The Pennsylvania State University

The Graduate School

College of Agricultural Sciences

THE MATING SYSTEMS OF THE EMERALD ASH BORER AND RELATED

BUPRESTID BEETLES

A Dissertation in

Entomology

Jonathan Peter Lelito

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

May 2009

The dissertation of Jonathan Peter Lelito was reviewed and approved* by the following:

Thomas C. Baker Professor of Entomology Dissertation Advisor Chair of Committee

James L. Frazier Professor of Entomology

James H. Marden Professor of Biology

James H. Tumlinson Professor of Entomology

Gary W. Felton Professor of Entomology Head of the Department of Entomology

*Signatures are on file in the Graduate School iii

ABSTRACT

The emerald ash borer, Agrilus planipennis (EAB), is a serious introduced invasive pest of North American ash trees in the genus Fraxinus. My research objectives

were to study the behavior of this beetle and other members of the genus Agrilus also

present in North America to find pre-mating behaviors that could be exploited to create a

more species-specific and effective trap for EAB than is currently available in the toolkit

for monitoring this invasive pest.

The first series of experiments I conducted were designed to elucidate activity

patterns and mate-finding behaviors of EAB in the field and laboratory. I conducted

behavioral observations of virgin pairs of EAB of known ages and recorded behaviors that might be related to mate-finding or courtship, such as wing-fanning, ‘juddering’ or

body-vibration, contact and mounting behavior by males. I also extensively observed

EAB in the field, looking specifically for gender-specific behaviors and diurnal activity

periods that might hint at how the beetles locate their mates in a natural setting. Males

are more active during the day, and both sexes strongly prefer sunlit perches on the host

plant. Male EAB perform pre-copulatory flights that end in rapid dives onto the backs of

stationary conspecifics, and are attracted to both dead male and dead female beetles

pinned to host plant leaves, suggesting the use of a visual cue to locate mates.

My next step was to take my work to the field and used the knowledge I gained

about the mating system of EAB to create a male-specific trap that may help to monitor

the spread of this pest. My first method of trap construction was to attach a dead EAB to

an ash leaflet and cover the leaflet in a spray-on adhesive; this simple strategy was iv

effective at capturing adult male EAB. I then tested these simple traps at both high and

low EAB population-density and at high and low heights in ash trees. As I had

hypothesized based upon my earlier behavioral observations and experiments, traps

captured more EAB at higher heights and higher population densities, and always

captured a strongly male-biased sample of adult EAB. I then performed a series of

trapping experiments to potentially improve the design of the trap, including using

synthetic materials with and without dead EAB and chemical cues as lures for adult EAB.

This series of experiments culminated in a broad comparison of my trap types against

USDA APHIS monitoring traps. I have now shown that on a surface-area basis, my traps are more effective than a plastic purple prism trap for capturing adult EAB in the field.

My research has also contributed to a change in APHIS’ monitoring regime from purple to green traps.

I have also performed investigations into the chemical cues used by EAB to assess a contacted conspecific for gender. By examining the cuticular chemistry of newly emerged and mature adult EAB of both genders, I have shown, in collaboration with other researchers, that female EAB cuticular chemistry changes as the beetle matures.

This change coincides with sexual activity in the laboratory. Further, use of a specific

cuticular hydrocarbon, 3-methyltricosane, can increase the time spent investigating a

dead beetle by live, adult male EAB. This is a first step toward identification of the

contact sex pheromone used by adult (presumably male) EAB to assess a contacted

conspecific for gender information, and has increased our understanding of the mating

system of this insect. v

Though the focus of this work is Agrilus planipennis, I also performed comparative studies on the mating behaviors of two other species in the target genus

Agrilus: A. subcinctus, a native ash-twig borer, and A. cyanescens, an introduced insect that feeds upon invasive plants in the genus Lonicera. My work on these insects shows that males of both species also appear to use visual cues to locate mates in a way similar to that of male EAB. Indeed, male A. cyanescens are even attracted to non-conspecific insects of similar color and can be induced to attempt to copulate with these non- conspecific lures, even off of the primary host plant. My experimental results strongly suggested that vision is the primary mode of mate location in my three target species.

These comparative studies helped to further the depth of understanding we have about mating behaviors in both EAB and Buprestid beetles in general.

In summary, my work has increased scientific understanding of the mating systems of three species of Agrilus beetles in the family Buprestidae. I have assessed the use of vision and contact chemistry in the mating systems of these beetles, and I have tested and improved a novel trap using a visual cue as a lure for a pest insect. This knowledge has allowed us to ask new questions regarding the roles of vision and cuticular chemistry in insects, and has added to the information available to researchers and regulatory officials concerned with slowing the spread of an invasive insect, the emerald ash borer.

TABLE OF CONTENTS

LIST OF FIGURES ...... ix

LIST OF TABLES ...... xi

ACKNOWLEDGEMENTS ...... xiii

CHAPTER 1: INTRODUCTION ...... 1

Topic and Scope of this Dissertation ...... 1

Buprestidae ...... 1

General ...... 1

Host-plant Location ...... 5

Mate-finding Behaviors ...... 7

Structural Coloration in Insects ...... 9

Insect Cuticular Lipids ...... 16

Agrilus planipennis, the Emerald Ash Borer ...... 18

Dissertation Chapters ...... 22

References ...... 25

CHAPTER 2: VISUALLY MEDIATED ‘PARATROOPER COPULATIONS’ IN THE MATING BEHAVIOR OFAGRILUS PLANIPENNIS (COLEOPTERA: BUPRESTIDAE), A HIGHLY DESTRUCTIVE PEST OF NORTH AMERICAN ASH TREES ...... 38

Abstract ...... 38

Introduction ...... 39

Methods ...... 40

Insects ...... 41

Laboratory Behavioral Observations ...... 41

Field Behavioral Observations ...... 42 vii

Mate-Finding...... 43

EAB Sticky Trapping Using Dummy Beetles ...... 48

Behavioral Analyses ...... 48

Statistical Analyses ...... 51

Results ...... 51

Laboratory Behavioral Observations ...... 51

Field Behavioral Observations ...... 54

Mate-Finding...... 56

EAB Sticky Trapping Using Dummy Beetles ...... 57

Female Ovipositor Pulsation ...... 61

Discussion ...... 61

References ...... 66

CHAPTER 3: NOVEL VISUAL-CUE-BASED STICKY TRAPS FOR MONITORING OF EMERALD ASH BORERS, AGRILUS PLANIPENNIS (COL., BUPRESTIDAE) ...... 68

Abstract ...... 68

Introduction ...... 69

Methods ...... 71

EAB Sticky-Leaf-Trapping ...... 71

Colored-Card Sticky-Trapping ...... 73

Statistical Analyses ...... 75

Results ...... 75

EAB Sticky-Leaf-Trapping ...... 75

Colored-Card Sticky-Trapping ...... 76

Discussion ...... 81 viii

References ...... 84

CHAPTER 4: BEHAVIORAL EVIDENCE FOR A CONTACT SEX PHEROMONE COMPONENT OF THE EMERALD ASH BORER, AGRILUS PLANIPENNIS FAIRMAIRE (COLEOPTERA: BUPRESTIDAE) ...... 88

Abstract ...... 88

Introduction ...... 89

Methods ...... 90

Insects ...... 90

Solvent Dipping and SPME Sampling ...... 91

Chemical Analysis ...... 91

Synthesis of 3-methyltricosane ...... 92

Field Behavior ...... 93

Laboratory Behavior ...... 95

Statistical Analyses ...... 99

Results ...... 100

Solvent Dipping and SPME Sampling ...... 100

Field Behavior ...... 101

Laboratory Behavior ...... 105

Discussion ...... 108

References ...... 110

CHAPTER 5: FIELD INVESTIGATION OF THE MATING BEHAVIORS OF AGRILUS CYANESCENS AND AGRILUS SUBCINCTUS ...... 112

Abstract ...... 112

Introduction ...... 113

Methods ...... 114 ix

Agrilus subcinctus ...... 114

Agrilus cyanescens ...... 116

Statistical Analyses ...... 118

Results ...... 119

Agrilus subcinctus ...... 119

Agrilus cyanescens ...... 123

Discussion ...... 129

References ...... 133

CHAPTER 6: A COMPARISON OF VISUAL- AND CHEMICAL-LURE STICKY TRAPS FOR MONITORING AGRILUS PLANIPENNIS FAIRMAIRE (COLEOPTERA: BUPRESTIDAE) ...... 135

Abstract ...... 135

Introduction ...... 136

Methods ...... 137

Visual Cue Test ...... 137

540 nm Sticky Cards ...... 139

Red Sticky Cards...... 140

Dichroic Glass Lures...... 140

Manuka Oil Lures ...... 141

Phoebe Oil Lures ...... 143

Statistical Analyses ...... 143

Results ...... 144

Visual Cue Test ...... 144

540 nm Sticky Cards ...... 144

Red Sticky Cards...... 146 x

Dichroic Glass Lures...... 149

Manuka Oil Lures ...... 151

Phoebe Oil Lures ...... 153

Discussion ...... 156

References ...... 159

CHAPTER 7: SUMMARY AND CONCLUSIONS ...... 162

Visually mediated mate-finding behavior ...... 162

Contact-cue mediated sex discrimination ...... 164

Trapping buprestids using visual lures ...... 166

Chemically mediated host-finding behavior ...... 168

LIST OF FIGURES

Fig. 1-1 Current status of the phylogeny of the Buprestidae ...... 3

Fig. 1-2 Closeup of emerald ash borer adult in the basking posture ...... 11

Fig. 1-3 Closeup photograph of emerald ash borer adult showing cuticular dimpling pattern and irridescent coloration ...... 13

Fig. 1-4 Photographic sequence of the development of adult coloration by EAB subsequent to pupal eclosion ...... 14

Fig. 2-1 Dead, pinned EAB adults in the three postures used to test male responses in the field to dead conspecifics with their elytra in different positions ...... 44

Fig. 2-2 Closeup photograph of EAB adult pinned to leaf with elytra closed, which is the lure used to study male pre-copulatory behavior in the field ...... 45

Fig. 2-3 Response of feral male EAB in the field to the varying posture of lure used in intial mate-finding experiements ...... 47

Fig. 2-4 Flight tracks prepared from video recording of male EAB during approach and landing on dead conspecifics in the field ...... 50

Fig. 2-5 Mean number of copulation attempts to each treatment of dead conspecific, as well as blank leaves and pins during field mate-finding experiments ...... 58

Fig. 2-6 Mean investigation time, by lure treatment, of EAB males in the field mate-finding experiment ...... 59

Fig. 2-7 Numbers of EAB adults captured in sticky-trapping experiments using dead, pinned EAB as a lure on an ash leaflet covered in Tangle-Trap ...... 62

Fig. 2-8 Closeup photograph of female EAB performing ‘ovipositor pulsation’ behavior in the field ...... 63

Fig. 3-1 Effect of sticky-trap age on the capture of EAB adult in the field during 2007 sticky-trapping experiments ...... 74

Fig. 3-2 Mean total capture of feral male EAB on EAB-SLTs during 2007 field experiments, by treatment ...... 77 xii

Fig. 3-3 Mean capture of male EAB per colored-card sticky trap during 2007 field trapping experiments ...... 78

Fig. 3-4 Mean capture of Agrilus cyanescens on colored-card sticky traps during the 2007 EAB field trapping experiments ...... 80

Fig. 4-1 A photograph showing a live male EAB in the center of the arena used for the ‘beetle-lure’ laboratory cuticular lipid bioassay ...... 98

Fig. 4-2 Gas chromatographic profiles of young and mature male and female EAB, with 3-methyltricosane indicated by an asterisk ...... 102

Fig. 4-3 Mean invesitgation time of beetle-lures by feral male EAB in the field, by cuticular lipid treatment ...... 104

Fig. 4-4 Number of copulation attempts being performed by male EAB to dead beetle-lures in the laboratory cuticular lipid bioassay ...... 107

Fig. 5-1 The probability of a male A. subcinctus performing either antennation only, ‘pounce’ behavior, or an airborne approach and copulation attempt to the four classes of beetle-lure used in the A. subcinctus field mate-finding experiment ...... 122

Fig. 5-2 Photograph of a live feral male A. cyanescens attempting copulation with an elytron of the tiger beetle Cicindela sexguttata that had been treated with a solvent wash from female A. cyanescens ...... 124

xiii

LIST OF TABLES

Table 2.1 Principal component analysis of EAB laboratory behaviors ...... 53

Table 2.2 Position of feral EAB on trees during dawn behavioral observations ...... 55

Table 2.3 Position of feral EAB on trees during nighttime behavioral observations .. 55

Table 2.4 Statistical table of pairwise comparions between lure treatments used during EAB mate-finding experiments ...... 60

Table 4.1 Summary of beetle-equivalent rates, chemical treatments of lures, and dosage of compound by treatment, for the EAB laboratory cuticular lipid bioassay ...... 96

Table 4.2 Summary of results from cuticular lipid extraction and analysis of young and mature adult EAB of both sexes ...... 103

Table 4.3 Results of the EAB laboratory cuticular lipid bioassay by beetle- equivalent rate, lure type, and chemical treatment of the lure ...... 106

Table 5.1 Mean duration in seconds of pre-copulatory behaviors performed by feral male A. subcinctus to washed and unwashed, male and female dead conspecifics used as lures in the field behavior experiment performed on A. subcinctus ...... 121

Table 5.2 Mean durations, in seconds, of behaviors performed by feral male A. cyanescens after coming into contact with dead, washed and unwashed, male and female A. cyanescens pinned to Lonicera host plants and nearby non-host plants during field experiments ...... 126

Table 5.3 Mean durations, in seconds, of behaviors performed by feral male A. cyanescens after coming into contact with the eight classes of heterospecifc and conspecific lures used during heterospecific lure field experiments both on and off Lonicera host plants ...... 127

Table 6.1 Summary of adult EAB capture data resulting from the ‘Visual Cue Test’ field experiment ...... 145

Table 6.2 Summary of adult EAB capture data resulting from the ‘540 nm Sticky Card’ field experiment ...... 147

Table 6.3 Summary of adult EAB capture data resulting from the ‘Red Sticky Card’ field experiment ...... 148 xiv

Table 6.4 Summary of adult EAB capture data resulting from the ‘Dichroic Glass Lures’ field experiment ...... 150

Table 6.5 Summary of adult EAB capture data resulting from the ‘Manuka Oil Lures’ field experiment ...... 152

Table 6.6 Summary of adult EAB capture data resulting from the ‘Phoebe Oil Lures’ field experiment ...... 154

Table 6.7 Effect of the presence or absence of Phoebe Oil on whole-tree capture of adult EAB during the ‘Phoebe Oil Lures’ field experiment ...... 155

ACKNOWLEDGEMENTS

I am indebted most of all to Dr. Thomas C. Baker, my advisor, my mentor, and my friend. Without his support, patience, input, and guidance – and his sense of humor – my work would certainly not have been as meaningful, or as enjoyable, as it has been.

I must also extend my thanks to the members of my dissertation committee: to

Dr. James Tumlinson, who, by always asking ‘What else have you got for us?’ helped me to make the most of my education; to Dr. James Frazier, whose advice and guidance helped me in many ways to become a better scientist and professional, and to Dr. James

Marden, who lent an outside ear to my work and offered many helpful suggestions along the way.

Thanks are also due to the colleagues whom I have been blessed with. Dr.

Katalin Böröczky has taught me more about chemistry than I ever learned as a student, and her kind advice has helped me overcome many obstacles. Mr. Bryan Banks has helped me to design so many of my traps and lures that he is surely due a degree in entomology by association. Drs. Andrew Myrick, Michael Domingue, and Seong-Gyu

Lee have been the best lab mates and friends one could ask for.

My family has been extremely important in supporting me throughout my life and career – my grandparents always encouraged me to learn everything I could about the natural world. I must extend by gratitude to my brother Jacob, who has always been supportive of me, patiently enduring hikes and outdoor fun where I played the role of entomology instructor. Above all others my father, Brian, and my mother, Mary, are to be thanked for their love, for giving me my first insect net at the age of four, and for xvi

letting me wander off into the field to make great discoveries – and not getting too upset when those discoveries escaped all over the dining room.

CHAPTER 1

INTRODUCTION

The emerald ash borer, Agrilus planipennis Fairmaire (EAB), is an introduced, invasive and highly destructive beetle whose larvae feed on and rapidly kill all native

North American ash trees in the genus Fraxinus. I have conducted research on EAB for the duration of my dissertation work and have opened up several important lines of investigation into EAB and buprestids in general. Below I provide brief overviews concerning topics related to some of my dissertation research findings. These include aspects of structurally generated insect coloration, insect cuticular hydrocarbons and the roles they play in insect behavior, and finally, aspects of the phylogeny and biology of the Buprestidae, the coleopteran family containing the genus Agrilus. I also provide an overview of what little is currently known about EAB as well as a brief synopsis of the methods by which phytophagous insects use olfaction to locate host plants. This body of knowledge may help put into context the work I present on the mating system of EAB and its relatives and on experiments to develop novel methods of trapping this invasive pest.

Buprestidae

General.

Beetles in the family Buprestidae, variously known as Metallic Wood-Boring

Beetles, Jewel Beetles, or Flat-Headed Borers (after the larvae), are usually brightly-

2 colored, iridescent and/or boldly patterned beetles. They are members of the monotypic coleopteran superfamily Buprestoidea, whose larvae feed on plant tissue (Bellamy and

Nelson 2002; White 1983). Larval feeding is most often conducted in the sapwood of trees and shrubs and some of these species are serious pests of wood. Others mine leaves and a few create galls in stems or else feed on root tissue (White 1983).

The Buprestoidea (Figure 1-1) lie within the Elateriformia, which includes the

Elateridae (click beetles) and their nearer relatives (Kukalová-Peck and Lawrence 1993).

The Buprestoidea do not have a clear sister group, and resolving taxonomic levels within the group has thus far been contentious (Bellamy 2003). Clear differences between the

Elateroidea and the Buprestoidea (beyond the obvious morphological considerations – buprestids do not possess the ‘click’ mechanism) include the description of sex pheromones from some members of the former, often pestiferous group (Tolasch et al.

2007; Tamaki et al. 1990; Borg-Karlson et al. 1988; Butler et al. 1975). Pheromones of many elaterids studied to date are made of multi-component blends, with up to 24 chemicals being secreted from female pheromone glands (Yatsynin and Rubanova 2002).

Within some species in the genus Agriotes there are even pheromone ‘dialects’, wherein the complicated pheromone blends of each species vary in a predictable way between populations (Yatsynin et al. 1996). Antennal morphology has also been studied in a few species of elaterids, notably Limonius aeruginosus (Merivee et al. 1998) and Melanotus villosus (Merivee et al. 1999). In both of these species, antennal morphology is similar between the genders, with the exception of type II sensilla basiconica in L. aeruginosus and type II trichoid sensillae in M. villosus. Males possess a significantly larger number of the latter sensilla type than do females, and neurons housed within these putatively

Figure 1-1. This figure shows a basic phylogeny of the Polyphaga showing the position of the Superfamily Buprestoidea (shown in red). Adapted from Bellamy 2003 and Kukalová- Peck and Lawrence 1993.

4 respond to the sex pheromone components of each species (Merivee et al. 1998, 1999).

Pheromones have been identified from other less related beetle groups as well, including the families Scarabaeidae, Curculionidae, and Scolytidae (Tóth et al. 2007; Miller et al.

2005; Petrice et al. 2005; Leal et al. 1994, 1993; Tumlinson et al. 1977, 1971; Henzell and Lowe 1970).

The family Buprestidae is divided into four subfamilies and numerous tribes and subtribes that vary in their taxonomic recognition (Bellamy 2003). The subfamily

Agrilinae, which contains the genus Agrilus, is known from all biogeographical areas of the planet and contains a sizeable proportion of the total species (almost 3000 described thus far) in the Buprestoidea (Bellamy 1985). Adults in the genus Agrilus such as EAB typically have metallic coloration of the adults and larvae are phloem-feeders (White

1983). Adults are alert, fast-moving and quick to take flight. Many seem to prefer sunny situations and exposed perches (Bellamy and Nelson 2002). Other common members of this genus in North America that are considered pests include the Two-Lined Chestnut

Borer, A. bilineatus Weber, which attacks oak, chestnut, and related trees, and the Bronze

Birch Borer, A. anxius Gory, which often kills introduced ornamental birch trees but can also harm native birch species. The behavior of A. cyanescens and A. subcinctus, an introduced and a native species, respectively, will be addressed in my comparative work in Chapter 5.

Host-plant Location.

Past research has indicated that buprestids appear to first locate a host plant and then secondarily seek mates by visual, tactile, and other non-pheromonal cues (Carlson and Knight 1969; Matthews and Matthews 1978; Gwynne and Rentz 1983). Much of my

5 research has shown directly and indirectly that it is the host plant that brings conspecifics together for mating.

Several Agrilus species appear to be preferentially attracted to damaged hosts.

Among these are: A. planipennis (McCullough et al. 2006; Poland et al. 2005); A. bilineatus (Cote and Allen 1980, and references therein); A. difficilis Gory (Akers et al.

1986; Westcott 1973); A. liragus Barter and Brown (Barter 1965), and A. anxius (Barter

1957; Anderson 1944). “Damaged hosts” in such cases variously means trees girdled for experimental purposes, or else naturally broken branches on otherwise healthy trees, but the effect is likely the same: the damaged plants emit a volatile chemical profile that indicates stress, and this volatile emission can function as a long-distance cue to attract herbivores and their natural enemies (Dicke and van Loon 2000). Terpenoids, which are given off by many plants after attack by insects (Van Den Boom et al. 2004), are a class of volatile organic compounds emitted by stressed plants in addition to those volatiles that are emitted normally or as the result of mechanical damage to tissue (Peñuelas and

Llusiá 2004). Induced plant volatiles from stressed plants may directly permit plants to reduce immediate attack by insects and resist future damage indirectly by serving as volatile cues by which natural enemies of the herbivore are drawn into the vicinity

(Thaler et al. 2002; Dicke and van Loon 2000; Paré and Tumlinson 1999, 1997; De

Moreas et al. 1998; Turlings and Tumlinson 1992). In addition to these roles, induced plant volatiles such as terpenoids may also indicate a plant that is actively being attacked by conspecific herbivores, and thus may facilitate attraction of additional herbivorous insects, including beetles in the families Chrysomelidae and Scarabaeidae (Bolter et al.

1997; Loughrin et al. 1995; Harari et al. 1994). The EAB has been shown to be more

6 attracted to prism-shaped traps emitting tree-distillate oils containing sesquiterpenes than to traps lacking these oils (Crook et al. 2008). The hypothesized advantage to the insect is the location of a favorable host-food source, as indicated by the release of large amounts of volatiles into the environment (Vet and Dicke 1992). For a resource the size of a mature ash tree, competition for access to food is likely to be minimal between individual EAB. For EAB, it is also possible that cueing in on the plant volatile signal aids in location of conspecifics for mating.

Akers et al. (1986) reported that in a nursery setting, both large and small honeylocust trees (Gleditsia triacanthos) especially those in drought-stressed condition, were colonized by A. difficilis. Westcott (1973) reported that drought stress attracted buprestids and other boring insects to trees and predisposed them to subsequent damage, although the mechanism of attraction was unclear.

De Groot et al. (2008) have shown that EAB, especially the males, show antennal responses to the green leaf volatile Z-3-hexenol, which is emitted from ash trees upon feeding by EAB. Female antennal responses were weaker and less consistent than those of males. Females were not preferentially attracted to traps containing Z-3-hexenol (De

Groot et al. 2008). A series of experiments conducted on A. bilineatus and its oak host tree yielded interesting results, including a strong preference of this species for artificially girdled trees over non-girdled, otherwise healthy trees (Dunn et al. 1986). Another study of A. bilineatus showed that beetles were attracted to conspecifics in the presence of host leaves that were used as food within cages in preference to the presence of only host leaves (Dunn and Potter 1988). However, male and female A. bilineatus arriving at cages did not prefer host-tree logs over empty control cages in both seasons, indicating logs of

7 the host-tree alone may not emit an attractive signal to A. bilineatus (Dunn and Potter

1988). Therefore, studies indicate thus far that it is possible that in conjunction with damaged or stressed host-plant material conspecific buprestids increase the attraction of additional beetles, although this remains to be tested more vigorously.

Mate-Finding Behaviors.

Little direct evidence exists in the literature concerning actual mating behaviors of

Agrilus or other buprestids. What little information is available is largely of general scope concerning broad groups of species (Carlson and Knight 1969; Matthews and

Matthews 1978). In some species of buprestids, such as the widespread pest of fruit and other trees Chrysobothris femorata Olivier, males are able to attract females by drumming on the substrate (Fenton 1942). Such behavior has not been observed in

Agrilus. In other buprestid genera, males possess pectinate antennae that have been hypothesized to function in olfaction, presumably to locate females in the habitat (Wellso

1966). Members of the genus Agrilus do not typically show sexual dimorphism in antennal structure at the macroscopic level. They also do not appear to differ significantly between the sexes when examined under scanning electron microscopy (D.

Crook, personal communication).

These few reports involving the mating behavior of beetles in the genus Agrilus

(e.g. Rodriguez-Saona et al. 2008; Lelito et al. 2007; Akers and Nielsen 1992; Gwynne and Rentz 1983) have generally indicated the use of visual and tactile cues for location of conspecifics. However, field studies conducted on A. bilineatus by Dunn and Potter

(1988) yielded an interesting result: feral males arriving at cages preferentially arrived at those cages that contained female beetles compared with those that did not, although this

8 result was not duplicated in both of the field seasons in which this experiment was performed. The authors themselves advised caution in the interpretation of these results and no subsequent report has been published showing clear attraction of one gender to another over distance in an Agrilus species.

In some cases buprestids have been reported to be attracted to human-made objects such as glass bottles, as was observed for a buprestid species in Australia

(Gwynne and Rentz 1983). Chapter 2 of this dissertation involving the role of vision in

A. planipennis mate-finding (Lelito et al. 2007) represents one of the very few experimental studies directly observing and manipulating mating behavior of any member of the genus Agrilus in the field. A second study in a later publication by other investigators supported several of my conclusions regarding the role of vision in the EAB mating system (Rodriguez-Saona et al. 2008).

Akers and Nielsen (1992) examined captive individuals of A. anxius in the laboratory and recorded numbers of copulations and age of onset of sexual behaviors. To date this represents one of the few laboratory studies of buprestid mating behavior. The main conclusions of this particular work were that naïve male A. anxius took longer to copulate, and copulated for a shorter duration, than ‘experienced’ males who had previously copulated with a female. Interrupting copulation caused a shorter latency-to- copulation when the beetles were reintroduced to one another, but female A. anxius became unreceptive to males after being inseminated. No significant effect on fecundity of females being multiply mated was found, implying that for females, one mating is sufficient for future reproductive success and a single mating by females may occur in the wild (Akers and Nielsen 1992).

Structural Coloration in Insects

Because the Buprestidae are known for their brilliant metallic coloration and I have shown that there is a strong visual attraction of male EAB to dead EAB models pinned to leaves, it may be instructive to look at the structural color generating mechanisms used by selected other insects as examples. Similar mechanisms may be used by EAB, but have not as yet been investigated. First experimentally assessed by

Anderson and Richards in 1942, the wings of Morpho butterflies were shown to have complicated repeating structures on the scales of the wing that appeared to confer the iridescent color. Morpho butterfly wings, which usually emit a striking blue flash of color possess multiple micro-lamellar structures of varying heights on the scales that produce the interference blue color. The blue sheen comes from the combination of interference of off-color wavelengths and diffraction from the micro-lamellae of the scales themselves, as well as from a further ‘tuning’ of emitted light by means of selective absorption of unwanted background wavelengths by an underlying pigment layer on the wing (Kinoshita et al. 2002). There have been numerous other studies on the scale micro-structures of various Lepidoptera that emit brilliant structural interference colors as well (Ghiradella 1991, 1989, 1986, 1984; Ghiradella et al. 1972; Huxley 1976;

Morris 1975).

The production of structural colors involves some form of differential reflectance between layers of material having different heights that causes most wavelengths of reflected light to interfere with each other and cancel out. One narrow set of wavelengths, however, is reinforced during reflection, enhancing the intensity of the

10 reflectance of this wavelength, creating a pure color (Land 1972). The most physically

‘optimal’ formation, from a mathematical point of view, is the ‘quarterwave stack’ (Land

1972; Macleod 1969), that involves material, such as the cuticle of the insect, that is layered at regular intervals equal to one-quarter the wavelength of the desired reflected light. This principle can be modified not only by varying the interval between the layers to create different, pure colors, but also by varying the layers within the ‘stack’ into a

‘chirped’ pattern (Ouellette et al. 1995). The latter pattern creates the metallic broadband reflectance of silver or gold seen in some beetle species as well (Parker et al. 1998).

In addition to Lepidoptera, there have been investigations into structural coloration in other groups of insects, most notably the beetle family Scarabaeidae (Parker et al. 1998; Neville 1977; Caveney 1971). Research into the physics and optics of the

‘jewel’ scarab beetles, in particular, has elucidated many of the physical processes through which scarabs create different coloration (Parker et al. 1998). In an examination of green and gold scarabs, those authors found that both the lamellar structure and the underlying pigment layer (or its absence) work together to create a specific color of adaptive value to the insect. By varying the interlayer distance as the depth within the cuticle as a whole increases, a higher degree of monochromatic reflectance is achieved

(Land 1972). There is also evidence that a basal layer of pigmentation can enhance the interference coloration produced by the upper layers by absorbing light that would otherwise reflect up through the lamellae and reduce the purity of the desired color

(Parker et al. 1998).

It is possible that the intense metallic coloration of the EAB (Figure 1-2) is produced by processes similar to those of the scarab jewel beetles. I have observed,

Figure 1-2. This figure shows an image of an adult emerald ash borer, with its elytra partially open in a posture the insect assumes when basking in direct sunlight or immediately preceding flight.

12 through careful observation of the pupae and newly eclosed adults of EAB, a process that suggests that both pigment and structure play a role in adult color. The elytra and wings at the time of eclosion are clear, and upon expansion of the wings, myriad dimples

(Figure 1-3) appear on the elytral cuticle that are highly reflective to steady light sources and flash photography. Also at this time, the rest of the beetle is already a brilliant iridescent green or green-gold color. Within 24 hours of eclosion, the elytra have taken on their characteristic emerald color (Figure 1-4). The reflective dimples resemble those of several species of metallic scarab beetles (Chrysophora chrysochlora, Pyronota festiva and Chrysina woodi) described by Lowrey et al. (2007). My observations of the rapid color change following the full formation of the physical structure of the elytra suggests that a basal pigment is involved, or else the material that ultimately fills the cuticular dimples may be involved in color production by EAB.

Figure 1-3. A dorsal (A) and lateral (B) view of individual EAB, showing the reflective dimples that cover the cuticular surface of the beetle and may impart the brilliant color of the insect.

15 Figure 1-4. The first panel shows a male emerald ash borer pupa just prior to adult emergence (A) with the green iridescence of the body clearly visible through the pupal cuticle. Following eclosion of the adult, the wings and elytra quickly expand and take on a shining silver-white coloration after approximately one to two hours (B). Four hours after eclosion (C), some green iridescence is visible, especially along the midline of the abdomen at the junction of the elytra. At 24 hours post-eclosion (D), the adult is assuming its final coloration, although the cuticle is still soft and pliable and further hardening will occur for approximately another 24 hours.

Insect Cuticular Lipids

The primary role of the cuticular surface lipids that coat the insect cuticle is to protect insects from water loss (Hadley 1984) and attack by microorganisms and other pathogens (Howard and Blomquist 2005, and references therein). Because these roles for cuticular lipids are not specifically examined by my dissertation work, I refer the reader to two excellent reviews of those subjects and others concerning the myriad functions of insect cuticular lipids (Howard and Blomquist 1982, 2005). My main research focus on the cuticular lipids of EAB was to investigate their role as potential sex

attractants and in gender discrimination by individual insects upon contact.

Although a total-body cuticular lipid profile has been often shown to be species- specific (Howard 1993), profiles may vary between the genders within an insect species

(Lockey 1991). Moreover, many insects utilize surface-lipid-based contact cues to identify each other as conspecifics once initial contact has been made (Smith and Breed

1995; Scott 1994; Howard 1993; Jallon 1984). In the case of certain insects such as the house fly, Musca domestica, Z-9-tricosene was identified as a cuticular hydrocarbon that also functioned as a sex attractant and aggregation pheromone (Carlson et al. 1971). My first examination of EAB in the field led to the conclusion that a contact chemical cue was involved in gender recognition by male EAB (Lelito et al. 2007), and I began to pursue cuticular chemistry as a potential new source for chemical lures (see Chapter 4).

Some of the many roles for cuticular lipids in insect behavior can include the mediation of social functions and to elicit mating behavior, depending on the circumstances

(Howard and Blomquist 2005). In light of previous work on the M. domestica sex

17 pheromone (Carlson et al. 1971) chemical analysis of EAB cuticular coatings seemed like a logical course to pursue.

Cuticular lipids can play a key role in nestmate recognition in the social

Hymenoptera (Breed 1998; Singer et al. 1998; Vander Meer and Morel 1998) and

Isoptera (Clément and Bagnères 1998). Social status can also affect the cuticular lipid profile of an individual. A change in social status from subordinate worker to dominant reproductive female subsequently can result in a corresponding change in the lipid profile

(Peeters et al. 1999; Monnin et al. 1998). Other insect species have evolved the ability to exploit nestmate communication systems. Staphylinid beetles that live as inquilines within termite colonies, for example, have evolved a profile of cuticular lipids that the beetles themselves synthesize to match the average profile of termites of their host species (Howard et al. 1980).

Cuticular lipids have been shown in many studies on many insect species of the years to play significant roles in courtship and gender recognition. Once individual insects locate each other and have made contact, courtship behaviors can ensue that depend on cuticular lipids to affect the outcome of the encounter (Van den Assem et al.

1980; Matthews 1975). Male insects such as those of the wasp Cardiochiles nigriceps will sometimes respond with sexual behavior to both male and female dead conspecifics if female cuticular lipids are applied to the cuticle of an experimental ‘dummy’

(Syversten et al. 1995). Additionally, data reported by Page et al. (1990) suggest that cuticular lipids are likely to play a role in the recognition of conspecifics for aggregation and mating by cone beetles (Coleoptera: Scolytidae), because many morphologically similar species are distinct in their lipid profiles.

18 In D. melanogaster, newly emerged adult flies can have identical male and female cuticular profiles, but sexual dimorphism of lipid composition can develop as the flies age and become sexually active (Péchiné et al. 1988). I thus chose to investigate both newly eclosed and mature EAB of both genders for any differences in their cuticular lipid profiles that might play a role in courtship behavior after the initial visually-guided male approach to females. My results have shown that females and males are at first very similar with respect to cuticular lipid composition but this characteristic changes as the adults approach and attain sexual maturity, typically between five and ten days of age

(Lelito et al. 2007). Further, one of the compounds that increases in quantity in females over time, 3-methyltricosane, was shown to release pre-copulatory activity in male EAB in both field and laboratory experiments (see Chapter 4).

Agrilus planipennis, The Emerald Ash Borer

The emerald ash borer, Agrilus planipennis Fairmaire, is a serious non-native pest of ash (Fraxinus sp., Oleaceae) trees in parts of North America including Michigan,

USA, and the adjacent area of Ontario, Canada (Haack et al. 2002). It is not a pest within the native range of China, Japan, and surrounding countries (Herms et al. 2005); indeed, it is only found in any abundance when attacking reforested areas planted with North

American ash species (Liu et al. 2003; Bauer et al. 2005; Gould et al. 2005). On this

continent, the beetle is able to feed readily on green ash (F. pennsylvanica Marsh.), white

ash (F. americana L.), and black ash (F. nigra Marsh.), and the larval damage to phloem

often kills the tree within two to three years (Liu et al. 2003). Other native species are

attacked somewhat less readily, but are subsequently destroyed as well when the favored

19 hosts are removed by heavy infestations (Agius et al. 2005). EAB larvae are not currently known to complete development in plant genera other than Fraxinus, but oviposition will occur in no-choice tests on walnut and privet, among others (Agius et al.

2005).

Evidence suggests the beetle was established for 6-10 years before initial detection (McCullough and Katovich 2004), and likely arrived in the United States in wood packing material from Asia (Herms et al. 2004). After its initial discovery near

Detroit, Michigan, the beetle has subsequently turned up in Ohio and Indiana as well

(Ohio State University 2006; Indiana Department of Natural Resources 2006). In 2006, new infestations were identified in the area of Kane County, Illinois (Illinois Department of Agriculture 2006), and the Upper Peninsula of Michigan (Michigan Department of

Agriculture 2006), indicating further spread was likely. The beetle spreads by both adult dispersal and the transport of larvae and pupae in firewood. Adults are active and disperse on their own during the extent of their 3-4 week life span (Lyons et al. 2004), but the movement of infested nursery stock has also contributed to the spread of EAB

(Marchant 2005). Adult females are able to fly up to 4 km daily, but the modal distance traveled is only 0.8 km in 24 hours (Taylor et al. 2004). Currently, it is estimated that without human intervention (movement of firewood, etc.), EAB populations expand at approximately 1 km/year (McCullough et al. 2005). Nonetheless, infestations must be eliminated before the beetle is able to colonize the surrounding area and therefore efficient detection and trapping of EAB is of paramount importance in the control of this pest.

20 EAB infestations are insidious in that they often go undetected in the early stages of tree destruction. Dieback occurs first at the top of the tree, and the characteristic D- shaped exit holes are only noticeable at ground level by the time the beetle has significantly damaged the tree (Haack et al. 2002). To further complicate matters, recent evidence has come to light that suggests EAB larvae can take multiple years to develop in the tree, lengthening the time it takes to produce the visible symptoms commonly used for detection (Siegert et al. 2005). Currently, trapping ability for early detection is limited to the use of purple colored sticky traps that capture both sexes of EAB, and ‘trap trees’ that have been artificially girdled and then sampled later for any larvae that might have resulted from females ovipositing on the damaged tree (Cappaert et al. 2005).

Girdled trees appear to be attractive to EAB females as oviposition sites, and this suggests the use of some form of stress-response plant volatile compound in host-finding

(Poland et al. 2004, 2005). Testing has been undertaken to find these volatile cues

(Crook et al. 2005, 2007) and these volatiles may help to increase trap effectiveness.

The use of purple traps and girdled trees is often concurrent. It had been noted that EAB and other buprestids are attracted to the color violet (Oliver et al. 2002), and this gave rise to the original use of purple-colored plastic prism traps. A more recent study indicates that purple traps are more attractive to EAB compared to red, yellow, green, white, and other colors (Francese et al. 2005). Therefore, at present, purple traps are the only available tool for detecting EAB through trapping of adults. The trap may become even more useful if attractive host plant volatile compounds can be incorporated.

Girdled trees, although effective, come at a huge cost of time and labor and therefore are not optimal for wide-scale detection efforts (Cappaert et al. 2005). Purple traps may be

21 less reliable at extremely low density outlier sites (Metzger et al. 2007) and therefore a species-specific trap would be highly beneficial as a component of the toolkit used against EAB.

Some promise for luring EAB is currently offered by plant volatile blends distilled from a commercially available pair of tree distillates, Manuka Oil and Phoebe

Oil (Crook et al. 2005, 2007, 2008; Anulewicz et al. 2007). These oils contain compounds similar to the blend of induced volatiles emitted by ash bark and ash leaves, respectively (Crook et al. 2005, 2007), and are particularly rich in the abundance of many terpenoid compounds. Some evidence from the current regime of trapping devices suggests that the addition of certain blends of host-bark and host-leaf odor result in higher trap catch, depending on trap design (Poland and McCullough 2007). Locating host trees is critical for EAB: conspecifics are located on the host, and thus the host ash tree represents a resource for both food and potential reproduction.

Almost nothing is known specifically about the mating habits of EAB in the U.S. or in its native range aside from the work presented here. Some researchers report that more males are captured higher in the tree (Lance et al. 2007) which may be related to the site of mating behavior or to the higher level of male activity compared to females. EAB may be much like other buprestids studied to date, and thus there is no evidence to suggest that EAB uses a long range pheromone to locate conspecifics. The only potential pheromone-related compound that has any sex-specificity of production is a macrolide identified by Bartelt et al. (2007). However, no reports have yet been published that show this compound has any behavioral activity in the field or laboratory. Researchers

22 have pointed out that the lack of a long-distance pheromonal communication in EAB is likely to make the development of a species-specific trap more difficult (Otis et al. 2005).

Dissertation Chapters

As outlined above, the scientific knowledge concerning EAB is limited, and this

is true of the family Buprestidae as well. My research focuses on examining the behavior

of EAB and its close relatives under natural conditions in order to allow future

researchers to examine in more detail some of the areas my research has begun to

elucidate. As such I have attempted to investigate in the field the general mate-location

and mating behaviors of my target species and its relatives. These studies have indicated

the use of visual and contact chemical cues by these beetles to discriminate conspecifics

and have helped begin the development of a potentially improved trapping system based

on the visual cues used by EAB to locate prospective mates.

Chapter 2 begins my dissertation research by exploring the behavior of adult

emerald ash borers in the laboratory and in the field. Here I present the results of a

preliminary trapping experiment at the beginning of my first field season. This work

directly led to all later research on using visual cues to trap EAB. I characterized

‘paratrooper copulations’, the sequence of behaviors that brings flying male EAB into

direct contact with perched females and begins the mating sequence. This first field

exploration of natural EAB mating behavior was novel and helped to focus efforts to

learn more about this invasive insect and improved trap design. The chapter is entirely

my own work, and has been published in the Journal of Insect Behavior (Lelito et al.

2007). Chapter 2 generated two important lines of inquiry that formed the focus of later

23 work: 1) whether visual cues alone could trap adult EAB; 2) the elucidation of contact pheromone components in the cuticular lipids of females that had initially been indicated by the investigations described in Chapter 2.

Chapters 3 and 4 diverge somewhat in focus. Chapter 3 details my first wide- scale efforts at producing a novel device that uses a solely visual cue (specifically, a dead conspecific insect) to lure and trap adult EAB. I tested a novel sticky-leaflet trap design

(EAB-SLTs) at different heights in the tree and at different population densities of EAB in an effort to understand how these factors would influence trapping efficacy. I also performed these experiments to perhaps shed light on how other trap types might be influenced by these variables as well. Chapter 3 is entirely my own work and has been published in the Journal of Applied Entomology (Lelito et al. 2008).

Chapter 4 focuses on a second aspect generated from the results reported in

Chapter 2, namely, the role of cuticular lipids in the mating system of EAB. I attempted to investigate this issue from both a laboratory and a field perspective. With the assistance of Dr. Katalin Böröczky in Dr. James Tumlinson’s laboratory at Pennsylvania

State University, and using their GC-MS and associated equipment, we analyzed the cuticular lipid profiles of immature and mature EAB of both genders. We discovered characteristic differences in the female lipid profile that coincide with sexual maturity.

One branched cuticular compound, 3-methyltricosane, was tested in the field for behavioral activity. The application of this compound to a dead EAB lure significantly increased the time feral male EAB spent in attempted copulation with that lure compared to solvent-washed controls. However, dead female EAB with their native cuticular coating left intact elicited a significantly longer duration of investigation when contacted

24 by feral males. This led to the obvious conclusion that there may be other as yet unidentified compounds on the cuticle of female EAB that mediate sex recognition. All fieldwork was performed by me. This work has been accepted for publication, and will appear in the February 2009 issue of the Journal of Chemical Ecology.

Chapter 5 extends my investigation of aspects of the research reported in Chapters

2 and 4 by taking a comparative approach and examining the behaviors of two related

Agrilus species in the Buprestidae, A. subcinctus and A. cyanescens. I used pinned, dead beetles as lures to explore the visual responses to such stimuli in A. subcinctus and A. cyanescens. In both species, I found that as in EAB, males will respond to dead conspecifics of either gender with a copulation attempt. In addition, I performed a series of experiments with A cyanescens to test whether visual lures such as insects having a color or shape similar to conspecifics (in this case, blue-green elytra from an unrelated beetle species) could function effectively as a visual lure for male beetles. Indeed, I found that some of these heterospecific lures could be just as attractive to male A. cyanescens as conspecific lures. I found that as in EAB, a contact pheromone seems to be involved in courtship. Application of crude cuticular solvent washes containing conspecific female compounds increased the duration of male A. cyanescens investigation of heterospecific lures. Conversely, solvent washing a conspecific beetle lure to strip it of cuticular lipids decreased the duration of investigation by male beetles.

Finally, I also examined the behavior of A. cyanescens on plants other than its natural host species. I found that males will respond with attempted copulations to lures placed on non-host plants; however, the rate at which they do so is much lower than that observed on the host plant, suggesting that host-plant cues serve to attract and arrest the

25 beetles, after which they search for mates visually. This research was performed entirely by me and has been submitted to Physiological Entomology for publication.

In Chapter 6, I perform a final investigation of trap efficacy based on my previous work on trapping, detailed in Chapters 2 and 3. I compared a range of plastic prism trap types and chemical lures used by USDA APHIS to monitor EAB in the field with my own visual-lure traps and visual-lure traps enhanced with chemical lures identical to those used by USDA APHIS. These studies showed that exploiting the visually-mediated male EAB pre-copulatory dive is an effective method of trapping male EAB that outperforms purple prism traps on a surface area basis. In addition, I showed that the new APHIS green prism traps inspired by the results of my field research (Lelito et al.

2007) and the EAB-SLT traps could be made to trap more EAB by the addition of sesquiterpenes-rich Phoebe Oil extracts. I was provided with the chemical lures used for this work by ChemTica Internacional in Costa Rica, and the fieldwork and trap construction itself was performed entirely by me. This chapter has been submitted to the

Journal of Applied Entomology for publication.

REFERENCES

Agius AC, McCullough DM, Cappaert DA (2005) Host range and preference of the emerald ash borer in North America: preliminary results. In Mastro, V.C. and Reardon, R. (eds.), Emerald ash borer research and technology development meeting, FHTET-2004-15, USDA Forest Service, Morgantown, WV, pp. 28-29

Akers RC, Herms DA, Nielsen DG (1986) Emergence and adult biology of Agrilus difficilis (Coleoptera: Buprestidae), a pest of honeylocust, Gleditsia triacanthos. Great Lakes Entomologist 19:27-30

Akers RC and Nielsen DG (1992) Mating behavior of the bronze birch borer,

26 (Coleoptera: Buprestidae). Journal of Entomological Science 27:44-49

Anderson RF (1944) The relation between host condition and attacks by the bronze birch borer. J. Econ. Ent. 37:588-596

Anderson TF and Richards AG (1942) An electron microscope study of some structural colors of insects. Journal of Applied Physics 13:746-758

Anulewicz AC, McCullough DG, Poland TM, Cappaert DL (2007) Attraction of emerald ash borer to trap trees: can meja or manuka oil compete with girdling? In Mastro, V., Lance, D., Reardon, R. and Parra, G. (eds.), Emerald ash borer research and technology development meeting, FHTET-2007-04, USDA Forest Service, Morgantown, WV, p. 83

Bartelt RJ, Cossé AA, Zilkowski BW, Fraser I. (2007) Antennally Active Macrolide from Emerald Ash Borer Agrilus planipennis Emitted Predominantly by Females. J. Chem. Ecol. 33:1299-1302

Barter GW (1957) Studies of the bronze birch borer, Agrilus anxius Gory, in New Brunswick. Canad. Ent. 8912-36

Barter GW (1965) Survival and development of the bronze poplar borer Agrilus liragus Barter and Brown (Coleoptera: Buprestidae). Canad. Ent. 97:1063-1068

Bauer LS, Liu H, Haack RA, Gao R, Miller DL, Petrice TR (2005) Update on emerald ash borer natural enemy surveys in Michigan and China. In Mastro, V.C. and Reardon, R. (eds.), Emerald ash borer research and technology development meeting, FHTET 2004-15, USDA Forest Service, Morgantown, WV, p. 8

Bellamy CL (2003) An illustrated summary of the higher classification of the superfamily Buprestoidea (Coleoptera). Folia Heyrovskyana, Supplementum 10, Kabourek, Czech Republic

Bellamy CL (1985) A catalogue of the higher taxa of the family Buprestida (Coleoptera). Navorsinge van die Nasionale Museum, Bloemfontein 4:405-472

Bellamy CL and Nelson GH (2002) Buprestidae. In: Arnett, R. H. and Thomas, M. C.: American Beetles (Vol. 2). CRC Press LLC, Boca Raton, Florida, U.S.A.

Bolter CJ, Dicke M, van Loon JJA, Visser JH, Posthumus MA (1997) Attraction of Colorado potato beetle to herbivore damaged plants during herbivory

27 and after its termination. J. Chem. Ecol. 23:1003-1023

Breed MD (1998) Chemical cues in kin recognition: criteria for identification, experimental approaches, and the honey bee as an example. In Pheromone Communication in Social Insects: Ants, Wasps, Bees, and \ Termites. Eds: Vander Meer, R.K., Breed, M.D., Espelie, K.E., and Winston, M.L. p. 57-78. Westview, Boulder, Colorado, U.S.A.

Borg-Karlson AK, Agren L, Dobson H, Bergstrom G (1988) Identification and electroantennographic activity of sex-specific geranyl esters in an abdominal gland of female Agriotes obscurus (L.) and Agriotes lineatus (L.) (Coleoptera: Buprestidae). Experientia 44:531-534

Butler, L.I., McDonough, L.M., Onsager, J.A., and Landis, B.J. 1975. Sex- pheromones of Pacific Coast wireworm, Limonius canus (Coleoptera: Elateridae). Environmental Entomology 4: 229-230

Cappaert D, McCullough DG, Poland TM, Siegert NW (2005) Emerald ash borer in North America: A research and regulatory challenge. Am. Entomol. 51:152-165

Carlson DA, Mayer MS, Silhacek DL, James JD, Beroza M, Bierl BA (1971) Sex attractant pheromone of the housefly: isolation, identification, and synthesis. Science 174:76-77

Carlson RW and Knight FB (1969) Biology, taxonomy, and evolution of four sympatric Agrilus beetles (Coleoptera: Buprestidae). Contrib. Am. Entomol. Inst. 4:1-105

Caveney S (1971) Cuticle reflectivity and optical activity in scarab beetles. Proc. R. Soc. Lond. B 178:205-225

Clément J-L Bagnères A-G (1998) Nestmate recognition in termites. In Pheromone Communication in Social Insects: Ants, Wasps, Bees, and Termites. Eds: Vander Meer, R.K., Breed, M.D., Espelie, K.E., and Winston, M.L. p. 126-155. Westview, Boulder, Colorado, U.S.A.

Cote WA and Allen DC (1980) Biology of the two-lined chestnut borer, Agrilus bilineatus, in Pennsylvania and New York. Ann. Ent. Soc. Am. 73:409- 413

Crook DJ, Francese J, Fraser I, Mastro VC (2005) Chemical ecology studies on the emerald ash borer. In Mastro, V.C. and Reardon, R. (eds.), Emerald ash borer research and technology development meeting, FHTET 2004- 15, USDA Forest Service, Morgantown, WV, p. 55

Crook DJ. Khrimian A, Francese JA, Fraser I, Poland TM, Sawyer AJ, Mastro VC (2008) Development of a host-based semiochemical lure for trapping emerald ash borer Agrilus planipennis (Coleoptera: Buprestidae). Environmental Entomology 37:356-365

Crook DJ, Khrimian A, Francese JA, Fraser I, Poland TM, MastroVC (2007) Chemical ecology of the emerald ash borer. In Mastro, V., Lance, D., Reardon, R. and Parra, G. (eds.), Emerald ash borer research and technology development meeting, FHTET-2007-04, USDA Forest Service, Morgantown, WV, p. 79

De Groot P, Grant GG, Poland TM, Scharbach R, Buchan L, Nott RW, Macdonald L, Pitt D (2008) Electrophysiological response and attraction of emerald ash borer to green leaf volatiles (GLVs) emitted by host foliage. J. Chem. Ecol. 34:1170-1179

De Moraes CM, Lewis WJ, Paré PW, Alborn HT, Tumlinson JH (1998) Herbivore-infested plants selectively attract parasitoids. Nature 393:570- 573

Dicke M and van Loon, JJA (2000) Multitrophic effects of herbivore-induced plant volatiles in an evolutionary context. Entomol. Exp. Appl. 97:237- 249

Dunn JP and Potter DA (1988) Evidence for sexual attraction by the twolined chestnut borer, Agrilus bilineatus (Weber) (Coleoptera: Buprestidae). Canad. Ent. 120:1037-1039

Dunn JP, Kimmerer TW, and Potter DA (1986) Attraction of the twolined chestnut borer, Agrilus bilineatus (Weber) (Coleoptera: Buprestidae), and associated borers to volatiles of stressed white oak. Canadian \ Entomologist 118, 503-509

Fenton FA (1942) The Flatheaded Apple Tree Borer (Chrysobothris femorata (Olivier)). Oklahoma Agricultural Exp. Sta. Bull. No. B-259. 31 pages

Francese JA, Mastro VC, Oliver JB, Lance DR, Youssef N, Lavallee SG (2005) Evaluation of colors for trapping Agrilus planipennis (Coleoptera: Buprestidae). J. Entomol. Sci. 40:93-95

Ghiradella H (1991) Light and color on the wing: structural colors in butterflies and moths. Applied Optics 30:3492-3500

Ghiradella H (1989) Structure and development of iridescent butterfly scales:

29 lattices and laminae. J. Morphol. 202:69-88

Ghiradella H (1986) Structure and development of iridescent Lepidopteran scales: the Papilionidae as a showcase family. Ann. Entmol. Soc. Am. 78:252- 264

Ghiradella H (1984) Structure of iridescent Lepidopteran scales: variations on several themes. Ann. Entmol. Soc. Am. 77:637-645

Ghiradella H, Aneshensley D, Eisner T, Silberglied R, Hinton HE (1972) Ultraviolet reflection of a male butterfly: interference color caused by thin-layer elaboration of wing scales. Science 178:1214-1217

Gould J, Tanner J, Winograd D, Lane S(2005) Initial studies on the laboratory rearing of emerald ash borer and foreign exploration for natural enemies. In: Mastro, V.C. and Reardon, R. (eds.), Emerald ash borer research and technology development meeting, FHTET 2004-15, USDA Forest Service, Morgantown, WV, pp. 73-74

Gwynne DT and Rentz DCF (1983) Beetles on the bottle: male buprestids mistake stubbies for females (Coleoptera). J. Aust. Entomol. Soc. 23:79- 80

Haack RA, Jendek E, Liu H, Marchant KR, Petrice TR, Poland TM, Ye H (2002) The Emerald ash borer: a new exotic pest in North America. Newsletter of the Michigan Entomological Society. 47:1-5

Hadley NF (1984) Cuticle: Ecological significance. In J. Bereiter-Hahn, A.G. Matoltsy, and K.S. Richards (eds.), Biology of the Integument, Vol. 1, pp. 685-702. Spring-Verlag, Berlin

Henzell RF and Lowe MD (1970) Sex attractant of the grass grub beetle. Science 168: 1005-1006

Harari AR, Ben-Yakir D, Rosen D (1994) Mechanism of aggregation behavior in Maladera matrida Argaman (Coleoptera: Scarabaeidae). J. Chem. Ecol. 20:361-371

Herms DA, Stone AK, Chatfield JA (2004) Emerald ash borer: the beginning of the end of ash in North America? In Chatfield, J.A., Draper, E.A., Mathers, H.M., Dyke, D.E., Bennett, P.J., and Boggs, J.F. (eds.), Ornamental plants: annual reports and research reviews, 2003, OARDC- OSU Extension Special Circular 193, pp. 62-71

Howard RW (1993) Cuticular hydrocarbons and chemical communication. In D.

30 W. Stanley-Samuelson and D. R. Nelson (eds.), Insect lipids: chemistry biochemistry, and biology, pp. 179-226. University of Nebraska Press, Lincoln

Howard RW and Blomquist GJ (1982) Chemical Ecology and Biochemistry of Insect Hydrocarbons. Annual Review of Entomology 27:149-172

Howard RW and Blomquist GJ (2005) Ecological, behavioral, and biochemical aspects of insect hydrocarbons. Annual Review of Entomology 50:371- 393

Howard RW, McDaniel CA, Blomquist GJ (1980) Chemical Mimicry as an Integrating Mechanism: Cuticular Hydrocarbons of a Termitophile and Its Host. Science 210:431-433

Huxley J (1976) The coloration of Papilio zalmoxis and P. antimachus and the discovery of Tyndall blue in butterflies. Proc. R. Soc. Lond. Ser. B 193:441-453

Illinois Department of Agriculture. (2006) Emerald ash borer confirmed in Illinois. Available online at http://www.agr.state.il.us/newsrels/r0613061.html; last accessed on July 31, 2006

Indiana Department of Natural Resources. (2006) Division of Entomology and Plant Pathology: Emerald Ash Borer. Available online at http://www.in.gov/dnr/entomolo/pestinfo/ashborer.htm; last accessed on August 7, 2006

Jallon JM (1984) A few chemical words exchanged by Drosophila during courtship and mating. Behavioral Genetics 14:441-478

Kinoshita S, Yoshioka S, Fujii Y, Okamoto N (2002) Photophysics of structural color in the Morpho butterflies. Forma 17:103-121

Kukalová-Peck J and Lawrence JF (1993) Evolution of the hind wing in Coleoptera. The Canadian Entomologist 125:181-258

Lance DR, Fraser I, Mastro VC (2007) Activity and microhabitat-selection patterns for emerald ash borer and their implications for the development of trapping systems. In Mastro, V.C., Lance, D., Reardon, R., and Parra, G., (comps.), Emerald Ash Borer and Asian Longhorned Beetle Research and Technology Development Meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, pp. 73-74

31 Land MF (1972) The physics and biology of animal reflectors. Progr. Biophys. Molec. Biol. 24:75-106

Leal WS, Kawamura F, Ono M (1994) The scarab beetle Anomala albopilosa sakishimana utilizes the same sex pheromone blend as a closely related and geographically isolated species, Anomala cuprea. J. Chem. Ecol. 20:1667-1676

Leal WS, Sawada M, Hasegawa M (1993) The scarab beetle Anomala cuprea utilizes the sex pheromone of Popillia japonica as a minor component. J. Chem.Ecol. 19:1303-1313

Lelito JP, Fraser I, Mastro VC, Tumlinson JH, Böröczky K, Baker TC (2007) Visually mediated ‘paratrooper copulations’ in the mating behavior of Agrilus planipennis (Coleoptera: Buprestidae), a highly destructive invasive pest of North American ash trees. J. Insect Behav. 20:537-552

Lelito JP, Fraser I, Mastro VC, Tumlinson JH, Baker TC (2008) Novel visual- cue-based sticky traps for detection of emerald ash borers, Agrilus planipennis (Coleoptera: Buprestidae). J. Appl. Entomol. 132:668-674

Liu H, Bauer LS, Gao R, Zhao T, Petrice TR, Haack RA (2003) Exploratory survey for the emerald ash borer, Agrilus planipennis (Coleoptera: Buprestidae), and its natural enemies in China. The Great Lakes Entomol. 36: 91-204

Lockey KH (1991) Insect hydrocarbon classes: implications for chemotaxonomy. Insect Biochemistry 21:91-97

Loughrin JH, Potter DA, Hamilton-Kemp TR (2005) Volatile compounds induced by herbivory act as aggregation kairomones for the Japanese beetle (Popilia japonica Newman). J. Chem. Ecol. 21:1457-1467

Lowrey S, De Silva L, Hodgkinson I, Leader J (2007) Observation and modeling of polarized light from scarab beetles. J. Opt. Soc. Am. A 24:2418-2425

Lyons DB, Jones GC, Wainio-Keizer K (2004) The biology and phenology of the emerald ash borer, Agrilus planipennis. In Mastro, V.C. and Reardon, R. (eds.), Emerald ash borer research and technology development meeting, FHTET 2004-02, USDA Forest Service, Morgantown, WV, p. 5

Macleod HA (1969) Thin Film Optical Filters. Adam Hilger, London

Marchant KR (2005) Managing the emerald ash borer in Canada. In: Mastro,

32 V.C., Reardon, R., and Parra, G. (eds.), Emerald ash borer research and technology development meeting, FHTET 2005-16, USDA Forest Service, Morgantown, WV, p. 3

Matthews RW (1975) Courtship in parasitic wasps. In: Evolutionary strategies of parasitic insects and mites. Ed: P.W. Price, p. 66-111. Plenum Press, New York

Matthews RW and Matthews JR (1978) Insect Behavior. Wiley, New York.

McCullough DG, Poland TM, Cappaert DL (2006) Attraction of emerald ash borer to trap trees: effects of stress agents and tree type. In Mastro, V., Reardon, R., and Parra, G. (eds.), Emerald ash borer research and technology development meeting, FHTET-2005-16, USDA Forest Service, Morgantown, WV, pp. 61-62

McCullough DG, Siegert NW, Poland TM, Cappaert DI, Fraser I, Williams D (2005) Dispersal of emerald ash borer at outlier sites: three case studies. In: Mastro, V.C. and Reardon, R. (eds.), Emerald ash borer research and technology development meeting, FHTET 2004-15, USDA Forest Service, Morgantown, WV, pp 58-59

McCullough DG and Katovich SA (2004) Pest Alert: Emerald Ash Borer. USDA Forest Service Publication No. NA-PR-02-04

Merivee E, Rahi M, Bresciani J, Ravn HP, Luik A (1998) Antennal sensilla of the click beetle, Limonius aeruginosus (Olivier) (Coleoptera : Elateridae). International Journal of Insect Morphology and Embryology 27:311-318

Merivee E, Rahi M Luik A (1999) Antennal sensilla of the click beetle, Melanotus villosus (Geoffroy) (Coleoptera : Elateridae). International Journal of Insect Morphology and Embryology 28:41-51

Metzger JA, Fraser I, Storer AJ, Crook DJ, Francese JA, Mastro VC (2007) A multistate comparison of emerald ash borer (Agrilus planipennis Fairmaire) (Coleoptera: Buprestidae) detection tools. In: Mastro, V.C., Lance, D., Reardon, R., and Parra, G., (comps.), Emerald Ash Borer and Asian Longhorned Beetle Research and Technology Development Meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, 73- 74

Michigan Department of Agriculture. (2006) Emerald ash borer quarantine area maps. Available online at http://www.michigan.gov/mda/0,1607,7-125- _6860_30046---,00.html; last accessed on 7 August 7, 2006

Miller DR, Asaro C, Berisford CW (2005) Attraction of southern pine engravers and associated bark beetles (Coleoptera: Scolytidae) to ipsenol, ipsdienol, and lanierone in southeastern United States. J. Econ. Entomol. 98:2058- 2066

Monnin T, Malosse C, Peeters C (1998) Solid-phase microextraction and cuticular hydrocarbon differences related to reproductive activity in the queenless ant Dinoponera quadriceps. J. Chem. Ecol. 24:473-490

Morris RB (1975) Iridescence from diffraction structures in the wing scales of Callophrys rubi, the green hairstreak. J. Entomol. Ser. A 49:149-154

Neville AC (1977) Metallic gold and silver colours in some insect cuticles. J. Insect Physiol. 23:1267-1274

Ohio State University. (2006) Emerald Ash Borer: Ash Alert. Available online at http://ashalert.osu.edu/; last accessed on August 7, 2006

Oliver JB, Youseef Y, Fare D, Halcomb M, Scholl S, Klingeman W, Flanagan P (2002) Monitoring buprestid borers in production nursery areas. In: Haun, G. (ed.), Proceedings of the 29th Annual Meeting of the Tennessee Entomological Society, pp 17-23

Otis GW, Youngs ME Umphrey G (2005) Effects of colored objects and purple background on emerald ash borer trapping. In: Mastro, V., Reardon, R (eds.), Emerald ash borer research and technology development meeting, FHTET-2004-15, USDA Forest Service, Morgantown, WV, pp. 31-32

Ouellette F, Krug PA, Stephens T, Dhosi G, Eggleton BJ (995) ispersion compensation using chirped sampled fibre Bragg gratings. Electronics Letters 31:899-901

Page M, Nelson LJ, Haverty MI, Blomquist GJ (1990) Cuticular hydrocarbons of eight species of North American cone beetle, Conophthorus Hopkins. J. Chem. Ecol. 16:1173-1198

Paré PW and Tumlinson JH (1999) Plant volatiles as a defense against insect herbivores. Plant Physiol. 121:325-332

Paré PW and Tumlinson JH (1997) Induced synthesis of plant volatiles. Nature 385:30-31

Parker AR, McKenzie DR, Large MCJ (1998) Multilayer reflectors in animals using green and gold beetles as contrasting examples. J. Exp. Biol.

34 201:1307-1313

Péchiné JM, Antony C, Jallon JM (1988) Precise characterization of cuticular compounds in young Drosophila by mass spectrometry. J. Chem. Ecol. 14:1071-1085

Peeters C, Monnin T, Malosse C (1999) Cuticular hydrocarbons correlated with reproductive status in a queenless ant. Proc. R. Soc. London 266:1323- 1327

Peñuelas J and Llusiá J (2004) Plant VOC emissions: making use of the unavoidable. Trends Ecol. Evol. 19: 402-404

Petrice TR, Haack RA, Poland TM (2005) Evaluation of three trap types and five lures for monitoring Hylurgus ligniperda (Coleoptera: Scolytidae) and other local scolytids in New York. Great Lakes Entomol. 37:1-9

Poland TM, McCullough DG (2007) Evaluation of a multicomponent trap for emerald ash borer incorporating color, silhouette, height, texture, and both ash leaf and ash bark volatiles. In Mastro, V., Lance, D., Reardon, R. and Parra, G. (eds.), Emerald ash borer research and technology development meeting, FHTET-2007-04, USDA Forest Service, Morgantown, WV, pp. 74-76

Poland TM, McCullough DG, De Groot P, Grant G, Macdonald L, Cappaert DL (2005) Progress toward developing trapping techniques for the emerald ash borer. In Mastro, V., Reardon, R (eds.), Emerald ash borer research and technology development meeting, FHTET-2004-15, USDA Forest Service, Morgantown, WV, pp. 53-54

Poland TM, De Groot P, Grant G, McDonald L, McCullough DG (2004) Developing attractants and trapping techniques for the emerald ash borer. In Mastro, V., Reardon, R (eds.), Emerald ash borer research and technology development meeting, FHTET-2004-02, USDA Forest Service, Morgantown, WV, pp. 15-16

Rodriguez-Saona CR, Miller JR, Poland TM, Kuhn TM, Otis GW, Turk T, Ward DL (2007) Behaviors of adult Agrilus planipennis (Coleoptera: Buprestidae). Great Lakes Entomologist 40:1-16

Scott D (1994) Genetic variation for female mate discrimination in Drosophila melanogaster. Evolution 48:112-121

Siegert NW, McCullough DG, Liebhold AM, Telewski FW (2005) Reconstructing the temporal and spatial dynamics of emerald ash borer in

35 black ash: A case study of an outlier site in Roscommon County, Michigan. In Mastro, V., Reardon, R (eds.), Emerald ash borer research and technology development meeting, FHTET-2004-15, USDA Forest Service, Morgantown, WV, pp. 21-22

Singer TL, Espelie KE, Gamboa GJ (1998) Nest and nestmate discrimination in independent-founding paper wasps. In Pheromone Communication in Social Insects: Ants, Wasps, Bees, and Termites. Eds: Vander Meer, R.K., Breed, M.D., Espelie, K.E., and Winston, M.L. p. 104-125. Westview, Boulder, Colorado, U.S.A.

Smith BH and Breed MD (1995) The chemical basis for nestmate recognition and mate discrimination in social insects. In R. T. Cardé, and Bell, W. J. (eds.), Chemical ecology in Insects 2, pp. 287-317. Chapman and Hall, New York

Syversten TC, Jackson LL, Blomquist GJ, Vinson SB (1995) Alkadienes meditating courtship in the parasitoid Cardiochiles nigriceps (Hymenoptera: Braconidae). J. Chem. Ecol. 21:1971-1989

Tamaki Y, Sugie H, Nagamine M, and Kinjo, M (1990) 9,11-dodecadienyl- butyrate and 9,11-dodecadienyl-hexanoate female sex pheromone of the sugarcane wireworm Melanotus sakishimensis Ohira (Coleoptera: Elateridae). Jpn. Kokai Tokkyo Koho. 61:12601

Taylor RAJ, Bauer LS, Miller DL, Haack RA (2004) Emerald ash borer flight potential. In: Mastro, V., Reardon, R (eds.), Emerald ash borer research and technology development meeting, FHTET-2004-02, USDA Forest Service, Morgantown, WV, pp. 31-32

Thaler JS, Farag MA, Paré PW, Dicke, M (2002) Jasmonate-deficient plants have reduced direct and indirect defenses against herbivores. Ecol. Lett. 5:764- 74

Tillman JA, Seybold SJ, Jurenka RA, Blomquist GJ (1999) Insect pheromones: an overview of biosynthesis and endocrine regulation. Insect Biochemistry and Molecular Biology 29:481-514

Tolasch T, von Fragstein M, Steidle JLM (2007) Sex Pheromone of Elater ferrugineus L. (Coleoptera: Elateridae) J. Chem. Ecol. 33: 2156-2166

Tóth T, Furlan L, Campagna G (2007) Attractant for the sugar-beet weevil Conorrhynchus (Cleonus) mendicus (Col.: Curculionidae). J. Appl. Entomol. 131:569-572

36 Tumlinson JH, Glein MG, Doolittle RE, Ladd TL, Proveaux AT (1977) Identification of the female Japanese beetle sex pheromone: inhibition of male response by an enantiomer. Science 197:789-792

Tumlinson JH, Gueldner RC, Hardee DD, Thompson AC, Hedin PA, Minyard JP (1971) Identification and synthesis of the four compounds comprising the boll weevil sex attractant. J. Org. Chem. 36:2616-2621

Turlings TC and Tumlinson JH (1992) Systemic release of chemical signals by herbivore-injured corn. Proc. Natl. Acad. Sci. 89:8399-8402

Van den Assem J, Jackmann F, Simbolotti P (1980) Courtship behavior of Nasonia vitripennis (Hymenoptera: Pteromalidae): some qualitative, experimental evidence for the role of pheromones. Behaviour 75:301-307

Van Den Boom CEM, Van Beek TA, Posthumus MA, De Groot A, Dicke M (2004) Qualitative and quantitative variation among volatile profiles induced by Tetranychus urticae feeding on plants from various families. J. Chem. Ecol. 30: 69-89

Vander Meer RK and Morel L (1998) Nestmate recognition in ants. In Pheromone Communication in Social Insects: Ants, Wasps, Bees, and Termites. Eds: Vander Meer, R.K., Breed, M.D., Espelie, K.E., and Winston, M.L. p. 79-103. Westview, Boulder, Colorado, U.S.A.

Vet LEM and Dicke M (1992) Ecology of infochemical use by natural enemies in a tritrophic context. Annual Review of Entomology 37:141-172

Wellso SG (1966) Sexual attraction and biology of Xenorhipis brendeli (Coleoptera: Buprestidae) J. Kan. Ent. Soc. 39:242-245

Westcott C (1973) The gardner’s bug book. 4th Edition, Doubleday and Co., Garden City, New York

White RE (1978) A field guide to the beetles of North America. Houghton- Mifflin, New York

Wicker C and Jallon JM (1995a) Hormonal control of sex pheromone biosynthesis in Drosophila melanogaster. Journal of Insect Physiology 41: 65-70

Wicker C and Jallon JM (1995b) Influence of ovary and ecdysteroids on pheromone biosynthesis in Drosophila melanogaster (Diptera: Drosophilidae). European Journal of Entomology 92:197-202

37 Yatsynin VG and Rubanova EV (2002) Analysis of pheromones of snapping beetle (Coleoptera, Elateridae). Agrokhimiya 8:77-81

Yatsynin VG, Rubanova EV, Okhrimenko NV (1996) Identification of female- produced sex pheromones and their geographical differences in pheromone gland extract composition from click beetles (Col., Elateridae). J. Appl. Ent. 120:463-466

CHAPTER 2

Visually mediated ‘paratrooper copulations’ in the mating behavior of

Agrilus planipennis (Coleoptera: Buprestidae), a highly destructive

pest of North American ash trees.

Jonathan P. Lelito, Ivich Fraser, Victor C. Mastro, James H. Tumlinson, Katalin Böröczky and Thomas C. Baker

ABSTRACT

The emerald ash borer, Agrilus planipennis, is a serious invasive pest of North

American ash (Fraxinus) trees. In captivity, mating is initiated by beetles at least ten days old, and appears to be based simply on random contact with a member of the opposite sex. In the field, male A. planipennis search the tree during flight, and attempt

to copulate with dead beetles of both sexes pinned to leaves, after descending rapidly

straight down onto the pinned beetles from a height of from 30 to 100 cm. All evidence

suggests that males find potential mates using visual cues. Equal numbers of feral males approach all ‘dummy’ beetles; however, considerably more time is spent attempting

copulation with dead females rather than males, suggesting a contact chemical cue.

Sticky traps prepared from dead, pinned EAB capture crawling insects as well as male A.

39 planipennis, at a rate similar to that at which small purple sticky traps of similar overall area capture crawling insects and both sexes of feral EAB.

INTRODUCTION

The emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera:

Buprestidae), is a serious non-native pest of ash trees (Fraxinus sp., Oleaceae). EAB populations are spreading rapidly in the Midwest and Mid-Atlantic states of the U.S., as well as in some adjacent areas of Ontario, Canada. The beetle was first identified near

Detroit, Michigan and Windsor, Ontario in 2002 (Haack et al. 2002), and has subsequently been detected in Ohio and Maryland in 2003, Indiana in 2004, and Illinois in 2006. A thorough review of the timeline of introduction, host plants, means of spread, and other relevant information is provided by Poland and McCullough (2006). In June

2007, EAB was identified in Pennsylvania as well (Pennsylvania DCNR 2007).

EAB infestations are insidious in that they often go undetected during initial colonization. The characteristic D-shaped exit holes left by emerging adults are only noticeable at ground level by the time the beetle has significantly damaged the tree

(Haack et al. 2002). To further complicate matters, EAB larvae can take two years to develop in healthy trees, lengthening the time it takes to produce the visible symptoms commonly used for detection (Poland and McCullough 2006; Cappaert et al. 2005;

Siegert et al. 2005). Currently, delimiting new infestations involves the use of ‘detection trees’ (also widely referred to as ‘trap trees’) that have been artificially girdled and subsequently sampled for any larvae present. Girdled trees, while effective, come at a

40 huge cost of time and labor and therefore are not optimal for wide-scale detection efforts

(Cappaert et al. 2005). A purple panel trap for capturing EAB adults is currently undergoing testing but this trap is not yet being used operationally (Crook et al. 2006;

Francese et al. 2005, 2006; Metzger et al. 2006).

Finally, little is known about the mating habits of EAB. There is no evidence to suggest that EAB uses a long range pheromone to locate conspecifics (Otis et al. 2005).

Past research has indicated that buprestids appear to first locate their host and then secondarily seek mates by visual, tactile, and other non-pheromonal cues (Carlson and

Knight 1969; Matthews and Matthews 1978; Gwynne and Rentz 1983). With the urgent need for early detection of new infestations in mind, we sought to identify any precopulatory behaviors in feral and laboratory-reared EAB that might be significant to either short- or long-range mate attraction. Dead, pinned EAB males and females were used as ‘dummies’ to test for precopulatory behaviors in the field. Subsets of both male and female beetles were washed in dichloromethane to remove chemical cues; we expected that all types of pinned beetles would be approached by feral males if EAB depends heavily on vision for mate finding. If one type of ‘dummy’ were to be favored by males over the other, this could indicate that there are olfactory, visual, or contact chemical differences between the sexes. We also utilized the dead beetles themselves as a trap, and tested the detection effectiveness of these ‘dummy’ beetles compared to small purple traps.

METHODS

41 Insects

All beetles (Agrilus planipennis) were kept on a 14:10 light cycle and fed green ash leaves, Fraxinus pennsylvanica, collected from outdoors. Beetles were provided by

USDA APHIS PPQ personnel at the experimental station in Brighton, Michigan, 48116.

Beetles were reared from infested ash logs collected locally and kept in sealed barrels at ambient room temperature, with a cone at one end leading to an opening. This opening was capped with a removable plastic bottle. Beetles, attracted to the light at the opening, fell into the bottle and were collected every twenty-four hours. Beetles were separated by sex on the day they emerged from the rearing barrels.

Laboratory Behavioral Observations

Individual beetles were removed from sex-specific containers with soft forceps.

A male and a female beetle were placed into a small plastic food-storage tub with paper toweling as a substrate as well as ash leaves in a florist’s vial. The tub and vial were washed and new toweling and leaves were provided after each trial. Each beetle was randomly chosen from among the age cohorts (1-, 5-, 10-, 15-, and 20-day-old beetles) to form random combinations of beetles in each pairing. Four or five pairs were set up in a similar manner to be observed simultaneously. Beetles were given fifteen minutes to acclimate to their cage and then were observed for thirty minutes. The following behaviors were noted: contact between individuals, wing fanning, open-wing basking, flight, vibratory interactions, mounting attempts, and copulation. All pairs were observed

42 for 30 minutes, and the number of instances in which the beetles performed each behavior was noted. Beetles were frozen and discarded after mating trials, thus preventing any introduction of contact compounds into the sex-segregated cages or through the reuse of individual beetles in mating trials.

Field Behavioral Observations

On 7 and 10 June 2006, at night between 9:00 and 11:15 PM EDT, 30 feral beetles were counted and observed on each occasion in the USDA/APHIS quarantined area around Brighton, MI. Beetles were located on ash trees by manual inspection while wearing a headlamp equipped with a red LED (Rayovac, Inc.). The position of each beetle on the tree and its activity and sex were recorded before beetles were released back in to the tree nearby. Similarly, on 12 and 13 June 2006, in the morning between 6:45 and 8:20 AM EDT, 30 beetles were counted and observed on each occasion, and their sex, activity and position on the tree were recorded. On 8, 9, 10, 13, and 18 June 2006, from 9:00 AM until 5:00 PM EDT, ten, random, one-meter sections of branch on green ash trees were assessed for the presence of EAB at 30-minute intervals. If only one or two beetles were present on the branch, they were simply counted without being removed from the tree. Beetles in groups of three or more individuals (within the one meter section of branch examined) were collected and sexed using a hand lens, then returned to a nearby tree. During each day of the mate-finding experiment detailed below, beetles present on the tree trunk up to 2.5 meters from the ground were collected and sexed, and then released back onto the trunk.

Mate-Finding

Lab-reared beetles of both sexes were killed in sex-specific vials by freezing, and then, segregated by gender, were immediately pinned through the thorax with a size 2

Monarch insect pin to a Styrofoam board. The beetles were randomly chosen to be pinned in one of the following three postures (Figure 2-1): elytra shut (their normal posture on the leaf); elytra pinned at approximately 30 degrees to the body with the hind wings pinned at a 45 degree angle relative to the body (this mimics the behavior they perform before flight and while they bask); and with both elytra and wings positioned at an approximately 90 degree angle to the body (an exaggerated version of the basking posture). Beetles were then allowed to dry for 3 days at standard room temperature, at which point they had assumed these positions permanently.

After three days, half of the beetles of each gender and posture were randomly selected to be washed for 10 minutes in dichloromethane and then returned to dry for one day, at which point they were washed again. Washed beetles were not used until at least

24 hours after the final washing. All washed or unwashed dried ‘dummy’ beetles were then taken to ash trees with EAB infestations and pinned onto the terminal leaflet of a leaf facing the sun (Figure 2-2). All beetles were pinned to leaflets of approximately the same height (2 m) in a given replicate.

The identity of each ‘dummy’ beetle was inconspicuously coded by means of a small piece of adhesive tape using one of four colors placed on the underside of the leaflet to which the beetle was pinned. Colors were used as follows: red, unwashed

Figure 2-1

This figure shows the three positions tested for each sex in order to determine which was most attractive to feral EAB males. The model in the center is pinned with elytra shut, and elicits the most frequent aerial approaches by feral males.

Figure 2-2

This is the setup for the pinned beetles used to attract wild male EAB. The pin was inserted through the thorax of the beetle, and then used to attach the beetle to the midvein of an ash leaflet.

46 female; orange, washed female; yellow, unwashed male; green, washed male. Insect pins containing no beetles were placed out on sunny terminal leaflets as well. Any beetles arriving on leaves with any of these treatments were sexed and recorded.

To judge overall EAB activity on the tree for comparison, five empty terminal leaf clusters were marked with a wrapping of Teflon tape around the base of the twig.

Leaflets with pins only and empty leaflets each had a randomly colored piece of tape placed underneath the leaflet. All six treatments in a replicate were monitored for the presence of EAB for two hours at a time. Beetles that arrived at any ‘dummy’ beetle or control leaf cluster were captured by hand or sweep net, sexed and released back into the tree, with the time of attraction and gender of each feral beetle being recorded.

Initially, all three postures of beetles were used. However, responses by feral males to open-winged ‘dummy’ beetles were very low (Figure 2-3), as were responses to those models with a 30 degree elytral angle. In further observations, therefore, only pinned beetles (of either sex) with elytra closed were used. After two hours, used

‘dummy’ beetles were replaced and the locations of each were randomly chosen in the portion of the tree currently facing the sun. In this way, the position of the experiment changed each day depending on the time.

Three infested sites in the area of Brighton, Michigan were used for this experiment, and each was used in a regular rotation on any day in which the weather was suitable. Experimental sites included one isolated tree in a business park; several dead and dying trees surrounding a commercial parking lot; and a combination of dead, dying, and relatively healthy infested trees in a forest edge setting. ‘Dummy’ beetles were used

Figure 2-3

This figure shows the effect of model beetle posture on the number of paratrooper copulation approaches observed in feral male EAB. Models of either gender with fully open elytra and wings elicited no paratrooper copulation attempts. Models of either gender with fully closed elytra resulted in the highest observed number of paratrooper copulation attempts.

48 between 9:00 AM and 5:00 PM EDT from 20 June 2006 until 29 June 2006. Together, the visual model experiments comprised a total of ca. 40 observation-hours.

EAB Sticky Trapping Using Dummy Beetles

Beetles prepared identically to those used in the visual stimuli assessment were placed on terminal leaflets of ash tree leaves and coated with a layer of Tanglefoot by hand- application while wearing rubber gloves. In addition, other traps were made from:

1) 2.5 cm X 2.5 cm squares of purple trap material; 2) 2.5 cm X 2.5 cm squares of green metallic paper (HOTP-10371 ‘Emerald Glimmer’, www.paperpizazz.com); 3) ovals of

green metallic paper the size of an EAB; and 4) ovals of green metallic paper twice the

length and width of an EAB. All such traps were displayed randomly on the terminal

leaflets of ash trees in ten replicates of each trap type at each of two field sites.

Placement was completed on 30 June 2006. These sticky traps were monitored for the

presence of trapped feral EAB on 4 July 2006 and again on 8 July 2006. Any beetles

trapped were removed with forceps, rinsed in Histo-Clear (HS-200, National Diagnostics)

and sexed under a dissecting scope.

Behavioral Analyses

The approaches of feral male beetles onto leaves containing pinned ‘dummy’

beetles were recorded for illustrative purposes using a SONY HandyCam digital

camcorder (Model DCR-TRV350, Sony Corporation). Video was transferred via an

49 IEEE 1394 connection to a Dell Inspiron 8600 laptop computer using the Windows XP

Home operating system and Windows Movie Maker 2.0 (Microsoft Corp.), and the beetle images were captured at 512 kbps at a size of 320 by 240 pixels. Flight tracks (Figure 2-

4) were prepared by placing transparency sheets over a computer monitor while the video was played frame-by-frame in Movie Maker. At each frame (1/16 s), a marking pen was used to place a dot on the transparency sheet at the center of the feral beetle’s image. The final position of the feral male on the model was recorded as a dot within an oval. The dots were connected in sequence and an outline of the leaf was then traced at the position it was in when the feral beetle landed.

Figure 2-4

This figure shows various approach tracks of feral male EAB approaching pinned, closed-elytra models on leaves. The scale bar at left indicates 4 cm in each frame. The small oval on each leaflet depicted is the position of the pinned EAB on the leaf. The span of time between each dot is 1/16 second.

51 Statistical Analyses

Descriptive statistics for lab and field behavioral data were obtained with SPSS version 13 (SPSS, Inc. 2004) on a Dell Inspiron 8600 laptop computer using the

Windows XP (Microsoft Corp.) operating system. Analysis of lab behavioral data was performed using PROC PRINCOMP in SAS version 9.1 (SAS Institute 2005). Analysis of feral male approaches by pinned beetle treatment was performed using PROC GLM

Type III sum of squares in SAS, including Tukey’s Studentized range test. Male approach data was log transformed prior to this analysis. The number of males approaching each pinned beetle treatment and the controls was analyzed using PROC

GLM in SAS, again including Tukey’s Studentized Range Test for multiple comparisons.

RESULTS

Laboratory Behavioral Observations

During the 260 trials conducted in the laboratory, rates of copulation were

extremely low. Principal component analysis revealed no strong correlations between

any two behaviors. Among the stronger, although still weak, correlations were male wing fanning to male flight (correlation matrix value = 0.5365), female wing fanning to female flight (correlation matrix value = 0.5294), and contact to female juddering (a vibration of the body while stilting up on the legs) (correlation matrix value = 0.4166).

52 No behavior was strongly correlated with copulation; even male mounting of the female only correlated with a value of 0.5308. The complete correlation matrix is given in Table 2-1. Contact between the male and female beetles occurred in 85.8% of the trials. Following contact, females clung to males in 2.3% of trials. Males performed wing fanning behavior in 37.7% of the trials, whereas females performed wing fanning in

24.6% of the trials. Basking behavior (open wings) was performed by males in 20.4% of trials and by females in 24.3% of trials. Juddering by males occurred in 38.4% of trials and in 56.2% of trials by females. Flight occurred in 35.4% of the trials for males, and in

22.7% of the trials for females. Males mounted females in 5.3% of trials. For comparison, copulation only occurred in 2.3% of trials. One male mounted a female at one day of age (but was kicked off), and another mounted a female at five days of age, and was also unsuccessful at achieving a mating. However, most mounting was performed by males of at least ten days of age (11 out of 14 cases), and copulation was not observed until both the male and female were at least ten days old.

We terminated all trials at 30 minutes; therefore it is possible that more matings would have occurred given a longer interval. The lack of any obvious and consistent precopulatory behavior performed by either sex in our trials suggested that some condition necessary for efficiently getting the sexes together was missing in the laboratory. Increasing the density of EAB in a container did increase the number of mating pairs that formed (we used twenty of each sex in one trial and observed four matings in 30 minutes), but they occurred at the same frequency and in the same way in which single pairs were observed forming: beetles simply contacted one another, and the male quickly attempted to mount the female. To test the possibility that the beetles may

53 Table 2-1 PRINCOMP Correlation Matrix of EAB Lab Behavior

a. Variables: ‘cnt’, contact between sexes; ‘mwf’, male wing fanning; ‘fwf’, female wing

fanning; ‘mow’, male opens wings; ‘fow’, female opens wings; ‘mj’, male juddering; ‘fj’,

female juddering; ‘mf’, male flight; ‘ff’, female flight; ‘fcm’, female clings to male;

‘mm’, male mounts female; and ‘cop’, copulation occurs.

54 use acoustic cues to make contact, we set up a bat detector (Model #T-7407, Acorn

Naturalists) approximately 30 cm from the mating arenas, and no ultrasound emissions were detected as having been produced by the beetles during the course of this experiment.

Field Behavioral Observations

In the early morning around sunrise, the 60 beetles examined were found primarily sitting on leaflets (95%) as compared to those found on petioles (5%) (Table 2-

2). Of the 95% of EAB on leaflets, 29.8% were found on terminal leaflets, 3.5% on terminal leaflet tips, 21.1% on non-terminal leaflet tips, and 45.6% were found on a leaflet other than the terminal (Table 2-2). Of those beetles on terminal leaflets or terminal leaflet tips, only 26.3% were male. At this time of day, 58.3% of the beetles observed on any tree location were female. Feeding on leaves was observed in 45% of beetles, while mating pairs comprised 3.3% of the total EAB observed. All other beetles were apparently inactive.

At night, beetles were also found primarily on leaflets (86.7% of 60 EAB observed) (Table 2-3). Just over forty-six percent (46.7%) of the 60 beetles found on the leaflets at night were male. At night, feeding on leaflets was performed by 8.3% of the beetles, with 91.7% of the beetles being inactive. The five beetles observed to be feeding initiated this behavior after the headlamp was brought to bear on their location. Of the 60 beetles observed, 8.3% were located on petioles, and 5% were located on a branch of the tree rather than a leaflet. We further divided the 86.7 % of total beetles observed (52/60)

55 Table 2-2. Position of Feral EAB on Ash Trees at Sunrise

a. L = leaflet; LT = leaflet tip; TL = terminal leaflet; TLT = terminal leaflet tip;

P = petiole.

Table 2-3. Position of Feral EAB on Ash Trees After Sunset

a. B = branch; L = leaflet; LT = leaflet tip; TL = terminal leaflet; TLT = terminal leaflet

tip; P = petiole.

56 on leaflets into the same categories used above. We observed 15.4% of these to be on terminal leaflets, 5.8% on terminal leaflet tips, 21.2% on leaflet tips, and 57.7% on leaflets other than the terminal. In this case, beetles found on terminal leaflets or their tips, only 33.3% were male (Table 2-3). This pattern is similar to that noted during the day for leaflet tips.

Many beetles were observed flying near the canopy of the tree as the temperature warmed during the morning observations. We captured 30 flying beetles on the second morning of observation; of the beetles caught in flight, 28 were male. The shift to male bias at night may be a result of those actively flying beetles settling on the tree.

Of the 91 total beetles observed on tree trunks during the day, 70 were female

(77% of total), while only 21 (23%) were male. No mating pairs were observed forming on trunks, but nine pairs were observed on trunks already in copula. Highest activity on tree trunks, quantified as number of beetles observed per hour, occurred between 1500 and 1800 PM EDT during which time a total of 47 female and 16 male beetles were observed on the trunks. This period appeared to be associated with the highest daytime temperatures (greater than 75°F).

Mate-Finding

Feral male EAB flying rapidly around the foliage suddenly hovered 0.3 – 1.0 m above beetles pinned to leaves and then rapidly and accurately dove onto the backs of the

‘dummy’ beetles. We term this behavior ‘paratrooper copulation’, because of the rapid,

57 often straight descent by males (Figure 2-4). Probing movements with the aedeagus typically followed landing by at most a few seconds, especially when the male landed on an unwashed female. ‘Dummy’ beetles pinned with the elytra closed elicited frequent paratrooper approaches compared to those pinned with wings splayed. ‘Dummy’ beetles with open wings were very poor elicitors of feral male behavior (Figure 2-3).

Equivalent numbers of feral males were observed on the leaflets containing each of the four types of closed-elytra beetles (ANOVA; F = 0.47, p = 0.7024) and these leaflets had significantly more males on them than did either leaflets with an empty pin or completely blank leaves (ANOVA; F = 14.65, p < 0.0001).

All four types of closed-elytra beetles evoked equivalent numbers of paratrooper copulation attempts (Figure 2-5). No paratrooper approaches were observed in response to blank leaflets either with, or without, a pin (Figure 2-5). Once the males had completed their approach, there were significant differences in the amount of time they spent in contact with the dead beetles related to their sex and whether or not they had been washed with dichloromethane. We include mounting, copulation attempts, antennation, and remaining in constant contact with the pinned EAB in the broad category of ‘contact’ by male EAB. Feral males spent significantly more time (ANOVA, F = 50.87, P <

0.0001) in contact with unwashed female EAB than with any other type (Figure 2-6,

Table 2-4). Also, males spent significantly less time in contact with unwashed male EAB than with any other type. Washed male and washed female beetles evoked equivalent, intermediate durations of contact time by the males (Figure 2-6).

EAB Sticky Trapping Using Dummy Beetles

Figure 2-5

This figure shows the mean number of paratrooper copulation attempts observed per replicate (blue bars) and the total number of paratrooper attempts (red bars) observed in response to the different models during the eight replicates of our experiment. ‘FW’ = washed female; ‘FU’ = unwashed female; ‘MW’ = washed male; ‘MU’ = unwashed male; ‘P’ = leaflet with pin; ‘EL’ = empty leaflet. Eight replicates of this experiment were conducted, each lasting for two hours. The number of feral males observed near any of the four types of beetle models is not statistically different (ANOVA, F = 0.47, p =

0.7024) but all four of the models had significantly more males than either of the two controls (ANOVA, F = 14.65, P < 0.0001).

Figure 2-6

Bars indicate two standard errors about the mean (squares). The treatment type of pinned

EAB is shown on the X-axis, with ‘W’ representing ‘washed’ beetles and ‘U’ representing ‘unwashed’ beetles. The time in seconds spent on the pinned beetle is shown on the Y-axis. Bars having no letters in common are significantly different from

one another (ANOVA, F = 50.87, P < .0001).

60 Table 2-4. GLM Analysis of Time Spent on Pinned Beetle Treatments by Male EAB

a. Pinned beetle treatments are as follows: ‘FW’, washed female; ‘FU’, unwashed female;

‘MW’, washed male; ‘MU’, unwashed male. b. Asterisks indicate a significant difference between treatments.

61 Figure 2-7 shows the relative captures of feral beetles in response to the five types of traps on leaflets used during summer 2006. During the course of this experiment, only males were captured in response to metallic green ovals or to pinned EAB on leaves.

Purple squares and green metallic squares captured both male and female EAB. Some of the trapped beetles managed to crawl short distances through the Tanglefoot layer, and thus we assume that some beetles may have escaped.

Female Ovipositor Pulsation

Finally, we observed a behavior performed by female EAB in which the ovipositor and its surrounding soft tissue was extended outward at intervals while the female was stationary on a leaf. This soft tissue was extended for between 0.5 and 1 second at a time, interspersed in an apparently random manner by the entire ovipositor being extended as well. Individual females performed this behavior for several to many minutes, with a pulsation of the soft terminal integumental tissue generally continuing throughout this time. Figure 2-8 depicts a close-up posterior view of a feral female EAB in this posture. No feral EAB adults were observed to approach such females during the greater than three hours of observation of these EAB females.

DISCUSSION

EAB males appear to seek their mates using visual cues once prospective mates

are already present on the host tree. Males initiate approaches equally to all treatments of

Figure 2-7

Number and sex of wild EAB caught on sticky-leaf traps containing different visual cues in June and July of 2006. Trap types used included one-inch by one-inch metallic green squares, one-inch by one-inch purple squares, pinned female EAB, small EAB-sized metallic green ovals and large metallic green ovals approximately double the size of an

EAB. Pinned female EAB attracted only males.

Figure 2-8

This image shows a posterior view of the abdominal tip of a feral female EAB performing ovipositor pulsation.

64 pinned model beetles having their elytra closed and wings concealed, but the males spend a significantly longer time mounting and attempting to copulate with unwashed female pinned beetles than they do to pinned males, or female washed beetles (Figure 2-6). All treatments show significant differences from one another with the exception of washed males as compared to washed females; the lack of difference here suggests our washing method was effective at removing a contact cue (either antagonist or attractant). It appears that a contact pheromone is involved in copulation and mating after the visual stage of mate-location is finished.

When we washed the dead beetles with dichloromethane, we removed the chemical contact cues that males apparently use to judge whether what they’ve landed on is a male or a female beetle. The washed models were contacted equivalent amounts of time regardless of their sex. Therefore a contact cue also appears to be involved in male- male repellency, because males that landed on unwashed pinned males spent significantly less time on these beetles than in any of the other treatments (Figure 2-6).

Our results indicate that the initial short-range attraction of flying males to sedentary females is visual: males will drop onto and attempt to copulate with male, as well as female, beetles pinned to a leaf. They persist the shortest amount of time when they have contacted an unwashed male beetle and for the longest time when contacting an unwashed female beetle.

In our trapping experiments using visual models pinned to leaves, our preliminary results with a low sample size preclude any statistical analysis. However, if the results hold true at higher sample sizes, the significance for EAB monitoring following this preliminary experiment may be important. Due to the success of sticky traps based on

65 pinned beetles in such a short time during the decline of naturally flying beetles, we suggest that using dead EAB visual lures in this way may be quite effective at trapping male EAB in low-density populations. The specificity of these traps for EAB appears to be higher than the purple traps used currently (i.e. beetle sticky traps caught fewer ants, flies, etc.); we also used purple traps of comparable area to the sticky surface created by leaflets and beetles.

Our observations indicate that males are the more active sex in the canopy, and the observations of others support this (Lance et al. 2006). Therefore, male EAB may be a better target for a trapping program simply because they are highly vagile, fly rapidly around the tree canopies, and are more likely to encounter the ‘attractant’ visual lure on ash leaflets. It is possible that in this way the EAB mating system could be effectively exploited for monitoring, despite the apparent lack of a long distance sex pheromone.

Further, if pinned female EAB were added to the current purple trap this may also enhance this EAB trapping system by visually inducing males to land forcefully on the traps. This concept will be tested during the 2007 EAB flight.

During our observations, an unexpected and striking behavior observed was what we have termed ‘ovipositor pulsation’. This behavior was performed only by female

EAB, typically while sitting on the epicormic shoots that arise from EAB-damaged ash trees, and was exclusively observed in the afternoon (after 2PM EDT). We note that although this behavior is relatively rarely observed, it is individually persistent. That is, a given female will tend to continue this behavior for many minutes once she has begun to perform it. Despite the superficial resemblance of this behavior to that of a “calling”

66 lepidopteran, we observed no evidence of any attraction of feral male EAB to such females in the many hours of observation that we undertook.

REFERENCES

Cappaert D, McCullough DG, Poland TM, Siegert NW (2005) Emerald ash borer in North America: A research and regulatory challenge. Am. Entomol. 51: 152-165

Carlson, RW and Knight FB(1969) Biology, taxonomy, and evolution of four sympatric Agrilus beetles (Coleoptera: Buprestidae). Contrib. Am. Entomol. Inst. 4:1-105

Crook D, Khrimian A, Francese J, Fraser I, Poland TM, Mastro V (2006) Chemical ecology of emerald ash borer. In Mastro, V.C. Lance, D., Reardon, R., and Parra, G. (eds.), Emerald ash borer and Asian longhorned beetle research and technology development meeting, FHTET-2007-04, USDA Forest Service, Morgantown, WV, p 79

Francese JA, Mastro VC, Oliver JB, Lance DR, Youssef N, and Lavallee SG (2005) Evaluation of colors for trapping Agrilus planipennis (Coleoptera: Buprestidae). J. Entomol. Sci. 40: 93-95

Francese JA, Fraser I, Lance DR, Mastro VC (2006) Developing survey techniques for emerald ash borer: the role of trap height and design. In Mastro, V.C. Lance, D., Reardon, R., and Parra, G. (eds.), Emerald ash borer and Asian longhorned beetle research and technology development meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, p. 72

Gwynne DT and Rentz DCF (1983) Beetles on the bottle: male buprestids mistake stubbies for females (Coleoptera). J. Aust. Entomol. Soc. 23: 79- 80

Haack RA, Jendek E, Liu H, Marchant KR, Petrice TR, Poland TM, and Ye H (2002) The Emerald ash borer: a new exotic pest in North America. Newsletter of the Michigan Entomological Society. 47:1-5

Lance DR, Fraser I, Mastro VC (2006) Activity and microhabitat-selection patterns for emerald ash borer and their implications for the development of trapping systems. In Mastro, V.C. Lance, D., Reardon, R., and Parra,

67 G. (eds.), Emerald ash borer and Asian longhorned beetle research and technology development meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, p. 77

Matthews RW and Matthews JR (1978) Insect Behavior. Wiley, New York

Metzger JA, Fraser I, Storer AJ, Crook DJ, Francese JA, and Mastro VC (2006) A multistate comparison of emerald ash borer (Agrilus planipennis Fairmaire) (Coleoptera: Buprestidae) detection tools. In Mastro, V.C. Lance, D., Reardon, R., and Parra, G. (eds.), Emerald ash borer and Asian longhorned beetle research and technology development meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, p. 73

Otis GW, Youngs ME, and Umphrey G (2005) Effects of colored objects and purple background on emerald ash borer trapping. In Mastro, V., Reardon, R (eds.), Emerald ash borer research and technology development meeting, FHTET-2004-15, USDA Forest Service, Morgantown, WV, pp. 31-32

Pennsylvania Department of Conservation and Natural Resources (2007) Emerald Ash Borer. Available online at http://www.dcnr.state.pa.us/forestry/fpm_invasives_EAB.aspx; last accessed on July 5, 2007

Poland TM and McCullough DG (2006) Emerald ash borer: invasion of the urban forest and the threat to North America’s ash resource. J. Forestry 104:118-124

Siegert NW, McCullough DG, Liebhold AM, Telewski FW (2005) Reconstructing the temporal and spatial dynamics of emerald ash borer in black ash: A case study of an outlier site in Roscommon County, Michigan. In Mastro, V., Reardon, R (eds.), Emerald ash borer research and technology development meeting, FHTET-2004-15, USDA Forest Service, Morgantown, WV, pp. 21-22

CHAPTER 3

Novel visual-cue-based sticky traps for monitoring of emerald ash

borers, Agrilus planipennis (Col., Buprestidae)

Jonathan P. Lelito, Ivich Fraser, Victor C. Mastro, James H. Tumlinson, and Thomas C. Baker

ABSTRACT

We examined various methods of trapping emerald ash borers (EAB), Agrilus

planipennis Fairmaire, using solely visual cues based on previous work that has documented the importance of visual cues in EAB mate location. Here, we give the results of two of these methods, colored sticky cards (yellow or blue), or live ash leaves covered with spray-on adhesive to which dead male EAB visual lures had been pinned.

Feral male beetles were captured effectively on the sticky traps made from dead male

EAB on ash leaves. These sticky-leaf-traps captured more male EAB when deployed in high-population-density areas than low-density areas, but did capture EAB even at lower population-densities. More feral males were captured on these traps when they were placed higher in the trees, regardless of the population-density of EAB. Very few feral female EAB were captured using the sticky-leaf-traps. This novel method of EAB trapping may allow ‘real-time’ population detection and monitoring of EAB adults during the active flight period rather than locating larval galleries during the fall and winter after

69 adult flight and attack. Feral male beetles were also captured using standard yellow- or blue-colored sticky cards to which male EAB had been affixed with adhesive; however this type of trap was much less effective overall than using the sticky-leaf-traps. Further,

A. cyanescens, a species similar in color to EAB but smaller in size, showed a strong response to blue-colored sticky traps to which dead male EAB had been affixed with adhesive, suggesting a general use of visual cues in the mating systems of some of the other Buprestidae as well.

INTRODUCTION

The emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera:

Buprestidae), an invasive pest currently threatening the complete destruction of North

America’s ash resource, was first detected in the Midwestern U.S. and adjacent Canada in

2002 (Haack et al. 2002). Effective detection and monitoring of this pest has so far

remained elusive (Poland and McCullough 2006). Identifying a reliable species-specific

lure has proved to be relatively difficult thus far, despite the elucidation of antennally

active plant volatiles (Rodriguez-Saona et al. 2006, Poland et al. 2005, 2006; Crook et al.

2005, 2006, 2007), as well as a sex-specific macrolide produced by female beetles

(Bartelt et al. 2007). The USDA Animal and Plant Health Inspection Service (APHIS)

has created purple sticky traps that are still under research and development (Francese et

al. 2005, 2007; McCullough et al. 2006; Poland et al. 2006, 2007), but these are generally

effective at detecting EAB adults only at high population-densities. Girdling ash trees

and stripping the bark to reveal larvae during the following fall and winter remains the

70 most reliable method of EAB detection, especially at low population-densities (Cappaert et al. 2005; Poland et al. 2005, 2007; Fraser and Mastro 2007). One of the key shortcomings of relying on the girdled-tree method is that EAB detection is delayed until eggs have hatched and larvae have developed until the point that their galleries are evident. Detection is also delayed by the necessarily time-consuming cutting, and stripping of the logs cut from girdled trees. Detecting EAB infestations by the girdled- tree method therefore results in reliable, but delayed and costly, detection (Cappaert et al.

2005). Current efforts are also underway to determine the effects of ash tree densities and EAB population-densities on the efficacy of trap types across the entire area of the

U.S. that is under APHIS-mandated quarantine (Metzger et al. 2007).

Identification of the expanding ‘edge’ of a given infestation is critical to the subsequent removal of infested trees in an effort to prevent further spread of EAB. Early studies of outlier sites suggested that EAB could infest trees at least 600 m from a point- source (McCullough, et al. 2005). Sawyer (2007) describes an incident in which a tree containing EAB in Maryland was left standing just outside the estimated edge of an infestation (in this case, trees had been removed out to 800 m from a point-source focus).

It was originally declared that EAB had been eliminated from the area, but the infested tree and several others nearby were subsequently discovered, and this omission subsequently resulted in re-quarantine of the area. Based on population simulations, careful monitoring for EAB must continue for several years to ensure eradication

(Sawyer 2007).

We have previously shown that male EAB locate conspecifics predominantly using visual cues once the beetles are near or on a host tree (Lelito et al. 2007). The role

71 of visual cues has also been investigated via changes in trap color (Otis et al. 2005) and silhouette (Francese et al. 2007; Poland and McCullough 2007). Our previous work had also described preliminary tests of the use of these visual mate-finding cues in order to create a selective and sensitive trap for EAB in the wild (Lelito et al. 2007). The goal of the present study was to explore the utilization of visual mate-location behavior of male

EAB in capturing beetles at both high and low population densities. With potentially improved visual-cue-based methods of trapping, we sought to monitor EAB infestations in ‘real-time’, i.e. detecting invading active EAB adults rather than their progeny, or evidence thereof in the form of galleries, the following fall and winter.

METHODS

EAB Sticky-Leaf-Trapping

Our low density EAB population density sites were to the west and southwest of

the town of South Lyon, in Oakland County, Michigan. No survey traps were present in

this area during the course of the study; however, this area is characterized by nearly

complete ash mortality, with mature trees having died at least two seasons previously and

thus unlikely to support a high density of EAB. The only live ash remaining consisted of

the epicormic shoots arising from dead stumps. We also used two high population

density EAB populations: one to the south and another to the northwest of the town of

Howell, both in Livingston County, Michigan. The high density sites were characterized

by live ash trees in a state of moderate to severe dieback from borer damage. Unbaited

purple survey traps were present at both high density sites, and captured a mean total (±

72 S.E.) of 243.67±108.43 EAB northwest of Howell, and a mean total of 118.00±60.19 south of Howell. All four of our sites were dominated by green ash, Fraxinus pennsylvanica, but white ash (F. americana) was found at both high density sites as well.

A small number of the trees at the South Lyon sites were blue ash (Fraxinus quadrangulata).

Dead EAB males were pinned to the terminal leaflets of ash leaves (one EAB per leaflet) in the field, and then covered with Spray-On Tangle-Trap (The Tanglefoot

Company, Grand Rapids, Michigan, USA). We hereafter will refer to this trap type as an

EAB-Sticky-Leaf-Trap (EAB-SLT). We also placed control traps, which consisted of a terminal ash leaflet sprayed with Tangle-Trap, without a pinned EAB. Our experimental configuration was as follows: two sites at each EAB population density, each of which contained six ash trees, at least 10 m apart and of at least 10 cm DBH (diameter at breast height), onto which EAB-SLTs were deployed. On each tree, we placed three EAB-SLTs and three control traps at each of two heights in the tree (2 m and 4 m from the ground), for a total of 72 EAB-SLTs at each population density. All of the EAB-SLT deployments occurred over the course of a single day (June 16, 2007). Traps were subsequently monitored every 2 days after placement. If EAB males were found stuck to traps they were counted and then removed with forceps, bagged by trap type, and then returned to the laboratory where they were frozen. These captured EAB were then washed in Histo-

Clear (HS-200, National Diagnostics, Atlanta, Georgia, USA), and their sex was determined under a binocular microscope. Other buprestids were also found on the traps, and these were also collected and returned to the laboratory where they were identified and tallied, but not sexed. By the eighth day after placement, many traps showed serious

73 deterioration due to phytotoxicity from the propellants in the Tanglefoot application and/or the blockage of leaf respiration. Trap catch declined rapidly as traps aged (Figure

3-1); therefore, on the twelfth day after deployment, traps were examined for beetles, none were found, and all the remaining traps were removed.

Colored-Card Sticky-Trapping

Blue- plus yellow-colored sticky cards (10 by 25 cm) were purchased from

Hummert International (Earth City, Missouri, USA, Cat. Nos. 0136051 and 0136001).

Two experimental treatments and one set of controls (Blue-Control, ‘B-C’ and Yellow-

Control, ‘Y-C’ traps) were prepared from these base sticky cards. Experimental traps consisted of sticky cards with one vertical column of four dead adult male EAB [Blue 1-

Column (‘B-1’) and Yellow 1-Column (‘Y-1’) traps] evenly spaced down the centerline of the card or two vertical columns of four dead adult male EAB [Blue 2-Column (‘B-2’) and Yellow 2-Column (‘Y-2’) traps] evenly spaced down the length of the card. Dead male EAB were attached to the trap by gentle pressure against the adhesive. A total of 24 cards of each treatment (color X number of beetles affixed) were prepared. These sticky cards were hung from outer branches of ash trees at 4 m height in the tree, at a site of high EAB population density south of the town of Howell, Michigan. They were allowed to rotate freely, such that the side of the trap with beetles on it was not bound in a certain position. Traps were placed on May 31 2007, and monitored every four days through

July 2 2007. Any EAB and other buprestids found stuck to the traps were removed, returned to the laboratory, and frozen. As above, EAB were then sexed, while non-EAB buprestids were not. In a manner similar to EAB-SLTs, trap catch declined with trap age,

Figure 3-1

Mean per-trap capture of feral EAB on EAB-SLTs pooled across all trap treatments at our four field sites in SE Michigan during June and July of 2007.

75 and in this case it was most likely due to the accumulation of dust, debris, and dead insects on the cards.

Statistical Analyses

Mean capture, per 48-h period, of feral EAB and other buprestids caught on EAB-

SLTs was log-transformed to achieve normality. The transformed data were then compared by height and EAB population density treatments, as well as by an interaction effect between these two factors, using PROC GLM, SAS 9.1.3 (SAS Institute, 2006), and including Tukey’s Studentized Range Test for multiple comparisons. We used a

CONTRAST statement within PROC GLM to perform an orthogonal contrast of the number of EAB males captured on those colored-cards without EAB affixed to those with EAB affixed. Trap capture data for each 48-h period, of each species of buprestid sampled on colored-card traps, were log-transformed to achieve normality and compared by trap-color and beetle-lure treatments, including Tukey’s Studentized Range Test, using PROC GLM, SAS 9.1.3 (SAS Institute, 2006).

RESULTS

EAB Sticky-Leaf Trapping

EAB-SLTs placed high in the tree at high density sites outperformed the other

three experimental treatments overall (ANOVA, F=10.18, P<0.0001). Control traps

caught no beetles at low EAB population density, and a total of only 3 female and 1 male

EAB at high EAB population density. Both height in the tree (ANOVA, F=14.56,

76 P=0.0005) and EAB population density (ANOVA, F=13.31, P=0.0008) had significant effects on experimental trap success (Figure 3-2). At high EAB population densities,

2.64 beetles were captured on average per EAB-SLT when the trap was placed at 4 m height, and 0.94 beetles were captured on average per trap when the trap was placed treat

2 m height. At low EAB population-densities, an average of 1 beetle was captured per trap at 4 m height in the ash tree, and an average of 0.28 beetles were captured per trap when the trap was placed at 2 m height. Individuals of three non-target buprestid species were captured on these traps as well, almost always in close proximity to the dead EAB pinned to the leaf, as was also typical for the male EAB stuck to the EAB-SLT. Seventy- two individuals of Agrilus cyanescens Ratzeburg were captured in this manner on EAB-

SLTs. The height of the trap in the tree significantly influenced trap catch of A. cyanescens, with more individuals being caught at 4 m height than at 2 m height

(ANOVA, F=15.80, P=0.0003). EAB population density had no effect on the capture rate of A. cyanescens (ANOVA, F=1.76, P=0.1935). Forty-eight individuals of Agrilus subcinctus Gory were found on EAB-SLTs in total and overall there were significant differences in A. subcinctus capture between treatments (ANOVA, F=9.85, P<0.0001).

Significantly more A. subcinctus were captured on the higher traps in the tree (ANOVA,

F=21.76, P<0.0001), and at the higher EAB population density (ANOVA, F=6.72,

P=0.0137).

Colored-Card Sticky-Trapping

In total, 40 feral EAB males were captured on colored card traps (Figure 3-3).

Male EAB did not show a preference for trap color (ANOVA, F=0.00, P=1.00), but

Figure 3-2

Mean total capture per trap of feral male EAB, per EAB-SLT, by treatment.

Significantly more feral male EAB were captured on 4 m high traps at high EAB population density sites (ANOVA, F=10.18, P<0.0001). Means having no letters in common are significantly different according to Fisher’s LSD (P<0.05). Bars represent one standard error about the mean.

Figure 3-3

Mean total capture, per colored sticky trap, of feral male EAB by trap treatment. More feral male EAB were captured on colored sticky traps to which male EAB had been affixed than on traps without affixed EAB according to an orthogonal contrast (df=2,

F=4.80, P=0.0198). Bars represent one standard error about the mean.

79 seemed to be influenced within a given color treatment by the presence of affixed conspecifics (df=2, F=4.80, P=0.0198). An average of 0.38 male EAB were captured on each B-1 trap. The average capture of male EAB on other trap treatments were: 0.42 per

B-2 trap, 0.04 per B-C trap, 0.33 per Y-1 trap, 0.46 per Y-2 trap, and 0.04 per Y-C trap.

Eleven female EAB were also captured in this experiment, but these did not show a preference for the color of the sticky-card or the presence of dead male EAB affixed to the trap (Adjusted χ2, d.f.=1, P=0.55).

Greater numbers of both A. subcinctus and A. cyanescens were captured on the colored card traps than were EAB. Two-hundred fifty-one A. subcinctus were captured in total, but neither the trap color nor the presence of affixed EAB on the trap influenced the trap catch (ANOVA, F=1.53, P=0.2298). Sixty A. cyanescens in total were collected from colored card traps, with significantly more A. cyanescens captured on B-1 traps than on all other treatments (Figure 4; ANOVA, F=3.13, P=0.0330). B-2 and Y-1 traps caught more A. cyanescens than Y-2, B-C, and Y-C traps (Figure 3-4; ANOVA, F=3.13,

P=0.0330).

Figure 3-4

Mean number of A. cyanescens captured, in total per trap, on colored sticky traps by treatment. All traps were hung at 4 m height. Significantly more A. cyanescens were captured on B-1 traps than all other treatments. B-2 and Y-1 traps captured significantly more A. cyanescens than the remaining treatments (ANOVA, F=3.13, P=0.0330). Means having no letters in common are significantly different according to Fisher’s LSD test

(P<0.05). Bars represent one standard error about the mean.

81 DISCUSSION

The attraction of feral male EAB to conspecifics on adhesive surfaces for trapping

purposes had previously been demonstrated on a limited basis (Lelito et al. 2007) and we now confirm these results on a broader scale. If we could produce a longer period of trapping effectiveness (i.e., extending the period before leaf decay or dust accumulation),

it is likely that more EAB could be captured and trap efficacy at low EAB population-

densities could be increased. We are currently exploring alternative adhesives that are

designed to reduce dust accumulation and still retain trapping efficacy for EAB. Testing

these improved traps will take place during the 2008 EAB flight.

In terms of surface area, colored cards were a much less efficient trapping method than EAB-SLTs, considering that 183 EAB were captured on approximately 1.11 m2

(1728 in.2) of sticky surface for EAB-SLTs, and 51 EAB were captured on 7.43 m2

(11,520 in.2) of colored card traps, given here by generously estimating each terminal ash

leaflet as 0.1016 m by 0.0762 m (4 in. by 3 in.). This amounts to one EAB captured for

every 0.006 m2 (9.44 in.2) of EAB-SLT surface area, and one EAB captured for every

0.146 m2 (225.8 in.2) of colored card surface area, even when females are included in the

total colored-card trap capture but not included in the total EAB-SLT trap capture. EAB-

SLTs also outperform colored cards on a per-trap basis even at high EAB population densities at 4 m height. EAB-SLTs captured an average of 2.64 EAB per trap, more than a 5-fold increase over Y-2 traps, the most effective colored card treatment (with an average capture of 0.46 EAB per trap).

82 Species-specificity of trap-capture was also greater for EAB-SLTs than for colored traps: EAB represented 44.4% of the total number of buprestids captured on

EAB-SLTs versus 13.7% of buprestids captured on colored card traps, when counting both male and female EAB captured on each type of trap. This may be critical, as the capture of similarly colored non-EAB buprestids such as A. cyanescens on traps may lead to false-positive EAB detection. However, our results suggest that due to the attractive nature of objects resembling conspecifics to the buprestids studied here, some cross- attraction is likely to result from any trap design incorporating a visual lure. In addition to buprestids, a variety of other insects were captured on our traps. The most numerous of these were Diptera, followed by Coleoptera, Hemiptera, and Hymenoptera. We did not pursue the identification of these insects beyond the level of Order.

Sex specificity of trap-capture was also greater for EAB-SLTs: 97.8% of EAB captured on EAB-SLTs were male, compared to 78.4% of EAB on colored card traps.

Although a more male-specific trap may not be useful for slowing the spread of this species, this approach does represent an increase in trap-specificity over standard colored card traps. Also, the greater apparent vagility of males (Lelito et al. 2007) makes them a better target for detection of spreading populations, and the EAB-SLTs are well-suited for this effort.

Other researchers have used colored sticky traps (usually purple) when testing potential volatile lures for EAB (Francese et al. 2005; Crook et al. 2006; Poland et al.

2006) and our data suggest such colored card approaches may predispose these efforts to low rates of EAB capture. In fact, purple panel traps had been originally conjectured to be inadequately attractive to EAB (Otis et al. 2005). Our experiments suggest that

83 finding the most efficient method of trapping EAB may first hinge on designing a trap that can catch EAB at low population densities even without volatile lures. Based on our current results, EAB-SLTs appear to be a useful initial design around which to improve this type of synthetic trap.

Our data provide support for the possibility that EAB might be able to be successfully monitored using a hand-deployable, low-cost, and mass-produced ‘visual- lure’ trap. The success of these traps may possibly be further enhanced by the emission of ash volatiles, specifically those found in ash bark or foliage already shown to increase the capture of EAB on purple traps (Crook et al. 2006; Poland et al. 2006, 2007).

However, work by others suggests that even traps baited with plant compounds in this manner cannot outperform girdled trees as detection agents for EAB (Anulewicz et al.

2007).

Our current results also agree with those of others who have noted increased EAB activity (Lelito et al. 2007) and greater trap catch (Fraser et al. 2006; Lance et al. 2007) higher in ash trees. Despite this, it remains a possibility that we can capture EAB adults with EAB-SLTs for monitoring purposes, even at low heights on ash trees.

The capture of A. cyanescens on colored card traps with EAB affixed suggests that objects similar in color and size to a conspecific can act as a functional visual lure for buprestids, including EAB. This also agrees with previous work regarding male buprestid beetles and their attraction to objects by visual means (Gwynne and Rentz

1983). The implications of these findings are clear: a species- or sex-specific trap for

EAB will likely require further research and outside-the-box thinking, beyond merely adding plant volatiles to an arbitrarily colored sticky card. Our future experiments will

84 aim to examine the effects of visual lures on EAB, and perhaps other buprestids as well, in the context of providing a useful detection tool for monitoring expanding populations of EAB.

REFERENCES

Anulewicz AC, McCullough DG, Poland TM, Cappaert DL (2007) Attraction of emerald ash borer to trap trees: can meja or manuka oil compete with girdling? In: Mastro VC, Lance D, Reardon R, Parra G, (comps.), Emerald Ash Borer and Asian Longhorned Beetle Research and Technology Development Meeting, FHTET-2007-04, USDA Forest Service, Morgantown, WV, pp. 83-84

Bartelt RJ, Cossé AA, Zilkowski BW, Fraser I (2007) Antennally active macrolide from the emerald ash borer Agrilus planipennis emitted predominantly by females. J. Chem. Ecol. 33: 1299-1302

Cappaert D, McCullough DG, Poland TM, Siegert NW (2005) Emerald ash borer in North America: A research and regulatory challenge. Am. Entomol. 51: 152-165

Crook DJ, Francese JA, Fraser I, Mastro V (2005) Chemical ecology studies on the emerald ash borer. In: Mastro VC, Reardon R, (eds.), Emerald ash borer research and technology development meeting, FHTET-2004- 15,USDA Forest Service, Morgantown, WV, p. 55

Crook DJ, Fraser I, Francese JA, Mastro VC (2006) Chemical ecology of the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), in relation to tree volatiles. In: Mastro VC, Reardon R, Parra G, (comps.), Emerald Ash Borer Research and Technology Development Meeting, FHTET 2005-16, USDA Forest Service, Morgantown, WV, p. 63

Crook DJ, Khrimian A, Francese JA, Fraser I, Poland TM, Mastro VC (2007) Chemical ecology of emerald ash borer. In: Mastro VC, Lance D, Reardon R, Parra G, (comps.), Emerald Ash Borer and Asian Longhorned Beetle Research and Technology Development Meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, p. 79

Francese JA, Fraser I, Lance DR, Mastro VC, Oliver JB, Youssef N (2005) Studies to develop an emerald ash borer survey trap: II. Comparison of

85 colors. In: Mastro VC, Reardon R, (eds.), Emerald ash borer research and technology development meeting, FHTET 2004-15, USDA Forest Service, Morgantown, WV, p. 62

Francese JA, Fraser I, Lance DR, Mastro VC (2007) Developing survey techniques for emerald ash borer: the role of trap height and design. In: Mastro VC, Lance D, Reardon R, Parra G, (comps.), Emerald Ash borer and Asian Longhorned Beetle Research and Technology Development Meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, pp. 72-73

Francese JA, Mastro VC, Oliver JB, Lance DR, Youssef N, Lavallee SG (2005) Evaluation of colors for trapping Agrilus planipennis (Coleoptera: Buprestidae). J. Entomol. Sci. 40: 93-95

Fraser I, Mastro VC (2007) Emerald ash borer attraction to girdled trees: effect of placement and timing on attraction. In: Mastro VC Lance D, Reardon R, Parra G, (comps.), Emerald Ash borer and Asian Longhorned Beetle Research and Technology Development Meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, pp. 77-78

Fraser I, Mastro VC, Lance DR (2006) Emerald ash borer dispersal – a release and recapture study. In: Mastro VC, Reardon R, Parra G, (comps.), Emerald Ash Borer Research and Technology Development Meeting, FHTET-2005-16, USDA Forest Service, Morgantown, WV, p. 9

Gwynne DT, Rentz DCF (1983) Beetles on the bottle: male buprestids mistake stubbies for females (Coleoptera). J. Aust. Entomol. Soc. 23: 79-80

Haack RA, Jendek E, Liu H, Marchant KR, Petrice TR, Poland TM, Ye H (2002) The Emerald ash borer: a new exotic pest in North America. Newsletter of the Michigan Entomological Society 47:1-5

Lance DR, Fraser I, Mastro VC (2007) Activity and microhabitat-selection patterns for emerald ash borer and their implications for the development of trapping systems. In: Mastro VC, Lance D, Reardon R, Parra G, (comps.), Emerald Ash Borer and Asian Longhorned Beetle Research and Technology Development Meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, p. 77

86 McCullough DG, Siegert NW, Poland TM, Cappaert DL, Fraser I, Williams D (2005) Dispersal of emerald ash borer at outlier sites: three case studies. In: Mastro VC, Reardon R, (eds.), Emerald ash borer research and technology development meeting, FHTET 2004-15, USDA Forest Service, Morgantown, WV, pp. 58-59

McCullough DG, Poland TM, Cappaert DL (2006) Attraction of emerald ash borer to trap trees: effects of stress agents and trap height. In: Mastro VC, Reardon R, Parra G, (comps.), Emerald Ash Borer Research and Technology Development Meeting, FHTET-2005-16, USDA Forest Service, Morgantown, WV, pp. 61-62

Metzger JA, Fraser I, Storer AJ, Crook DJ, Francese JA, Mastro VC (2007) A multistate comparison of emerald ash borer (Agrilus planipennis Fairmaire) (Coleoptera: Buprestidae) detection tools. In; Mastro VC, Lance D, Reardon R, Parra G, (comps.), Emerald Ash Borer and Asian Longhorned Beetle Research and Technology Development Meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, pp. 73-74

Otis GW, Youngs ME, Umphrey G (2005) Effects of colored objects and purple background on emerald ash borer trapping. In: Mastro VC, Reardon R, (eds.), Emerald ash borer research and technology development meeting, FHTET-2004-15, USDA Forest Service, Morgantown, WV, pp. 31-32

Poland TM, McCullough DG (2006) Emerald ash borer: invasion of the urban forest and the threat to North America’s ash resource. J. Forest. 104: 118- 124

Poland TM, McCullough DG (2007) Evaluation of a multicomponent trap for emerald ash borer incorporating color, silhouette, height, texture, and ash leaf and bark volatiles. In: Mastro VC, Lance D, Reardon R, Parra G, (comps.), Emerald Ash Borer and Asian Longhorned Beetle Research and Technology Development Meeting, FHTET-2007-04, USDA Forest Service, Morgantown, WV, pp. 74-76

Poland TM, McCullough, DG, de Groot P, Grant G, Macdonald L, Cappaert DL (2005) Progress toward developing trapping techniques for the emerald ash borer. In: Mastro VC, Reardon R, (eds.), Emerald ash borer research and technology development meeting, FHTET-2004-15, USDA Forest Service, Morgantown, WV, pp. 53-54

Poland TM, Rodriguez-Saona C, Grant G, Buchan L, de Groot P, Miller J, McCullough DG (2006) Trapping and detection of emerald ash borer: identification of stress-induced volatiles and tests of attraction in the lab and field. In: Mastro VC, Reardon R, Parra G, (comps.), Emerald Ash

87 Borer Research and Technology Development Meeting, FHTET 2005-16, USDA Forest Service, Morgantown, WV, pp. 64-65

Rodriguez-Saona C, Poland TM, Miller JR, Stelinski LL, Grant GG, de Groot P, Buchan L, MacDonald L (2006) Behavioral and electrophysiological responses of the emerald ash borer, Agrilus planipennis, to induced volatiles of Manchurian ash, Fraxinus mandshurica. Chemoecology 16: 75-86

SAS Software, Version 9.1.3 (2006) SAS Institute, Cary, NC, USA.

Sawyer A (2007) Defining the ‘edge’ of isolated emerald ash borer infestations: simulation results and implications for survey and host tree removal. In: Mastro VC, Lance D, Reardon R, Parra G, (comps.), Emerald Ash Borer and Asian Longhorned Beetle Research and Technology Development Meeting, FHTET-2007-04, USDA Forest Service, Morgantown, WV, pp. 16-18

CHAPTER 4

Behavioral evidence for a contact sex pheromone component of the

emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera:

Buprestidae)

Jonathan P. Lelito, Katalin Böröczky, Tappey H. Jones, Ivich Fraser, Victor C. Mastro, James H. Tumlinson, and Thomas C. Baker

ABSTRACT

Emerald ash borers, Agrilus planipennis, were sampled for cuticular hydrocarbon

profile using solvent dipping to determine if there are differences in these compounds

between the sexes. We then assessed feral male EAB in the field for behavioral changes

based on the application of a female-specific compound to dead, solvent washed beetles.

Males in the field spent significantly more time attempting copulation with dead, pinned female beetles coated with a three-beetle-equivalent dose of 3-methyltricosane than with solvent washed beetles or those coated in 3-methyltricosane at lower concentrations.

Males in the field spent the most time investigating dead, unwashed female pinned beetles. In the laboratory, sexually mature males were presented with one of several mixtures applied in hexane to filter paper disks or to the elytra of dead female beetles first washed in solvent. Male EAB also spent more time investigating dead beetles treated

89 with, and solution applications that contained 3-methyltricosane than dead beetles and filter paper disks treated with male body wash or a straight-chain hydrocarbon not found on the cuticle of EAB.

INTRODUCTION

The emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) is a growing threat to the ash (Fraxinus sp.) resource of North America (reviewed in Poland and McCullough 2006). The beetle is spreading rapidly, and efforts to detect new infestations are of paramount importance to slowing further spread. Thus far, no species-

specific trap is available for wide deployment. The use of girdled ‘trap trees’ has proven

to be an effective but cost- and labor-intensive method of detecting new infestations

(Cappaert et al. 2005). Progress toward development of a widely deployable trap has

been made on the grounds of trap color (Francese et al. 2005), with purple traps being the

most effective at capturing adult EAB. Induced volatiles from ash trees were found to be

antennally active in adult EAB (Rodriguez-Saona et al. 2006) and incorporated into

purple prism traps for a significant gain in trapping effectiveness (Crook et al. 2008).

Green leafy volatiles derived from ash trees have also been identified and shown to

improve the capture of adult EAB on traps (De Groot et al. 2008).

Previously, the mating system of this beetle was examined in the field and the

laboratory, and vision appears to play a key role in how males locate potential mates

(Lelito et al. 2007). This is similar to the mating systems of other buprestids examined to

date (Carlson and Knight 1969; Matthews and Matthews 1978; Gwynne and Rentz 1983).

Further, males appear to discriminate between the sexes once contact is made; feral males

90 spend significantly more time attempting copulation with females than males or solvent washed beetles of both sexes, suggesting the use of a contact cue (Lelito et al. 2007).

In an effort to understand the role of any sex-specific compounds the beetle may employ in mate recognition, we conducted solvent dipping and SPME sampling of the cuticles of both immature and mature male and female beetles. These samples reveal characteristic differences between the sexes once they are mature. Here, we examine the behavioral role of 3-methyltricosane, a long-chain hydrocarbon found to be present on the cuticles of mature females, but only in traces on the cuticles of male or immature female EAB. The compound was tested for behavioral activity in the field, using dead adult EAB as lures in a manner similar to that used in the past to identify precopulatory behavior (Lelito et al. 2007), as well as in a laboratory bioassay to assess the arrestant and/or attractant properties of the compound.

METHODS

Insects

Newly emerged adult beetles were provided by the staff of the Brighton, MI (US

48116) USDA APHIS PPQ laboratory. These were segregated by sex upon emergence

from rearing barrels, maintained in separate rearing tubs, and fed on ash foliage obtained

from trees grown indoors. Beetles used for solvent and SPME sampling were either 3

days post-eclosion (‘young’) or at least 10-12 days post-eclosion (‘mature’). Live beetles

used in laboratory behavior assessments were between at least 8 and 10, but not more

than 18 days of age, and were considered ‘mature’ as well (Bauer et al. 2004; Lyons et al.

91 2004). The beetles that we used as field lures were similarly allowed to mature, killed by freezing, and were then pinned through the thorax and allowed to dry.

Solvent Dipping and SPME Sampling

Young (three days post-eclosion) and mature (12 days post-eclosion) beetles of both sexes were extracted in 3 aliquots of 300 μl of dichloromethane (B&J, High Purity

Solvent) or hexane (B&J, Ultra Resi-Analyzed) and the fractions were combined. The solvent was evaporated in a gentle stream of nitrogen. The samples were redissolved in 3 aliquots of 20 μl of an internal standard solution containing 50 ng/μl of 16-methyl hexatriacontane in hexane. We used three beetles for each sample and we had four replicates of each group. We did not detect significant differences between the dichloromethane and hexane washes of the beetles.

The spatial distribution of cuticular compounds on the surface of mature males and females was investigated using SPME fibers coated with 7 μm of polydimethylsiloxane (PDMS) (Supelco, Bellefonte, PA, USA). Body tagmata (head, thorax, and abdomen) of three mature EAB of each sex were sampled for two minutes each by rubbing a SPME fiber gently against the cuticle of the beetle. We prepared three replicate SPME samples of each body part. Beetles were held in place with soft forceps during fiber application.

Chemical Analysis

All samples were analyzed in an Agilent 6890 GC-FID system equipped with an

Equity-5 column (30m x 0.2mm x 0.2μm; Supelco, Bellefonte, PA) for quantification

92 purposes. To identify components, selected samples were analyzed on an identical column in an Agilent 6890N GC coupled with a 5973N MSD system in EI mode

(+70eV). The oven temperature program was 50°C (1 min) - 20°C/min - 210°C -

3°C/min - 320°C (10 min) for all GC analyses. The temperature of the injector was held at 280°C, the FID in the GC and the transfer line in the GC-MS were kept at 300°C.

Samples were injected splitless (0.75 min) and run at an average linear flow velocity of

25 cm/s in the GC and 30 cm/s in the GC-MS. SPME samples were analyzed using the same settings.

Identification of the compounds was based on their MS spectra (NIST05,

Masslib) and their Kovats indices on the Equity-5 column described elsewhere (Böröczky et al. 2008).

Quantification of hydrocarbons was based on their peak area values obtained from our data acquisition and analysis software (Chemstation, Agilent). Peak area values were corrected with the relative response factors described elsewhere (Böröczky et al. 2008).

Percent composition was calculated as percentage of the sum of all identified compounds.

Absolute amounts were calculated relative to the internal standard. The FID response was linear in the concentration range of the compounds we analyzed.

Synthesis of 3-methyltricosane

A solution containing 0.78g (2.8mmol) of eicosanal in 30mL of anhydrous ether was added to an excess of ethereal 2-butyl magnesium bromide under an argon atmosphere. The mixture was stirred overnight and after careful addition of 10% HCl, the ether layer was separated, washed with saturated NaHCO3, and dried over anhydrous

MgSO4. After filtration, the residue was taken up in 7mL of pyridine and treated with

0.31mL of methane sulfonyl chloride at 0oC and stirred overnight. After the addition of

50mL of ether, the mixture was washed with 10% HCl, and the ether layer was separated,

washed with saturated NaHCO3, and dried over anhydrous MgSO4. After filtration, the

solvent was removed in vacuo and the residue was taken up in 50mL of ethyl acetate and

hydrogenated over 100mg of PtO2 under 3 Atm of hydrogen overnight. After the mixture

was filtered and the solvent was removed, flash chromatography (silica gel/ hexane)

provided 521mg of 3-methyltricosane, m/z 338 [M+](0.2), 309(10), 281(2), 25312),

239(2), 225(3), 211(3), 197(3), 183(3), 169(4), 155(5), 141(7), 127(9), 113(13), 99(19),

97(9), 85(41), 83(9), 71(65), 69(14), 57(100), 56(36), 55(22), 43(57), 41(24). GC-MS

was carried out in the EI mode using a Shimadzu QP-2010 GC-MS equipped with an

RTX-5, 30 m x 0.25 mm i.d. column. The instrument was programmed from 60°C to

250°C at 10°C/min and held at 250°C for 40 minutes.

The synthetic compound had a Kovats index (2371 on the Equity-5 column used

above) and MS spectrum identical to that of the compound found in the body washes.

Field Behavior

The dead, pinned female ‘beetle-lures’ used for these experiments were either:

unwashed, ‘U’; washed in dichloromethane for ten minutes and then allowed to dry for

24 h, ‘W’; or solvent-washed, dried, and then coated with one of our experimental

treatments. Experimental treatments involved the application of 10 μL of one of the

following solutions to the dorsal cuticle of a dead, dichloromethane-washed, and dried

female EAB: 4 ng/μL n-eicosane (‘E’) (an impurity resulting from our synthesis of 3-

94 methyltricosane); 2 ng/μL 80%/20% 3-methyltricosane/n-eicosane mixture (‘T1’); 6 ng/μL 80%/20% 3-methyltricosane/n-eicosane mixture, (‘T2’); or 20 ng/μL 80%/20% 3- methyltricosane/n-eicosane mixture, (‘T3’). Thus, T1, T2, and T3 represented 16, 48, and 160 ng of 3-methyltricosane, respectively. All applications were made with a 10 μL glass pipette, which was cleaned between applications by three separate washes with 10

μL of hexane. We prepared fresh beetle-lures for each replicate of the field experiment.

Field experiments took place between 7 June and 5 July 2007 between the hours of 1000 AM and 1600 PM EST, on days without heavy rain. Each two-hour replicate

(one per suitable day, for a total of 17 replicates) of the field experiment was performed in an area of high-density EAB population located in Livingston County, Michigan,

U.S.A., south of the town of Howell. The site used was private agricultural and forest land, containing a large number of green ash trees (F. pennsylvanica), as well as several oak and other tree species. Our experiments were conducted prior to and through the peak EAB flight period. Individual ash trees selected for these experiments were between 10 and 20 m tall and had healthy branches accessible at 2-3 m height from ground level for the pinning of beetle-lures. Each tree used in this experiment was selected randomly on each day, with the precondition that at least five live EAB could be seen on that tree from ground level (i.e. a presumably infested tree).

For each replicate of field experiment, we pinned three beetle-lures of each treatment (U, W, E, T1, T2, and T3), each to its own terminal leaflet of a compound ash leaf, on the sunny side of a selected ash tree, between 2 and 3 m from ground level.

Beetle-lures were positioned randomly (not blocked by treatment), not less than 10 cm

95 apart but not more than 30 cm apart within each replicate. We then observed these lures from the ground for a two hour period. We timed the duration of behavior whenever any feral EAB attempted copulation with a pinned beetle-lure and subsequently persisted in investigation of that lure. We defined ‘investigation’ of a beetle-lure to broadly mean a feral EAB coming into, and remaining in contact with a pinned beetle-lure subsequent to an airborne approach and copulation attempt.

Laboratory Behavior

We prepared experimental arenas using a plastic 100 mm diameter Petri dish (Cat.

No. 08-757-12, Fisher Scientific) lined with a 100 mm diameter filter paper insert (Cat.

No. 1001-100, Whatman). For each experiment, we applied 10 µL of hexane as a control to the filter paper at each of two opposite points along the diameter of the dish, both 40 mm from the center of the filter paper. We then applied 10 µL of one of the experimental solutions (in hexane) to the paper at the other two opposite points along the perpendicular diameter. Experimental solutions are listed in Table 4-1.

We prepared both male and female body washes by placing twelve mature individual EAB into 100 μL of hexane in a 2 mL glass vial. We gently agitated the vial for two minutes, and extracted the remaining liquid. For the lower dosage of one beetle- equivalent (1BE), we prepared both male and female body washes as above, with the difference that we only used four mature beetles in 100 μL of hexane to create the experimental solution. We prepared fresh body wash solution at both dosages as needed and stored any remaining solution in a capped 2 mL glass vial in a standard freezer.

Table 4-1. The beetle-equivalent (BE) rates, compounds used as lure treatments, dosage of those compounds in ng/, and abbreviations used in the text for all combinations of BE rate, lure treatment, and dosages in the laboratory bioassay.

97 We first performed a series of trials, 25 of each treatment, using the above experimental solutions at a dosage of approximately three beetle-equivalents (3BE) for a total of 150 trials. Dosages at the 3BE rate are listed in the top half of Table 4-1.

Subsequently, we performed an identical series of 25 trials to each treatment with a dosage of approximately one beetle-equivalent of compound in 10 μL of hexane, again for a total of 150 trials. Dosages at the 1 BE rate are listed in the lower half of Table 4-1.

Fresh filter paper inserts in new Petri dishes were used for each trial and were prepared immediately before placing a live EAB in the arena. Each trial began when we placed individual mature (8-18 days post-eclosion) male EAB into the dish at the center- point of the filter paper. Each EAB male was then observed for a period of 10 minutes, during which time we recorded the number and duration of entries into a 1-cm diameter area centered on the application point.

Subsequent to the filter paper trials, we performed another identical set of trials using the same suite of experimental solutions and the two dosages as above, but instead of the solutions being applied directly to the filter paper, they were applied to the dorsal cuticle of a dead, solvent-washed female EAB that was hot-glued to the filter paper at the typical solution application points (Fig. 4-1). Beetle-lure filter-paper setups were prepared by hot-gluing solvent washed dead female EAB to the four application points on the filter paper disk. Such setups were prepared not more than 24 hours before being used in a trial, and were stored in a freezer individually within closed Petri dishes prior to use. For these beetle-lure trials, we recorded the number of direct male to female-beetle- lure contacts, the duration of each contact, and the number of times the male attempted to

Figure 4-1. The experimental arena used for the laboratory behavioral assay; the beetle in the center is a live male at the release point.

99 copulate with the beetle-lure. The BE rates and dosages used for these trials are listed in

Table 4-1.

Finally, we conducted a series of trials testing mature female EAB for their response to beetle-lures in the arena bioassay, using the same experimental treatments as above for mature males at the 3BE dosage. Again, each trial lasted for ten minutes and utilized one individual live female EAB and four beetle-lures (two control and two experimental applications). We recorded the number and duration of female- to beetle- lure contacts for each treatment.

A total of 25 individual 10-minute trials were conducted for each treatment/lure combination (e.g. 3 BE dosage, filter paper application) between 10 February and 15

March 2008, between 900 AM and 1400 PM EST. Three trials were run concurrently, with arenas approximately 10 cm apart under full-spectrum fluorescent lighting at 25°C.

The lighting was situated on wooden supports such that all arenas were lit from directly overhead the center of the arena from a height of approximately 40 cm. Within each experiment, treatments were completely randomized within a day, so that all treatments had an equal chance of occurring at a given time of day. Male EAB were frozen and discarded after experiencing a single trial.

For these laboratory experiments and the field experiments outlined above we purchased n-eicosane and n-tetracosane from Aldrich Chemical Co. (99% purity, St.

Louis, MO, USA) and synthesized the 3-methyltricosane mixtures as outlined above.

Statistical Analyses

100 We compared the mean investigation time of each treatment of beetle-lure in the field experiment time using a two-way ANOVA (analysis of variance), with replicate and treatment of the lure as factors. Treatment means were separated by in pair-wise comparisons by Tukey’s Honestly Significant Differences (HSD) test. We employed a binomial test of proportions to detect any treatments that were contacted more often than their paired hexane control (a significant deviation from a 50-50 ratio could indicate either attraction to, or avoidance of, a given treatment). Laboratory bioassay data were log-transformed prior to analysis to achieve a normal distribution. We utilized PROC

GLM in SAS for comparisons of mean times of investigation between treatments by male

EAB, using both the lure type and the chemical treatment of the lure as factors with investigation time as the dependent variable. We performed all statistical analyses in

SAS Version 9.1.3 (SAS Institute, 2006) on a Dell Inspiron Laptop running Windows XP

Home Edition.

RESULTS

Solvent Dipping and SPME Sampling

Major components of the cuticle of both male and female EAB were found to be

saturated and monomethyl branched odd-chain hydrocarbons in the range of chain-length

C23-C29. The methyl branch was typically in the middle of the chain. Terminally

branched monomethyl alkanes, dimethyl alkanes, and unsaturated hydrocarbons were

minor components. More polar lipids, such as fatty acids, ethers, and acetate and

101 butyrate esters of long-chain alcohols, were also detected in the body wash samples of both sexes.

Solvent dipping experiments revealed characteristic differences between the cuticular chemistry of male and female beetles (Figure 4-2). The hydrocarbon 3- methyltricosane is a minor component of the female body wash, but occurs only in traces in the male body wash. Furthermore, it was found in limited quantity on the cuticle of the young female EAB, but it increased in abundance with the age of the beetle, coinciding with sexual maturity. The estimated amount of 3-methyltricosane on a mature female is approximately 50 ng of material. Other terminally branched mono- methylalkanes showed a similar trend but not as strongly as 3-methyltricosane (Table 4-

2). We did not detect any variation in expression of 3-methyltricosane between the tagmata of the females with SPME.

Field Behavior

Male EAB in the field remained in contact for a significantly longer duration with beetle-lures that were unwashed in solvent (X¯ = 209.9 s) than they did with the other treatments (ANOVA, df=5, F=20.66, P<0.0001; Fig. 4-3). Experimental treatments T3 and E elicited somewhat shorter durations of investigation (X¯ = 71.2 and 41.1 s, respectively) from male EAB than did unwashed beetle-lures (ANOVA, df=5, F=20.66,

P<0.0001; Fig. 4-3). However, these durations of investigation were still significantly longer than that elicited by the washed female control (X¯ = 14.5 s). Experimental treatments T1 and T2 elicited durations of investigation (X¯ = 16 and 20.7 s, respectively) not statistically different from the washed female beetle-lure (ANOVA, df=5, F=20.66,

102

Figure 4-2. Gas chromatographic profiles of the solvent wash of mature females, a), young females, b), young males, c), and mature males, d). The peak marked with an asterisk is 3- methyltricosane.

103

Table 4-2. The amount of terminally branched mono-methylalkanes and the total alkanes identified on young and mature male and female emerald ash borers. Means and S.E.M.s shown are derived from four replicates of this analysis for each sex, each containing material from three beetles.

104

Figure 4-3. Columns represent the mean time (in seconds) spent by feral male EAB in investigation of beetle-lures in the field in response to different treatments during each two-hour period of observation, with a total of 17 replicates. Treatments are listed in the following order on the x-axis: ‘W’, a solvent-washed dead female EAB; ‘T1’, 2 ng/μL 3-methyltricosane mixture application onto a washed female EAB; ‘T2’, 6 ng/μL 3-methyltricosane mixture application; ‘E’, 4 ng/μL eicosane application; ‘T3’, 20 ng/μL 3-methyltricosane mixture application; ‘U’, a non-solvent-washed dead female EAB. Bars represent one standard error of the mean. Columns having no letters in common are significantly different (df=5, F=19.73, P<0.0001). This experiment was performed between 7 June and 5 July 2007 south of Howell, Michigan.

105 P<0.0001; Fig. 4-3). Replicate was not a significant source of variation (ANOVA, df=16, F=1.65, P=0.1038). Male EAB approached in flight and landed on all types of lures in the field at statistically equivalent rates (ANOVA, df=5, F=0.536, P=0.739).

Laboratory Behavior

The differences in investigation time by male EAB based on lure type and chemical treatments in the laboratory behavioral trials were somewhat similar to the results of our field behavior experiment above and are summarized in Table 4-3. Using both lure type and treatment as factors, both treatment and lure type had a significant effect on male behavior (ANOVA, df=25, F=4.34, P<0.0001) and thus data were separated by lure type and analyzed separately for treatment effects. The chemical treatment applied to the lure had a significant effect on male behavior for both beetle- lures (Table 4-3; ANOVA, df=12, F=4.79, P<0.0001) and filter paper applications (Table

4-3; ANOVA, df=12, F=2.72, P=0.0017). A greater number of cuticular lipid applications onto dead female EAB (treatments FBW3, TRM3, TRP3; Table 4-3) evoked significant differences in male behavior over their respective controls than did the corresponding lipid applications directly onto filter paper (treatment FBW3 only; Table

4-3).

Male EAB in the laboratory trials made significantly more total copulation attempts to beetle-lures in response to treatments FBW3 and TRP3 at 3BE dosage than to the other treatments and the hexane-only controls (ANOVA, df=12, F=6.032, P<0.0001;

Fig. 4-4, black columns). At 1BE dosage, no significant variation in the number of copulations between treatments occurred (Fig. 4-4, grey columns).

106

Table 4-3. The results of the laboratory bioassay, showing beetle-equivalent (BE) rates and mean durations of contact by treatment. A total of 25 male EAB were used in each combination of lure and treatment. Treatment abbreviations are the same as given in Table 1.

107

Figure 4-4. Columns represent the total number of copulation attempts performed by male EAB to each experimental treatment of beetle-lure. Black columns indicate 3BE dosage, grey columns indicate 1 BE dosage. Treatment abbreviations on the x-axis are the same as in Figure 4. Bars represent two standard errors of the mean. Columns having no letters in common are significantly different from one another (df=12, F=6.032, P<0.001).

108

The number of male beetles contacting a given treatment applied to a beetle-lure was never significantly different from the number of contacts with its paired hexane control (Binomial test of proportions, P > 0.05 for all treatment-control pairs); similarly, the number of male EAB coming into contact with areas of the filter paper to which solutions had been applied was not significantly different than contacts with control solution applications (Binomial test of proportions, P > 0. 05 for all treatment-control pairs).

Female EAB did not exhibit a significant preference for remaining in contact with a given treatment over any other (ANOVA, df=6, F=0.472, P=0.829). Female EAB also did not significantly prefer to initially contact any treatment over its paired control during the course of the experiment (Binomial test of proportions, P > 0. 05 for all treatment- control pairs).

DISCUSSION

Although the approach phase of the EAB mating system is highly visually

mediated, previous work provided evidence for the involvement of a contact sex

pheromone on females that influenced male behavior after they landed on females (Lelito

et al. 2007). Our results are the first step in identifying the components of this pheromone

and support the pheromonal role of the cuticular hydrocarbon 3-methyltricosane when it

is applied to the surface of a dead conspecific. It is likely that other lipids present on the

female cuticle play a role in the contact sex pheromone as well. Further, 3-

methyltricosane has a chiral center at position 3, and we cannot assume that only one

109 isomer occurs on the cuticle of the beetle and mediates behavior. To date, the only way to determine the configuration of chiral hydrocarbons is to synthesize both enantiomers, which was not part of the current study. The higher 3BE dosage needed to elicit a change in behavior may be related to our synthetic mixture of 3-methyltricosane. The dosage of the correct enantiomer of 3-methyltricosane perceived by the beetle may be different from the overall applied dosage of both enantiomers due to the presence of both enantiomers in our synthetic mixture.

Males in the laboratory spent more time investigating beetle-lures to which we had reapplied female body wash or synthetic mixtures containing 3-methyltricosane, a preference we observed among feral male EAB tested in the field as well. Given that the total amount of the various lipids that are present on an unwashed female beetle, or are contained in the solvent wash from a female beetle may be 600 times higher than the amount of 3-methyltricosane in our synthetic mixture (Table 4-2), it is actually quite remarkable that such an effect was observed. Therefore, it is likely that the behavioral effect of 3-methyltricosane may be synergized by the perception of other compounds naturally secreted onto the cuticular surface of the unwashed beetle-lures, and this may also play a role in the higher dosage necessary to elicit a difference in response by males.

Males appeared to detect the contact sex pheromone or the sex pheromone component 3-methyltricosane through their antennae, as indicated by a continuous antennation of the treated substrate; this was especially obvious when the male encountered an affixed beetle to which female extract or 3-methyltricosane had been applied. Male chemoreception may not be limited to the antennae: males were also observed to ‘scratch’ their tarsi against both the filter paper and the affixed beetles in

110 many cases. This behavior was often followed by renewed bouts of vigorous antennation. Although it is possible that males might be able to detect the presence of females from outside their visual range either directly or indirectly by olfactory cues such as volatiles given off by the damaged host (Crook et al. 2008), 3-methyltricosane is highly unlikely to serve as a long-range attractant, as shown by a lack of differential approach to our treatments over just a few centimeters.

REFERENCES

Bauer LS, Haack RA, Miller DL, Petrice TR, Liu H (2004) Emerald ash borer life cycle. In: Emerald ash borer research and technology development meeting. Mastro VC and Reardon R (comps.). FHTET-2004-02, USDA Forest Service, Morgantown, WV, p. 8

Böröczky K, Minard RD, Park K-C, Jones TJ, Baker TC, Tumlinson JH (2008) Differences in cuticular lipid composition of the antennae of Helicoverpa zea, Heliothis virescens, and Manduca sexta. J. Ins. Physiol. 54:1385- 1391

Cappaert D, McCullough DG, Poland TM, Siegert NW (2005) Emerald ash borer in North America: A research and regulatory challenge. Am. Entomol. 51: 152-165

Carlson, RW and Knight FB (1969) Biology, taxonomy, and evolution of four sympatric Agrilus beetles (Coleoptera: Buprestidae). Contrib. Am. Entomol. Inst. 4:1-105

Crook DJ, Khrimian A, Francese JA, Fraser I, Poland TM, Sawyer AJ, Mastro VC (2008) Development of a Host-Based Semiochemical Lure for Trapping Emerald Ash Borer Agrilus planipennis (Coleoptera: Buprestidae). Envir. Ent. 37: 356-365

111

Francese JA, Mastro VC, Oliver JB, Lance DR, Youssef N, and Lavallee SG (2005) Evaluation of colors for trapping Agrilus planipennis (Coleoptera: Buprestidae). J. Entomol. Sci. 40: 93-95

Gwynne DT and Rentz DCF (1983) Beetles on the bottle: male buprestids mistake stubbies for females (Coleoptera). J. Aust. Entomol. Soc. 23: 79- 80

Lelito JP, Fraser I, Mastro VC, Tumlinson JH, Böröczky K, Baker, TC (2007) Visually mediated ‘paratrooper copulations’ in the mating behavior of Agrilus planipennis (Coleoptera: Buprestidae), a highly destructive invasive pest of North American ash trees. J. Insect Behav. 20:537-552

Lyons DB, Jones GC, Wainio-Keizer K (2004) The biology and phenology of the emerald ash borer. In: Emerald ash borer research and technology development meeting. Mastro VC and Reardon R (comps.). FHTET- 2004-02, USDA Forest Service, Morgantown, WV, p. 5

Matthews RW and Matthews JR (1978) Insect Behavior. Wiley, New York

Poland TM and McCullough DG (2006) Emerald ash borer: invasion of the urban forest and the threat to North America’s ash resource. J. Forestry 104:118-124

Rodriguez-Saona CR, Poland TM, Miller JR, Stelinski LL, Grant GG, De Groot P, Buchan L, Macdonald L (2007) Behavioral and electrophysiological responses of the emerald ash borer, Agrilus planipennis, to induced volatiles of Manchurian ash, Fraxinus mandshurica. Chemoecology 16:75-86

SAS Institute (2006) SAS Software Version 9.1.3. SAS Institute, Cary, NC, USA.

CHAPTER 5

Field investigations of the mating behaviors of Agrilus cyanescens and Agrilus subcinctus.

Jonathan P. Lelito, Ivich Fraser, Victor C. Mastro, James H. Tumlinson, and Thomas C. Baker

ABSTRACT

We examined two species of buprestids in the genus Agrilus, A. subcinctus Gory and A. cyanescens Ratzeburg (Coleoptera: Buprestidae), for behaviors involved in mate- finding by using dead conspecifics of both sexes and non-conspecific insects affixed to foliage as elicitors of behavior. The lures we used were either washed in dichloromethane to remove contact hydrocarbon cues, or remained unwashed. Male beetles of both species approached, antennated, and/or attempted to copulate with dead beetles of either sex, but investigated unwashed conspecific females for significantly longer durations. Male A. subcinctus did not prefer male or female lures for initial approach, but did prefer to remain in contact with female lures. When A. subcinctus approached dead-conspecific lures by crawling into contact with them via the substrate, unwashed female beetles were preferred for mounting over all other treatments, further suggesting that a contact pheromone is involved in mate choice. Male A. cyanescens also differentiated between cuticular wash treatments, but dead females were significantly

113 preferred over males for both initial airborne approach and the duration of attempted copulation. Male A. cyanescens will also attempt copulation with the elytra and whole bodies of green-blue colored tiger beetles pinned to leaves, and will remain in contact longer if these non-conspecific lures have had conspecific female cuticular compounds applied to them. Our results indicate the use of primarily visual cues, and subsequently contact chemosensory information, in the mate-location and sex-discrimination by two species in the genus Agrilus.

INTRODUCTION

The body of work involving the mating behavior of buprestids (e.g. Rodriguez-

Saona et al. 2008; Lelito et al. 2007; Akers and Nielsen 1992; Gwynne and Rentz 1983;

Carlson and Knight 1969) has thus far indicated the primary use of visual and tactile cues

for location and discrimination of conspecifics, in some cases mediating attraction of

beetles to man-made objects. It has been suggested that buprestids, particularly those in

the genus Agrilus, may first locate the host by olfactory or other means, and then locate

conspecifics visually, or by vibratory and tactile cues (Carlson and Knight 1969).

However, Dunn and Potter (1988) indicated preferential attraction of males to cages

containing females compared to host-logs only in A. bilineatus Weber, suggesting the use

of a female-produced pheromone. Because female A. bilineatus tended to arrive first at host-volatile-based lures, and the sex-ratio of beetles thus attracted was skewed toward females at these lures as well (Dunn and Potter 1986), males were deemed unlikely to rely solely on vision to locate conspecifics. However, with the potential exception of a

114 putative sex-pheromone component recently identified as being produced by female emerald ash borers, A. planipennis Fairmaire (Bartelt et al. 2007), no pheromones have been conclusively identified from the Buprestidae.

Here we report the examination of the behavior of two buprestids, A. subcinctus

Gory and A. cyanescens Ratzeburg, in the field for evidence of their use of visual and/or chemical cues in mate-location and gender discrimination. We utilized conspecific and non-conspecific insects, both on and off the host plant, as elicitors of behavior to determine the roles of vision and chemistry in the mating system of these two beetles.

METHODS

Agrilus subcinctus

Individual A. subcinctus to be used as lures were captured in the wild, placed into

separate glass vials for transport to the laboratory, and then separated by gender with the

aid of a binocular microscope. While a specimen was held with forceps, we applied

gentle pressure to the lateral portions of the abdomen to cause extension of the genitalia,

allowing gender identification to be made. Males of this species also have tell-tale green

iridescence on the frons, a narrower abdomen, and are generally smaller than female A.

subcinctus. If a definitive identification of gender could not be made or the specimen

was damaged extensively by extension of the genitalia, the specimen was discarded.

After gender identification, we killed beetles by freezing and randomly assigned beetles

of each gender into two treatments: to be washed for five minutes by gentle agitation in a

115 5 mL vial containing 1 mL of dichloromethane, or to remain chemically unwashed. Once we had washed some of the beetles, we placed the beetles into vials segregated by wash treatment and gender and stored these in a freezer until use.

To create the lure setups for this experiment, we harvested undamaged ash leaves growing from epicormic shoots on ash tree stumps. We then cut each leaf such that only the lowest pair of leaflets remained attached to the petiole, which we then placed into a plant wick with fresh water. We placed two equally-spaced drops of Tangle-Trap (The

Tanglefoot Company, Grand Rapids, MI, U.S.A.) onto the midvein of each of the two live ash leaflets. We then affixed one individual A. subcinctus to each of the Tangle-Trap droplets, for a total of four affixed conspecifics per setup. Each of these setups contained one individual from each of the following four treatments: 1. unwashed female; 2. unwashed male; 3. female washed for five minutes in dichloromethane, and 4. male washed for five minutes in dichloromethane. Individual treatments of beetles were randomly assigned to be affixed to one of the four Tangle-Trap droplets on the ash leaf.

Finally, we pinned each setup to a live ash twig adjacent to a leaf and observed the behavior of feral A. subcinctus individuals. We used each setup only once, for two hours at a time. In three replicates of the experiment, an observer was actively present to monitor the experimental setup; in a further seven replicates, we positioned a video camera (Model DCR-TRV350, Sony Corporation of America, New York City, U.S.A.) on a tripod and focused on the lure setup to record the approaches and behaviors of individual A. subcinctus. To establish the gender of beetles performing a given behavior, when an observer was present to monitor the dead conspecifics used as lures, we captured, with a small net, those live beetles observed interacting with affixed

116 conspecifics, and identified their gender as above. These experiments were conducted during the hours of 0800 – 1400 EST between 24 May 2007 and 6 June 2007.

Agrilus cyanescens

Individual A. cyanescens were captured by hand from the foliage of infested

Lonicera bushes near Pinckney, Michigan, on 23-31 May 2008, and separated by gender based on examination of the genitalia as described above. The beetles were then placed into gender-segregated vials, frozen for one hour, and pinned through the elytra with a steel insect pin (Size 0, BioQuip, Rancho Dominguez, CA, U.S.A.) and either gently washed for five minutes in dichloromethane and dried for 30 minutes, or left unwashed but similarly allowed to stand for 30 minutes. All pinned insects were stored in small specimen boxes by gender and wash-treatment in a standard freezer until their use as lures in a given experiment.

Each of ten replicates of this experiment were performed as follows: one of each of the four treatments (male and female, washed and unwashed) of dead A. cyanescens was pinned to the midvein of a leaf on a Lonicera bush in random order, no less than 10 cm but no more than 15 cm distant from any other dead, pinned beetle, at approximately

1.5 m height from the ground. The dead beetles were then observed for one hour, during which time the number of approaching A. cyanescens was recorded, as was the number and duration of copulation attempts to each of the pinned beetles. At the end of each hour of observation, the dead, pinned beetles were removed and the entire setup was repeated on another Lonicera bush. In addition, we performed six replicates of this

117 experiment identical to those above, with the exception of the location: we instead pinned our four beetle-lures to Rubus and Eleaganus plants growing within two and four meters from infested Lonicera bushes. All beetle-lures were discarded at the end of each day of experiments.

We then employed videotaping (Model DCR-TRV350, Sony Corporation) to record the responses of individual A. cyanescens to insects other than conspecifics. We used the following insects and insect parts as lures for videotaped experiments: an intact

Green Immigrant Leaf Weevil, Polydrusus sericeus; an intact male six-spotted tiger beetle, Cicindela sexguttata; a single elytron of a male C. sexguttata; a pinned head of a male C. sexguttata; the dorsal thorax and abdomen (with wings and legs removed) of a blue-bottle fly, Calliphora sp.; an intact metallic wood boring beetle, Brachys ovatus; and an intact male emerald ash borer, Agrilus planipennis. We placed one of each of the above lures onto an individual leaf of an A. cyanescens-infested Lonicera bush, focused the camera on the lures, and recorded the behavior of any approaching live A. cyanescens for approximately one hour. This observational setup was performed four times.

Subsequent to videotaping the responses of A. cyanescens to heterospecific insects, we performed twelve additional one-hour experiments using the same spacing between lures and placement guidelines as above. Six replicates of this experiment were performed on infested Lonicera bushes and six on adjacent Rubus and Eleaganus. We used dichloromethane cuticular washes of male and female A. cyanescens to be reapplied to our lures as potential contact cues. A wash was prepared from each gender of A. cyanescens by placing ten A. cyanescens of the same gender into 150 μL of dichloromethane in a 4 mL glass vial, and gently agitating the vial for five minutes. The

118 lures used for this setup were as follows: one of each of the four treatments of A. cyanescens used as above; an unwashed C. sexguttata elytron; a C. sexguttata elytron washed in dichloromethane, dried for 30 minutes and then treated with 10 μL of the female A. cyanescens wash, and a C. sexguttata elytron washed in dichloromethane, dried for 30 minutes, and then treated with 10 μL of the male A. cyanescens wash. The dead insects used as lures were discarded at the end of each day of experiments.

Finally, we performed six one-hour replicates using the following setup: four individual pinned C. sexguttata elytra, placed as above on Rubus and Eleaganus in proximity to infested Lonicera. We used one of each of the following treatments during every replicate: an unwashed C. sexguttata elytron; a dichloromethane-washed elytron; an elytron first washed in dichloromethane, dried for 30 minutes, and then treated with 10

μL of the female A. cyanescens wash, and a C. sexguttata elytron first washed in dichloromethane, dried for 30 minutes, and then treated with 10 μL of the male A. cyanescens wash. Left or right C. sexguttata elytra were randomly assigned to each treatment, and the elytra were discarded at the end of each day.

All of these experiments took place on a given day between 0900 and 1400 hours

EST, and were conducted between 29 May 2008 and 23 June 2008.

Statistical Analyses

A binomial test of proportions was employed to detect any preference for male or female lures being approached by live male conspecifics, and a t-test was used to compare the mean size of male and female lures of each species. In both species, we

119 assessed the duration of copulation following an airborne approach (a ‘paratrooper’ copulation; Lelito et al. 2007) with the gender and wash treatment (and the species, when using tiger beetle elytra) of the lure as factors in ANOVA, using PROC GLM within SAS

Statistical Software, version 9.1.3 (SAS Institute 2006). In the case of A. subcinctus only,

we also compared the mean duration of the following behaviors: ‘pounce’ approaches, in

which a live male approaches a lure by crawling along the leaf and then rapidly faces and

jumps onto the lure, and ‘antennation’, in which a live beetle approaches and antennates a

lure but does not make any other contact with it. We used gender and wash treatments as factors in ANOVA using PROC GLM. Data were log transformed (log10(x+1)) prior to

analysis where necessary to satisfy conditions of normality. Finally, we employed a

Fisher’s Exact Test (Fisher 1922) to compare the number of feral A. subcinctus performing ‘pounce’ and ‘antennation’ behaviors when contacting either gender of unwashed pinned conspecifics by walking up to them on a leaf, and again to compare the number of feral A. subcinctus performing these behaviors when contacting either gender of solvent-washed pinned conspecifics.

RESULTS

Agrilus subcinctus

All feral A. subcinctus that were captured after performing ‘paratrooper

copulation’ attempts were identified as males (N = 9). Of those feral beetles that were

captured after a ‘pounce’ behavior, in which a feral beetle approached, faced, and jumped

120 or climbed onto the back of an affixed conspecific, 91% were identified to be male (30 males; N = 33 captured). The mean length in mm (± SE) of the dead beetles we used as lures varied significantly between male (X¯ = 3.839 ± 0.046) and female (X¯ = 3.954 ±

0.046) beetles (t-test, t = -1.757, d.f. = 78, P = 0.083). However, all four beetle-lure types elicited ‘paratrooper’ approaches by feral beetles at equivalent rates (binomial test of proportions, two-sided P = 0.396, CI: 0.47 - 0.57).

The duration of ‘paratrooper copulation’ behavior was significantly longer for unwashed females than for either of the washed beetle lures; unwashed male lures elicited the shortest duration of behavior (Table 5-1; F = 17.75, d.f. = 3, P < 0.0001).

Similarly, the duration of ‘pounce’ behavior was significantly longer for unwashed females than for any other treatment combination (Table 5-1; F = 21.81, d.f. = 3, P <

0.0001), all of which elicited equivalent lower responses. When an affixed lure elicited only antennation by a feral beetle, the relationship of behavioral durations to the lure treatment was similar to that observed during ‘pounce’ behavior, except that washed male conspecifics were not antennated for a significantly longer or shorter duration than either unwashed male or washed female dead conspecifics (Table 5-1; F = 15.84, d.f. = 3, P <

0.0001).

Although approaches from the air resulted in equivalent rates of approach to all treatment of lure, the proportions of these behaviors that were elicited when feral beetles approached on the substrate were quite different (Fig. 5-1). When we examined ‘pounce’ and ‘antennation’ behaviors together, 62 of 93 (67%) substrate-based approaches (i.e. those approaches not from flight) by feral beetles to unwashed female conspecifics were

‘pounce’ behaviors. In contrast, 76 of 85 (89%) of approaches by feral beetles to

121

122

Figure 5-1

Probability of a given behavior being performed by a feral A. subcinctus when coming into contact with a lure. Behaviors are grouped into two categories: live beetles performing antennation of the affixed conspecific and live beetles performing a ‘pounce’ onto the affixed beetle. Unwashed female lures were more likely to elicit a ‘pounce’ behavior than unwashed male lures (Fisher’s Exact Test, P < 0.0001), whereas live beetles were equally likely to perform a ‘pounce’ onto washed lures of either gender

(Fisher’s Exact Test, P = 0.2492). Unwashed male models were significantly more likely to elicit only an antennation by live A. subcinctus than they were to elicit a ‘pounce’ behavior (Fisher’s Exact Test, P < 0.0001).

123 unwashed male conspecifics resulted only in ‘antennation’ of the affixed beetle, with

‘pounce’ behavior occurring during the other nine (11%) substrate-based contacts with unwashed male conspecifics. This difference is statistically significant (Fisher’s Exact

Test, P < 0.0001). Feral males showed no statistically significant preference for performing a ‘pounce’ behavior to either gender of washed beetle-lure (Fisher’s Exact

Test, P = 0.2492), performing ‘pounce’ behavior to washed female and washed male conspecifics in 35 out of 107 approaches (33%), and 41 out of 100 (41%) approaches, respectively.

Agrilus cyanescens

In the field, live male A. cyanescens performed aerial approaches to dead, pinned conspecifics, followed by ‘paratrooper’ copulation attempts (Fig. 5-2). The mean length, in mm, of the dead A. cyanescens we used as lures was significantly greater for female

(X¯ = 6.893 ± 0.070) than for male (X¯ = 6. 346 ± 0.073) lures (ANOVA, F = 480.379, d.f. =

3, P < 0.001). The tiger beetle elytra used for heterospecific attraction tests were also significantly longer than both male and female A. cyanescens (X¯ = 7.685 ± 0.082 mm,

ANOVA, F = 480.379, d.f. = 3, P < 0.001).

When we used only the four treatments of dead, pinned conspecifics as lures on

Lonicera host plants, live male A. cyanescens preferred to perform aerial approaches to

pinned female (N = 154) conspecifics over pinned male (N = 115) conspecifics (binomial

test of proportions, two-sided P = 0.0160, CI: 0.51 - 0.63). Male A. cyanescens also

showed a strong preference for remaining in contact with unwashed female conspecifics

124

Figure 5-2

A live male A. cyanescens attempting copulation with a C. sexguttata elytron treated with female A. cyanescens cuticular wash, lateral (A) and dorsal view (B).

125 pinned to leaves following an airborne approach and copulation attempt compared to the other three treatments (Table 5-2; ANOVA, F = 9.79, d.f. = 3, P < 0.001).

When the four treatments of dead conspecifics were present on non-host plants in

proximity to infested Lonicera bushes, male A. cyanescens preferred to perform aerial

approaches to pinned female (N = 39) conspecifics over pinned male (N = 22)

conspecifics (binomial test of proportions, two-sided P = 0.0252, CI: 0.51 - 0.76). Male

A. cyanescens also attempted copulation for a significantly longer duration with those

pinned female conspecifics that were unwashed in solvent compared to the other

treatments (Table 5-2; ANOVA, F = 2.98, d.f. = 3, P = 0.0391). The mean distance from

non-host plants used in these experiments to the nearest Lonicera bush was 2.32 m.

In total, 86 feral male A. cyanescens responded with a copulation attempt

(following an aerial approach) to the heterospecific lures we videotaped in the field. Live

male A. cyanescens made one copulation attempt to pinned P. sericeus, 19 copulation

attempts to pinned whole C. sexguttata tiger beetles, 42 copulation attempts to the pinned tiger beetle elytron, 7 copulation attempts to the pinned tiger beetle head, zero copulation attempts to the pinned Calliphora fly, three copulation attempts to pinned B. ovatus, and

14 copulation attempts to the pinned A. planipennis. We chose C. sexguttata elytra as the heterospecific lures to be used in subsequent field experiments.

When the four treatments of both dead conspecifics and C. sexguttata elytra were pinned to Lonicera leaves, live male A. cyanescens showed a preference for remaining in contact, and attempting copulation with, unwashed female conspecifics and C. sexguttata elytra treated with female A. cyanescens cuticular wash over all the other treatments

(Table 5-3; ANOVA, F = 17.90, d.f. = 7, P < 0.0001). Male A. cyanescens did not prefer

126

127

128 conspecifics (N = 61) over heterospecifics (N = 59) for aerial approaches and subsequent copulation attempts (binomial test of proportions, two-sided P = 0.8558, CI: 0.42 - 0.60).

When the combined conspecific and heterospecific lure setup was observed on non-host plants in proximity to Lonicera bushes, live male A. cyanescens again spent a significantly longer time in contact and attempted copulation with both unwashed and washed female conspecifics as well as C. sexguttata elytra treated with female A. cyanescens body wash versus all other treatments (Table 5-3; ANOVA, F = 7.38, df = 7, P

< 0.0001). Male A. cyanescens did not prefer conspecifics (N = 81) over heterospecifics

(N = 41) for aerial approaches and subsequent copulation attempts (binomial test of

proportions, two-sided P = 0.9122, CI: 0.40 - 0.62). The mean distance from non-host

plants used in these experiments to the nearest Lonicera bush was 2.05 m.

Male A. cyanescens only showed a significant preference for remaining in contact

with C. sexguttata elytra to which female body wash had been applied, compared to the

other three treatments of C. sexguttata elytra (X¯ = 28.067 ± 8.396 sec; ANOVA, F = 7.89,

d.f. = 3, P = 0.0003), when these elytra were placed on non-host plants in proximity to

infested Lonicera. The mean duration, in seconds, of attempted copulation by live male

A. cyanescens to unwashed C. sexguttata elytra (X¯ = 8.667 ± 1.768), dichloromethane

washed elytra (X¯ = 8.667 ± 1.293), and the male body wash application to a washed

elytron (X¯ = 7.222 ± 1.402) was not significantly different (ANOVA, F = 7.89, d.f. = 3, P =

0.0003). The mean distance from the nearest Lonicera bush to the plants used for this

experiment was 2.7 m.

For comparison, we recorded the duration of any copulation observed to occur

between live male and female A. cyanescens present on Lonicera in the field during these

129 experiments. We observed a total of 22 such copulations, which lasted for a mean of

531.682 ± 57.050 seconds. We observed the initiation of 10 of these 22 copulations, and these lasted for a mean duration of 726.100 ± 58.368 seconds; those 12 copulations for which we did not observe the initiation of the behavior lasted for a mean duration of

369.667 ± 61.796 seconds.

DISCUSSION

In both species used in these experiments, our data strongly support the role of

vision in mate-finding. Males of both A. subcinctus and A. cyanescens fly over the host

plant, and execute abrupt dives onto the dorsal surfaces of pinned conspecifics (and non-

conspecific insects in the latter buprestid). Following the aerial approach and dive, a

copulation attempt frequently follows, especially when the pinned conspecific is a female

(Figure 5-2). Males conduct their aerial approaches to other insects as well, especially if

those insects are blue-green in color and iridescent (in other words, similar in color to a

conspecific). If these insects have been treated with a solvent wash from a female

conspecific, male A. cyanescens are more likely to remain in contact with and attempt

copulation with that insect. This leads to our conclusion that males of these two species

initially conduct a visual search for mates during flight near the host plant, and utilize

size and/or contact chemical cues to assess gender information.

Our results regarding the probability of ‘antennation’ and ‘pounce’ behavior suggest that once contact is made on a leaf, an individual live male A. subcinctus is more likely to continue to investigate and attempt to copulate with an affixed dead conspecific

130 if that conspecific is a female beetle that has not been washed in solvent. This lends strong evidence to the hypothesis that males utilize a contact chemical cue to discriminate female from male conspecifics once contact has been established. In addition, when the approach is made from the air, where contact cues are not available, both genders and wash-treatments of beetle are approached equally by A. subcinctus males. This suggests that long-range mate-location on a host plant is visual, and gender discrimination occurs once contact with a conspecific has been made. This mating system is strikingly similar to that of the emerald ash borer, although we observed a much lower rate of aerial approaches directly onto the dead beetle-lure than we had previously observed in the emerald ash borer mating system (Lelito et al. 2007).

Male A. cyanescens show a similar pattern of mating behaviors to those of A. subcinctus, in that they also approach from the air and spend more time investigating female conspecifics once in contact with them. However, in this species, more live males in the field aerially approached pinned female conspecifics than they did dead, pinned male conspecifics. Our results do not necessarily indicate the use of a female-specific odor, however, because our beetle-lures had been washed in solvent and dried, which would likely have removed any odorant cues involved. Additionally, C. sexguttata elytra, emerald ash borers (A. planipennis), and other blue-green colored insects elicited attempted copulations by live male A. cyanescens, suggesting that size and color may be key factors in the elicitation of these aerial approaches by male A. cyanescens. The tendency for male A. cyanescens to approach and land on any like-colored insect also likely explains the coincident capture of A. cyanescens on visual-lure traps using emerald ash borer beetle-lures noted by Lelito et al. (2008).

131 We cannot completely rule out that females of either species also perform

‘paratrooper’ and ‘pounce’ behaviors under circumstances other than those we observed.

However, no female individuals of A. subcinctus or A. cyanescens were ever captured after a descent from flight onto a pinned conspecific during our experiments. In addition, the few female A. subcinctus captured subsequent to a ‘pounce’ behavior may have been simply crawling over the lure; on several occasions up to a dozen individual live beetles could be observed on other leaflets of the same compound leaf. Additionally, during the

A. cyanescens experiments, the density of individual A. cyanescens during these experiments was much lower than we observed during the A. subcinctus experiments.

We did not observe any contacts to occur between a live male A. cyanescens crawling on a Lonicera leaf and one of our pinned lures, save for males that first descended onto a lure from the air, and then subsequently walked off that lure and back onto it again.

Our experiments show that these two species share a strong visual component in their mating systems with A. planipennis, and this raises the possibility that other Agrilus species may also have this trait. However, we agree with the assessment of Rodriguez-

Saona et al. (2007), in that a dual mating strategy may be used whereby at high population density, visual cues dominate and at lower population density, pheromone use is possible. Under very low population-density conditions, it seems unlikely that vision alone would serve to bring the sexes together in an efficient manner, although it is certainly possible that feeding-induced host plant volatiles and then visual search by males may serve this role. Host plant odors are likely to play a role in the location of host ash trees by the emerald ash borer, as evidenced by the significant increase in trap catch of adult emerald ash borers shown by Crook et al. (2008) when ash-bark derived volatiles

132 are added to purple prism traps. In addition, during our A. cyanescens experiments both on and off the host, we recorded a greater number of attempted copulations by males to lures placed on the host (24.3 per hour, on average) compared to lures on non-host plants, which averaged 11.8 male A. cyanescens visits per hour (a significant difference; t-test, t

= 6.154, df = 26, P < 0.001). However, host-plant odors are not necessary prerequisites to mating, because the beetle species we examined here will attempt to mate off the natural host. In the two species we examined, the beetles exist at a high population- density; for A. subcinctus this was due to an overwhelming abundance of dead host material. For A. cyanescens, there may be a relative freedom from natural enemies (A. cyanescens is not a native insect in North America) as well as an abundance of host plant.

We suggest that performing similar experiments on a native North American member of the genus Agrilus, especially one that is not likely to be undergoing a population augmentation, might provide valuable insight into the behavior of this group of insects.

In summary, we have shown here that two species of buprestids in the genus

Agrilus, A. cyanescens and A. subcinctus, appear to first utilize visual cues to locate prospective mates, and then subsequently assess a conspecific using a contact chemical cue, as evidenced by a difference in the duration of attempted copulation with the conspecific insect. In some cases, buprestids can be induced to attempt copulation with non-conspecific insects and still exhibit a difference in the duration of attempted copulation based on the manipulation of the contact chemical cue present. Our results provide the groundwork for future work on visual and contact chemical cues in other buprestid species, many of which remain largely uninvestigated under field conditions.

133 REFERENCES

Akers RC and Nielsen DG (1992) Mating behavior of the bronze birch borer, (Coleoptera: Buprestidae). Journal of Entomological Science 27:44-49

Bartelt RJ, Cossé AA, Zilkowski BW, Fraser, I (2007) Antennally Active Macrolide from Emerald Ash Borer Agrilus planipennis Emitted Predominantly by Females. J. Chem. Ecol. 33:1299-1302

Carlson RW and Knight FB (1969) Biology, taxonomy, and evolution of sympatric Agrilus beetles (Coleoptera: Buprestidae). Contributions to the American Entomological Institute 3:1-105

Crook DJ, Khrimian A, Francese JA, Fraser I, Poland TM, Sawyer AJ, Mastro VC (2008) Development of a host-based semiochemical lure for trapping emerald ash borer Agrilus planipennis (Coleoptera: Buprestidae). Environmental Entomology 37:356-365

Dunn JP and Potter DA (1988) Evidence for sexual attraction by the twolined chestnut borer, Agrilus bilineatus (Weber) (Coleoptera: Buprestidae). Canadian Entomologist 120:1037-1039

Dunn JP, Kimmerer TW, Potter DA (1986) Attraction of the twolined chestnut borer, Agrilus bilineatus (Weber) (Coleoptera: Buprestidae), and associated borers to volatiles of stressed white oak. Canadian Entomologist 118:503-509

Fisher RA (1922) On the interpretation of χ2 from contingency tables, and the calculation of P. Journal of the Royal Statistical Society 85:87-94

Gwynne DT and Rentz DCF (1983) Beetles on the bottle: male buprestids mistake stubbies for females (Coleoptera). Journal of the Australian Entomological Society 23:79-80

Lelito JP, Fraser I, Mastro VC, Tumlinson JH Baker TC (2008) Novel visual-cue- based sticky traps for monitoring of emerald ash borers, Agrilus planipennis (Col., Buprestidae). Journal of Applied Entomology 132:668- 674

134

Rodriguez-Saona CR, Miller JR, Poland TM, Kuhn TM, Otis GW, Turk T, Ward DL (2007) Behaviors of adult Agrilus planipennis (Coleoptera: Buprestidae). Great Lakes Entomologist 40:1-16

SAS Institute (2006) SAS Software Version 9.1.3. SAS Institute, Cary, NC, U.S.A.

CHAPTER 6

A comparison of visual- and chemical-lure sticky traps for monitoring

Agrilus planipennis Fairmaire (Coleoptera: Buprestidae).

Jonathan P. Lelito, Ivich Fraser, Victor C. Mastro, James H. Tumlinson, and Thomas C. Baker

ABSTRACT

We compared the efficacy of chemical and visual lures for monitoring emerald ash borer (EAB) populations alone and in combination using modifications of current methods of trapping for this invasive pest. Placing dead adult EAB as visual lures on both ash leaves and green plastic cards, both coated in Tangle-Trap, significantly increased the capture of adult male EAB over their respective control lacking dead EAB as a lure. Red plastic cards coated in Tangle-Trap, with and without adult EAB added as lures, performed poorly compared to similar traps using EAB on green surfaces. Green, blue, and red dichroic glass lures did not result in the capture of large numbers of adult

EAB when affixed to the green plastic cards. Purple prism traps baited with Manuka oil lures consistently had higher trap-catch of adult EAB than unbaited purple prism traps.

Green prism traps baited with Phoebe oil lures caught significantly more EAB than unbaited green prism traps, and when used at the same site, caught significantly more

EAB than unbaited and Manuka oil baited purple traps. The use of Phoebe oil on a tree

136 significantly increased the trap catch of visually baited sticky traps even when the Phoebe oil was not directly emitted by the trap, suggesting the odor is useful in bringing more adult EAB into the vicinity of a tree.

INTRODUCTION

The emerald ash borer, Agrilus planipennis Fairmaire, is an invasive insect that is an increasingly serious threat to North America’s native ash tree resource (Haack et al.

2002). The emerald ash borer, ‘EAB’, attacks all North American ash tree species in the genus Fraxinus and is likely to cause mortality of nearly all ash trees in areas into which the beetle spreads (Poland and McCullough 2006). Early detection is critical to controlling the spread of EAB, but efforts thus far to come up with an easily deployable, inexpensive, and effective means of detecting EAB have been stymied by the lack of an identified sex pheromone or other species-specific lure (Poland and McCullough 2006;

Cappaert et al. 2005) and also by the difficulty of placing traps high in the tree, which has been shown to increase effectiveness (Lance et al. 2007).

Originally, research into improving the effectiveness of sticky traps for EAB largely focused on the color of the trap (Francese, et al. 2005). Recent advances in two important areas have shown promise for increasing the efficacy of available trapping tools. First, the mating system of EAB has been shown to be visually-based (Lelito et al.

2007; Rodriguez-Saona et al. 2007) and the visual stimulus of a conspecific has been used to capture beetles in the field, even at only 4 m height within a tree (Lelito et al.

2007, 2008). Second, the addition of Manuka and Phoebe oil to plastic purple prism traps has been conclusively shown to increase the number of EAB caught on the trap (Crook et

137 al. 2008). These oils contain host-plant volatiles, including sesquiterpenes that have been shown to be antennally active in adult EAB. Certain components of these essential oils include volatiles that had previously been shown to be antennally active and thus potentially attractive to adult EAB (Rodriguez-Saona et al. 2006; Poland et al. 2005,

2006; Crook et al. 2005, 2006, 2007).

Our goal was to test many of the methods previously shown to be effective at trapping EAB and to compare these with visual, chemical, and a combination of visual- and chemical-lure-based traps in an effort to provide an improved trap for use in monitoring programs for this pest. We also tested new types of visual lure-based traps incorporating synthetic visual lures and novel adhesives. The goal here was to create a longer-lasting, visually baited EAB trap based on our earlier studies showing that male

EAB locate females on ash leaves by visual means (Lelito et al. 2007), and that males can be lured to and captured on sticky leaves by pinned dead EAB models (Lelito et al.

2008).

METHODS

Visual Cue Test

We compared several methods of using dead adult EAB as a visual lure by

pinning one of the following to a terminal leaflet of an ash leaf and then covering the

visual lure and the leaf with spray-on Tangle-Trap (The Tanglefoot Company, Grand

Rapids, Michigan): 1) a dead adult EAB; 2) a dead EAB with both elytra removed to

expose the red dorsal abdominal surface; 3) two EAB elytra pinned in contact with each

other to create a lure slightly reduced in length but similar in width to a whole EAB; 4) a

single EAB elytron; and 5) two EAB elytra pinned one behind the other to create a visual

138 stimulus one-half the width of an EAB but twice the length. A dead EAB pinned to a leaflet and covered with Tangle-Trap has been called a sticky leaf trap, or SLT (Lelito et al. 2008). A terminal ash leaflet sprayed with Tangle-Trap but not containing a visual lure served as an SLT control. A complete set of each of these SLTs was placed on terminal leaflets of ash leaves on the south side of an ash tree at 4 m height. The SLTs were removed and replaced with new ones twice during this experiment, at 8-day intervals, because the leaflets tend to decay over time (Lelito et al. 2008). When replacing an SLT, the next nearest ash leaf from the one removed was chosen to be used for the new SLT. We replicated this experiment on four ash trees that were between 20 and 25 m apart, each tree having visible EAB emergence holes but still having live foliage on at least half the tree. Also in each tree, one unbaited purple prism trap and one unbaited green prism trap were hung from a rope at 4 m height, separated by at least 2 m from one another. Individual SLTs were also at least 2 m from the next nearest trap (SLT or prism trap). The prism traps were scraped to remove the Tangle-Trap and any captured insects, and then were re-glued at the same time as the SLTs were replaced. The

SLTs and prism traps used for this experiment were placed in trees on 14 June 2008, and subsequently redeployed on 20 June 2008 and 28 June 2008. We checked all traps for the presence of adult EAB and other insects twice a week. Adult EAB found on the traps were removed, sexed, counted, and discarded. Other insects similar in size to EAB were collected from the traps and identified to order level. Any buprestids, regardless of size, were also recorded and keyed to genus (species where possible) level. This experiment was performed at two EAB-infested sites near the town of Williamston in Ingham

County, Michigan.

139 540 nm Sticky Cards

To test whether the visual stimulus of a conspecific is attractive when placed on a synthetic surface designed to mimic the color of ash foliage, we used plastic, 10 X 10 cm sticky cards having peak reflectance at 540 nm, similar to ash foliage, obtained from

ChemTica Internacional S.A., Costa Rica. We prepared a total of 64 sticky cards which were painted with a “dry” adhesive, also obtained from ChemTica. After melting the adhesive at 100°C and applying it to the cards with a paint scraper, the sticky substance cools to result in a surface tackier to the touch than Tangle-Trap. Thirty-two experimental sticky card traps had a single, whole adult female EAB added as a visual lure. We prepared an equal number of control traps, which were painted with the glue but did not have an EAB added to the trap. Both sets of traps were wrapped in wax paper and stored until use.

We chose four ash trees in which to place our traps that were between 20 and 25 m from one another and that had visible EAB exit holes but with foliage remaining. We placed the following four trap treatments into each tree at 2 and 4 m height: 1) one EAB- baited SLT; 2) one control SLT; 3) one sticky card with an EAB affixed, and 4) one sticky card without an EAB affixed. Sticky card traps were placed over several ash leaflets using metal twist ties run through holes punched in each corner of the card. Once the sticky-card was tied to leaves, we sprayed the dead EAB affixed to the center of each trap with spray-on Tangle-Trap to ensure the insect was covered in adhesive. In each tree, we also hung one unbaited purple prism trap from a rope over a branch at 4 m height.

140 We placed all traps associated with this experiment into trees on 6 and 7 June

2008, and subsequently replaced all traps on 20 June 2008. We checked all traps for the presence of adult EAB and similar-sized insects twice weekly, recording the number and gender of adult EAB captured and keying the other insects to order level. We checked all traps and removed them from the ash trees on 7 and 8 July 2008. This experiment was performed at two EAB-infested sites, one near the town of Pinckney in Washtenaw

County, Michigan, and the other near the town of Williamston in Ingham County,

Michigan.

Red Sticky Cards

The setup of traps on trees and the choice of trees for this experiment was identical to that outlined for the green sticky cards outlined above, with the exception that these sticky cards were deep reddish-green in color, similar to that of newly flushing ash foliage. We purchased the red-colored plastic stock from The Sign Stop (State College,

Pennsylvania) and cut individual cards that were 10 X 10 cm. The red cards were glued to the surface of an identically sized green sticky card to provide support for the thinner material from which the red plastic was constructed. We then painted the red surfaces with the same high-temperature glue as we used for the green sticky cards. Dead EAB were affixed or not (controls) to the surface as in the previous experiment. All traps were placed on 14 June 2008 and then removed on 8 July 2008. Traps were checked twice weekly, and all EAB were removed, sexed, counted, and discarded. This experiment was performed at one EAB-infested site west of the town of Pinckney, Washtenaw County,

Michigan, and another south of the town of Williamston, Ingham County, Michigan.

Dichroic Glass Lures

141 For this experiment, we employed the green sticky cards used above, but instead of an adult EAB as the visual lure, we used glass lures of several colors and shaped similar to EAB adults to see if EAB males could be attracted by a synthetic visual stimulus. The glass lures we used were manufactured by Essential Glass Works (Boise,

Idaho). We used one each of the following six types of glass lures for this experiment: a

3.0 cm long oval, reflective light blue in color; a 1.5 cm long oval, reflective light blue in color; a 3.0 cm long red oval; a 1.5 cm long red oval; a 3.0 cm long green and gold oval, and a 1.5 cm long green and gold oval. All of these lures were 0.5 cm wide.

We similarly chose four ash trees with live foliage but also having visible EAB emergence holes on the trunk for each of two sites at which we performed this experiment. Into each ash tree at 4 m height, we placed the following traps: an unbaited purple prism trap, one of each of the above glass lures affixed to its own green sticky card, an EAB-baited SLT, and a control SLT (with no beetle affixed to it). We placed all traps into ash trees on 6 and 7 June 2008, and checked them twice weekly through 6 July

2008. Traps were redeployed on these dates as per the above experiments, and captured

EAB were removed, sexed, counted, and discarded. We performed this experiment at two EAB-infested sites in Ingham County, Michigan.

Manuka Oil Lures

Manuka oil dispensers were provided as pre-made plastic packets, by ChemTica

Internacional in Costa Rica. The dispensers had been measured to release 25 mg/day for

45 days. We chose six ash trees for this experiment, each having visible EAB exit holes while still retaining some healthy foliage (i.e. an infested but live tree). For this experiment, and the next involving Phoebe Oil lures, we set up two each of three

142 ‘classes’ of experimental trees at each location used for our experiments. The three classes of trees were set up in such a way that we could test multiple hypotheses concerning both visual and chemical luring of EAB, as well as test the effect of the presence vs. absence of the lures themselves on overall attraction of EAB to host trees by site, and on a tree-by-tree basis, to gain better resolution of EAB behaviors.

We set up two trees as ‘Type 1’, and these contained the following six types of traps: 1) a purple prism trap with a Manuka oil lure hung from the spreader in the center of the trap; 2) an unbaited purple prism trap; 3) an EAB-baited SLT with a Manuka oil lure hung directly below it from the petiole of the leaf; 4) a terminal ash leaflet sprayed with Tangle-Trap and having a Manuka oil lure hung directly below it from the petiole;

5) an EAB-baited SLT lacking Manuka oil, and 6) an unbaited leaflet sprayed with

Tangle-Trap. Two ‘Type 2’ trees contained two unbaited purple prism traps, two EAB- baited SLTs, and two SLTs without an EAB lure, and these trees served as a control for

EAB attraction at each site, in the absence of chemical lures. Two ‘Type 3’ trees contained: 1) a Manuka baited purple prism trap; 2) an unbaited purple prism trap; 3) two EAB-baited SLTs; 4) two SLTs without an EAB lure, 5) and two Manuka oil lures, each hung from the petiole of a separate, individual ash leaf at least 1 m from the nearest trap. Type 3 trees

All traps were hung at 4 m height in the tree, and each tree that contained traps was between 20 and 25 m from the nearest trap-containing tree. All volatile lure packets were rotated randomly between traps at the end of the first trap-check of each week. The two sites used for these experiments were EAB-infested forest/agricultural edge habitat, one in Ingham and one in Washtenaw County in southeastern Michigan. All traps were

143 placed into ash trees on 9 and 10 June 2008 and were checked for EAB as above twice weekly through 6 July 2008. We removed all traps from the trees after the final check between 10 and 14 July 2008.

Phoebe Oil Lures

Phoebe oil dispensers were obtained as manufactured plastic packets that released

25 mg/day from ChemTica Internacional. The setup for this experiment was identical to that used in the Manuka oil lure experiment outlined above, with the exception that the volatile lure packets associated with the appropriate traps contained the Phoebe oil lure, and the prism traps that were used in this experiment were green rather than purple. We used two EAB-infested sites: a roadside forested area in Ingham Township and an agricultural forest-edge habitat near the town of Williamston, in Ingham County,

Michigan. All traps were placed into ash trees on 9 and 10 June 2008 and were checked twice weekly through 7 July 2008. We removed all traps from ash trees between 10 and

14 July 2008.

Statistical Analyses

We performed all statistical analyses using ANOVA in the PROC GLM procedure in SAS Software Version 9.1.3 (SAS Institute, 2006), including Tukey’s LSD test for multiple comparisons. We based our comparison of each trap type on the surface- area adjusted quantity of beetles/m2/day, calculated by averaging the number of adult

EAB of each gender captured per m2 of trap surface over the number of days elapsed

since the previous trap check. We performed our analysis on total beetles captured, as

well as separately analyzing the number of male and female EAB captured to examine

potential gender-specific responses to our lures. For each analysis, we used

144 beetles/m2/day as the dependent variable, and site, individual tree, and trap type as

factors. For the Manuka and Phoebe oil experiments, we also used the tree setup as a

factor. Values of beetles/m2/day for a given experiment were log-transformed

(log10(x+1)) prior to analysis.

RESULTS

Visual Cue Test

Overall, the type of trap used was the only significant factor in determining trap

capture for the combined capture of both genders of EAB (Table 6-1; ANOVA, df=11,

F=28.51, P<0.0001).

Trap type was a significant factor influencing the capture of male EAB, with

EAB-baited SLTs capturing more male beetles/m2/day when compared to all other trap

types tested (Table 6-1; ANOVA, df=11, F=37.32, P<0.0001). More male EAB (70 in

total) were captured on EAB-baited SLTs than on any other type of SLT, although SLTs

with two elytra side by side captured a total of 33 male EAB during the course of this

experiment. Other SLTs captured far fewer total male EAB: 11 on SLTs with red

abdomen lures, 8 on SLTs baited with one EAB elytron, 2 on SLTs with two EAB elytra

arranged end-to-end, and 1 male EAB in total on unbaited SLTs.

Adult female EAB were uninfluenced by site, individual tree, or trap type during

this experiment (Table 6-1; ANOVA, df=11, F=0.75, P=0.6853).

540 nm Sticky Cards

145

146 Height and trap type were significant factors influencing the combined capture of both genders of EAB during this experiment, with the 4 m trap height being preferred over 2 m height and EAB-SLTs capturing the most adult beetles (Table 6-2; ANOVA, df=12, F=38.42, P<0.0001).

Similarly, height of the trap and trap type were significant factors influencing the capture of adult male EAB (Table 6-2; ANOVA, df=12, F=54.93, P<0.0001).

Significantly more (78) males were captured on EAB-baited SLTs at 4 m height than any other trap type on the basis of surface area, although in terms of total male EAB captured, purple traps, which captured 77 male EAB, are equivalent to EAB-baited SLTs. Purple traps captured 166 EAB in total during this experiment. Green sticky cards at 4 m height captured a total of 41 male EAB, while green sticky cards at 2 m height captured only 4 male EAB. EAB-baited SLTs at 2 m height captured 22 male EAB.

In contrast to male EAB, for female EAB experiment site and trap type were the significant factors influencing trap capture, with females preferring green sticky cards without an EAB affixed (total capture of females being 11) over the other trap types on the basis of surface area at both sites (Table 6-2; ANOVA, df=12, F=2.15, P=0.0130).

Purple prism traps captured the most female EAB in total, with 89 female EAB being captured on this type of trap.

Red Sticky Cards

In overall capture of both genders of EAB, the height of the trap and the trap type itself were significant factors contributing to trap capture on red sticky cards (Table 6-3;

ANOVA, df=12, F=24.98, P<0.0001). In this case EAB-SLTs, not red sticky cards, at 4 m height were preferred overall. Red sticky cards at 4 m height and EAB-SLTs at 2 m

147

148

149 height captured equivalent numbers of total EAB at a rate significantly less than EAB-

SLTs at 4 m height, and significantly greater than the trap capture of the remaining trap types.

Adult male EAB showed the same pattern of preference as the total of both genders of EAB (Table 6-3; ANOVA, df=12, F=45.41, P<0.0001). Significantly more total male EAB were captured, on the basis of surface area, on EAB-baited SLTs at 4 m height (67), versus red sticky cards at 4 m height (17 male EAB). Purple traps captured

52 male EAB in total.

Capture of adult female EAB was only influenced significantly by trap type, with females preferring blank red sticky cards over all other treatments on the basis of surface area (Table 6-3; ANOVA, df=12, F=3.80, P<0.0001). Purple traps captured the most female EAB in total (99) during the course of this experiment.

Dichroic Glass Lures

The sum total of the capture of both genders of adult EAB was only significantly influenced by trap type in this experiment, with EAB-baited SLTs significantly preferred over the other trap types on the basis of surface area (Table 6-4; ANOVA, df=12,

F=17.54, P<0.0001). Purple prism traps captured the most total adult EAB numerically

(115), with EAB-baited SLTs capturing a total of 40 adult EAB during the course of this experiment.

Adult male EAB preferred EAB-SLTs over other trap types on the basis of surface area (Table 6-4; ANOVA, df=12, F=20.43, P<0.0001); these traps captured a total of 35 male EAB in total. Large and small green dichroic glass lures affixed to green sticky cards captured a total of 8 and 7, respectively, adult male EAB, with the other

150

151 dichroic glass lures capturing very few (2 or fewer) adult male EAB in total. Purple prism traps captured a total of 73 male EAB during the course of this experiment.

Surprisingly, adult female EAB also significantly preferred EAB-SLTs over the other trap types on the basis of surface area (Table 6-4; ANOVA, df=12, F=1.96,

P=0.0271). Dichroic glass lures on green sticky cards captured three or fewer female

EAB in total during the course of this experiment, while prism traps captured a total of 42 female EAB.

Manuka Oil Lures

Overall, adult EAB of both genders responded at a statistically equivalent rate to

EAB-baited SLTs with and without Manuka Oil lures, and control SLTs with a Manuka

Oil lure (Table 6-5; ANOVA, df=11, F=7.57, P<0.0001). Site was a significant factor to capture of EAB as well; we analyzed total trap capture separately according to each site and found the same relationships between trap types at both sites.

When we examined the capture of only male EAB, we captured significantly greater numbers of male EAB on EAB-baited SLTs with Manuka Oil lures compared to all other trap types on the basis of trap surface area (Table 6-5; ANOVA, df=11, F=,

P<0.0001). A total number of 128 male EAB were captured on purple prism traps with

Manuka Oil lures, whereas unbaited purple prism traps and EAB-baited SLTs captured

106 and 105, respectively, adult male EAB.

Female EAB were captured in significantly higher numbers on control SLTs

(those not baited with an adult EAB) which contained a Manuka Oil lure than the other trap types on the basis of surface area (Table 6-5; ANOVA, df=11, F=2.86, P=0.0012).

Unlike the positive response of male EAB to the EAB-SLTs, females were captured at

152

153 significantly lower rates on SLTs containing the EAB model plus Manuka oil than to

SLTs plus Manuka oil that lacked this visual lure (Table 6-5). Numerically, the greatest capture of female EAB occurred on purple prism traps baited with Manuka Oil (158) and unbaited purple prism traps (170).

Phoebe Oil Lures

In the Phoebe Oil experiment, overall capture of adult EAB, adjusted for surface area, was significantly influenced by both trap type and tree type (Table 6-6; ANOVA, df=12, F=33.12, P<0.0001). Total adult capture was greater on trees containing Phoebe

Oil lures versus those that had no such lures (Table 6-7; ANOVA, df=2, F=15.70,

P<0.0001); adult EAB also were captured in greater numbers on trap types that had

Phoebe Oil lures in direct association with them, when compared to their respective controls not containing Phoebe Oil lures (Table 6-6; ANOVA, df=5, F=66.38, P<0.0001).

Male EAB were likewise influenced both by tree type and trap type, and were captured in greater numbers on the same tree and trap types as both genders combined

(Tables 6-6 and 6-7; ANOVA, df=11, F=42.69, P<0.0001).

The capture of EAB females was also significantly influenced by tree type and trap type (Tables 6-6 and 6-7; ANOVA, df=11, F=11.64, P<0.0001). Greater numbers of females were captured on Type 1 trees and on control SLTs with Phoebe Oil lures present, than on Type 2 and 3 trees and control SLTs without Phoebe Oil lures, respectively. Similar to the female response to Manuka oil above, female EAB were significantly less attracted to SLTs with Phoebe Oil lures containing an EAB visual lure, than to SLTs with a Phoebe Oil lure that did not contain the dead EAB (Table 6-6).

154

155

156

DISCUSSION

Our data show that in areas of high EAB population density a dead adult EAB pinned to a terminal ash leaflet that is covered in adhesive captures greater numbers of male EAB on a surface-area basis than does a plastic prism trap. In any given experiment, no chemical lure or synthetic plastic trap by itself outperformed this simple trapping strategy. Capture levels were further increased for this EAB-SLT trap when ash-based volatile lures were added to the trap. However, we think it unlikely that making an EAB-SLT as large as a green prism trap would retain its trapping efficacy on the basis of surface area. The advantage of an EAB-SLT is that it exploits male pre- mating behavior in a reliable way; taking this stimulus out of the appropriate context for the insect has the potential to result in a decrease in attractiveness to patrolling males.

We interpret our results to suggest that current trapping technology could be further improved by more closely mimicking the color or other qualities of an ash leaf, and perhaps incorporating some form of visual lure to attract male EAB to allow a synthetic trap with improved attractiveness to be deployed. Our present data and the results of others (Crook et al. 2008) support the conclusion that the use of Phoebe Oil or an improved synthetic blend of host volatiles is likely to increase capture of adult EAB on a trap as well.

While EAB-SLTs are successful at capturing adult EAB, these traps are small and must be positioned carefully to allow a human observer to locate and check the trap quickly. Thus they are perhaps more suited for research purposes than for wide-scale use as a monitoring device. Nonetheless, we have effectively exploited the visually-based

157 mating system of EAB to trap this insect, as shown by the capture of a highly male- biased sample of adult EAB on EAB-SLTs. Engineering efforts that will increase trap longevity or ease of maintenance and monitoring may yet yield a useful and effective trap for adult EAB that is based on the SLT design. There is some evidence that effectiveness of a given trap type is related to the population density of EAB (Metzger et al. 2007), and because we tested traps only at high population density, we cannot speculate on how these traps may perform in other areas.

In general, our results support our previous conclusions regarding the use of

EAB-SLTs for monitoring of adult EAB (Lelito et al. 2008), with a few notable differences. In our present experiments, we captured far more adult EAB on control

SLTs lacking the dead EAB visual lure than previously. Only in the red sticky-card experiment did control SLTs capture no adult male EAB during the duration of the experiment, and some adult female EAB were captured on control SLTs in each experiment. The variation in response to both EAB-SLTs and control SLTs by adult

EAB between experiments may be due to site characteristics. These would include death of host trees during the field season, local population density variations, changes in operational sex ratio, localized severe weather (e.g. hail that strips leaves from trees and damages SLTs) or other factors such as an every-other-year EAB emergence peak in the initial phases of EAB infestation (Siegert et al. 2007).

Our results also support the hypothesis that emerald ash borers locate host trees using stress-related volatile cues and agree with similar studies that show that the use of stress-related volatile lures increase adult EAB trap capture (Crook et al. 2008; Poland and McCullough, 2007). Our results showed that the addition of Phoebe oil to a trap

158 itself or to the tree in general (as in Type 3 trees) increased the total capture of adult EAB on traps in the tree compared to trees that contained no volatile emitters. EAB adults thus appear to be drawn not only to the trap itself but also to the tree as a whole. Male and female EAB appear to respond to our manipulation of tree-based olfactory cues in a subtly different way. More male EAB were captured in both Type 1 and Type 3 trees, whereas more female EAB were captured in Type 1 trees than in Type 2 trees. This suggests that male EAB may be generally attracted to a damaged tree that is likely to harbor conspecifics, whereas female EAB may be attracted to more localized odor sources that indicate a damaged area of the tree favorable for oviposition.

This suggestion is supported by our results with SLTs that showed that female

EAB were captured in significantly greater numbers on blank SLTs and sticky cards in the presence of either Manuka or Phoebe Oil ash tree volatiles, than they were on EAB-

SLTs and sticky cards containing the visual cue of a dead EAB. It can be inferred from these results that both the Manuka and the Phoebe Oil volatiles attracted females to the leaflets, but the presence of an EAB visual cue on the leaflet deterred them from landing.

In contrast, male captures on blank SLTs were not increased by the presence of either

Manuka or Phoebe Oil volatiles.

We can conclude from this series of experiments that male EAB can be trapped effectively using a dead EAB on an ash leaflet as a visual lure, highlighting again the role of vision in male EAB behavior (Lelito et al. 2007). Dichroic glass lures of the types we used here were not optimal stimuli for EAB males, although this should not rule out the use of synthetic EAB-mimic objects for trapping in the future. This result serves to highlight again that the size and shape of the visual lure is clearly of importance. Males

159 are more likely to be trapped by an SLT containing a whole dead EAB or two elytra placed horizontally together than by other arrangements, such as the exposure of the red dorsal surface of the EAB abdomen or two elytra positioned lengthwise along the midvein of the leaf. The color of the trap itself appears to play a role, with male EAB being trapped in greater numbers on green, rather than red, colored sticky cards to which dead EAB have been affixed. Thus, the background context against which a motionless

EAB appears is important in influencing male EAB attraction. Thus, the combination of a live EAB and its ‘perch’ seems to play a role in the visual signal that patrolling males perceive during an aerial approach and landing.

REFERENCES

Cappaert D, McCullough DG, Poland TM, Siegert NW (2005) Emerald ash borer in North America: A research and regulatory challenge. Am. Entomol. 51:152-165

Crook DJ, Khrimian A, Francese JA, Fraser I, Poland TM, Sawyer AJ, and Mastro VC (2008) Development of a host-based semiochemical lure for trapping emerald ash borer Agrilus planipennis (Coleoptera: Buprestidae). Environ. Entomol. 37:356-365

Crook DJ, Francese JA, Fraser I, Mastro VC (2005) Chemical ecology studies on the emerald ash borer. In: Mastro VC, Reardon R, (eds.), Emerald ash borer research and technology development meeting, FHTET 2004-15, USDA Forest Service, Morgantown, WV, p. 55

Crook DJ, Khrimian A, Francese JA, Fraser I, Poland TM, Mastro VC (2007) Chemical ecology of emerald ash borer. In: Mastro VC, Lance D,

160 Reardon R, Parra G, (comps.), Emerald Ash Borer and Asian Longhorned Beetle Research and Technology Development Meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, p. 79

Francese JA, Mastro VC, Oliver JB, Lance DR, Youssef N, Lavallee SG (2005) Evaluation of colors for trapping Agrilus planipennis (Coleoptera: Buprestidae). J. Entomol. Sci. 40:93-95

Haack RA, Jendek E, Liu H, Marchant KR, Petrice TR, Poland TM, Ye H (2002) The Emerald ash borer: a new exotic pest in North America. Newsletter of the Michigan Entomological Society 47:1-5.

Lelito JP, Fraser I, Mastro VC, Tumlinson JH, and Baker TC (2008) Novel visual-cue-based sticky traps for monitoring of emerald ash borers, Agrilus planipennis (Col., Buprestidae). J. Appl. Ent. 132:668-674

Lelito JP, Fraser I, Mastro VC, Tumlinson JH, Böröczky K, and Baker TC (2007) Visually mediated ‘paratrooper copulations’ in the mating behavior of Agrilus planipennis (Coleoptera: Buprestidae), a highly destructive invasive pest of North American ash trees. J. Insect Behav. 20:537-552

Metzger JA, Fraser I, Storer AJ, Crook DJ, Francese JA, Mastro VC (2007) A multistate comparison of emerald ash borer (Agrilus planipennis Fairmaire) (Coleoptera: Buprestidae) detection tools. In: Mastro VC, Lance D, Reardon R, Parra G, (comps.), Emerald Ash Borer and Asia Longhorned Beetle Research and Technology Development Meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, pp. 73-74

Poland TM and McCullough DG (2006) Emerald ash borer: invasion of the urban forest and the threat to North America’s ash resource. J. Forest. 104:118- 124

Poland TM and McCullough DG (2007) Evaluation of a multicomponent trap for emerald ash borer incorporating color, silhouette, height, texture, and ash leaf and bark volatiles. In: Mastro VC, Lance D, Reardon R, Parra G, (comps.), Emerald Ash Borer and Asian Longhorned Beetle Research and Technology Development Meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, pp. 74-76

161

Rodriguez-Saona CR, Miller JR, Poland TM, Kuhn TM, Otis GW, Turk T, and Ward DL (2007) Behaviors of adult Agrilus planipennis (Coleoptera: Buprestidae). Great Lakes Ent. 40:1-16

SAS Institute. 2006. SAS Software Version 9.1.3. SAS Institute, Cary, NC, USA

Siegert NW, McCullough DG, Tluczek, AR (2007) Two years under the bark: towards understanding multiple-year development of emerald ash borer larvae. In: Mastro VC, Lance D, Reardon R, Parra G, (comps.), Emerald Ash Borer and Asian Longhorned Beetle Research and Technology Development Meeting, FHTET 2007-04, USDA Forest Service, Morgantown, WV, p. 20

CHAPTER 7

SUMMARY AND CONCLUSIONS

The previous chapters detail the results of my experiments regarding mate-finding in the Buprestidae, especially EAB. I have shown that male EAB actively use visual cues to locate potential conspecifics on the host tree. Rapid aerial approaches and dives followed by copulation attempts are performed in response to the presence of individuals of either sex of EAB on ash leaflets. However, females are far more likely to elicit a longer duration of attention by males, due to the presence of a cuticular lipid contact cue.

I have contributed to an investigation of the cuticular lipids that has resulted in the identification of a methylalkane, 3-methyltricosane, that is the first contact-sex- pheromone component to be identified in the Buprestidae. It is a behaviorally active and female-specific compound that elicits longer durations of investigation and attempted copulation by male EAB than do solvent-only controls in both laboratory and field bioassays. I have also shown that it is possible to trap adult male EAB using a dead conspecific as a lure on a trap, and that such visual traps can be enhanced by incorporating host plant odors derived from tree oils. Visual trapping may hold promise for monitoring EAB, perhaps even in low-density populations where current trapping systems are less effective.

Visually mediated mate-finding behavior

My first goal in studying adults of the emerald ash borer was to determine if the beetles performed any behaviors that could indicate the method by which they located prospective mates. I observed EAB in the field throughout the day and recorded diel

163 patterns of activity, typical position on the tree, and other behaviors. I also paired individuals of known age in the laboratory in small plastic arenas to more carefully observe contact between individual EAB previously denied access to the opposite sex.

In the laboratory, male EAB may attempt to initiate mating behavior as early as five days of age post-eclosion, whereas females do not allow mating to actually proceed until they are at least 10 days of age post-eclosion. The number of successful matings achieved in my laboratory bioassay was very low. Although many behaviors were performed by both sexes and occurred in many pairings, such as wing fanning, flight, and

‘juddering’ (a rapid vibration of the body against the substrate), no behavior significantly correlated with a successful mating. Bright light increased the number of instances of wing-fanning and other behaviors, but did not increase the number of successful matings achieved.

In the field, EAB are most active during sunny, warm weather, especially in the morning. Males are more likely to be found flying, whereas females are more likely to be found feeding on foliage or searching tree trunks for oviposition sites. Males fly in search of mates and rapidly dive onto the backs of females and quickly attempt to mate.

They will approach and perform aerial dives, or ‘paratrooper copulations’, in response to dead beetles of either sex, suggesting the primacy of visual information in long range mate location in EAB. After contact, males spend significantly more time investigating and attempting to mate with dead, non-solvent washed females than with solvent washed beetles of either sex. Males terminate contact with dead, non-solvent washed males more quickly than with other models. All evidence thus points to the use of female- and male- specific contact cues for sex discrimination in this species.

164 I also performed my experiments with dead conspecifics as visual lures with two other species in the genus Agrilus, A. cyanescens and A. subcinctus, and in each case I showed that males in the field are attracted to the visual stimulus of a conspecific on a leaf. In addition, males of A. cyanescens, a medium-sized buprestid that is blue-green in color, are attracted to the elytra of green tiger beetles (Cicindela sexguttata) and will attempt copulation with them just as they would with a conspecific female. These experiments provided me with further evidence that visual signals play a dominant role in the mating system of Agrilus beetles, and suggest that the behaviors seen in EAB are unlikely to be restricted to that species.

Based on my field and laboratory experiments, I have concluded that adult EAB require a maturation period and begin sexual activity after approximately ten days of age in the laboratory. In the field, they are most active in sunlight and on warm, calm days.

Females are more likely to be found sitting on foliage, whereas males are more likely to fly between different areas on the host tree. Males perform aerial dives upon visual detection of a perched conspecific of either sex, and subsequently appear to assess the contacted conspecific based on a contact chemical cue. Visual cues appear to be the primary stimuli used by male EAB for locating females for mating.

Contact-cue mediated sex discrimination

Subsequent to my investigation of the visually-mediated initial stages of mating behavior in Agrilus beetles, I pursued the role of contact chemical cues in the mating system of these insects. My first goal was to identify what, if any, sex-specific compounds might be present on the cuticle of adult insects, with the idea that any such

165 compounds would be the most likely discriminatory cue used by adults to assess gender.

It had become apparent during my field work on mate-finding with EAB that solvent- washing the dead beetles used as lures caused feral male EAB to spend significantly less time investigating these washed lures compared to unwashed lures. Therefore, I collected solvent washes from freshly emerged as well as from older, mature male and female EAB and compared these samples through gas chromatography. With the help of my collaborators, one particular compound, 3-methyltricosane, was identified that was present as a trace compound in the cuticular lipids of males and freshly emerged females, but that was more abundant in the washes taken from mature female EAB. This change in cuticular chemistry occurred at the same time as the onset of female sexual maturity, as

I had previously assessed in adult EAB in the laboratory.

I performed field trials and laboratory bioassays to determine the behavioral responses of EAB males and females to 3-methyltricosane. The application of 3- methyltricosane to solvent-washed dead beetles in the field caused a significant increase in the time feral males spent in attempted copulation with these models, as compared to beetle-lures treated with other solvent or with other compounds not present on the cuticle of female EAB. However, these results clearly indicated that more EAB-derived compounds are involved in the contact pheromone blend. A three-beetle-equivalent dose of 3-methyltricosane was still investigated by male EAB for a significantly shorter duration than an unwashed female beetle. This is a promising area for future research on

EAB and other buprestids.

Field experiments on Agrilus subcinctus and Agrilus cyanescens (reported in

Chapter 5) also suggest a role for cuticular lipids in the mating systems of these two

166 beetle species as well. In experiments with A. subcinctus, washed beetle-lures of both sexes were contacted by male A. subcinctus in the field for a significantly shorter duration than unwashed female beetle-lures. The same was found for A. cyanescens during field experiments, strongly suggesting that like EAB, these two species utilize a contact cue to assess contacted conspecifics. In further field experiments on A. cyanescens, I showed that the re-application of a solvent wash prepared from mature female conspecifics caused a longer duration of investigation by male A. cyanescens than solvent controls in the field when applied to the elytra of a tiger beetle (which has a similar color to A. cyanescens). That males can be chemically manipulated in this way strongly supports my hypothesis that male Agrilus beetles use contact chemical information to assess the sex of a contacted conspecific insect. In addition, my experiments with visual models, especially those involving heterospecific insects as functional lures for copulation attempts by male A. cyanescens in the field, argue for the primary use of vision in long-distance location of such conspecifics.

Based upon the results of my experiments with cuticular lipids, I can conclude that these three species in the genus Agrilus use a contact pheromone cue, specifically some key component(s) of the cuticular lipid profile, to identify the sex of a contacted conspecific. In the case of EAB, the mature-female-specific cuticular methylalkane 3- methyltricosane is one of these pheromone components, and it has significant behavioral effects on male EAB in the field and laboratory.

Trapping buprestids using visual lures

167 Using the ‘paratrooper copulation’ behavior to capture EAB adults in the field became the focus of field work for two seasons of my research, especially after it became apparent from my previous work that EAB did not utilize a long-range sex pheromone in mate-finding. The first and simplest design for an EAB trap incorporated a dead EAB adult pinned to the terminal leaflet of an ash leaf. This whole leaflet was then covered in a spray-on formulation of Tangle-Trap adhesive.

Trap capture on these EAB-SLTs (EAB Sticky-Leaflet-Traps) was typically highly male-biased, and declined as the trap aged and the adhesive dried and/or became covered in debris. EAB-SLTs captured adult male EAB at both high and low EAB population densities, but were more effective at the higher population density. EAB-

SLTs also captured more male EAB when they were placed higher in the tree. EAB-

SLTs incorporating only one EAB elytron, two EAB elytra arranged end-to-end, or else just the red dorsal surface of the abdomen were not as effective at capturing adult EAB as

EAB-SLTs that used one entire EAB adult or two EAB elytra side-by-side. These results suggest that shape, size and color are the primary cues associated with visual recognition of a conspecific by male EAB.

Thus, I have shown that male EAB can be lured with a visual stimulus and trapped in this way for monitoring purposes. It remains a distinct possibility that with improved trap design and longevity, newly developing EAB infestations and extremely low density populations could be detected using these methods. On the basis of trap surface area, EAB-SLTs are a more efficient method of capturing adult EAB than purple prism traps, which is the current USDA APHIS standard method of monitoring EAB populations.

168

Chemically mediated host-finding behavior

Although buprestids may use visual cues to locate conspecifics for mating purposes, volatile chemicals do appear to play a significant role in the location of the host plant. The higher number of copulation attempts that I recorded on host plants (versus on nearby non-host plants) during my field experiments with A. cyanescens and A. subcinctus suggest that buprestids aggregate around the host.

In order to examine the influence of host-produced volatiles on EAB attraction, I used Manuka and Phoebe Oil emitters on several trap types to compare these ‘enhanced’ traps against standard traps without lures. Manuka and Phoebe Oils are tree distillates that contain a variety of induced plant compounds and are especially rich in sesquiterpenes commonly produced by ash when under attack by EAB. I showed that traps with dead EAB adults as visual lures (e.g. EAB-SLTs) and those without visual lures (e.g. a standard purple prism trap) will capture more adult EAB when either type of emitter is placed on the trap than will the same trap without an emitter. In addition, the presence of Phoebe Oil emitters in a tree significantly increased the total adult capture of

EAB in the tree in which emitters were placed (even if not associated directly with a trap) compared to a tree that had no emitters but that contained the same trap types.

Based on my volatile-lure experiments, I demonstrated that plant-based volatiles can increase the capture of EAB adults on a variety of trap types, and might therefore be recommended to be included as part of an EAB monitoring program. In addition, the increase in trap capture on trees with Phoebe Oil compared to those without the oil hints

169 at the underlying, chemically-mediated mechanism by which EAB might gather on a stressed host tree.

My experiments and observations have answered several questions regarding the mate-finding behaviors of buprestids, particularly EAB. I have shown that EAB and other members of the genus Agrilus utilize visual cues to locate conspecifics on the host plant and subsequently determine gender by means of a contact pheromone. However, the buprestids are still a largely uninvestigated group of insects that will provide rich rewards to those researchers who pursue answers to new questions involving this fascinating group of insects.

VITA

Jonathan Peter Lelito

Education The Pennsylvania State University, Fall 2005-Present Pursuing a PhD, Entomology Advisor: Dr. Thomas C. Baker State University of New York College at Fredonia, Fall 2003-Spring 2005 Master of Science, Biology Advisor: Dr. William D. Brown Thesis: ‘Do Hungry Females Increase Sexual Signaling in a Sexually Cannibalistic Praying Mantid?’ State University of New York College at Fredonia, Fall 1999-Spring 2003 Bachelor of Science, Biology

Professional Experience Graduate Teaching Assistant, The Pennsylvania State University, 2005-Present Graduate Teaching Assistant, SUNY Fredonia, 2003-2005; Instructor for Plant Diversity & Ecology (Fall) and Animal Biology & Evolution (Spring) each year. Library Computer Assistant, SUNY Fredonia, 1999-2005

Publications Lelito, J.P. and Brown, W.D. 2008. Mate attraction by females in a sexually cannibalistic praying mantis. Behav. Ecol. Sociobiol. 63: 313-320.

Lelito, J.P., Fraser, I., Mastro, V.C., Tumlinson, J.H., and Baker, T.C. 2008. Novel visual-cue-based sticky traps for detection of emerald ash borers, Agrilus planipennis (Coleoptera: Buprestidae). J. Appl. Entomol. 132: 668-674.

Lelito, J.P, Myrick, A.J. and Baker, T.C. 2008. Interspecific pheromone-plume interference among sympatric heliothine moths: a wind tunnel test using live, calling females. J. Chem. Ecol. 34: 725-733.

Lelito, J.P., Fraser, I., Mastro, V.C., Tumlinson, J.H., Böröczky, K., and Baker, T.C. 2007. Visually mediated ‘paratrooper copulations’ in the mating behavior of Agrilus planipennis (Coleoptera: Buprestidae), a highly destructive invasive pest of North American ash trees. J. Insect Behav. 20: 537-552.

Lelito, J.P. and Brown, W.D. 2006. Complicity or conflict over sexual cannibalism? Male risk-taking in the praying mantis Tenodera aridifolia sinensis. Am. Nat. 168: 263-269.