(Orchestes Fagi L.) and Its Novel Host American Beech (Fagus

Home , Anthonomus musculus, Aterpini, Curculioninae, Curculio sayi, Fagus grandifolia, Heilipus

Comparative analyses on the phenology, electrophysiology, and chemistry of the beech

leaf-mining weevil (Orchestes fagi L.) and its novel host American beech (Fagus

grandifolia Ehrh.)

Simon P. Pawlowski

Thesis

submitted in partial fulfillment of the requirements for

the Degree of Master of Science (Biology)

Acadia University

Fall Graduation 2017

The examining committee for the thesis was:

______Dr. Bobby Ellis, Chair

______Dr. Suzanne Blatt, External Reader

______Dr. Glenys Gibson, Internal Reader

______Dr. Kirk Hillier, Supervisor

______Dr. Jon Sweeney, Supervisor

______Dr. Michael Stokesbury, Acting Head

This thesis is accepted in its present form by the Division of Research and Graduate Studies as satisfying the thesis requirements for the degree of Master of Science (Biology).

I, Simon P. Pawlowski, grant permission to the University Librarian at Acadia University to reproduce, loan, or distribute copies of my thesis in microform, paper, or electronic formats on a non-profit basis. I, however, retain the copyright in my thesis.

______Author

______Supervisor

______Date

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Table of Contents

List of tables ...... vii List of figures ...... ix Abstract ...... xii List of abbreviations ...... xiii Acknowledgements ...... xv CHAPTER 1.0 INTRODUCTION ...... 1 1.1 Background information ...... 1 1.1.1 Beech leaf-mining weevil (Orchestes fagi L.) ...... 1 1.1.2 American beech (Fagus grandifolia Ehrh.) ...... 4 1.2 Significance...... 6 1.3 Figures and tables ...... 8 1.4 References ...... 14 CHAPTER 2.0 DOES ORCHESTES FAGI HAVE A PIONEER SEX?...... 18 2.1 Introduction ...... 18 2.2 Materials and methods ...... 20 2.2.1 Field trapping bioassays ...... 20 2.2.2 Sex ratios within an Orchestes fagi population and the validation of a non- invasive sexing technique ...... 21 2.2.3 Y-tube olfactometer bioassays ...... 22 2.2.4 Statistical analyses ...... 23 2.3 Results ...... 23 2.3.1 Field trapping bioassays ...... 23 2.3.2 Sex ratios within an Orchestes fagi population ...... 24 2.3.3 Y-tube olfactometer bioassays ...... 25 2.4 Discussion ...... 25 2.5 Figures and tables ...... 30 2.6 References ...... 35 CHAPTER 3.0 BEHAVIOURAL BIOASSAYS ...... 39 3.1 Introduction ...... 39 3.2 Materials and methods ...... 43 3.2.1 Animals ...... 43

3.2.2 Colour-tube bioassays ...... 43 3.2.3 Y-tube olfactometer bioassays ...... 44 3.2.4 Field trapping bioassays ...... 45 3.2.5 Statistical analyses ...... 47 3.3 Results ...... 48 3.3.1 Colour-tube bioassays ...... 48 3.3.2 Y-tube olfactometer bioassays ...... 48 3.3.3 Field trapping bioassays ...... 49 3.4 Discussion ...... 50 3.5 Figures and Tables ...... 57 3.6 References ...... 63 CHAPTER 4.0 A NOTE ON A NOVEL ELECTROANTENNOGRAM METHOD FOR ORCHESTES FAGI L...... 70 4.1 Introduction ...... 70 4.2 Materials and methods ...... 71 4.3 Figures and tables ...... 74 4.3 References ...... 77 CHAPTER 5.0 CHEMICAL ANALYSES AND ELECTROPHYSIOLOGY ...... 81 5.1 Introduction ...... 81 5.2 Methods...... 82 5.2.1 Volatile collection from Fagus grandifolia Ehrh...... 82 5.2.2 Animals ...... 84 5.2.3 Electrophysiological responses to F. grandifolia Ehrh. volatiles ...... 84 5.2.4 Chemical isolation and identification ...... 85 5.2.5 Data analyses ...... 85 5.3 Results ...... 86 5.3.1 Volatile profiles of Fagus grandifolia Ehrh. at five developmental stages ..... 86 5.3.2 Electrophysiological responses to Fagus grandifolia Ehrh. volatiles ...... 87 5.3.3 Presence of unknown compounds ...... 88 5.4 Discussion ...... 88 5.4.1 Volatile profiles of Fagus grandifolia Ehrh. at five developmental stages ..... 88 5.4.2 Electrophysiological responses to Fagus grandifolia volatiles ...... 90

5.4.3 Presence of unknown compounds ...... 93 5.5 Figures and Tables ...... 94 5.6 References ...... 100 CHAPTER 6.0 CONCLUSIONS ...... 104 6.1 Summary ...... 104 6.2 Future directions ...... 105 6.3 References ...... 107 APPENDIX A ...... 108 APPENDIX B ...... 108

List of tables

Table 1.1. A list of all known predator and parasitoid species on Orchestes fagi L.in its native range with special consideration of the developmental stage affected and presence in introduced range. I = idiobiont; K = koinobiont; L = larval stage; P = pupal stage. Grey boxes indicate no record ......

Table 3.1 Chemical composition of beech leaf blend lures and weevil cuticular hydrocarbon lures used in Orchestes fagi L. trapping bioassay 3 conducted near Halifax, Nova Scotia in 2014...... 63

Table 4.1 List of successfully tested electroantennogram preparations for curculionid species with special consideration of adult body length. All preparations shown failed to produced reliable results for Orchestes fagi L......

Table 5.1 VOCs produced by American beech, Fagus grandifolia Ehrh., at five developmental stages assessed with GC-FID. Compounds selected for analysis were based on VOCs produced by European beech, Fagus sylvatica L. Presence or absence of VOCs in F. grandifolia samples was assessed by comparison to retention times of 1µg of external standard......

Table 5.2 Ranked VOCs produced by Fagus grandifolia Ehrh. in the largest quantity at each stage of development in descending order; A = largest...... 99

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viii

List of figures

Figure 1.1 (A) Orchestes fagi L. adult female; ventral and side view under light microscope (400X); (B) live O. fagi L2 larva dorsal view; extracted from leaf mine under light microscope (400X); (C) O. fagi mining pattern: egg laid on central vein; I – L1 feeding and movement toward leaf margin creating a linear mine; II – L2 lateral feeding and movement begins; III – L3 lateral feeding and movement creating a blotch mine; P – pupation chamber (Pullin 1985)...... 8

Figure 1.2 Native range of American beech, Fagus grandifolia Ehrh. (Tubbs and Houston 1990)...... 9

Figure 1.3 Historical spread of beech bark disease in Canada and the United States of America from 1911 to 2003. Solid lines indicate the spread of the complex; dotted lines indicate adjunct populations or areas of expansion limited by the range of F. grandifolia (Morin et al. 2007)...... 10

Figure 2.1 Example of a lure containing live adult Orchestes fagi L. to be attached to a yellow sticky card for use in field trapping bioassays. White arrow indicates plastic mesh cage used to house live animals. Black arrow indicates supply of sugar water...... 30

Figure 2.2 Terminal abdominal segments in adult Orchestes fagi L.. Left = male; Right = female. Arrows indicate edge of 5th sternite: laterally concave in males; convex in females...... 30

Figure 2.3 Y-tube olfactometer with 2-channel air delivery system. Dotted black lines indicate choice threshold. Arrows indicate direction of air flow...... 31

Figure 2.4 Number of adult Orchestes fagi L. captures on yellow sticky cards using live adult O. fagi as lures. Boxes indicate median value with upper and lower quartiles; whiskers indicate minimum and maximum values; · indicates outliers; * indicates significant difference...... 32

Figure 2.5 Orchestes fagi L. daily counts at weekly intervals at Ashburn Golf Club as measured through branch-beat sampling on Fagus grandifolia branches in summer 2015. Red line indicates beginning of emergence period of pre-overwintered adults. Dots indicate sampling days ......

Figure 2.6 Mean daily temperature and total precipitation from 1 May – 31 July 2015 at Halifax Airport, NS, Canada (Environment Canada). Dotted lines indicate beat-sampling dates......

Figure 2.7 Precent response of Orchestes fagi L. (adult male and female) to different stimuli presented in a Y-tube olfactometer bioassay. Bursting bud refers to beech buds which have freshly eclosed with some scales still intact. N = 20 positive responses; NR = Non-responders...... 35

Figure 3.1 Schematic diagram displaying the tenants of „appropriate/inappropriate landings‟ theory exemplified by the cabbage maggot (Delia radicum L.) and a suitable Brassica oleraceae L. and antagonist clover plant (Trifolium subterraneum L.). Numbers represent insect actions: 1-2: D. radicum detects host plant odours; 3: Selection of green (i.e. plant) material; 4-7: Flight interspersed with repeated host location (see text for more detail). Image adapted from Finch and Collier (2000)...... 57

Figure 3.2 Percent reflectance of Creatology™ foam assessed in comparison to a sheet of white paper control using a spectrometer. Image courtesy of Catherine Little (Memorial University)...... 57

Figure 3.3 Colour-tube choice assay for Orchestes fagi L.. Left: interior of the tube with 6 colour choices and band of sticky material along upper edge. Right: Complete experimental set-up...... 58

Figure 3.4 Mean ± SEM number of landings of Orchestes fagi L. (adult female and male) in a colour-tube bioassay. Letters „a‟ and „b‟ represent significant differences in adult male colour preference; * indicates significant differences between male and female colour preference...... 59

Figure 3.5 Precent response of pre-overwintered Orchestes fagi L. (female and male) to stimuli presented in a Y-tube olfactometer bioassay. N = 20 positive responses; NR = Non-responders...... 60

Figure 3.6 Percent response of post-overwintered Orchestes fagi L. (female and male) to stimuli presented in a Y-tube olfactometer bioassay. N = 20 positive responses; NR = Non-responders...... 61

Experiment 2: June 2014; LB2 = new leaf blend. Experiment 3: July 2014; LB3 = 8:1 blend of β-caryophellene and (Z)-3-hexenyl acetate. Letters „a‟ and „b‟ indicate significant differences between mean control trap catch. Boxes indicate median value with upper and lower quartiles; whiskers indicate maximum and minimum values; · indicates outliers...... 62

Figure 4.1 Common EAG insect preparations for recording sensory responses from antennae. All images depict the use of dual electrodes (Ag/AgCl wires) within glass capillaries: ground electrode (left) and recording electrode (right). (A) Single excised antenna; (B) Head mount. Commonly used for insects with small antennae, depicted here as a club antenna; (C) Whole mount or body mount. Used to prolong life of preparation at the expense of increase musculature movement. All images adapted from Syntech (2004)...... 74

Figure 4.2 Novel electroantennogram preparation for Orchestes fagi L. Adult female (~2.0 mm body length) shown. (A) Antennal club is secured to glass with wax; (B) a piezo-electric saw is placed near the distal club segment and incised; (C) antennal club is stabilized with an insect pin; (D) a glass capillary containing Ag/AgCl electrode is inserted into the head and a tungsten electrode is inserted into the antennal club incision...... 75

Figure 5.1 Five stages of Fagus grandifolia Ehrh. leaf development: (1) closed and elongated bud; (2) newly bursting bud; (3) newly emerged leaves; (4) fully developed leaf; and (5) senescing leaf (Image 5 adapted from Famartin 2014, https://commons.wikimedia.org/w/index.php?curid=36975055) ...... 94

Figure 5.2 Volatile collection setup using PVAS22 to collect two simultaneous volatile samples from stage 4 Fagus grandifolia Ehrh. leaves. Black arrow indicates HayeSep® Q volatile trap, second trap is obscured from view. Dotted white arrows indicate direction of air flow...... 95

Figure 5.3 EAD and GC-FID output showing response of a female Orchestes fagi L. to volatiles collected from stage 5 Fagus grandifolia Ehrh. leaves. Top spectrum: EAD output; lower spectrum: GC-FID output...... 96

Figure 5.4 Mean ± SEM electroantennographic response amplitudes of Orchestes fagi L. to identified Fagus grandifolia Ehrh. VOCs assessed using GC-EAD. Nmale and Nfemale = 4; *mixture of α- and ß-pinenes...... 97

Abstract

The beech leaf-mining weevil, Orchestes fagi L. (Coleoptera: Curculionidae) is a common pest of European beech, Fagus sylvatica L. (Fagales: Fagaceae), in its native

European range which has recently (2012) become established in Nova Scotia, Canada where it similarly damages American beech, F. grandifolia Ehrh. The objective these studies are to develop effective strategies to monitor and control of this new invasive species. Chapters focus on the life history and phenology of both O. fagi and its novel host, F. grandifolia. Evidence is presented to support the existence of a male-produced repellent pheromone and for males to have trichromatic vision. Female O. fagi had larger electroantennal responses to host volatile organic compounds. GC-FID analyses of F. grandifolia leaf development indicated fluctuating volatile profiles throughout the growing season. The studies in this thesis provide phenological and biochemical context for future studies on O. fagi host location tactics and the development of integrated pest management strategies.

xii

List of abbreviations

AGC: Ashburn Golf Club, Fairmont, NS, Canada (44°38‟43.0”N, 63°38‟10.2”W)

CABL: Chemical Analysis and Bioimaging Laboratory

CFS: Canadian Forest Service

EAD: electroantennal detection

EAG: electroantennogram

ERG: electroretinogram

FID: flame ionization detector

GC: gas chromatogram

I: idiobiont

IPM: integrate pest management

K: koinobiont

KCIC: K.C. Irving Environmental Science Centre (45°05‟12.7”N, 64°22‟05.8”W)

L: larval instar

LB: leaf blend

MB: mushroom body

MS: mass spectrometer

NIST: National Institute of Standards and Technology

NR: non-responder

P: pupal stage

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PVAS: portable volatile assay system

RH: relative humidity

RO: reverse osmosis

SL: Sandy Lake, Bedford, NS, Canada (44°43‟41.8”N, 63°41‟08.3”W)

SPME: solid-phase micro-extraction

UV: ultraviolet

VOC: volatile organic compound

WB: weevil blend

xiv

Acknowledgements

I wish to thank Dr. Kirk Hillier for all his help and patience. Your guidance has been invaluable and it has been a pleasure trying to work my way through this project with you. To Jon Sweeney, I will forever appreciate your positivity even in the face of months of frustrating data collection and the chance to take my mind off of work with Lego®

Lord of the Rings™ and bocce ball. Thank you both for all your hard work editing and discussing this thesis. I would also like to thank Dr. Peter Silk for providing chemical compounds and the reminder that, “weevils are notoriously difficult to work with”. A special thank you goes out to Colin MacKay who has been my field work mentor for going on four years now. Thank you to the KCIC staff Dr. Dave Kristie, Peter Romkey, and Adrien Greene for the use of their facilities. Thank you to Andrew Hebda at the

Natural History Museum for your assistance with your collections. Thank you to

Canadian Forest Service staff who helped me during my time at the Atlantic Forestry

Centre: Rob Johns; Eric Moise; Cory Hughes; Peter Mayo; Kate van Rooyen; and Glen

Forbes. Thank you to all the supporters of this project including: Natural Resources

Canada; SERG-International; Atlantic Canada Opportunities Agency – Atlantic

Innovation Fund; and Acadia University. I will be forever grateful to the Hillier lab members: Cate Little; Rebecca Rizzato; Lise Charbonneau; Jesse Saroli; Lara Thomas;

Heather Crozier; Katrin Sommerfeld; and Loay Jabre for all the moral and technical support. A final thank you is extended to my friends and family and to Emily Cann who have put up with my groaning throughout this project.

CHAPTER 1.0 INTRODUCTION

The beech leaf mining weevil, Orchestes fagi L. (Curculionidae: Curculioninae:

Ramphini), also commonly referred to as beech flea weevil, and previously of the genus

Rhynchaenus, is a common pest of European beech, Fagus sylvatica L. (Fagales:

Fagaceae) in Europe. It has recently been recorded as established in North America where it affects American beech, F. grandifolia Ehrh. (Sweeney et al. 2012). This invasive species represents a serious threat to already declining populations of American beech across North America. The purpose of this project was to assess aspects of life history, chemical ecology, and the interaction between O. fagi and F. grandifolia to enable development of a monitoring system for use in early detection of O. fagi.

1.1 Background information

1.1.1 Beech leaf-mining weevil (Orchestes fagi L.)

Orchestes fagi is the predominant insect pest of European beech, Fagus sylvatica

L., across its native European range (Grimm 1973; Nielsen 1974a; Nielsen 1974b;

Nielsen and Ejlersen 1977). Adults are 2.2-2.8 mm long, black with golden pubescence, and have robust hind femora used for jumping (Figure 1.1A; Sweeney et al. 2012). Eggs are laid singly in the internal tissue of beech leaves along the mid-rib on the underside of developing leaves at the time of budburst. Larvae feed and pupate within the palisade and spongy parenchyma tissues creating characteristic linear-blotch mines with a brown and scorched appearance (Nielsen 1966; Nielsen 1978; Pullin 1985; Sweeney et al.

2012). Eggs are 0.6–0.7 mm x 0.3 mm and require between 8–10 days to develop

(Nielsen 1966; Bale 1984). There are three larval instars: L1 (head capsule ≈ 0.25 mm) feed directionally toward the leaf margin, away from the petiole following eclosion; L2

(head capsule ≈ 0.4 mm) begin feeding laterally; L3 (head capsule ≈ 0.5 mm) continue to feed along the leaf edge and pupate upon maturation forming a pupation chamber within the parenchyma (Figure 1.1B and C; Nielsen 1966; Pullin 1985). Single linear and blotch mines can consume approximately 15 mm3 of leaf tissue and up to seven developing larvae can co-occur on a single leaf (Nielsen 1966). Larval mortality, however, is high (~

85%) with most death occurring at L3 (~ 40–50%; Nielsen 1968; Watt and McFarlane

1992; Woodcock and Vanbergen 2008). This high level of mortality is attributed to three main causes: (1) predation; (2) parasitoids; and (3) „coincidence factor‟. O. fagi has many predator and parasitoid species in its native range which affect different stages of development and increase mortality by 26–61% (Table 1.1; Nielsen 1968; Watt and

MacFarlane 1992; Woodcock and Vanbergen 2008). The phrase „coincidence factor‟, coined by Nielsen (1968), refers to the time-sensitive oviposition of O. fagi eggs occurring during the period of beech bud burst and flush in early May (Nielsen 1966;

Nielsen 1968; Nielsen and Ejlersen 1977; Phillipson and Thompson 1983; Pullin 1985;

Day and Watt 1989). The aging and lignification of beech leaf vascular tissue that begins as early as one week following budburst reduces the ability of first instar larvae to feed and develop (Nielsen 1966; Nielsen 1968; Pullin 1985; Day and Watt 1989). The egg- laying period is thus limited to approximately two weeks following bud burst with the majority of eggs being laid within the first week (Phillipson and Thompson 1983; Bale

1984). Adult emergence occurs 11–16 days following eclosion from the egg (Nielsen

1966). Newly emerged adults are not fully developed and require an obligate overwintering period of diapause to complete sexual maturation (Bale 1979). Although males may begin developing sperm prior to overwintering, ovarian development in

females remains dormant until post-overwintered feeding (Bale 1979). Several habitats have been documented as important for O. fagi overwintering including: moss on beech stems; young beech; spruce; yew; under soil, stumps, and stones; leaf litter, grass tussocks and stems; ferns; and umbelliferous plants (Nielsen 1970; Grimm 1973; Bale

1981; Grimm 1990). In Nova Scotia, Morrison et al. (2017) observed most O. fagi overwintering in cracks, crevices and under bark scales on the stems of beech cankered by beech bark disease and on adjacent red spruce, Picea rubens Sarg. and red maple,

Acer rubrum L., with much lower numbers in the leaf litter and none in the soil.

Following reproductive diapause, feeding and mating may begin; O. fagi is thus univoltine and regulated by obligate diapause.

The feeding cycle of adult O. fagi has been documented in Europe as containing three distinct phases: (1) initial feeding in early spring on hawthorn (Crataegus sp.

Tourn. [Rosales: Rosaceae]) and beet (Beta vulgaris L. [Caryophyllales:

Amaranthaceae]); (2) summer feeding and oviposition on beech; and (3) post- emergence/pre-overwintering feeding on non-host plants (Dieter 1964; Bale and Luff

1978). The third phase contains a large variety of non-host plants including: apricot; cherry; apple; lime; sycamore; dog rose; blackberry; rowan; blackcurrant; redcurrant;

English elm; birch, hazel; common oak; crack willow; dark leaved willow; and beech nuts (Dieter 1964; Bale and Luff 1978; Pajares et al.1990), and is marked by an almost complete rejection of beech foliage (Bale and Luff 1978; Phillipson and Thompson

1983). Bale and Luff (1978) found that young beech leaves were preferred over any alternate food source including old beech leaves. Newly emerged, pre-overwintered adults engage in feeding activity in Europe (Dieter 1964; Grimm 1973; Bale and Luff

1978; Pajares et al. 1990) but it is likely that this feeding is minimal compared to post- overwintered feeding activity (Bale and Luff 1978). Bale and Luff (1978) concluded that

O. fagi oviposited only on F. sylvatica but in surveys of British arboreta, Welch (1994) demonstrated that O. fagi had successfully completed its life cycle on leaves of the

European oak species Quercus frainetto Ten. [Fagales: Fagaceae] and the North

American species Q. phellos L., though the incidence was relatively rare.

In its introduced range, Moise et al. (2015) observed very little feeding by O. fagi on host plants other than American beech in no-choice bioassays conducted in both the lab and field and suggested the population established in Nova Scotia may reflect a genetic bottleneck resulting from local adaptation or that F. grandifolia may provide a superior food source than European beech.

1.1.2 American beech (Fagus grandifolia Ehrh.)

American beech, Fagus grandifolia Ehrh., is a broad-leaved, deciduous hard- wood which lives up to 300–400 years (Tubbs and Houston 1990). It is the only species of beech native to North America and is distributed throughout most of the eastern region of the United States and southern Canada (Figure 1.2). This range encompasses a variety of subspecies of F. grandifolia (Weathington 2014). Fagus sylvatica is also grown in

North America as an urban decorative tree (Weathington 2014). Defoliation and possible mortality of high value beech trees caused by several consecutive years of O. fagi infestation may reduce aesthetic and property values, and cost residents for tree removal.

Flowering occurs between April and May and the majority (~90%) of tree growth occurs between May and June (Tubbs and Houston 1990). Triangular beechnuts grow once a season, maturing and ripening between September and November at which time they are

a food source for a variety of birds and mammals including humans (Homo sapiens L.

[Primata: Hominidae]), mice (Mus L. [Rodentia: Muridae]), squirrels (Rodentia:

Sciuridae), chipmunks (Tamias Illiger [Rodentia: Sciuridae]), black bears (Ursus americanus Pallas [Carnivora: Ursidae]), deer (Artiodactyla: Cervidae), foxes (Carnivora:

Canidae), ruffed grouse (Bonasa umbellus L. [Galliformes: Phasianidae]), ducks

(Anseriformes: Anatidae), and blue jays (Cyanocitta cristata L. [Passeriformes:

Corvidae]; Tubbs and Houston 1990). Beech wood is excellent for turning and steam bending, wears well, and is easily treated with preservatives making it a common lumber source for flooring, furniture, veneer, containers, railroad ties, and novelties (Tubbs and

Houston 1990). Beech wood is also commonly used as rough lumber, fuel wood, charcoal, pulp, and creosote (Tubbs and Houston 1990). Beech reproduction can occur both sexually and vegetatively through flower pollination and root suckers, respectively

(Tubbs and Houston 1990; Beaudet and Messier 2008).

Populations of F. grandifolia across North America are currently in decline due to a variety of factors including insect pests and fungal pathogens, the most damaging of which is beech bark disease, a complex of an introduced scale insect and a fungus

(Houston et al. 1979; Houston and O‟Brien 1983; Tubbs and Houston 1990; Houston

1994; Davis and Meyer 2004; Jha et al. 2004). Of concern in Nova Scotia are the scale insect Cryptococcus fagisuga Lind. (Hemiptera: Eriococcidae) introduced to Nova Scotia from Europe in 1890 (Houston et al. 1979) and the native Xylococculus betulae (Perg.)

Morrison (Hemiptera: Margarodidae; Houston and O‟Brien 1983). This pest damages beech causing susceptibility to the ascomycete bark canker fungi, Neonectria faginata

Castlebury (Hypocreales: Nectriaceae; formerly Nectria coccinea var. faginata Lohman,

Watson, and Ayers) and Neonectria ditissima (Tul. and C.Tul.) Samuels and Rossman

(formerly Nectria galligena Bres; Castlebury et al., 2006; Houston and O‟Brien, 1983), forming the complex (Houston et al. 1979; Tubbs and Houston 1990; Davis and Meyer

2004). The indigenous N. ditissima is a significantly less aggressive bark parasite than the

European introduced N. faginata (Mahoney et al., 1999). Beech bark disease has been spread by biotic vectors and anthropomorphic transport and is a major factor in the decline of American beech populations (Figure 1.3; Tubbs and Houston 1990; Davis and

Meyers 2004; Jha et al. 2004; Morin et al. 2007).

1.2 Significance

Orchestes fagi is a recent invasive pest in North America which infests F. grandifolia, damaging leaf tissues and thus lowering photosynthetic potential.

Populations of F. grandifolia in decline from beech bark disease now face another highly damaging pest species. The predator and parasitoid fauna of O. fagi is well known in

Europe (Nielsen 1968; Askew and Shaw 1974; Pullin 1985; Day and Watt 1989;

Woodcock and Vanbergen 2008) but only three species have been previously recorded in

Nova Scotia (Table 1.1). Large O. fagi populations present in infested areas may cause almost complete defoliation of beech trees within peak growth season (personal observation, Halifax, NS). Additionally, O. fagi is listed as one of several potential insect vectors of N. galligena in Europe (Mihál et al. 2014) and it is possible that O. fagi might increase the spread of beech bark disease in North America but this remains to be verified. Defoliation weakens American beech and increases their susceptibility to bark canker fungi and root diseases such as Armillaria sp. Staudt (Agaricales:

Physalacriaceae), and other pathogens (Houston 1994; Beaudet and Messier 2008).

Heavy insect damage can furthermore lower beechnut production thus negatively affecting animal foraging. During high density O. fagi infestations in Europe, significant damage has occasionally occurred to fruits like cherries and apples growing in orchards near beech forests (Chauvin et al 1976; Bale and Luff 1978). The purpose of this project is to begin developments toward an effective monitoring system which will allow for confirmation of presence and quantification of abundance of O. fagi as it expands its introduced range. An effective monitoring system can help manage populations and reduce its spread in North America. Chemical, neurophysiological, and behavioural analyses were used to determine the relative attraction to host plant and conspecific volatiles by O. fagi for evaluation of a lure-based trapping and monitoring system.

1.3 Figures and tables

Figure 1.2 Native range of American beech, Fagus grandifolia Ehrh. (Tubbs and Houston 1990).

Grey Grey

ge.

consideration of the the of consideration

in its native range special with its in range native

Orchestes Orchestes fagi

A list of all known predator and parasitoid species on on parasitoid species and predator known list A all of

developmental stage affected and presence in introduced range. I = idiobiont; K = koinobiont; L = larval stage; P pupal = stage; larval sta = idiobiont; range. koinobiont; = = K introduced in presence L I stage and affected developmental boxesrecord no indicate Table Table 11

names from Universal Chalcidoidea Database (Noyes 2014). (Noyes Chalcidoidea namesDatabase from Universal

*Valid species species *Valid the at Natural of Museum collections and 2014) samples on Collection National (Gibson in present Canadian the **Based personal Hebda, (Andrew Halifax, communication). Canada NS, History,

1.4 References

Askew RR, Shaw MR (1974) An account of the Chalcidoidea (Hymenoptera) parasitising leaf-mining insects of the deciduous trees in Britain. Biol J Linn Soc 6: 289-335.

Bale JS (1979) The occurrence of an adult reproductive diapause in the univoltine life cycle of the beech leaf mining weevil, Rhynchaenus fagi L. Int J Invert Rep 1: 57- 66.

Bale JS (1981) Seasonal distribution and migratory behaviour of the beech leaf mining weevil, Rhynchaenus fagi L. Ecol Entomol 6: 109-118.

Bale JS (1984) Bud burst and success of the beech weevil, Rhynchaenus fagi: feeding and oviposition. Ecol Entomol 9: 139-148.

Bale JS, Luff ML (1978) The food plants and feeding preference of the beech leaf mining weevil, Rhynchaenus fagi L. Ecol Entomol 3: 245-249.

Beaudet M, Messier C (2008) Beech regeneration of seed and root sucker origin: A comparison of morphology, growth, survival, and response to defoliation. Forest Ecol Manag 255: 3659-3666.

Castlebury LA, Rossman AY, Hyten AS (2006) Phylogenetic relationships of Neonectria/Cylindrocarpon on Fagus in North America. Can J Bot 84: 1417- 1433.

Chauvin G, Gueguen A, Strullu DG (1976) A propos d‟une infestation des hêtres en Bretagne par l‟Orchestes fagi L. (Coléoptère Curculionidae). Biologie et forêt 453:174 343-348.

Day KR, Watt AD (1989) Population studies of the beech leaf mining weevil (Rhynchaenus fagi) in Ireland and Scotland. Ecol Entomol 14: 23-30.

Davis C, Meyer T (2004) Field guide to tree diseases of Ontario. Nat Resour Can, Canadian Forest Service, Great Lakes Forestry Centre, Sault St. Marie, ON. NODA/NFP Tech Rep TR-46 pp. 92-93.

Dieter VA (1964) Beitrag zur epidemiologie und biologie des Buchenspringrüßlers Rhynchaenus (Orchestes) fagi L. an Obstgewächsen. Anz Schädl 37: 161-163.

Gibson GAP (2014) Lists of CNC chalcid taxa by family and their regional distributions. Available from: http://www.canacoll.org/Hymenoptera/Staff/Gibson/Gibson_Lists.htm#Refs. Accessed 18 November 2014.

Grimm R (1973) Food and energy turnover of phytophagous insects in beech forests. I. Oecologia 11: 187-262.

Grimm R (1990) Flugverhalten und wirtdfindung des Buchenspringrüßlers Rhynchaenus fagi L. (Col.: Curculionidae) beim einflug in den buchenwald nach der überwinterung. Mitt Dtsch Ges Allg Angew Ent 7: 395-404.

Houston DR (1994) Major new tree disease epidemics: Beech bark disease. Annu Rev Phytopathol 32: 75-87.

Houston DR, O‟Brien, JT (1983) Beech bark disease. Forest insect & disease leaflet 75, U.S. department of agriculture forest service.

Houston DR, Parker EJ, Lonsdale D (1979) Beech bark disease: patterns of spread and development of the initiating agent Cryptococcus fagisuga. Can J For Res 9: 336- 344.

Jha S, Harcombe PA, Fulton MR, Elsik IS (2004) Potential causes of decline in American beech (Fagus grandifolia Ehrh.) in Weir Woods, Texas. Tex J Sci 56: 285-298.

Mahoney EM, Milgroom MG, Sinclair WA (1999) Origin, genetic diversity, and population structure of Nectria coccinea var. faginiata in North America. Mycologia 91: 583-592.

Mihál I, Cicák A, Tsakov H (2014) Selected biotic vectors transmitting beech bark necrotic disease in Central and South-Eastern Europe. Folia Oecologica 41: 62- 74.

Moise ERD, Forbes GB, Morrison A, Sweeney JD, Hillier NK, John RC (2015) Evidence for a substantial host-use bottleneck following the invasion of an exotic, polyphagous weevil. Ecol Entomol 40: 796-804.

Morin RS, Liebhold AM, Tobin PC, Gottschalk KW, Luzader E (2007) Spread of beech bark disease in the eastern United States and its relationship to the regional forest composition. Can J For Res 37: 726-736.

Morrison A, Sweeney J, Hughes C, Johns R (2017) Hitching a ride: firewood as a potential pathway for range expansion of an exotic beech leaf-mining weevil, Orchestes fagi (Coleoptera: Curculionidae). Can Entomol (published online 16 December 2016, pp. 129–137, DOI: https://doi.org/10.4039/tce.2016.42)

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Nielsen O (1968) Studies on the fauna of beech foliage 2. Observations on the mortality and mortality factors of the beech weevil [Rhynchaenus (Orchestes) fagi L.] (Coleoptera: Curculionidae. Natura Jutlandica 14: 1011-1021.

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Nielsen BO (1974b) A study on the weevil fauna (Curculionidae) in a Danish beech forest. Ent Medd 42: 169-188.

Nielsen BO (1978) Food resource partition in the beech leaf-feeding guild. Ecol Entomol 3: 193-201

Nielsen BO, Ejlersen A (1977) The distribution pattern of herbivory in a beech canopy. Ecol Entomol 2: 293-299.

Noyes JS (2014) Universal Chalcidoidea Database. World Wide Web electronic publication. Available from http://www.nhm.ac.uk/chalcidoids. Accessed 18 November 2014.

Pajares JA, Allue M, Hernandez E (1990) Rhynchaenus fagi L., un curcliónido minador foliar del haya. Bol San Veg Plaga 16: 411-418.

Péré C, Bell R, Turlings TCJ, Kenis M (2011) Does the invasive horse-chestnut leaf mining moth, Cameraria ohridella, affect the native beech leaf mining weevil, Orchestes fagi, through apparent competition? Biodivers Conserv 20: 3003-3016.

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CHAPTER 2.0 DOES ORCHESTES FAGI HAVE A PIONEER SEX?

2.1 Introduction

Weevils (Curculionidae) utilize a wide range of host species and conspecific- locating mechanisms. By understanding the complex phenological relationships of a study organism, its host, and its conspecifics, insight into the production and use of pheromones can be gleaned. Most pheromones discovered in weevils are male-produced and attract both sexes but female-produced sex pheromones and oviposition deterrent pheromones have been discovered in some species (Bartelt 1999). Within the subfamily

Curculioninae, all species for which pheromones have been documented use male- produced pheromones in some respect. The male boll weevil, Anthonomus grandis

Boheman (Curculioninae: Anthonomini), emits a pheromone that is highly attractive to virgin females and is amplified by the presence of multiple males (i.e. there is no inhibitory effect on other males; Hardee et al. 1967). The primary pheromone components, collectively referred to as grandlure, were documented by Tumlinson et al.

(1969) and have been used to monitor and suppress A. grandis populations, replacing harmful control methods such as DDT sprays and mechanical destruction of infested cotton stalks (Cross 1973; Bartelt 1999). Male pepper weevils, A. eugenii Cano, produce a 6-compound blend attractive to both male and female conspecifics with major components including geraniol and geranic acid and sharing three additional compounds with grandlure (Eller et al. 1994). Cranberry weevil males, A. musculus Say, produce a 3- compound pheromone blend, all components of which have been previously documented in grandlure; males and females also produce geraniol but it did not attract conspecifics

(Szendrei et al. 2011). The pecan weevil, Curculio caryae Horn (Curculioninae:

Curculionini), uses a 4-compound male-produced pheromone blend that is identical to grandlure except in blend ratios and grandisol chirality (Hedin et al. 1997). The consistency in male-produced pheromones among the Curculionidae suggests that males may play a role in host location and attraction in host-obligate species such as Orchestes fagi (Curculioninae: Ramphini).

The presence of a male-produced aggregation pheromone often indicates a life history including a „pioneer‟. The term „pioneer‟ or „pioneer sex‟ in this chapter is adopted from bark beetles (Curculionidae: Scolytinae) and refers to initial colonization of host plant material by one sex and the subsequent production of aggregation pheromone to attract conspecifics. In Dendroctonus (tribe Hylesinini) and Ips (tribe Scolytini) species, the male pioneer locates a susceptible host and produces pheromone to attract females which initiate colonization through gallery construction and microorganism inoculation. Females produce additional aggregation pheromone on colonized trees to attract conspecifics to overwhelm host defenses and provide mating opportunities

(Coulson 1979; Foelker and Hofstetter 2014). It is currently unknown if species within the Curculioninae share a similar life history, however, personal observations (Halifax,

NS) of O. fagi have found large populations of males emerging early in the season ahead of the females (protandry). I hypothesize that O. fagi males act as the pioneer sex to locate and colonize hosts.

The objective of this chapter is to assess sex ratios within an introduced population of O. fagi to understand host-finding mechanisms and conspecific interactions

(i.e., provide evidence for an aggregation or sex pheromone). Identification of a pioneer sex would greatly narrow the scope of search for a pheromone and provide a more

detailed phenological timeline for O. fagi. This chapter focuses on conspecific interactions using live adults as potential attractants in both laboratory and field studies and a survey of sex ratios within a population over one cycle of activity. Additionally, a non-invasive technique for determining the sex of O. fagi was assessed.

2.2 Materials and methods

2.2.1 Field trapping bioassays

Field trapping bioassays using live O. fagi adults as lures were conducted 2 May–

1 August 2014 to assess potential attraction from either pheromone or cuticular volatiles.

All trapping took place at Sandy Lake, Bedford, NS, Canada (SL) off an industrial road owned by the Nova Scotia Water Commission (44°43‟41.8”N, 63°41‟08.3”W). Three treatments were deployed in a complete randomized block design with three replicate blocks. The treatments were: (1) three live adult females; (2) three live adult males; (3) empty cage control. Trials were based on effective trapping of A. grandis using live adult males as lures (Hardee et al. 1972). Adult O. fagi were housed within a 3.5 cm x 0.5 cm mesh cage and attached to a micropipette tip containing a supply of sugar water (Figure

2.1) and attached to a 15 cm x 9 cm yellow sticky card. Adult O. fagi used in the cages had been collected from beech trees at the forested border between Ashburn Golf Club

(AGC) and residential Fairmont, NS (44°38‟43.0”N, 63°38‟10.2”W) from April–June,

2014, sexed in the lab (see below) and stored in the dark at 5–10°C until used in field bioassays. Traps were hung in the lower canopy of Fagus grandifolia trees (~1–2 m high) ensuring a minimum of 5 m inter-tree and 10 m inter-block spacing where the natural arrangement of beech within the forest allowed. Care was taken to ensure cages were placed in shade to protect the weevils from heat and desiccation. Insect lures were

replaced weekly to ensure that they contained live adults; there was no adult mortality during this length of time (data not shown). Trapped O. fagi were counted weekly and yellow sticky cards were replaced biweekly.

2.2.2 Sex ratios within an Orchestes fagi population and the validation of a non-invasive sexing technique

To assess sex ratios within the population in Halifax, branch-beating samples were taken at AGC from 6 May–16 July 2015. A 1 m x 1 m canvas tarp was placed directly below a selected F. grandifolia branch in the lower canopy. The branch was given five sharp taps to dislodge any insects. O. fagi were collected using an aspirator and returned to the lab. Two branches from each of 10 trees were sampled weekly with a minimum of 5 m between sample trees. Sexes were separated upon collection based on the curvature of the 5th abdominal sternite; males being concave laterally and females being convex throughout (Figure 2.2). A random subset (N = 40) of sexed O. fagi were dissected to confirm the accuracy of this non-invasive technique.

Individuals selected for dissection were directed into a cut micropipette tip until the elytra became lodged in the opening, exposing the head and first set of legs. The exposed portion of the animal was cut and the body was immediately submerged in 4% gluteraldehyde fixative (in saline sucrose solution) and placed in an ice bath on a rocker for 24 hours. The reproductive organs were then dissected under stereomicroscope in saline solution, replaced in 4% gluteraldehyde and gently agitated for 2 hours before being stored at 5°C for 4 days. Organs were then dehydrated in ascending ethanol series and stored in 70% ethanol.

2.2.3 Y-tube olfactometer bioassays

Adult O. fagi behavioural responses to a potential calling pioneer sex were assessed using a Y-tube olfactometer (1 cm diameter) using methods modified from

DeSilva et al. (2013). Incoming air was passed through a two-channel air delivery system

(Analytical Research Systems Inc., Micanopy, FL), charcoal filtered and split to two adjoining bubblers to be humidified with distilled water. The air was then passed to two flow regulators which maintained a constant stream of 1 L min-1 entering each arm of the olfactometer. Light was monitored using a dual range light meter (Traceable®, Control

Company, TX, USA) to ensure a difference of < 20 lux between arms. The olfactometer was positioned vertically so each stimulus-bearing arm was at the apex of the Y-tube to assist in the natural upward movement of O. fagi due to positive phototactic tendencies

(Grimm 1990).

Stimuli presented to post-overwintered adult O. fagi included: (1) freshly bursting beech bud; (2) freshly bursting beech bud with 1 adult female; (3) freshly bursting beech bud with 1 adult male. All stimuli were presented opposite an unbaited arm to individuals of each sex. Individual O. fagi were placed at the bottom of the olfactometer and given 10 minutes to climb the central column and select an arm. A stimulus was deemed to have been selected when the individual crossed the choice threshold arbitrarily selected as a ridge in the connecting pieces of the Y-tube which presented an obstacle (~ 5 cm from junction; Figure 2.3). Crossing the obstacle was thus deemed a successful choice of the stimulus within that arm. Beetles failing to make a choice within 10 minutes were excluded from calculations but noted as non-responders. An experiment was complete when 20 O. fagi had successfully selected one of the stimuli given the above criteria.

Between trials (i.e. individual weevils) within an experiment, stimulus-free air was allowed to flow through the Y-tube to remove lingering odours. Stimuli within an experiment were used throughout the duration of the experiment and were exchanged in each arm between trials to minimize directional bias. Individual O. fagi were used only once and were frozen immediately following completion. Before and after each experiment (i.e., each different pair of treatment stimuli), the Y-tube components were washed with soap and water and scrubbed with a bottle brush, rinsed with acetone, and allowed to dry in a fume hood for a period of no less than 30 minutes to allow all acetone to evaporate.

2.2.4 Statistical analyses

All data processing and analyses were completed using R v. 3.1.2 (R Core Team

2015). Count data from field traps and beat samples were assessed using generalized linear models (loglink, family = negative binomial) and Tukey‟s contrasts (glht function, package multcomp) to assess the effect of sex on mean trap catch and sex and time of year on abundance, respectively. Data are presented as means ± SE. Y-tube olfactometer trials were converted to proportions of responding adults based on the total number used.

Analysis was conducted using a Chi-square test assuming an expected 1:1 ratio of stimulus choice (α = 0.05, df = 1).

2.3 Results

2.3.1 Field trapping bioassays

All traps baited with live adult O. fagi were successful in trapping O. fagi throughout the period of study (Figure 2.4). Female-baited traps had no significant effect on mean trap catch (N = 48; 8.31 ± 2.87) as compared with an unbaited control (N = 48;

8.69 ± 4.36; z = 0.166, P = 0.87) while traps using male lures significantly decreased catch (N = 48; 2.75 ± 1.15; z = -2.67, P < 0.01). Mean trap catch throughout the season was consistent except for a significant increase in July (z = 4.05, P < 0.0001; see Table

A.1 for pair-wise comparisons).

2.3.2 Sex ratios within an Orchestes fagi population

O. fagi collected with beat samples displayed clear fluctuations in population throughout the summer of 2015 (Figure 2.5) though none of these fluctuations were statistically significant between months (May: N = 7, 123.29 ± 25.24; June: N = 6, 95.25

± 29.03; July: N = 3 138.67 ± 34.15; see Table A.2 for pair-wise comparisons). There were no differences in male and female O. fagi emergence patterns and population abundance (z = -0.448, P = 0.654). Post-overwintered adults appeared on F. grandifolia trees mid-May and underwent two distinct population spikes separated by a rapid decline; pre-overwintered adults emerged late-June and underwent a similar emergence pattern

(Figure 2.5). Pre-overwintered adult emergence occurred one week earlier than in the

2014 field sticky trap trials. Weather data during the sampling period was collected by

Environment Canada at the Halifax Airport, NS, Canada (Figure 2.6).

Validation of a non-invasive technique for sexing adult Orchestes fagi

Dissected reproductive systems of both males and females were compared with O. fagi systems as documented by Aslam (1961) and cross-referenced with reproductive systems of other Curculionids: Heilipus lauri Boheman (Molytinae: Hylobiini); H. pittieri Barber; H. trifaciatus Fabricius; Pachyrhynchus mohagani (Entiminae:

Pachyrhynchini); P. tilikensis; P. lubanganus; and Larinodontes freidbergi (Lixinae:

Lixini; Castañeda-Vildózola et al. 2007; Bollino and Sandel 2015; Gültekin and

Friedman 2015). A total of 20 males and 20 females were separated based on external characteristics described previously and subsequently dissected to determine sex. Only

1/40 adults (a female) was sexed incorrectly using external characteristics.

2.3.3 Y-tube olfactometer bioassays

Adult male O. fagi in a Y-tube olfactometer bioassays showed a significant positive response to a bursting beech bud when compared against a clean air blank

(Figure 2.7; χ2 = 5.801, N = 31, df = 1, P < 0.05). This response shifted significantly towards a clean air blank when an adult male O. fagi was presented alongside a bursting beech bud stimulus (χ2 = 11.852, N = 27, df = 1, P < 0.001). The addition of a female had no effect on the response of males to bursting beech buds. Adult female O. fagi showed no significant attraction to any stimulus presented.

2.4 Discussion

It remains unclear if O. fagi use a pioneer sex to locate host material and aide in colonization. The results presented in this chapter offer no concrete evidence for either sex producing or using pheromones for host or conspecific location. The anecdotal observations on which this chapter was based (high male populations in the early summer) were not seen when a more in depth study was undertaken; indeed, sex ratios within the population were almost identical throughout the active period of 2015.

However, this cannot be confirmed mathematically due to experimental design error: all collected samples were pooled within each sampling period thus forcing a summation of count data as opposed to an averaging per day. Data could only be manipulated on a monthly basis, which while providing some information, was far less powerful than an assessment by sampling day.

The population underwent a series of fou distinct population peaks followed by immediate declines. Two population peaks were expected coinciding with emergence of adults in spring and the new generation a little over a month later. The cause of the deep dip in the adult numbers on the 22 May beat sample is unknown but may be due to: (1) vertical migration; (2) horizontal migration; or (3) inclement weather.

(1) In its native European range, O. fagi exhibits discrete niche partitioning in which larvae feed on upper canopy leaves and adults feed in the lower canopy (Nielsen and Ejlersen 1977; Nielsen 1978; Day and Watt 1989). If this also occurs in its new host and introduced range, then O. fagi adults may move to the upper canopy when food supplies decrease in the lower canopy, possibly due to over-crowding. Our branch beat samples only accessed the lower canopy. Thus, emerging adults would deplete the lower canopy and migrate to the upper canopy causing a plummet in abundance; the second peak would coincide with late emerging adults which subsequently migrate as well.

Vertical migration toward upper canopy feeding by O. fagi adults has been previously documented by Phillipson and Thompson (1983) though no explanation was given as to its origin. If overcrowding and resource depletion lead to vertical migration, it could indicate that intraspecific competition would occur between adults and larvae for feeding sites, contrary to previous documentation (Day and Watt 1989; Watt and McFarlane

1992). This may indicate that over-feeding on F. grandifolia is self-limiting due to initiation of intraspecific competition through vertical migration. None of this can be confirmed at this time.

(2) Behaviour of O. fagi post-oviposition is largely unknown, though feeding on host material has been observed up to 46 days post-emergence (Phillipson and Thompson

1983). Bale (1981) found that female O. fagi in Great Britain and Denmark migrate away from beech stands following oviposition to die. Most eggs are laid within 72 hours of emergence and few are laid > 1 week post-emergence (Bale 1984). This would indicate that females emerge, mate, lay eggs within 1 week, and then horizontally migrate from beech hosts. This follows the presented timeline with emergence following a decline after

~ 1 week. A second peak of late emerging adult O. fagi then followed the same pattern.

This, however, can only account for female horizontal migration, although it is likely that males have similar life spans and thus either migrate or perish alongside females.

(3) Activity and abundance of adult O. fagi on host foliage may be reduced in inclement weather. Orchestes fagi remain active down to -2°C, high activity (i.e. flight) is diminished below 10°C (Grimm 1990; Coulson and Bale 1995). The steep decrease in weevil abundance in mid-May 2015 occurred on 22 May when the average temperature was <10°C and it rained (Figure 2.6). However, the sudden decrease in numbers on 29

June did not coincide with cool temperatures, although the previous day was cool and rainy (Figure 2.6). This could indicate that the effects of low temperature and precipitation on O. fagi (i.e. reduced activity) require a period of at least 24 hrs to diminish.

These insights into O. fagi population dynamics give no indication of a pioneer sex and it is unclear if these fluctuations will occur in future field seasons. Laboratory and field trapping bioassays were also inconclusive in determining the status of a pioneer.

While no significant positive effect of live adults on mean trap catch was observed, there is a trend for higher trap catch in those traps using a female lure. While many Curculionid pheromones to date have been documented as male-produced, some species utilize

female-produced, often long-range, pheromone (Bartelt 1999). Anthonomus grandis female frass has been shown to elicit behavioural responses in males (McKibben et al.

1977; Hedin et al. 1979). Additional female-produced pheromones have been found in: the cabbage seed weevil, Ceutorhynchus assimillis Paykull (Ceutorhynchinae; Evans and

Bergeron 1994); the sweet potato weevil (Apioninae), Cylas formicarius Fabricius

(reviewed by Jansson et al. 1991), C. brunneus Fabricius, and C. puncticollis Boheman

(Smit et al. 1994); the West Indian sugarcane rootstock borer, Diaprepes abbreviatus L.

(Otiorrhynchinae; Beavers et al. 1982); and the pine weevil, Hylobius abietis L.

(Hylobiinae; Selander 1978; Tilles et al. 1988). At this time, the presence of a female- produced pheromone in O. fagi is merely speculation, but future studies on frass volatiles may prove useful to identify a pheromone.

Attraction of mature male O. fagi to bursting beech buds was expected and is likely adaptive for early feeding and mate-finding before leaf tissue lignification (see

Chapter 1.0). Lack of female attraction to beech buds was unexpected and may indicate the necessity of a male-produced pheromone (see above) in addition to host volatiles for a behavioural response to occur. A male‟s presence on a bursting bud did not support this hypothesis, however. It is possible that laboratory bioassay conditions were not suitable for female response or male pheromone emission. Hock et al. (2014) found that positive responses of female plum curculio, Conotrechalus nenuphar Herbst (Molytinae:

Conotrechelini), to males vs. blank controls in olfactometer bioassays varied with age

(mature > immature), mating status (virgin males > mated males), and number of males present (two males > one male). All male and female O. fagi were considered mature

(i.e., had undergone natural reproductive diapause and were collected from wild

populations), but their mating status could not be controlled. Egg development occurs within the first 48–96 hours post-overwintering emergence and mating can occur before egg development is complete (Bale 1984; personal observation) thus it is assumed that most collected specimens would have been previously mated. Presence of > 1 male on a bud or 1 male alone was not tested vs. a female, however the significant negative response of males to a bursting bud with another male indicates that there may be a male- produced pheromone or that feeding may alter the volatile profile of the bud, reducing desirability. This latter hypothesis is unlikely as Watt and McFarlane (1992) demonstrated that O. fagi were unhindered by any anti-herbivore damage-induced response in beech leaves and, in fact, found a significant positive relationship between the number of eggs laid and leaf damage. This chapter provides evidence, in field and laboratory tests, for a male-produced repellent pheromone in O. fagi.

Results of this study indicate that it is unlikely that O. fagi are protandrous or protogynous, however, pioneering cannot be confirmed or denied. Population fluctuations throughout the active period occur similarly between the sexes and appear to be highly correlated with the weather. Vertical and/or horizontal migration may play a role in population dynamics and sample collections in the upper canopy could aide in the resolution of these hypotheses. Y-tube olfactometer and field trapping bioassays indicate the presence of a male-produced anti-male pheromone though its effect on female behaviour is currently undetermined. This study did confirm a non-invasive method for sexing of live adult O. fagi with 98% efficiency. Based on these results, it is unlikely that

O. fagi has a pioneer, and the presence of male- and/or female-produced pheromones cannot be ruled out.

2.5 Figures and tables

Figure 2.2 Terminal abdominal segments in adult Orchestes fagi L.. Left = male; Right = female. Arrows indicate edge of 5th sternite: laterally concave in males; convex in females.

Figure 2.3 Y-tube olfactometer with 2-channel air delivery system. Dotted black lines indicate choice threshold. Arrows indicate direction of air flow.

* *

Dots Dots

beatsampling on

overwintered overwintered adults.

iod of pre of iod

as measured through branch through measured as

Ashburn Golf Ashburn Club

L. daily counts at weekly intervals at counts daily weekly at L.

branches in summer 2015. Red line indicates beginning of emergence per of beginning emergence indicates line Red summer in 2015. branches

Orchestes fagi Orchestes

Fagus grandifolia Fagus days sampling indicate Figure

31 July 2015 at Halifax Airport, NS, Canada Halifax NS, at 2015 31Canada Airport, July

–

sampling dates. sampling

e and total precipitation from 1 May May 1 precipitation e total from and

Meantemperatur daily

Figure Figure Canada). beat indicate lines Dotted (Environment

2.6 References

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Bale JS (1981) Seasonal distribution and migratory behaviour of the beech leaf mining weevil, Rhynchaenus fagi L. Ecol Entomol 6:109-118.

Bale JS (1984) Bud burst and success of the beech weevil, Rhynchaenus fagi: feeding and oviposition. Ecol Entomol 9:139-148.

Bartelt RJ (1999) Chapter 5: Weevils. In: Hardie J, Minks AK (ed) Pheromones of non- Lepidopterous insects associated with agricultural plants. CABI Publishing, Wallingford, UK, pp 91-112.

Beavers JB, McGovern TP, Adler VE (1982) Diaprepes abbreviates: laboratory and field behavioural and attractancy studies. Environ Entomol 13: 436-439.

Bollino M, Sandel F (2015) Three new species of the genus Pachyrhynchus Germar, 1824, from Lubang Island (Philippines) (Curculionidae: Entiminae: Pachyrhynchini). Mun Ent Zool 10:2 392-401.

Castañeda-Vildózola A, Valdez-Carrasco J, Equihua-Martínez A, González-Hernández H, Romero-Nápoles J, Solís-Aguilar JF, Ramírez-Alarcon S (2007) Genitalia de tres especies de Heilipus Germar (Coleoptera: Curculionidae) que dañan frutos de aguacate (Persea Americana Mill) en México y Costa Rica. Neotrop Entomol 36:6 914-918.

Coulson RN (1979) Population dynamics of bark beetles. Ann Rev Entomol 24: 417-447.

Coulson SJ, Bale JS (1995) Supercooling and survival of the beech leaf mining weevil Rhynchaenus fagi L. (Coleoptera: Curculionidae). J Insect Physiol 42:617-623.

Cross WH (1973) Biology, control, and eradication of the boll weevil. Ann Rev Entomol 18: 17-46.

Day KR, Watt AD (1989) Population studies of the beech leaf mining weevil (Rhynchaenus fagi) in Ireland and Scotland. Ecol Entomol 14:23-30.

DeSilva ECA, Silk PJ, Mayo P, Hillier K, Magee D, Cutler GC (2013) Identification of sex pheromone components of the blueberry spanworm Itame argillacearia (Lepidoptera: Geometridae). J Chem Ecol 39: 1169-1181.

Eller FJ, Bartelt RJ, Shasha BS, Schuster DJ, Riley DG, Stansly PA, Mueller TF, Shuler KD, Johnson B, Davis JH, Sutherland CA (1994) Aggregation pheromone for the pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae): identification and field activity. J Chem Ecol 20:7 1537-1555.

Evans KA, Bergeron J (1994) Behavioural and electrophysiological response of cabbage seed weevils (Ceutorhynchus assimilis) to conspecific odor. J Chem Ecol 8: 679- 687.

Foelker CJ, Hofstetter RW (2014) Heritability, fecundity, and sexual dimorphism in four species of bark beetles (Coleoptera: Curculionidae: Scolytinae). Ann Entomol Soc Am 107:1 143-151.

Gültekin L, Friedman ALL (2015) A new species Larinodontes freidbergi sp. nov. (Coleoptera: Curculionidae: Lixinae) from India. J Insect Biodiv 3:1 1-6.

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CHAPTER 3.0 BEHAVIOURAL BIOASSAYS

3.1 Introduction

The beech leaf-mining weevil, Orchestes fagi L., was identified as the cause of extensive damage to the foliage of American beech, Fagus grandifolia Ehrh., growing in and around Halifax, Nova Scotia, in 2012 but anecdotal reports of defoliation suggest the pest may have been established for several years prior to its discovery (Sweeney et al.

2012). Host plants play an important role in O. fagi development and population dynamics. Despite being a polyphagous feeder as an adult in its native Europe (Dieter

1964; Grimm 1973; Bale and Luff 1978; Phillipson and Thompson 1983; Pajares et al.

1990), its host range in North America appears to be exclusive to beech (Moise et al.

2015). The mechanisms of host location in this species are unknown but both semiochemicals (kairomone or pheromone) and visual cues are likely involved.

Many beetle species use chemical cues to locate a suitable host, attract or repel conspecifics, and engage in interspecies interactions. Chemical cues have been shown to elicit behavioural responses in many insect orders, of which most study has focused on

Lepidoptera (see reviews in Strausfeld 1976; Hansson 1995; Hansson and Anton 2000;

Martin et al. 2011; Strausfeld 2012). These compounds, detected predominantly through olfaction, play an important role in the behavioural responses of insects. Attraction or repulsion elicited by such compounds is the basis for many integrated pest management

(IPM) tactics which use either a push or a pull system or a combination of the two. In a push tactic, a semiochemical is placed on or near the host plant that repels or interferes with normal pest behaviour, e.g., reducing normal attraction to or alignment and oviposition on the host plant. Push systems have been effectively used to manage red

palm weevil, Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae), using non- host compounds including geraniol, 1-octen-3-ol, and α-pinene to disrupt feeding

(Guarino et al. 2015); and the European cherry fruit fly, Rhagoletis cerasi L. (Diptera:

Tephritidae), using host marking pheromone to deter oviposition (Katsoyannos and

Boller 1976; Katsoyannos and Boller 1980). A pull tactic uses attractant semiochemicals as trap baits for survey and monitoring or for population suppression (mass trapping) or placed directly on host plants to concentrate infestations of the target pest in smaller areas or on individual plants (e.g. trap trees). Effective management of the soybean stalk weevil, Sternechus subsignatus Boehman (Coleoptera: Curculionidae); the raspberry weevil, Aegorhinus superciliosus Guérin (Coleoptera: Curculionidae); the cranberry weevil, Anthonomus musculus Say (Coleoptera: Curculionidae); and the brown spruce long-horned beetle, Tetropium fuscum Fabricius (Coleoptera: Cerambycidae) have been documented using conspecific sex or aggregation pheromone (Ambrogi and Zarbin 2008;

Ambrogi et al. 2012; Mutis et al. 2009; Mutis et al 2010; Szendrei et al. 2009; Szendrei et al. 2011; Barbour et al. 2007; Lemay et al. 2010). A push/pull system, i.e. a combination of the two, can be used to simultaneously force an insect away from a target plant and attract it to a different stand of plants or a trap. The control of R. ferrugineus has been enhanced by the combination of antifeedant compounds (above) and aggregation pheromone baited molasses traps (Guarino et al. 2015).

It has become clear that olfactory cues are not the only sensory cues used by insects during host selection and that evidence for the importance of visual and colour cues is increasing (see reviews in Briscoe and Chittka 2001; Stavenga 2002; Reeves

2011). Visual cues eliciting a behavioural response can be simple, such as orientation of

the white pine weevil Pissodes strobi Peck (Coleoptera: Curculionidae) and the warren root collar weevil Hylobius warreni Wood (Coleoptera: Curculionidae) to vertical silhouettes of their respective host plants (VanderSar and Borden 1977; Machial et al.

2012), or complex interactions between shape and colour. Colour vision is the ability to distinguish different wavelengths of light but not necessarily their associated intensity

(Hilbert 1992). Insects have been shown to have tri-chromatic vision, being able to detect wavelengths in the ultraviolet (UV; 340–370 nm), blue (440–460 nm), and green (520–

550 nm) ranges (Kebler et al. 2003; Hausmann et al. 2004; Diclaro et al. 2012).

Photoreceptors for these wavelengths are likely an ancestral trait in the Insecta and are ubiquitous in insect eyes unless one or more has been secondarily lost due to selective pressure (Chittka 1996). Detection of colour likely represents an important step in host plant detection by insects: the differentiation between landing sites, namely plant matter and soil (Finch and Collier 2000).

Higher brain function (e.g. behavioural output) in insects is dictated by structures called mushroom bodies (MBs) that contain calyx-type structures which receive afferent neurons from discrete sensory processing units and produce efferent neurons which terminate collectively (Strausfeld 2002). This collection of afferent neurons through the

MB structure provides evidence for the integration of sensory modalities at a unified higher-level (i.e., behavioural) response (Strausfeld 2009). It is thus an oversight to simply acknowledge olfactory or visual cues in relation to behavioural response; though many insect studies have focused primarily on olfaction. An integrative theory, which applies both olfactory and visual cues in host location, proposed by Finch (1996), Finch and Kienegger (1997), and Finch and Collier (2000) is referred to as

„appropriate/inappropriate landings‟ (Figure 3.1). This theory combines three distinct phases of host-plant selection: (1) detection of suitable host-plant odours; (2) visual selection of green (i.e. plant) material; and (3) contact chemosensory acceptance of host- plant. A host-seeking insect first detects host volatile cues via olfaction and begins orientation towards the stimulus (Figure 3.1-1 and 2). Short-range host-finding is then stimulated by colour detection, with phytophagous insects showing a preference for landing on green (i.e., plant) surfaces as opposed to brown (i.e., soil or bark) surfaces

(Figure 3.1-3; Kostál and Finch 1994). In stands containing multiple species of plants, the insect then relies on contact chemoreception (olfactory and gustatory) to determine host suitability; landing on true host (i.e. „appropriate‟; Figure 3.1-3a) for feeding and/or oviposition, or non-host (i.e. „inappropriate‟, Figure 3.1-3b). Should the insect land on a non-host plant and/or interact with antagonistic compounds present, the insect will leave the site and the process repeats (Figure 3.1-4 to 7).

The co-opting of these natural host detection and orientation processes can be a useful mechanism for IPM. Trap and lure design can be heavily influenced by the visual and olfactory cues used by insects throughout these processes. It is thus the objective of this chapter to determine the behavioural responses of O. fagi in laboratory and field trials in an exploratory assessment of aspects of host-detection and -orientation. Due to the exploratory nature of this chapter, basic elements were examined including: colour preference in a simple multiple-choice assay; movement towards host tissue and associated volatiles in a Y-tube olfactometer; and the effect of host and conspecific volatiles on field trap catches. Collectively these experiments aim to elucidate the behavioural responses of O. fagi to volatiles from its host and conspecifics and provide

useful information on trap and lure design for survey, monitoring, and possible management tactics for integrated pest management of this invasive species.

3.2 Materials and methods

3.2.1 Animals

All O. fagi used in trials were collected from AGC in May-June of 2014 and 2015 by beating branches of F. grandifolia after buds had flushed, and were maintained at 5-

10°C. Sugar water was provided ad libitum. Mating status of adults was not determined but O. fagi mating and oviposition usually coincides with feeding in freshly burst beech buds (Bale 1979) so it is likely that most were mated. Sexes were separated upon collection based on the curvature of the 5th abdominal sternite; males being concave laterally and females being convex throughout. Individuals were maintained at ambient room temperature (18–21°C) at 10:14 light:dark cycle for 24 hrs prior to use in assays.

3.2.2 Colour-tube bioassays

Colour preference was assessed in a behavioural bioassay by allowing adult O. fagi to select a coloured sheet on which to climb or land. Assay chambers were constructed by placing two 10 cm x 45 cm strips of foam sheet (Creatology™, MSPC,

Irving, TX) of six different colour stimuli (black, green, red, white, blue, and yellow) vertically along the interior wall of a 40 cm diameter Premium spiral-wound paper tube

(Crown Fibre Tubing Inc., Kentville, NS). Wavelength reflectance curves were assessed using an ALTA II Reflectance Spectrometer (Vernier Software & Technology, OR;

Figure 3.2). Full sheets of foam were attached vertically in the order listed above in two complete series along the interior of the tube. The base of the tube was coated in white foam which was treated as a control colour. A 15 cm width band of sticky trap was

placed along the upper edge of the tube. A light source containing a VX-series high colour retention index (CRI), full spectrum LED light bulb (14 watt, 5600K, CRI > 95;

Yuji International Co., Ltd., Beijing, China) was placed 60 cm above the tube (Figure

3.3). During experimental trials all other light sources were removed.

A trial consisted of ten adult O. fagi held within a 17 cm x 11.5 cm clear plastic case in the centre of the tube base, in complete darkness. Sexes were tested separately.

The light source was then turned on and the lid of the case was removed without agitation. A clear Plexiglass® lid was then placed over the opening of the assay tube to seal in the O. fagi. A colour was deemed to be selected when an individual was trapped on a section of sticky band directly over the colour. Experiments were checked after 24 hours. The tube was rotated 90° between trials and all trapped beetles were removed. A total of 100 individuals (50 male and 50 female) were tested.

3.2.3 Y-tube olfactometer bioassays

Y-tube olfactometer experiments to assess behavioural differences between pre- and post-overwintered populations were conducted in a manner similar to that previously described (Chapter 2.0). All trials conducted on pre-overwintered populations were completed at the Canadian Forest Service (CFS; Natural Resources Canada, Fredericton,

NB) and all post-overwintered trials were conducted at the Chemical Analysis and

Bioimaging Laboratory (CABL; Acadia University, NS). Trials conducted on both populations included: (1) beech leaves; and (2) 15 mm x 5 mm beech wood slivers. Trials conducted exclusively on pre-overwintered populations additionally included: (1) 1 µg

(Z, E)-α farnesene; (2) 1 µg ß-caryophellene; (3) 1 µg (Z)-3-hexen-1-ol; (4) 1 µg (Z)-3- hexenyl acetate.. Trials conducted exclusively on post-overwintered populations

included: (1) 1 µg geranyl-p-cymene; (2) apple (Malus domestica Borkh) leaves.

Compounds were selected as those found via GC-MS analyses to be present in host tissues by Dr. Peter Silk (Insect Chemical Ecologist, CFS). Leaf samples of beech and apple used in bioassays were obtained by cutting branches from trees in the Acadia

Nature Woodlands Trail (Wolfville, NS; 45°04‟51.2” N, 64°21‟55.5” W) and immediately submerging the cut ends in reverse osmosis-purified water to reduce the impact of wounding on volatile emissions (Fall et al. 1999). Leaves were then excised at the petiole immediately prior to use in assays. Compounds were diluted in hexane and 1

µg was pipetted on a 15 mm x 5 mm strip of filter paper for use as a stimulus; all compounds tested were replaced with fresh strips every half hour to account for loss of volatility. All leaf samples were tested opposite a clean air blank and all chemical compounds were tested opposite a hexane blank.

3.2.4 Field trapping bioassays

Field trapping bioassays were completed in the summers of 2014 and 2015 at

AGC and SL using 15 cm x 9 cm yellow sticky cards (Contech Inc., Victoria, BC,

Canada) hung in the lower canopy (~ 2.0 m above ground) of beech trees. Lures were provided by Dr. Peter Silk (Natural Resources Canada, Atlantic Forestry Centre,

Fredericton, NB) and consisted of either a rubber septum or an impermeable membrane pouch with a polyethylene window impregnated or loaded with a given synthetic compound (or blend of compounds). The lures were attached to yellow sticky cards with a pin or paper clip. Due to the exploratory nature of the study, it was unclear whether compounds would elicit attractive or repulsive effects but all lures were predicted to be attractive due to their presence in host plant material and on the cuticle of adult O. fagi.

Treatments were replicated in randomized complete block designs with a minimum of 5 m spacing between treatments and blocks. Trapped O. fagi were counted weekly and yellow sticky cards were replaced biweekly. Volatility of blend components did not require replacement of lures within the trapping period.

Experiment 1

This was a 2 X 2 factorial trapping experiment testing the response of O. fagi adults to beech leaf volatiles and O. fagi cuticular hydrocarbons, presented separately vs. together on the same trap. The treatments included: (1) a blend of five compounds identified in leaves of American beech (LB); (2) a blend of four compounds tentatively identified from the cuticle of adult O. fagi (WB); (3) the LB and WB lures together on the same trap; and (4) unbaited control (Table 3.1). Blends were validated for composition and concentration by GC-MS analysis by Dr. Peter Silk (Insect Chemical Ecologist,

CFS). This experiment was conducted at AGC from 14 May–4 June 2014 with eight replicates per treatment.

Experiment 2

This was a 2 X 2 factorial trapping experiment testing the response of O. fagi adults to a new blend of beech leaf volatiles and the same WB as Experiment 1. The treatments included: (1) a new blend of beech leaf volatiles (LB2); (2) WB; (3) LB2 and

WB lures on the same trap; and (4) unbaited control. LB2 was made by soaking young leaves of American beech (picked shortly after budburst) in a 10:1 solution of dichloromethane:toluene which was subsequently filtered, rotovaped, and then treated with more dichloromethane and magnesium sulfate to extract water, and loaded onto rubber septa. This experiment was replicated eight times at AGC from 6–25 June 2014.

Experiment 3

This 2 X 2 factorial experiment was similar to experiments 1 and 2 except that the beech leaf blend now consisted of an 8:1 blend of β-caryophellene and (Z)-3-hexenyl acetate (8:1) (LB3). Treatments were: 1) LB3 (2) WB; (3) LB3 and WB lures on the same trap; and (4) unbaited control. It was replicated eight times at AGC, 26 June–18

July 2014.

3.2.5 Statistical analyses

Data processing and statistical analyses were completed using R v. 3.1.2 (R Core

Team 2015). Colour-tube bioassays were analyzed by two-way ANOVA (generalized linear model method, loglink, family = Poisson) and Tukey‟s contrasts (glht function, package multcomp) to assess colour stimuli effect on adult O. fagi presence. Sexes were assessed separately and pooled. Intersex differences were analyzed by contingency tables with log likelihood ratio tests (loglm function, package MASS) and G-tests (G.test function, package RVAideMemoire) according to Zar (1999). Y-tube olfactometer trials were converted to proportions of responding adults based on the total number used.

Analysis was conducted using a Chi-square test assuming an expected 1:1 response to stimulus (α = 0.05, df = 1). Percent response to beech leaves and wood between generations was assessed identically. All field trapping experiments were analysed by generalized linear models (loglink, family = negative binomial) and Tukey‟s contrast (α =

0.05; glht function, package multcomp) to assess lure effect on trap catch. Additionally, control traps were analyzed to assess trap catch between the 3 experiments; as lure type differed between experiments, catch data could not be pooled.

3.3 Results

3.3.1 Colour-tube bioassays

Colour-tube bioassays demonstrated significant differences in attraction of adult male and female O. fagi towards the colour stimuli presented (Figure 3.4). Of 100 total adults assayed (50 of each sex), 82 made a selection (Nmale = 46, Nfemale = 36). Both sexes were found significantly more frequently on blue and white (z = 2.69, P < 0.01; z = 2.81,

P < 0.01 respectively). Males were found significantly more frequently on blue (z = 2.42,

P < 0.05), green (z = 2.15, P < 0.05), and white (z = 2.55, P < 0.05) than other colours presented. Females were found less frequently on green and yellow than other colours but this trend was not significant (z = -1.10, P = 0.273). Sexes differed in their colour preference (G = 15.23, df = 5, P < 0.01). Male attraction to green was significant compared to females (G = 7.92, df = 1, P < 0.01) and female attraction to red was significant compared to males (G = 6.93, df = 1, P < 0.01). No other colours elicited a sex-specific response (G = 1.38, df = 3, P = 0.71).

3.3.2 Y-tube olfactometer bioassays

Response of O. fagi to various volatile stimuli differed between pre-overwintered and post-overwintered adults and between sexes.

Pre-overwintered weevils

Pre-overwintered females were attracted to (Z)-3-hexenyl acetate (χ2 = 3.97, N =

21, df = 1, P < 0.05) but preferred the hexane control to (Z,E)-α-farnesene (χ2 = 15.04, N

= 21, df = 1, P < 0.001; Figure 3.5). Females were significantly attracted towards beech leaves but only after a 24 hour period of starvation (χ2 = 8.011, N = 22, df = 1, P < 0.01).

No pre-overwintered males were attracted to any stimuli presented.

Post-overwintered weevils

Among post-overwintered adults, males were significantly attracted to geranyl-p- cymene (χ2 = 13.33, N = 21, df = 1, P < 0.001) and females were attracted to beech leaves without a period of starvation (χ2 = 18.52, N = 27, df = 1, P < 0.0001; Figure 3.6). No other stimuli were attractive to either sex.

Comparison between cohorts

Unstarved post-overwintered females were significantly more attracted to beech leaves than were pre-overwintered females when presented opposite a clean air blank (χ2

= 4.67, N = 53, df = 1, P < 0.05). No other significant differences in either sex between the generations were observed in replicated trials.

3.3.3 Field trapping bioassays

Control traps in Experiments 1 – 3 showed a significant increase in mean trap catch during Experiment 3 (z = 6.598, P < 0.0001; see Table B.1 for pairwise comparisons; Figure 3.7).

Experiment 1

None of the synthetic semiochemical blends tested in Experiment 1 increased mean trap catch when compared to an unbaited control (see Table B.2 for pairwise comparisons). The blend of LB and WB had a negative trend in trap catch but was not significant.

Experiment 2

None of the synthetic semiochemical blends tested in Experiment 2 increased mean trap catch when compared to an unbaited control (see Table B.3 for pairwise comparisons).

Experiment 3

None of the synthetic semiochemical blends tested in Experiment 3 increased mean trap catch when compared to an unbaited control (see Table B.4 for pairwise comparisons).

3.4 Discussion

Visual cues are important in the life history of O. fagi, as there were distinct preferences for certain colours as well as differences between the sexes. Males showed an affinity towards blue and green, indicating visual acuity between ~440 nm–600 nm

(Figure 3.2), consistent with insects in several other orders (Briscoe and Chittka 2001).

Male O. fagi may use trichromatic vision but this cannot be confirmed because we did not test their response to UV. Based on the findings of this study, and the fact that trichromatic vision is conserved across several insect orders (see reviews in Brisco and

Chittka 2001; Stavenga 2002; and Reeves 2011) I hypothesize that male O. fagi possess trichromatic vision.

Colour vision in beetles is poorly understood. Hausmann et al. (2004) provided some of the first evidence for colour detection in the apple blossom weevil, Anthonomus pomorum L. (Coleoptera: Curculionidae). When presented a colour choice vs. black control, female A. pomorum significantly preferred blue, green, and UV; males were only significantly attracted to UV. However, when given a choice between blue, green, or UV vs. a single opposing colour, male A. pomorum displayed significant attraction to blue in addition to UV indicating similar visual potential in both sexes. This provides support for specific colour preference as found in O. fagi, with males exhibiting potential trichromancy. Female O. fagi provide contrary results, being attracted to red

(wavelengths > 600 nm). Red receptors have developed independently in a number of insect orders (reviewed by Briscoe and Chittka 2001) including: the ruby meadowhawk,

Symptetrum rubicundulum Say (Odonata: Libellulidae; Meinertzhagen et al. 1983); tau emerald, Hemicordulia tau Selys (Odonata: Coculiidae; Yang and Osorio 1991); bronze carabid, Carabus nemoralis O.F. Müller and golden ground beetle, C. auratus L.

(Coleoptera: Carabidae; Hasselmann 1962); orchard swallowtail, Papilio aegus Donovan

(Lepidoptera: Papilionidae; Matic 1983); cabbage butterfly, Pieris brassicae L.

(Lepidoptera: Pieridae; Steiner et al. 1987); and comma, Polygonia c-album

(Lepidoptera: Nymphalidae; Eguchi et al. 1982). The development of red photo-receptors is difficult to understand though is often linked with host location (via flowers; reviewed by Briscoe and Chittka 2001). Attraction to red as seen in female O. fagi does not necessarily provide evidence for red photo-receptors as red light (up 650 nm) can stimulate green photo-receptors (~540 nm) provided sufficient light intensity causing insects to „see‟ red as green (Chittka and Waser 1997; Rodrígeuz-Gironés and Santamaría

2004). It is especially curious, then, that female O. fagi were not significantly attracted to green and thus red attraction may be evolutionarily adaptive however the cause and effect are currently unknown. The attraction to green, as seen in male O. fagi, is often associated with host location (Figure 3.1) which may provide evidence for the importance of males in initial host location and pioneering.

Attraction to white was also apparent in both sexes. Whether this was residual attraction due to the properties of „white‟ (i.e. containing all colours) or reflectance of adjacent colours is unknown. Hunt and Raffa (1991) observed preferential attraction to white traps in nocturnal Hylobius pales Herbst and Pachylobius picivorus Germar

(Coleoptera: Curculionidae), and suggested it may have been due to greater contrast of white vs. other colors tested for night-time host location.

Although colour vision has not been documented in many species of weevil, visual orientation to hosts is apparent. The aquatic milfoil weevil, Eurhychiopsis lecontei

Dietz (Coleoptera: Curculionidae) distinguished between different plant stems and locate its preferred host in the absence of chemical stimuli (Reeves and Lorch 2009). Hylobius warreni exhibits host-finding behaviour reliant on visual cues at short ranges, being attracted to cardboard cutouts in the shape of the trunk, crown, or tree of its host (Machial et al. 2012). This attraction was not enhanced or deterred by changing cue colour indicating highly specific shape detection in this species. Similarly, white pine weevil,

Pissodes strobe Peck (Coleoptera: Curculionidae), and the Chrysomelid Altica engstroemi Sahlberg (Coleoptera: Chrysomelidae) were found to be preferentially attracted to certain host shapes and silhouettes (VanderSar and Bordern 1977; Stenberg and Ericson 2007).

Visual cues should not be overlooked in future examination of IPM systems for

O. fagi. The species exhibits potential trichromatic vision in at least one of the sexes indicating specific visual acuities, particularly for wavelengths between 450–550 nm.

Further examination of the effect of host shape and colour on this species will be important in the creation of an effective monitoring and trapping system.

The likelihood of using visual stimuli in host-finding leads to an important question on the legitimacy of behavioural experiments in which visual stimuli are limited

(e.g. Y-tube olfactometer assays). In the Odonates Libellula depressa L. (Odonata:

Libellulidae) and Ischnura elegans Vander Linden (Odonata: Coenagrionidae),

olfactometer trials were enhanced by the presence of both olfactory and visual stimuli indicating that the systems are likely linked in prey detection and behavioural response even in insects with rudimentary olfactory capabilities (Piersanti et al. 2014a; Piersanti et al. 2014b). This may account in part for the relative low response levels of O. fagi in a Y- tube olfactometer assay.

Insect response to host plant tissues in a Y-tube olfactometer is of particular importance and interest. Host tissues elicit an attractive response from O. fagi, and the physiological state of the adult affects its behaviour. Females of both the parent generation (overwintered) and offspring generation (not yet overwintered) were significantly attracted to host leaves but neither generation was attracted to host wood.

However, the attraction to leaves was greater in females that had overwintered (i.e. were not in reproductive diapause); females which had not overwintered and were not sexually mature were not attracted to leaves unless they had been starved for 24 hr. Such an interaction is not unexpected from the ecology of this species, migrating away from beech after pupal eclosion (Bale 1981). Reliance on host tissues in the latter part of the season was expected to be diminished for two reasons: (1) previous observations describe feeding on alternative herbaceous plants during this part of the life cycle (Dieter 1964;

Bale and Luff 1978); and (2) leaf tissues of F. grandifolia are highly lignified in the late season and are thus difficult to feed upon (Phillipson and Thompson 1983). Here is presented evidence for the attraction and likely consumption of lignified host leaf tissue in times of starvation (i.e. if no alternate food source is available before entering diapause). The alternate host plant – O. fagi interaction in North America is currently unknown, though current attempts to initiate feeding on previously documented alternate

hosts has been unsuccessful (Moise et al. 2015). It is further important to note that this trend was seen only in adult females; male attraction to host tissues must occur by another means.

Male attraction to host material could be synergized by female presence on such material as in many Lepidoptera species but this was not supported by data from the olfactometer bioassay, where males did not respond to the stimulus of females on a bursting bud (Figure 2.6, Chapter 2) nor by the trapping experiments in which blends of

O. fagi cuticular hydrocarbons were unattractive when presented alone or combined with beech leaf volatiles. This, however, does not rule out active release of pheromone by either sex (i.e. calling behaviour). Contact and release pheromones have been documented in a number of Curculionid species including: female-produced pheromone in the sweet potato weevil, Cylas formicarius Fabricius (Brentidae: Cyladinae; Jansson et al. 1992); male-produced aggregation pheromone of the palm weevil, Rhynchophorus palmarus L. (Rhynchophorinae: Rhynchophorini; Oehlschlager et al. 1993) and the

Brazilian soybean stalk weevil, Sternechus subsignatus Boehman (Molytinae: Molytini;

Ambrogi and Zarbin 2008; Ambrogi et al. 2012); female-produced pheromone of the raspberry weevil, Aegorhinus superciliosus Guérin-Méneville (Cyclominae: Aterpini;

Mutis et al. 2009); and cuticular compounds of the rice water weevil, Oryzophagus oryzae Costa Lima (Erirhininae: Stenopelmini; Martins et al. 2013). Whether or not either male or female O. fagi produce a pheromone requires further study.

Male attraction to host material may be elicited through the presence of geranyl-p- cymene, a compound present in low quantities in beech bud emissions (Tollsten and

Müller 1996; Dindorf et al. 2006). Geranyl-p-cymene was the only stimulus to elicit a

behavioural response from male O. fagi, of which only adults who had undergone overwintering, obligate diapause, and sexual maturation responded. As such, this compound may be a behaviourally relevant attractant for mature male O. fagi to find host material and may thus represent an opportunity for use in future IPM studies. The use of this compound in host location and oviposition stimulation has been documented in the coffee leaf miner, Leucoptera coffeella (Lepidoptera: Lyonetiidae), a species with a similar life history (Magalhães et al. 2008).

Two additional compounds may be of interest for future analysis on behavioural response. These are the compounds eliciting immature (i.e. pre-overwintered) adult female activity: (Z)-3-hexenyl acetate and (Z, E)-α-farnesene. It is difficult to assess the importance of response to (Z)-3-hexenyl acetate, as this compound is produced almost ubiquitously by plants and is thus detected by almost all phytophagous insects. Attraction to this compound is often linked with attraction to damaged leaf material as this is one of the primary damage-induced volatile organic compounds (VOCs). It is known that there is little to no separation of O. fagi generations, with larvae and adults feeding concurrently on the same host (Day and Watt 1989; Watt and McFarlane 1992).

Attraction to (Z)-3-hexenyl acetate, then, may provide evidence for the positive attraction of female O. fagi toward host tissue via conspecific feeding. The latter compound, (Z, E)-

α-farnesene elicited less attraction than the hexane control. Such a response in a Y-tube olfactometer is curious, as it cannot be said to be a truly negative (i.e. repulsive) effect due to the nature of the assay (initial movement toward the compound through the central tube is required for a choice to be made). Thus, while (Z, E)-α-farnesene may or may not

have a repulsive effect, it can be stated with certainty that it is not an attractant for immature adult females.

While Y-tube olfactometer studies may have provided evidence of O. fagi behavioural patterns, little information can be derived from trapping experiments conducted to date. No compound blends tested influenced mean trap catch, with most tested compounds having a negative trend as compared to an unbaited control in 2014 and a slight positive trend in 2015. Future field studies should account for information provided throughout this chapter: (1) the importance of visual stimuli in attraction, namely trap colour ranging between 450–550 nm (shape discrimination by O. fagi is still unknown); (2) attraction to F. grandifolia may diminish in the late season; (3) the presence or absence of O. fagi pheromone is still unknown; (4) geranyl-p-cymene and

(Z)-3-hexenyl acetate elicit attractive behavioural responses in males and females respectively in a laboratory setting. The integration of visual and olfactory stimuli will be an important step in the creation and enhancement of an IPM strategy for the highly damaging O. fagi in North America.

3.5 Figures and Tables

Figure 3.2 Percent reflectance of Creatology™ foam assessed in comparison to a sheet of white paper control using a spectrometer. Image courtesy of Catherine Little (Memorial University).

Figure 3.3 Colour-tube choice assay for Orchestes fagi L.. Left: interior of the tube with 6 colour choices and band of sticky material along upper edge. Right: Complete experimental set-up.

Figure 3.5 Precent response of pre-overwintered Orchestes fagi L. (female and male) to stimuli presented in a Y-tube olfactometer bioassay. N = 20 positive responses; NR = Non-responders.

Figure 3.6 Percent response of post-overwintered Orchestes fagi L. (female and male) to stimuli presented in a Y-tube olfactometer bioassay. N = 20 positive responses; NR = Non-responders.

a a b

Figure 3.7 Number of adult Orchestes fagi L. captures on yellow sticky cards baited with various lure treatments in field experiments conducted at Ashburn Golf Club and Sandy Lake, NS in 2014. Experiment 1: May 2014; LB = leaf blend; WB = weevil blend. Experiment 2: June 2014; LB2 = new leaf blend. Experiment 3: July 2014; LB3 = 8:1 blend of β-caryophellene and (Z)-3-hexenyl acetate. Letters „a‟ and „b‟ indicate significant differences between mean control trap catch. Boxes indicate median value with upper and lower quartiles; whiskers indicate maximum and minimum values; · indicates outliers.

Table 3.1 Chemical composition of beech leaf blend lures and weevil cuticular hydrocarbon lures used in Orchestes fagi L. trapping bioassay 3 conducted near Halifax, Nova Scotia in 2014.

Lure Chemical* Load/ lure(μl pure) Leaf blend (pouches) (Z)-3-hexen-1-ol 100 (Z)-3-hexenyl acetate 100 (Z)-3-hexenyl acetate-butyrate (50/50) 250 (Z)-3-hexenyl acetate-isobutyrate 250 β-caryophellene 500 Weevil blend (septa) Z11-16Ald** 50 µg ethyl oleate 50 µg ethyl linoleate 50 µg ethyl linolenate 50µg * From Sigma Aldrich, used as received ** (Z)-11-hexadecenal; Bedoukian Research, >97% pure.

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CHAPTER 4.0 A NOTE ON A NOVEL ELECTROANTENNOGRAM METHOD FOR ORCHESTES FAGI L.

4.1 Introduction

Electroantennography (EAG) is a useful technique for identifying compounds detectable by the antennae of an insect, and their relative sensitivity (Schneider 1957). An electrophysiological response from the organism can be recorded and measured using a variety of insect or antennal preparations and software. All preparations require a specimen to be connected at one end to a ground or reference electrode and to a recording electrode at the other end. Electrical (i.e. neural) activity in the antennae in response to stimuli is amplified, filtered, and recorded by a computer. The most commonly used antennal preparation is a single excised antenna (Figure 4.1a). This method has been used in many orders of insects, and has been widely used for assaying Lepidoptera (DeSilva et al. 2013; Ma et al. 2015; Molnár et al. 2015), Diptera (Chen et al. 2015), Hymenoptera

(Villar et al. 2015; Artz and Pitts-Singer 2015), and Coleoptera (Dickens and Boldt 1985;

Hansen et al. 2015).

Although a simple and relatively consistent technique, the act of severing the antennal nerve may influence detection of compounds observed through the EAG. Hillier and Vickers (2011) found a reduction in spontaneous sensillar nerve firing in transected antennae as well as significantly lower responses to Z3-6:OH and significantly higher responses to Z3-6:OAc and ß-caryophellene. Due to such variation, some preparations opt to mount the entire head containing both antennae and an intact antennal nerve

(Figure 4.1b; Twidle et al. 2015). Additionally, some researchers opt to mount the entire body of the insect, allowing preparations to remain alive for long periods of time (Figure

4.1c; Karremans et al. 2015; Wan et al. 2015). It is important to note that any of the

above three preparation types may have intact or severed antennae. A severed antenna, e.g., the tip of the antenna has been cut away, may permit greater contact with antennal haemolymph and thus improve the conductivity between probes and specimen but may also have reduced response due to loss of some sensilla. Leaving the head or body of the insect intact may increase the background noise due to muscle movements which may obscure or mask olfactory responses to test compounds in the oscillogram (Schneider

1957).

EAGs have been performed successfully on a variety of Curculionid species

(Table 4.1) but none have been completed on Orchestes fagi L. One of the principal challenges in this species is its small size (~2.2–2.8 mm). Attempts to use previously documented EAG techniques on O. fagi individuals gave unreliable and inconsistent results. Here is presented a novel EAG preparation method for this species.

4.2 Materials and methods

Adult O. fagi were inserted head first into the cut tip of a 100 µL plastic pipette tip until the head was exposed, lodging the pronotum and thus limiting movement of the beetle. A piece of Kimwipe® or cotton was inserted behind the beetle, blocking the open pipette tip and securing the beetle in place. The preparation was then mounted on a glass microscope slide using dental wax. A glass microscope cover slip was broken and a small shard (~ 1 mm x 4 mm) was placed on the wax under the exposed head of the beetle.

Using forceps, both antennae were secured to the shard of coverslip using dental wax, ensuring that the club remained unencumbered (Figure 4.2A).

Due to poor conductivity in previous preparations attempted for O. fagi, a hole

(~2 µm in diameter) was cut in the tip of the club using a modified cut sensillum

technique (Hillier et al. 2006; Hillier and Kavanagh 2015; MacKay et al. 2015). A borosilicate capillary tube (1.0 mm x 0.5 mm; World Precision Instruments, Inc.,

Sarasota, FL, USA) was pulled to a 0.06 µm tip diameter with a 9–11 mm taper using a

Co P-97 Flaming/Brown Micropipette Puller (Program 0: ramp 525, pull 150, velocity

75, time 250, pressure 500; Sutter Instruments, Novato, CA). The capillary was mounted on a piezoelectric crystal using a small amount of dental wax. The piezo-crystal was controlled using a manual switch attached to a function generator (INSTEK© GOS-

620FG) and was placed directly adjacent to the distal segment of the antennal club using a manual micromanipulator (Figure 4.2B; World Precision Instruments, Sarasota, FL).

The capillary was made to resonate at high speed by alternating square wave sound frequencies (100K signal amplification) from the generator through the piezo-crystal, effectively creating a „piezo-electric saw‟. Micromanipulation of the saw toward the antenna allowed for precise removal of small amounts of cuticle.

EAG preparation was adapted from Ranger et al. (2012) due to the similarity in adult length (Table 4.1) and Leskey et al. (2009); forked probes were also attempted according to Keesey et al. (2012) and partly to Kendra et al. (2012), however this preparation did not yield consistently successful results. In preparation for mounting, the head of the insect was removed; ensuring the antennae remained intact, attached to the head, and embedded in wax. A 0.15 mm x 12 mm stainless steel insect pin (Minucie No.

15; Ento Sphinx®, Czech Republic) was embedded in wax along the proximal edge of the antennal club to secure the club in place (Figure 4.2C). Additional borosilicate capillaries were pulled to a 1 µm tip diameter with a 3–4 mm taper (Program 1: ramp

515, pull 0, velocity 30, delay 1, pressure 500), filled with saline with sucrose solution

(Insect Ringer; Kaissling 1974), and mounted over an Ag/AgCl wire for use as a reference electrode. The foramen of the head was attached to the reference electrode using conductive Signagel® (Parker Laboratories Inc., Fairfield, NJ). A portion of tungsten wire sharpened with KCl to an approximate tip diameter of 1 µm and rinsed with distilled water was used as a recording electrode. The recording electrode was mounted directly to a headstage probe through which the signal was amplified by an

IDAC-2® amplifier and data acquisition system (Syntech, Kirchzarten, Germany) and recorded using Data acquisition for Gas Chromatograph with EAD software (GcEad

2014 v.1.2.5, Syntech). The tungsten electrode was coated lightly in Signagel® and inserted directly into the incised hole in the antennal club (Figure 4.2D). The entire preparation was placed 10 mm from a continuous humidified air stream.

Electrophysiological results using this method are presented in Chapter 5.0. This provides the first evidence for the combined use of glass and tungsten electrodes in EAG preparation as a means to cope with small antennae which are difficult to manipulate using previously described techniques.

4.3 Figures and tables

Orchestes fagi Orchestes

attempted due to cost constraints cost to complexity. due and attempted

List of successfully tested electroantennogram preparations for special with of for consideration curculionidpreparations species tested electroantennogram successfully of List

*Preparation method not not method *Preparation adult body length. All preparations shown failed shown reliable to results All for preparations length. produced body adult Table

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CHAPTER 5.0 CHEMICAL ANALYSES AND ELECTROPHYSIOLOGY

5.1 Introduction

Virtually all plant species produce volatile organic compounds (VOCs) at some stage of development as a means of communication and protection (Fall et al. 1999; Tholl et al. 2006). Many studies have examined volatile profiles produced by a broad range of plant species as indicators of how plants interact with their environment, and of their metabolic and biosynthetic capabilities (Schuh et al. 1997; Fall et al. 1999). VOCs exhibit a wide range of chemical structures; however broad-leaved plants tend to produce terpenoid compounds in the highest volume followed by alcohols and esters (König et al.

1995; Tollsten and Müller 1996; Schuh et al. 1997; Dindorf et al. 2006). Most green plants also produce (Z)-3-hexen-1-ol and (Z)-3-hexenyl acetate, referred to as „green leaf alcohol‟ and „green leaf ester‟, respectively, due to their ubiquity in plants (reviewed by

Kesselmeir and Staudt 1999). VOC emission rates are highly variable between and within species and can be affected by factors including: developmental stage; plant physiology; light availability/intensity; temperature; time of day; wounding or infection; and water availability (König et al. 1995; Tollsten and Müller 1996; Schuh et al. 1997; Fall et al.

1999; Dindorf et al. 2006; Tholl et al. 2006). VOCs are commonly used for intra- and interspecies communication, and are often emitted because of wounding. Damage- induced volatiles can stimulate neighbouring plant conspecifics to produce protective compounds such as jasmonic acid and methyl jasmonate to act as antifeedants against herbivore or phytophagous insects (McCall et al. 1994; Engelberth et al. 2004).

Tritrophic interactions can also occur as predators and parasitoids detect these plant- produced wounding VOCs and co-opt them in the location of their prey or host species

(Dicke and Sabelis 1988). VOCs thus create a complex and fluid interaction between a plant and its environment, conspecifics, predators, and higher level fauna (Tollsten and

Müller 1996).

The role of plant-produced VOCs in host-location by pest species has been discussed in Chapter 3.0. It remains unclear how the highly invasive Orchestes fagi L. locates its host, Fagus grandifolia Ehrh., and which, if any, chemical cues are important in this process. Studies on beech volatiles to date have focused on European beech, Fagus sylvatica L., and little is known of the relationship between volatile profiles of this species and its American counterpart. The purpose of this chapter is to examine the volatile profiles produced by F. grandifolia based on the known profiles of its sister species F. sylvatica with a focus on development over the leaf-bearing period. These will be used to examine the electrophysiological sensitivity of O. fagi to compounds present in these profiles. In this chapter, I attempt to connect the phenological data of O. fagi gathered in Chapters 2.0 and 3.0 to phenological changes in its host. This can help narrow the scope of further research into how O. fagi locate their hosts and how that process may be co-opted or interrupted for management purposes, e.g., attractants as lures for survey and detection.

5.2 Methods

5.2.1 Volatile collection from Fagus grandifolia Ehrh.

Stages of development were selected based on visual and temporal characteristics:

(1) closed and elongated bud; (2) newly bursting bud (i.e. leaves beginning to show, scales still intact); (3) newly emerged leaves (~1–2 weeks old); (4) fully developed leaves

(>1 month old); (5) senescing leaves (i.e. leaves beginning to change colour and/or wilt,

>4 months old; Figure 5.1). All volatile collections were completed within the normal blooming season of 2016. Branch samples of F. grandifolia unifested with O. fagi and containing no fewer than 20 healthy and undamaged leaves were harvested from the K.C.

Irving Environmental Science Centre (KCIC) Botanical Gardens, Acadia University,

Wolfville, NS (45°05‟12.5”N, 64°22‟0.58”W) and the cut ends were immediately submerged in reverse osmosis (RO) water to reduce wound impact on volatile emissions

(Fall et al. 1999). Branch samples were brought to an onsite phytotron (KCIC, Acadia

University, Wolfville, NS, Canada) and maintained at 21°C and 75% relative humidity

(RH). VOCs from different stages of beech leaf development were collected using dynamic headspace trapping (solid phase extraction) and analyzed using gas chromatography (GC) coupled with a flame ionization detector (FID) or a mass spectrometer (MS). Samples were individually inserted into a 45 x 55 cm Look® Oven

Bag (Terinex Ltd., Bedford, England) and sealed around the lower branch. Volatile collection technique was adapted from Tollsten and Müller (1996). Charcoal filtered air was pumped into the headspace at a rate of 65 mL min-1 using a Portable Volatile Assay

System (PVAS22, VAS volatile assay systems, Rensselaer, NY; Figure 5.2). The air was then passed to the outlet and over a new solid adsorbent volatile trap (HayeSep® Q porous polymer adsorbent, Sigma-Aldrich) which had been rinsed with hexane and baked at 200°C for 2 hours to remove contaminants. The total collection period was over the course of 4 hours between 9:00 – 16:00 hrs. for a total collection volume of approximately 15.6 L. Collected compounds were eluted from the traps using 1 mL of hexane run through the trap in 150 µL aliquots under a pressure of non-reactive, ultra high purity N2. Samples were frozen until use in electrophysiological recordings with

GC-FID; and chemical identification with GC-MS. Samples were concentrated to 1 µL under a constant stream of N2 gas immediately prior to analyses.

5.2.2 Animals

All O. fagi used in electroantennal detection (EAD) trials were collected by branch beating from AGC and maintained at 5-10°C and 80% RH in the dark. Sugar water was provided ad libitum. All collections for GC-EAD trials were conducted in

August – September 2016 and thus specimens were considered pre-overwintered. Upon collection, sexes were separated based on external characteristics (see Chapter 2.0).

Individuals were maintained at ambient room temperature (18–21°C) at 10:14 light:dark cycle for 24 hrs prior to use in assays.

5.2.3 Electrophysiological responses to F. grandifolia Ehrh. volatiles

EAD recordings were completed coupled with a flame ionization detection Varian

450-gas chromatogram (GC-FID; Agilent Technologies Inc., Santa Clara, CA, USA) using a Stabilwax® Crossband® Carbowax® polyethylene glycol column (30 m x 0.25 mm x 0.25 µm; Chromatographic Specialties, Inc., Brockville, ON, Canada). One microlitre samples of all five stages of beech development were run independently according to methods adapted from Tollsten and Müller (1996), Moukhtar et al. (2005), and Molnár et al. (2015). The oven temperature was held at 50°C for 3 mins; increased at

4°C min-1 to 150°C and held for 10 mins; and then increased by 35°C min-1 to 220°C and held for 10 mins. Samples were injected into a split/splitless injector port at 220°C with the split closed for 1 min then at a split ratio of 20. Effluent was forced through the column using helium as carrier gas to a glass two-way splitter where it was split equally to the flame ionization detector at 300°C and into a heated EAD transfer line at 220°C

(Syntech, Kirchzarten, Germany). EAD effluent was delivered directly into a stream of charcoal-filtered humidified air directed at the antennal preparation. GC-FID signals were recorded simultaneously using the Galaxie Chromatography Data System v. 1.9.302.952

(Agilent Technologies Inc., Santa Clara, CA) and expressed as µV time-1.

Antennal preparation of adult O. fagi and EAD data acquisition was completed according to Chapter 4.0. Antennal responses and GC-FID signals were recorded simultaneously as µV amplitudes (Figure 5.3).

5.2.4 Chemical isolation and identification

Compounds detected by the GC-FID were identified by comparison with external standards of known volatiles found in F. sylvatica leaves run on the same method (Table

5.1). Peaks in GC-EAD recordings eluting at the same time as these standards were deemed to be these known compounds. Hexane blanks were run on identical methods to account for contamination.

Additionally, samples of stage 5 beech leaves were run on a Scion 456 GC with

SQ Mass Spectrometer (GC-MS; Bruker Daltonics Ltd., Coventry, UK) using the same

GC-method detailed above. First and last masses scanned were 60–350. This was used to confirm known compounds which elicited antennal responses. Compounds were identified by comparison with the National Institute of Standards and Technology (NIST) mass spectral library (version 2.0, USA) and external standard.

5.2.5 Data analyses

GC-FID peaks which corresponded with external standard retention times were recorded as µV (= elution amplitude). The mean of each amplitude was corrected using

hexane blanks. Corrected means were converted to a proportion of known F. sylvatica compounds based on those presented in the literature.

EAD responses were recorded as mV at each known retention time. Additional antennal responses were considered whenever the mV exceeded the normal fluctuations of the baseline (e.g. > 3 mV) and also at retention times at which there had previously been a response by a conspecific. Statistical analyses were completed using R v. 3.1.2 (R

Core Team 2015). Known compounds were analysed by two-way ANOVA (generalized linear model method, α = 0.05, loglink, family = Poisson) to assess the effect of compound and sex on amplitude (i.e. mV) of response. No compound X sex interaction was found and sexes were subsequently separated and one-way ANOVA was used to assess the effect of compound on amplitude of response. Amplitudes of response to unknown compounds were analysed as above.

5.3 Results

5.3.1 Volatile profiles of Fagus grandifolia Ehrh. at five developmental stages

All previously documented compounds from F. sylvatica were seen in at least one stage of F. grandifolia development, with the exception of germacrene D (Table 5.1). It is clear that volatile profiles change during phenological development and, importantly, over the lignification period. Closed, elongating buds (Stage 1) emitted high levels of (+)-

3-carene, α-terpinene, and geranyl-p-cymene, all three of which diminished to negligible levels in subsequent stages of development. ß-caryophellene was emitted in the highest quantity at stage 1 compared to other stages of development. Bursting buds (Stage 2) transitioned from primarily ß-caryophellene emitters to sabinene emitters along with increases in the pinenes and R-(+)-limonene. Stage 3 leaves emitted methyl jasmonate in

the highest quantity along with decreases in sabinene and R-(+)-limonene emissions, and increases in (Z)-3-hexenyl acetate and ß-caryophellene. Stage 4 (i.e. mature leaves) emitted sabinene in the highest quantity of any stage of development. Myrcene was also emitted in its largest quantity during this stage of development, though still at low levels relative to other identified compounds. This stage was thus characterized by the emission of sabinene, (Z)-3-hexenyl acetate, and methyl jasmonate. Stage 5 leaves displayed distinct differences from stage 4. While retaining high levels of sabinene emissions, stage

5 leaves had a resurgence in emission rates of α-terpinene and geranyl-p-cymene, which were also prevalent in stage 1 (elongating buds). Additionally, the leaf alcohol and leaf ester, (Z)-3-hexen-1-ol and (Z)-3-hexenyl acetate respectively, and the pinenes were emitted in the largest quantities in this late stage of development. Each stage of development has a distinct volatile profile characterized mainly by the emission of four primary VOCs: ß-caryophellene in stage 1; sabinene in stages 2 and 4; methyl jasmonate in stage 3; and (Z)-3-hexenyl acetate in stage 5 (Table 5.2).

5.3.2 Electrophysiological responses to Fagus grandifolia Ehrh. volatiles

Male and female O. fagi responded differentially to known F. grandifolia volatiles with females tending to have greater responses (P < 0.05; Figure 5.4). No compounds elicited significantly higher responses than others in males or females (F17, 62)

= 0.58, P = 0.89; F17, 78 = 0.563, P = 0.92 respectively). Males responded to all presented compounds except linalool and a trend toward larger antennal responses to myrcene, R-

(+)-limonene, and (Z)-3-hexenyl acetate was observed. Females responded to all presented compounds except R-(+)-limonene and a trend toward larger antennal

responses to germacrene D, α-terpineol, (Z)-3-hexenyl acetate, geranyl-p-cymene, and sabinene was observed.

5.3.3 Presence of unknown compounds

Several compounds were found in the chromatograms of F. grandifolia developmental stages which did not match any known F. sylvatica VOCs. Some of these were antennally active for O. fagi, producing significant responses between retention times of 22.30–23.28 minutes (Table 5.3). Identification of these compounds was not feasible over the course of this study.

5.4 Discussion

5.4.1 Volatile profiles of Fagus grandifolia Ehrh. at five developmental stages

Each developmental stage of F. grandifolia was marked by distinct changes in volatile profiles. Nothing has been published on the developmental VOCs of F. grandifolia or F. sylvatica to date and thus comparisons can only be made to single life stage profiles. It was assumed, therefore, that published research on beech leaf volatiles were done with mature leaves (i.e. stage 4) as developmental stage was often not stated.

Further evidence for this assumption is that previous studies found sabinene to be the major VOC emitted by F. sylvatica, comprising between 74–94% of total emissions

(König et al. 1995; Tollsten and Müller 1996; Schuh et al. 1997; Moukhtar et al. 2005;

Dindorf et al. 2006) and such high levels of sabinene were observed only in mature leaves in F. grandifolia. Relative concentration of sabinene fluctuated between other developmental stages and was completely absent from the elongating bud stage (i.e. stage

1). Stage 3 F. grandifolia leaves emitted less sabinene than any other post-flush stage.

This stage is also marked by a distinct increase in methyl jasmonate and (Z)-3-hexenyl

acetate, both of which are common damage-induced volatiles (McCall et al. 1994; Fall et al. 1999; Engelberth et al. 2004; see review by Keisselmeier and Staudt 1999 for more information). Young post-flush leaves, characteristic of stage 3, have yet to undergo lignification and are thus highly targetable by herbivores and phytophagous insects which would cause the production of such volatiles through feeding damage (Nielsen 1974;

Nielsen 1978). However, Fall et al. (1999) found that hexenal wound-induced VOCs were emitted from pools within the leaf tissue and thus localized to damage sites (i.e. these VOCs were not produced systemically) in F. sylvatica and the European aspen,

Populus tremula (Malpighiales: Salicaceae). Since care was taken to ensure only non- damaged leaves were sampled for volatile collection in this study, such an explanation is unlikely. Terpenoid emissions are induced by key enzymatic activities which in turn are highly correlated with developmental stage and rate of biosynthesis (Schuh et al. 1997;

Fall et al. 1999). It is unsurprising, then, that sabinene emissions display such dramatic fluctuations over development stages but whether there is an additional cause for the degree of fluctuation cannot be concluded.

ß-caryophellene emissions were high in stages 1–3. ß-caryophellene is a common sesquiterpene in plants and has been found to have both antimicrobial and antioxidant properties in nutmeg, Myristica fragrans Houtt (Magnoliales: Myristicaceae; Gupta et al.

2013). The large emissions of ß-caryophellene seen in this study may therefore be systemic responses to wounding either by herbivore damage or by the experimental design of branch sampling. To assess this, future studies should be conducted on F. grandifolia in natural conditions or sample trees grown in a greenhouse (however greenhouse-grown plants often emit low-levels of VOCs and can have completely

different volatile profiles; König et al. 1995). Dindorf et al. (2006) point out that F. sylvatica volatile profiles can fluctuate depending upon any number of factors including seasonality, drought, temperature, light, damage, and growth conditions. Thus, collections under „natural conditions‟ may be difficult and the results presented in this study represent a good basis for the types and relative volumes of VOC emissions by F. grandifolia foliage over one season of development.

5.4.2 Electrophysiological responses to Fagus grandifolia volatiles

Antennal responses gathered using a new insect preparation technique (see

Chapter 4.0) confirmed differential sex-biased sensitivity to F. grandifolia volatiles previously documented in behavioural trials by Pawlowski (2014). Females tend to have larger antennal responses to known host volatiles indicating a greater sensitivity to these compounds than their male conspecifics. Leskey et al. (2009) also found differing antennal responses between the sexes in the plum curculio, Conotrachelus nenuphar

Herbst (Coleoptera: Curculionidae), however this result is uncommon among other weevils. Sex-specific behavioural responses are common in the Curculionidae and have been seen in the boll weevil, Anthonomus grandis Bohman (Curculioninae; Dickens et al.

1991); the vine weevil, Otiorhynchus sulcatus Fabricius (Entiminae; van Tol and Visser

2002); the raspberry weevil, Aegorhinus superciliosus Guérin-Méneville (Cyclominae;

Mutis et al. 2010); the grey corn weevil, Tanymecus (Episomecus) dilaticollis Gyllenhal

(Entiminae; Toshova et al. 2010); and the osier weevil, Cryptorhynchus lapathi L.

(Cryptorhynchinae; Cao et al. 2015) to name a few. All of the above-mentioned weevils displayed no sex-specific antennal sensitivities to presented compounds through EAD or

EAG. Considering this, it is not surprising that male and female O. fagi display different

behavioural responses to host tissue and VOCs in a Y-tube olfactometer. The sex-specific electrophysiological responses found in this study provide evidence that such behavioural traits are founded in neurological responses. O. fagi males showed sex-specific attraction to geranyl-p-cymene and bursting F. grandifolia buds while females were attracted to adult F. grandifolia leaves (see Chapters 2.0 and 3.0). Unlike most weevils, O. fagi have sex-specific electrophysiological responses to host VOCs and thus future lure development must account for such specificity.

(Z)-3-hexenyl acetate elicited large antennal responses in both sexes. Similar antennal responses to (Z)-3-hexenyl acetate were seen in the cranberry weevil,

Anthonomus musculus Say, which corresponded to a repellent behavioural response in field trials (Szendrei et al. 2009; Szendrei et al. 2011). However, the reverse was true for

O. fagi as we observed (Z)-3-hexenyl acetate was significantly attractive to females in Y- tube olfactometer bioassays (Chapter 3.0). Attraction to (Z)-3-hexenyl acetate is common in other insects when used synergistically with pheromone and/or additional plant VOCs and has been seen in A. grandis (Dickens 1989); Colorado potato weevil, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae; Visser 1986); scarab beetle, Anomala octiescostata Burmeister (Coleoptera: Scarabaeidae; Leal et al. 1994); and diamondback moth, Plutella xyllostella L. (Lepidoptera: Plutellidae; Reddy and Guerrero 2000).

Antennal sensitivity and behavioural attraction likely indicate use of this compound in locating host material. Given the ubiquity of this compound in volatile profiles of green plants, it is further probable that (Z)-3-hexenyl acetate is used by O. fagi in phase 1 of host acceptance as dictated by the „appropriate/inappropriate landings‟ theory (see

Chapter 3.0 for more information).

R-(+)-limonene was among the compounds which elicited the greatest antennal response from male O. fagi while eliciting no response from females. This is of interest given its abundance in stage 2 F. grandifolia leaves and comparatively low emissions during other developmental stages. For these reasons, it is hypothesized that R-(+)- limonene plays a role in the attraction of male O. fagi and the surprising lack of response of females to bursting beech buds in olfactometer bioassays (Chapter 2.0). Similar results have been documented for A. superciliosus, where both males and females show significant attraction to R-(+)-limonene in field and EAG trials (Mutis et al. 2010).

Though no evidence can be confirmed, this could indicate a role of males in host location

(i.e. pioneering).

Results concerning geranyl-p-cymene are conflicting. Emission of this compound during the elongating bud stage (1) was higher than in any other stage of development, and occurred during the peak time for host location by the weevil. Behavioural responses by male O. fagi to geranyl-p-cymene in the olfactometer indicate it was attractive (see

Chapter 3.0) but the compound elicited minimal antennal response from males. Similar antennal responses to geranyl-p-cymene have been documented in the red palm weevil,

Rhyncophorus ferrugineus Olivier (Coleoptera: Curculiondae), however there has been no study on its behavioural implications (Guarino et al. 2012). Sabinene is also of interest given its abundance in emissions with no male antennal response. This, however, is not surprising as male host finding occurs in early F. grandifolia leaf development (i.e. pre- stage 3) and thus may hinge on other chemical cues (see above).

5.4.3 Presence of unknown compounds

Many compounds elicited antennal responses from O. fagi which were not among those selected as standards for this chapter (Table 5.3). Future identification of these unknowns will provide a more thorough understanding of both F. grandifolia VOC profiles in comparison with its sister F. sylvatica and the interaction between these profiles and O. fagi life history.

5.5 Figures and Tables

FID. FID.

L. Presence or absence absence Presence of or L.

Fagus sylvatica Fagus

g of of standard external g

Ehrh., at five developmental stages assessed with GC with stages assessed developmental five at Ehrh.,

Fagusgrandifolia

samples was assessed by comparison to retention times 1 of assessed to comparison by retention was samples

VOCs produced by American beech, beech, American produced by VOCs

F. grandifolia F.

Table Table VOCs in in VOCs European by VOCs on were beech, produced based for analysis Compoundsselected

*F. sylvatica mean percent total emissions from literature. Not all VOCs are represented due to incompatibility of literature data. Notes: Multiple retention times indicate multiple peaks in external standard. References: (1) König et al. 1995; (2) Tollsten and Müller 1996; (3) Schuh et al. 1997; (4) Fall et al. 1999; (5) Engelberth et al. 2004; (6) Moukhtar et al. 2005; (7) Dindorf et al. 2006; (8) Gossner et al. 2014.

Table 5.2 Ranked VOCs produced by Fagus grandifolia Ehrh. in the largest quantity at each stage of development in descending order; A = largest. Rank Stage A B C D 1 ß-caryophellene (+)-3-carene α-terpinene geranyl-p-cymene 2 sabinene R-(+)-limonene α-/ß-pinene ß-caryophellene 3 methyl jasmonate ß-caryophellene sabinene (Z)-3-hexenyl acetate 4 sabinene methyl jasmonate (Z)-3-hexenyl α-/ß-pinene acetate 5 (Z)-3-hexenyl sabinene α-/ß-pinene R-(+)-limonene acetate

Table 5.3 Orchestes fagi mean amplitude of responses to unknown VOCs from American beech, Fagus grandifolia Ehrh., based on repeated responses over multiple replicates within and/or between sexes. Results are presented for adult pre-overwintered male and female O. fagi as mean amplitude of response (mV) ± SEM. *indicates significance value within sex P < 0.05. Retention time Male (N=4) Female (N=4) 8.42 0.0 ± 0.00 3.9 ± 2.73 9.10 0.3 ± 0.25 2.3 ± 1.37 11.40 1.2 ± 1.01 0.0 ± 0.00 12.72 2.9 ± 1.60 1.6 ± 0.94 13.25 0.0 ± 0.00 4.1 ± 1.76 13.47 1.5 ± 1.47 3.6 ± 1.55 14.10 3.4 ± 3.21 2.4 ± 1.45 14.15 3.3 ± 3.25 2.3 ± 0.76 14.47 3.0 ± 3.00 2.2 ± 1.30 15.51 0.2 ± 0.17 2.0 ± 1.32 16.38 0.0 ± 0.00 2.3 ± 1.33 17.66 0.0 ± 0.00 3.9 ± 2.24 18.18 0.0 ± 0.00 1.4 ± 1.06 21.22 5.5 ± 4.56 14.0 ± 10.73 22.01 0.0 ± 0.00 12.8 ± 7.67 22.30 0.0 ± 0.00 *18.8 ± 16.43 22.45 0.0 ± 0.00 *16.8 ± 16.08 23.28 2.7 ± 2.43 *15.7 ± 12.44 23.87 1.3 ± 1.06 1.7 ± 1.03 25.26 0.2 ± 0.12 3.8 ± 0.79 26.97 5.8 ± 5.75 1.9 ± 1.19 29.51 0.0 ± 0.00 8.8 ± 6.47 29.96 0.1 ± 0.09 3.1 ± 1.81 32.75 4.4 ± 2.35 3.4 ± 1.30 33.24 3.4 ± 3.19 0.0 ± 0.00 34.80 0.2 ± 0.20 2.2 ± 1.36 36.01 2.5 ± 2.50 4.6 ± 2.90 38.22 1.9 ± 1.85 2.2 ± 1.37 39.77 0.2 ± 0.11 0.6 ± 0.60 40.77 2.5 ± 2.45 5.2 ± 2.29 41.83 0.2 ± 0.13 2.0 ± 1.14

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Mutis A, Parra L, Manosalva L, Palma R, Candia O, Lizama M, Pardo F, Perich F, Quiroz A (2010) Electroantennographic and behavioral responses of adults of raspberry weevil Aegorhinus superciliosus (Coleoptera: Curculionidae) to odors released from conspecific females. Environ Entomol 39:4 127-1282.

Nielsen BO (1974) A study on the weevil fauna (Curculionidae) in a Danish beech forest. Ent Medd 42:169-188.

Nielsen BO (1978) Food resource partition in the beech leaf-feeding guild. Ecol Entomol 3:193-201.

Pawlowski SP (2014) Chemical ecology of the invasive beech leaf mining weevil (Orchestes fagi L.) in Nova Scotia, Canada. B.Sc. Honours Thesis, Acadia University, April 2014.

R Core Team (2015) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Available from: http://www.R-project.org/.

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Reddy GVP, Guerrero A (2000) Behavioral responses of the diamondback moth, Plutella xyllostella to green leaf volatiles of Brassica oleracea subsp. capitata. J Agric Food Chem 48: 6025-6029.

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Szendrei Z, Averill A, Alborn H, Rodriguez-Saona C (2011) Identification and field evaluation of attractants for the cranberry weevil, Anthonomus musculus Say. J Chem Ecol 37: 387-397.

Szendrei Z, Malo E, Stelinski L, Rodriguez-Saona C (2009) Response of cranberry weevil (Coleoptera: Curculionidae) to host plant volatiles. Environ Entomol 38:3 861-869.

Tholl D, Boland W, Hansel A, Loreto F, Röse USR, Schnitzler JP (2006) Practical approaches to plant volatile analysis. Plant J 45: 540-560.

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CHAPTER 6.0 CONCLUSIONS

6.1 Summary

The invasive beech leaf-mining weevil, Orchestes fagi L., is a highly damaging pest to American beech, Fagus grandifolia Ehrh., in its introduced range. Studies presented in this thesis have provided foundational knowledge of O. fagi life history, host location, and electrophysiological response to host VOCs in addition to methodological approaches for future projects. Although no direct integrated pest management tactics were developed over the course of this thesis, many directions for future study have been determined.

It has become clear through the course of the studies presented here and by Moise et al. (2015) that O. fagi life history is slightly different in North America than in its native range. Population fluctuations appear to be more frequent in NA interspersed, potentially, with vertical or horizontal migration. Although neither protandrous nor protogynous, O. fagi pioneering has conflicting results and it is difficult to assess its role, if any, in host location and colonization. Male O. fagi appear to have a relationship with early stage beech leaves (i.e. stage 1–2 as presented in this thesis). There is behavioural attraction to bursting buds and geranyl-p-cymene, one of the dominant stage 1 volatiles, in a Y-tube olfactometer and electrophysiological response to R-(+)-limonene, a compound produced in largest volume during stage 2. These laboratory results did not translate to beat sampling, with males and females being present on beech in equal proportions throughout the season. It seems likely that males play a key role in host location which may occur quickly particularly reflecting on the apparent use of male-

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produced pheromone, common in the Curculionidae, to deter further male colonization seen in Chapter 2.0.

The role of visual stimuli in host location for O. fagi has become clear through this thesis. Males likely have trichromatic vision enabling them to see colours in the UV, blue, and green spectra. It remains unclear if females possess colour vision.

This thesis presents the first comprehensive study of Fagus grandifolia volatile profiles. There are clear fluctuations over the developmental period and results are relatively consistent with its European sister-species F. sylvatica. The terpenoid sabinene is the most abundant VOC emitted by F. grandifolia foliage though its presence and titre is highly variable throughout development. Use of these fluctuations by O. fagi is evident and suggests that attraction to young leaves and rejection of adult leaves documented by

Bale and Luff (1978) is a direct result of chemically-driven attraction. Specific compounds or blend of compounds involved in this process are still unknown.

6.2 Future directions

Further behavioural bioassays, including field trapping and Y-tube olfactometer or wind tunnel assays, on the roles of geranyl-p-cymene and R-(+)-limonene on male behaviour, and on the roles of (Z)-3-hexenyl acetate and (Z,E)-α-farnesene on female behaviour, may provide insight into how these compounds are used by O. fagi in host location.

Vision in Orchestes fagi represents a new field of study which will be of particular importance in the development of an effective IPM strategy. The presence of colour vision indicates strong visual acuity and therefore, future trapping studies using differently coloured and/or shaped trap designs may lead to better trap catch than seen in

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the studies presented in this thesis on yellow sticky cards. Additionally, electroretinograms (ERGS) can be used to measure the net electrical activity generated by neural and non-neural cells of the eye in response to light of varying wavelengths. This technique would allow for greater precision in the pursuit of understanding insect colour vision and the ability to assess the response to UV light to confirm the hypothesis of trichromatic vision in O. fagi.

Pioneering male O. fagi is still potentially the mechanism of host location and future examination of population dynamics will be important in assessing this. Of particular interest is the potential presence of a male-produced pheromone. Small-scale examination of this could include simple Petri dish experiments wherein a solid-phase microextraction (SPME) fibre is exposed to a male O. fagi on a bursting F. grandifolia bud. Collected volatiles could be run on GC-EAD and/or GC-MS to determine electrophysiological response and compounds produced by the male respectively.

A further field of study not discussed in this thesis is the putative role of sound production in O. fagi. Males and females produce „chirps‟ via stridulation using a file and scraper on the underside of the elytra and dorsal surface of the abdomen (Claridge 1968).

Claridge (1968) notes the use of stridulation during courtship, aggression, crowding, and disturbance. It is likely that stridulation is another important aspect of O. fagi life history which has yet to be examined and may play an important role in effective IPM strategies.

The large number of unidentified EAD and GC-MS peaks (see Chapter 5.0) also present an important area of future study. Although this thesis focused on the comparison of F. grandifolia and F. sylvatica, it is clear that there are many additional compounds present in the F. grandifolia VOC profile which may play a role in O. fagi life history.

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Further chemical analyses and identification of these peaks will provide a more robust examination of the complex relationship between this pest and its host.

6.3 References

Bale JS, Luff ML (1978) The food plants and feeding preference of the beech leaf mining weevil, Rhynchaenus fagi L. Ecol Entomol 3:245-249.

Claridge LC (1968) Sound production in species of Rhynchaenus ( = Orchestes) (Coleoptera: Curculionidae). Trans R Ent Soc Lond 120:287-296.

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APPENDIX A

Table A.1 Pair-wise comparisons between months for field trapping bioassays conducted from May 2nd–August 1st, 2014 assessed by generalized linear model (loglink, family = negative binomial) and Tukey‟s comparisons (glht function, package multcomp). * indicates significance. Pair-wise comparison Estimate Std. error z-value Pr (>|z|) June – May 0.5666 0.4002 1.416 0.4618 July – May 1.5622 0.3861 4.046 < 0.001 * August – May -2.2747 1.2035 -1.890 0.2104 July – June 0.9956 0.4279 2.327 0.0799 August – June -2.8413 1.2179 -2.333 0.0790 August – July -3.8369 1.2136 -3.162 0.0077 *

Table A.2 Pair-wise comparisons between months for branch-beat samples collected May 6th–July 16th, 2015 assessed by generalized linear model (loglink, family = negative binomial) and Tukey‟s comparisons (glht function, package multcomp). Pair-wise comparison Estimate Std. error z-value Pr (>|z|) June – May -0.2550 0.3628 -0.703 0.760 July – May 0.1310 0.4494 0.291 0.954 July – June 0.3860 0.4607 0.838 0.678

APPENDIX B

Table B.1 Pair-wise comparisons of control traps across field trapping bioassays. Experiments 1 – 3 (20 May–18 July, 2014) assessed by generalized linear model (loglink, family = negative binomial) and Tukey‟s comparisons (glht function, package multcomp). * indicates significance. Pair-wise comparison Estimate Std. error z-value Pr (>|z|) 2 – 1 -0.3238 0.2642 -1.226 0.4370 3 – 1 1.5792 0.2394 6.598 < 0.0001 * 3 – 2 1.9030 0.2477 7.682 < 0.0001 *

Table B.2 Pair-wise comparison of blends used in field trapping bioassay Experiment 1 (20 May–6 June, 2014) assessed by generalized linear model (loglink, family = negative binomial) and Tukey‟s comparisons (glht function, package multcomp). LB = leaf blend; WB = weevil blend. Pair-wise comparison Estimate Std. error z-value Pr (>|z|) LB – Control -0.36891 0.26078 -1.415 0.4898 LB + WB – Control -0.61147 0.26875 -2.275 0.1036 WB – Control 0.04167 0.25065 0.166 0.9984 LB + WB – LB -0.24256 0.27744 -0.874 0.8180 WB – LB 0.41058 0.25995 1.579 0.3900 WB – LB + WB 0.65314 0.26795 2.438 0.0701

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Table B.3 Pair-wise comparison of blends used in field trapping bioassay Experiment 2 (13 June – 25 June, 2014) assessed by generalized linear model (loglink, family = negative binomial) and Tukey‟s comparisons (glht function, package multcomp). LB2 = new leaf blend; WB = weevil blend. Pair-wise comparison Estimate Std. error z-value Pr (>|z|) LB2 – Control -0.72300 0.30057 -2.405 0.0759 LB2 + WB – Control -0.53063 0.29161 -1.820 0.2636 WB – Control -0.45831 0.28861 -1.588 0.3849 LB2 + WB – LB2 0.19237 0.31723 0.606 0.9300 WB – LB2 0.26469 0.31447 0.842 0.8343 WB – LB2 + WB 0.07232 0.30592 0.236 0.9953

Table B.4 Pair-wise comparisons of blends used in field trapping bioassay Experiment 3 (3 July – 18 July, 2014) assessed by generalized linear model (loglink, family = negative binomial) and Tukey‟s comparisons (glht function, package multcomp). LB3 = 8:1 ratio ß-caryophellene and (Z)-3-hexenyl acetate; WB = weevil blend. Pair-wise comparison Estimate Std. error z-value Pr (>|z|) LB3 – Control -0.34175 0.21862 -1.563 0.400 LB3 + WB – Control -0.39239 0.21899 -1.792 0.277 WB – Control -0.35417 0.21871 -1.619 0.368 LB3 + WB – LB3 -0.05064 0.22102 -0.229 0.996 WB – LB3 -0.01242 0.22074 -0.056 1.000 WB – LB3 + WB 0.03822 0.22110 0.173 0.998

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