD traps (n=17), MDT traps

(n=7) and BMSB traps (n=2) (Fig. 10B). All of the remaining tachinid species showed no significant pattern of attraction, but this may have been due to low sample sizes. Single specimens were captured for the species Euclytia flava

(MDD trap), Phasia sp. (MDT trap), and T. pennipes (MDT trap), and two specimens of C. fumipennis were captured at MDT traps. Finally, seven specimens of Gymnosoma par were captured. This species exhibited the widest range of attraction to the pheromone lures, as it is the only species that was captured at each type of trap, including a single individual that was captured at a control trap. No other tachinids were captured in any of the control traps.

When trap captures were lumped across treatments, all of the pentatomid species exhibited significant variation across sites (Figure 11A; Table 4). The only tachinid that showed a significant bias was the species Gymnoclytia occidua

(Figure 11B). By far, the most productive site was FULT-1 (Fulton Farms, Troy,

Ohio). A total of 45 pentatomids and 29 tachinids were attracted to traps placed at this location. The least productive site was CAHI (Carriage Hill MetroPark,

Huber Heights, Ohio), which had a total of five pentatomids and two tachinids attracted to traps.

OVIPOSITION ASSAYS

Model Host Trials

I used 3d-printed host models to investigate how host motion and pheromone presence influences host selection behaviors in Gymnoclytia occidua females (n=11). Out of 28 total trials, the parasitoids made first contact with the moving model 13 times, whereas the non-moving model was contacted first just three times. In the remaining 12 trials the females failed to make a choice during the duration of the trial. There was no significant difference in mean latency

(T=0.2145, df=2.481, P=0.85; Figure 13). Ignoring latency and only looking at trials in which the female made a choice, the result of a binomial probability test is significant (P=0.01). That is, the females made the choice to inspect the moving model in significantly greater frequency than the non-moving model.

When BMSB pheromones were present (n=19 trials) the female flies made a choice 12 times and failed to make a choice 7 times. When pheromones were not present (n=9 trials) the females made a choice in 4 of the trials and made no choice during the other 5 trials. The presence of host pheromone did not appear to influence attraction of flies to model hosts (χ2=0.2763, df=1, P=0.5991; Figure

12).

Live Host Trials

Variation in attack latency between native and non-native hosts were also measured using no-choice trials (n=19). Ten trials were conducted using only non-native hosts (BMSB) and nine trials were conducted using only native hosts (Euschistus spp.). In four of the trials (two for non-native and two for native) the parasitoid did not attack during the time period of the trial. Mean attack latency was virtually identical for both native and non-native hosts

(124.33s and 124.88s, respectively); (T=0.0091, df=11.253, P=0.99; Figure 13).

During the choice trials (n=31), I presented Gymnoclytia occidua females

(n=16) with live native hosts (Euschistus spp.) and live non-native hosts (BMSB) to determine host preference. In 16 trials the non-native hosts were the first to be attacked, native hosts were attacked first in 9 trials, and in the remaining 6 trials no choice was made during the trial duration (Figure 13). Mean latency was not significantly lower for the non-native hosts (124.25 s) versus the native hosts

(193.78 s; T=-1.0934, df=14.081, P=0.29). The result of binomial test looking only at the difference in host choice frequencies between native and non-native hosts was also not significant (P=0.11).

Host Suitability (Native vs. Non-native)

A total of 92 pentatomids were obtained that were either parasitized in the field or in the lab. They were kept alive to rear the tachinid larvae growing inside them, and by late 2016, all but eleven of these bugs had perished. Of those that died, successful development of Gymnoclytia occidua was observed on the

35 native hosts (Figure 14) but never on the non-native BMSB hosts. The success rate was 33% when using native hosts (13 surviving larvae out of 39 hosts) and

0% when using non-native hosts (0 surviving larvae out of 53 hosts; χ2=12.906, df=1, P=0.0003). The eleven bugs that remained alive were all BMSB, and parasitism of these bugs was deemed as failures because more than a month had passed since the time they were attacked; when successful, parasitoid larvae emerged from their hosts approximately 19 days after being attacked.

Additionally, two of the larvae that emerged from the field-parasitized native hosts ended up being other tachinid species (C. fumipennis and Euthera tentatrix). However, when field parasitized were excluded (Fig. 14B) the success rate on native hosts remained significantly higher (11 surviving larvae out of 33 hosts; χ2=8.925, df=1, P=0.003).

I received 142 stink bugs (101 BMSB and 41 native) in mid-autumn, the majority of which were donated to me by colleagues. Of these, I found evidence of parasitism (i.e., tachinid eggs) on 8 of the BMSB and 2 of the native bugs.

There was no significant difference in parasitism rates between BMSB and native pentatomids (χ2=0.064, df=1, P=0.801; Figure 15).

DISCUSSION

The purpose of this research was to investigate the use of kairomones by host-seeking tachinid flies, assess the importance of olfactory and visual stimuli during the host selection process, and determine the suitability of an invasive species as a novel host organism. Specifically, the main question that I sought to answer was if growing populations of invasive brown marmorated stink bugs are causing the fitness of some native tachinids to decrease by acting as an egg sink; or could tachinids actually contribute to the control of BMSB? Despite limited sample sizes in some of the experiments, the overall findings presented in this study support the hypothesis that BMSB are negatively affecting some species of Tachinidae.

The results obtained from the pheromone attraction study were informative. It confirmed the predictions that pentatomids would be more strongly attracted to traps baited with their own pheromone molecules, and that BMSB would also exhibit cross-attraction to the MDT pheromone lures. While none of the lures that were used contained C. hilaris pheromones, their attraction to

BMSB lures might be explained by the fact that they contained epoxide blends that are similar to C. hilaris pheromones. C. hilaris produce (Z)-α-bisabolene and

(4S)-cis- and (4S)-trans-(Z)- α -bisabolene epoxides (Aldrich et al., 1989).

Although all of the pentatomid taxa were attracted to MDT lures, the low numbers

37 of C. hilaris and Euschistus spp. bugs that were attracted to these traps could be due to chance and is not necessarily indicative of them being cross-attracted to this pheromone.

Attraction of tachinids to the pheromone lures was more scattered and not nearly as consistent as the pentatomids. Both of the species showing significant biases in attraction, Gymnoclytia occidua and Euthera tentatrix, are reported to primarily attack Euschistus spp. stink bugs (Arnaud, 1978) and the attraction of these flies to the MDD lures is consistent with use of these hosts. Despite the low numbers of Gymnosoma par, the pheromone these flies found to be the most attractive was the MDT pheromone of T. custator accerra stink bugs, which is one of their primary hosts (Jones et al., 1996). Gymnosoma par and Gymnoclytia occidua were the only two species attracted to traps containing BMSB pheromone lures. While this could potentially indicate attraction to the BMSB pheromones, the low numbers for both of these species suggests that these individuals arrived at BMSB traps by chance and not due to attraction. Some of the observed attractions may have been due to the fly being attracted to the trap structure itself, or so abundant that it is everywhere. Regardless, this confirmed the hypothesis that few, if any, native tachinids in the region are attracted to

BMSB pheromones.

The locations chosen for field sites included both semi-natural and agricultural areas (Figure 11). Although I did not specifically set out to compare and contrast these two types of sites, it is interesting to note that the most productive site was one of the agricultural sites (FULT-1), both in terms of total

38 number of species and numbers of individuals captured. This location corresponds to Fulton Farms in Troy, Ohio, which is a large semi-organic farm with more than 500 acres of various vegetable crops. It makes intuitive sense that a site like this would have large populations of both parasitoids and their hosts, as it represents an area of high food density for herbivorous insects.

Ironically however, traps that were placed at the other Fulton Farms site, FULT-2

(approximately 0.5 kilometers away from FULT-1) were not nearly as productive.

Communication with the owner, Jim Fulton, revealed that not all areas of the property are organic and insecticidal spraying is used for some crops. He further indicated that the rows of tomatoes and eggplants near the FULT-2 site had undergone insecticide treatments, while the strawberry and soybean plants near the FULT-1 site had not been sprayed. A third agricultural site at Patchwork

Gardens (PTWG) in Dayton, Ohio was not nearly as productive as FULT-1 in terms of bugs and flies. This location however, was substantially smaller at approximately 20 acres. It remains unclear exactly why the FULT-1 site was so productive, but it could possibly be a combination of its large area, abundant food supply for the pentatomids, and lack of insecticide spraying. In addition, traps at this location stood alongside a forest edge which provided abundant floral resources for adult flies.

During the model trials, moving hosts elicited more attraction by flies, but latency to attack did not differ between treatments. Mean attack latency on non- moving hosts was lower than on moving hosts; however, there was no significant difference, possibly due to low sample size. Non-moving hosts were chosen just

39 three times, and latency varied wildly. Two of the three occurrences averaged around 30 seconds of attack latency, whereas the third occurrence had a latency of 462 seconds. In trials where the moving host was chosen, the flies were often observed taking notice of and moving towards the moving host. In trials where an apparent choice was made early on, it was sometimes hard to discern whether the fly was making an actual choice or if they were just walking over the model without realizing that it was a potential host.

The effect of pheromone presence on host selection was unclear. Flies showed increased activity during trials where BMSB pheromones were present, and they were also more likely to make a host choice. The lack of significance was most likely due to there being considerably fewer trials that took place without the presence of pheromones. However, the observation that flies appeared to be more active and made choices more often in the presence of

BMSB pheromones, is suggestive that increased sampling would reveal a significant effect of pheromone presence on behavior. This would suggest that

Gymnoclytia occidua is at least partially sensitive to the pheromones of BMSB, and that the pheromones could elicit increased host selection behaviors in these flies.

In the choice trials using live hosts, Gymnoclytia occidua was observed attacking BMSB hosts in greater frequency than native hosts. Mean attack latency was also lower for attacks on BMSB, although low sample size and high variability limit the power to detect differential responses.. During the no-choice trials, mean attack latencies were essentially identical for native and non-native

40 hosts. The oviposition assays provided no evidence of avoidance of BMSB or preference for native hosts by Gymnoclytia occidua females. This is highly suggestive that females might prefer to choose BMSB host over native hosts, but more data are needed to confirm these results.

The results of the host suitability assays of BMSB were the most interesting. Despite the fact that Gymnoclytia occidua readily accepted BMSB hosts in the laboratory, not a single pupa or adult fly was ever recovered from this species. Dissections of deceased BMSB hosts also revealed no evidence of developing larvae. Conversely, successful parasitism of Euschistus hosts was observed occurring around 33 percent of the time, and in cases where development failed on these hosts, there was evidence that the parasitism had at least partially progressed. For example, eggs that were removed from deceased

Euschistus hosts usually revealed egg scars on the host’s integument, showing where the tachinid larvae had hatched out of the egg and bored into the host.

Each time that eggs were removed from deceased BMSB hosts, no such scars were ever observed. Furthermore, dissection of the abdomens of failed

Euschistus hosts never revealed any partially developed tachinid larvae, but were often found to be nearly empty, devoid of any viscera or internal organ structures. This was never the case when dissecting BMSB abdomens; internal structures remained intact. The fact that no evidence was found for even partial development of Gymnoclytia occidua larvae strongly suggests that BMSB is an unsuitable host for this tachinid. Finding no evidence of egg scars on BMSB suggests that their integumentary system possesses some properties that make

41 it an effective barrier against parasitoid entry. Determining exactly what those properties are will require further research.

This research illustrates one of the potential negative effects that an invasive brown marmorated stink bug can have on populations of tachinid parasitoids. While these results are not entirely conclusive, it appears as if BMSB may act as an egg sink for at least one tachinid species. As mentioned earlier, host records indicate that the tachinid Gymnoclytia occidua is a parasitoid of at least five species of pentatomid. Some of these pentatomids are known to be minor pests of both agricultural and ornamental plants, while others have the potential to be pests if their densities are high (McPherson, 1982; Schaefer &

Panizzi, 2000). Tachinid females produce a limited number of eggs during their lifecycle (Stireman et al., 2006). If mistaken oviposition events are a common occurrence for Gymnoclytia occidua, then this could result in decreased population density for this fly, potentially releasing populations of its typical hosts via reduced parasitism pressures. This could result in some of the host species reaching population levels where they attain significant pest status.

Although Gymnoclytia occidua was observed visiting traps containing

BMSB pheromones, the frequency of these occurrences was low. Therefore it remains uncertain if this parasitoid is being attracted by BMSB pheromones in the wild. It is also unclear if Gymnoclytia occidua accepts BMSB as a host in the wild. Individuals of BMSB were commonly encountered with tachinid eggs attached to their bodies, however the identity of tachinid species that laid these eggs is a mystery. Many phasiine tachinid flies produce eggs that look similar.

For this reason, the field-parasitism results (Figure 15) only confirm that BMSB are being used in the field by phasiines, but in order to determine which species are laying eggs more detailed research would have to be conducted to determine the what species. The parasitism rates in Fig. 15 are also likely underestimates of parasitism because some phasiine species (e.g., Cylindromyia spp.) inject eggs into hosts, and others lay eggs in concealed parts of the host (e.g.,

Gymnosoma par oviposits underneath elytra) where they may be overlooked

(Aldrich et al., 2006).

On one occasion I observed a Gymnoclytia occidua female attacking several BMSB adults and nymphs in the field, however this occurred under unnatural conditions. It happened on a residential property where a combination of pheromone lures were placed in close proximity to each other for the purpose of collecting stink bugs to bring back to the lab. This Gymnoclytia occidua female may have been attracted to the MDD or MDT pheromones of its native hosts, and upon arrival it encountered primarily BMSB hosts. Such events may also occur in the absence of pheromone traps; for example, in areas where BMSB occurs in high density and their food plants overlap with those of native

Euschistus parasitoids. This chance observation of Gymnoclytia occidua attacking an incorrect host after being drawn in by pheromones of its known hosts is consistent with the hypothesis that phasiine flies are less choosy at the host selection level because of their use of highly specific host-produced signals during host location.

FIGURES

Figure 1. The spread of the invasive species Halyomorpha halys, the brown marmorated stink bug (BMSB) through North America. Map courtesy of Tracy Leskey at USDA-APHIS and StopBMSB.org.

Figure 2. The life cycle of the tachinid parasitoid, Gymnoclytia occidua. A. A recently deceased, parasitized Euschistus servus female. The red circles highlight the locations of two tan colored eggs that had been oviposited by a Gymnoclytia occidua female several weeks prior. B. The same stink bug with the eggs removed. The red circle is highlighting the darkened area which is likely a scar left behind by the tachinid larva when it bored inside its host. The other egg revealed no such scar possibly indicating that it was a non-viable egg. C. Puparium of the larva that emerged from this host. D. The female Gymnoclytia occidua adult parasitoid that eclosed after approx. 14 days of pupation.

Figure 3. Sampling locations. A. Map of the state of Ohio. The red square shows the general area where sampling using pheromone-baited traps occurred. B. The area represented in the square zoomed in to show the individual sites. 1) Fulton Farms, Troy, Ohio; 2) Englewood MetroPark, Englewood, Ohio; 3) Carriage Hill MetroPark, Huber Heights, Ohio; 4) Patchwork Gardens, Dayton, Ohio; 5) Sugarcreek MetroPark, Bellbrook, Ohio. More details about the sampling area are given in Table 1.

Figure 4. A. An assembled pyramid trap. B. Assembled trap top with a pheromone lure hanging from the bottom of the lid.

Figure 5. Pentatomid pheromone molecules. A. The aggregation pheromone molecule of Euschistus spp. stink bugs; Methyl-2,4-decadienoate (MDD). B. The aggregation pheromone molecule of the stink bug Thyanta custator accerra; Methyl-2,4,6-decatrienoates (MDT). C. The two main components of the aggregation pheromone for H. halys (BMSB); (3S,6S,7R,10S)-10,11-epoxy-1- bisabolen-3-ol on left, and (3R,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol on right.

Figure 6. Photographs of collected tachinid species. A. Gymnoclytia occidua (Walker, 1849); B. Trichopoda pennipes (Fabricius, 1781); C. Cylindromyia fumipennis (Bigot, 1878); D. Euthera tentatrix (Loew, 1866); E. Euclytia flava (Townsend, 1891); F. Gymnosoma par (Walker, 1849). Not photographed: Phasia sp. (Latreille, 1804). Photograph ‘A’ courtesy of John Rosenfeld (Bugguide.net); all other photographs by M. W. Duncan.

Figure 7. Insect rearing chambers. A. One of the chambers used to house individual Gymnoclytia occidua females. B. Female parasitoid chambers and “bug-dorm” housing excess males.

Figure 8. 3D-printed stink bug models and the oviposition arena. A. Three of the models prior to being painted. A live H. halys (BMSB) male is also pictured. B. The same three models after being painted. C. The oviposition arena using model hosts. D. Configuration of the movement apparatus that was used to pull the moving model.

Figure 9. Containers used to house the parasitized hosts.

Figure 10. Insect attraction to pheromone lure treatments. A. The total number of individuals of the pentatomid species that were attracted to each pheromone treatment. B. The total number of individuals of the seven tachinid species that were attracted to the pheromone treatments. See Table 4.

Figure 11. Insect attraction to pheromone lure treatments lumped across field sites. A. The total number of individuals of the pentatomid species that were attracted to pheromone treatments at each site. B. The total number of individuals of the tachinid species that were attracted to pheromone treatments at each site. See Table 4.

Figure 12. The effect of pheromone presence vs. absence on host choice in Gymnoclytia occidua females during the model host trials. Chi-squared test: χ2=0.2763, df=1, P=0.5991.

Figure 13. Frequency of host choices made my Gymnoclytia occidua females during the oviposition assays. Number of trials is on the x-axis. A. Model host trials: Mean latency was 210.5 sec. for moving hosts and 178.3 for non-moving hosts; T-Test: (T=0.2145, df=2.481, P=0.8465). Binomial probability test of choice frequency: (P=0.01). B. (Live hosts) no-choice trials: Mean latency was 124.9 sec. for BMSB hosts and 124.4 for native hosts; T-Test: T=0.0091, df=11.253, P=0.9929). C. (Live host) choice trials: Mean latency was 124.3 sec. for BMSB hosts and 193.8 for native hosts; T-Test: (T=-1.0934, df=14.081, P=0.2926). Binomial probability test of choice frequency: (P=0.11).

Figure 14. Development success of Gymnoclytia occidua on native and non- native pentatomid host species. A. Results including all parasitized hosts. Chi- squared test: χ2=12.906, df=1, P=0.0003. B. Results after excluding hosts that were parasitized in the field. Chi-squared test: χ2=8.925, df=1, P=0.003.

Figure 15. Parasitism rates of field-collected pentatomids. Chi-squared test: χ2=0.064, df=1, P=0.801

TABLES

Table 1. Sampling locations for the pheromone attraction study.

Table 2. Number of individuals per species that were attracted to traps containing the three types of pheromone lures used (MDD, MDT and BMSB) and one negative control (CONT) containing no pheromones.

Table 3. Number of individuals per species that were attracted to traps containing the three types of pheromone lures used (MDD, MDT and BMSB) and one negative control (CONT) containing no pheromones.

Table 4. Summary of results of chi-squared tests to assess variation in pentatomid and tachinid attraction to pheromone traps.

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