Factors Affecting Defoliation of Eastern Hemlock (Tsuga Canadensis) by the Pale-Winged Gray Moth (Iridopsis Ephyraria)

FACTORS AFFECTING DEFOLIATION OF EASTERN HEMLOCK (TSUGA CANADENSIS) BY THE PALE-WINGED GRAY MOTH (IRIDOPSIS EPHYRARIA)

Meggy Hervieux

Department of Natural Resource Sciences McGill University Montréal, Québec, Canada

August 2011

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science

Table of Contents

LIST OF TABLES ...... iii LIST OF FIGURES ...... iv ACKNOWLEDGEMENT ...... vi PREFACE ...... vii CONTRIBUTION OF AUTHORS ...... viii ABSTRACT ...... ix RÉSUMÉ ...... x

CHAPTER 1: INTRODUCTION, LITTERATURE REVIEW, AND OBJECTIVES ...... 1 1.1 Introduction ...... 1 1.2 Literature review ...... 2 1.2.1 Natural history of the pale-winged gray moth ...... 2 1.2.2 Eastern Hemlock - Tsuga canadensis ...... 4 1.2.3 The PWG research context ...... 5 1.2.4 Factors affecting PWG feeding preference ...... 6 1.3 Objectives and Hypotheses ...... 10 1.4 Literature cited ...... 12

CHAPTER 2: EGG AND LARVAL DISTIBUTION OF THE PALE-WINGED GRAY MOTH (IRIDOPSIS EPHYRARIA) ON EASTERN HEMLOCK TREES IN NOVA SCOTIA...... 18 2.1 Introduction ...... 18 2.2 Material and Methods ...... 20 2.3 Results ...... 22 2.4 Discussion and Conclusion ...... 25 2.5 Literature cited ...... 26

CHAPTER 3: THE EFFECTS OF HYGROTHERMAL STRESS AND FOLIAGE QUALITY ON THE PALE-WINGED GRAY MOTH (IRIDOPSIS EPHYRARIA) IN HEMLOCK STANDS OF NOVA SCOTIA ...... 29 3.1 Abstract ...... 29 3.2 Introduction ...... 30 3.3 Material and Methods ...... 33 3.3.1 Description of study sites and insect ...... 33 3.3.2 Experimental design ...... 34 3.3.3 Statistical analyses ...... 37 3.4 Results ...... 40 3.5 Discussion ...... 55 3.5.1 Hygrothermal stress hypothesis ...... 55 3.5.2 Foliage quality hypothesis ...... 57 3.6 Conclusion ...... 61 3.7 Literature cited ...... 62

CHAPTER 4: GENERAL CONCLUSIONS ...... 66 APPENDICES ...... 68

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LIST OF TABLES

Table 3.1 Results of generalized linear mixed models evaluating the effects of crown location (crown level and sun exposure) on percent larval survival during each of two years (2009 and 2010) on mature eastern hemlock trees. Ushade = foliage in the shaded upper crown, Lshade = foliage in the shaded lower crown and Usun = foliage in the sun-exposed upper crown...... 46

Table 3.2 Results of a generalized linear mixed model evaluating the effects of crown location (crown level and sun exposure), month (June or July) and foliage age (current year and older) on the percentage of nitrogen in needles of eastern hemlock during summer 2010...... 49

Table 3.3 Results of generalized linear mixed models evaluating the effects of sun exposure of eastern hemlock saplings on percent larval survival during each of two years (2009 and 2010). SS = sapling trees previously exposed to sun but artificially shaded, Shade = shaded sapling trees and Sun = sun-exposed sapling trees...... 52

LIST OF FIGURES

Figure 2.1 Mean (±SE) percentage of eggs (estimated absolute number of eggs per section / estimated total number of eggs per tree) laid by PWG female for each tree section during summer 2010...... 23

Figure 2.2 Mean (±SE) number s of PWG larvae per beating a) per crown level (collection dates are merged) and b) on two different larval development dates during summer 2010 (crown levels are merged)...... 24

Figure 3.1 Map of Queens County in Southwest Nova Scotia with insert illustrating the location of the two study sites, Milton and Moose Hill...... 34

Figure 3.24a) Mean (± SE) percent defoliation caused by PWG larvae in the upper and lower crown of mature eastern hemlock trees at two study sites in 2010. Larvae in the upper crown were located either in the partial shade (Ushade) or in the sun (Usun), whereas all larvae in the lower crown were located on shaded branches (Lshade). b) Mean (± SE) percent defoliation for each foliage age-class of mature hemlock trees depicted in a). Abbreviations for yearly age-classes are CY, C+1, C+2 and C+3 for current-year, 1,2 and 3 year-old foliage...... 45

Figure 3.35Influence of crown location (crown level and sun exposure) in a) 2009 and b) 2010 on mean (±SE ) larval (dark bars) and total (i.e., up to adult emergence; light bars) survival of PWG larvae. Refer to Figure 3.2 for description of crown locations. Bars with different letters represent significantly different results...... 47

Figure 3.46a) Mean (±SE ) percentage of PWG adults that were female and b) mean (±SE ) subcostal vein length of adult females (dark bars) and males (light bars) that developed on branches of eastern hemlock in three crown locations (described in Figure 3.2) in 2009-2010. Bars with different letters represent significantly different results...... 48

Figure 3.57Mean (± SE) percent nitrogen (open bars) and water (closed bars) content of current-year (CY) and older (1 to 3 years old) foliage of eastern hemlock during summer 2010. Bars with different letters represent significantly different results...... 50

Figure 3.68a) Mean (± SE) percent defoliation caused by PWG larvae developing on sapling eastern hemlock trees at two study sites in 2010. Saplings were either located in the shade (Shade), in the sun but artificially shaded (SS) or in the sun (Sun). b) Mean (± SE) percent defoliation for each foliage age-class of sapling hemlock trees depicted in a). Abbreviations for yearly age-classes are CY, C+1, C+2 and C+3 for current-year, 1,2 and 3 year-old foliage...... 51

Figure 3.79Influence of sun exposure in a) 2009 and b) 2010 on mean (±SE ) larval (dark bars) and total (i.e., up to adult emergence; light bars) survival of PWG larvae. Refer to Figure 3.6 for description of sun exposure treatments. Bars with different letters represent significantly different results...... 53

Figure 3.810Influence of sun exposure on mean (± SE) b-pinene (dark gray bars), myrcene (light gray bars) and terpinolene (white bars) concentrations in eastern hemlock foliage during summer 2010. Refer to Figure 3.5 for a description of the treatments...... 54

ACKNOWLEDGEMENT

I would like to thank my two supervisors Chris Buddle and Dan Quiring for their inputs, encouragements and support. Their continuous help throughout the accomplishment of my thesis was well appreciated and precious to me. Over the two years, Chris also became a mentor and a friend. Thank you Chris.

Thank you to Dr. Rob Johns from the Canadian Forest Service in New- Brunswick who assisted me on the field in climbing trees and his advice and knowledge was very useful. A special thanks to all the field and lab assistants that helped me collect data and with whom, the accomplishment of my thesis has been possible: Dorothy Maguire, Drew Carleton, Louis-Philippe Rodier, Tristan Ménard, Maxime Chautard and Marcy Laity.

Biochemical analyses of the foliage were done under the supervision of Martin Charest, responsible for the practical work and research in Eric Bauce’s laboratory at Laval University. His help was indispensable and it was a pleasure to work with such a good friend.

A big thanks to Marc Mazerolle, at Université du Québec en Abitibi- Témiscamingue, who helped me with my statistical analyses.

A special thanks to my parents, my partner Sean Duffy and my immediate family who morally supported me over the past two years.

This thesis was possible because of the funding coming from the Natural Sciences and Engineering Research Council of Canada, iFOR and Le Fonds québécois de la recherche sur la nature et les technologies.

vii

PREFACE This thesis has four chapters.

Chapter 1

Chapter 1 contains a general introduction, a literature review and the objectives and hypotheses of this thesis.

Chapter 2

Chapter 2 is a short research paper about the egg and larval distribution of the pale-wing gray moth in mature eastern hemlocks. It will be submitted to The Canadian Entomologist as a research note.

Chapter 3

Chapter 3 is a manuscript to be submitted to the journal Oikos

Hervieux, M., Buddle, C.M., Quiring, D., Johns, R. and E. Bauce. The effects of hygrothermal stress and foliage quality on the pale-winged gray moth (Iridopsis ephyraria) in hemlock stands of Nova Scotia.

Chapter 4

Chapter 4 is a general conclusion on the thesis findings.

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CONTRIBUTION OF AUTHORS

Chris Buddle and Dan Quiring co-supervised Meggy Hervieux for her Master’s in Science at McGill University. Chris Buddle helped design the experiments, supervised the thesis progress (e.g., course load), helped collect data and edited the final thesis. Dan Quiring helped in the research design, supervised the two field seasons and edited the final thesis. Meggy Hervieux established the final experimental design and collected data during two consecutive summers of field experiments (2009-2010). Meggy Hervieux also analysed the data, prepared tables and figures, wrote the text of this thesis.

ABSTRACT

The main objective of this thesis was to study the possible factors that could explain the feeding preference of the pale-winged gray moth (PWG) (Iridopsis ephyraria) for the mid-lower crown of eastern hemlocks in south-west Nova Scotia. I first described egg and larval distribution between crown locations of mature hemlocks. I then focused on testing two hypotheses that could explain the pale-winged gray feeding preference for the foliage located in the mid-lower crown: (1) hygrothermal and (2) foliage quality hypotheses.

My research shows that eggs and larvae were principally located in the mid-lower crown. This larval preference could partly explain the greatest defoliation in the mid-lower crown of mature hemlocks. Experimental and observational data revealed that both hygrothermal stress and foliage quality varied between crown locations and thus affected the PWG feeding preference. The disadvantages of heat and water stress in the upper crown, however, outweighed the benefits to feed on higher foliage quality which explains the PWG feeding preference for foliage in the mid-lower crown. Additional data on foliage age showed that larvae prefer to feed on current-year foliage presumably because this age-class had higher water and nitrogen content. This feeding preference may have helped larvae to survive in the upper crown where temperatures are warmer.

This research adds to the literature on plant-insect interactions, allows a better understanding of the pale-winged gray moth natural history but mostly, informs us on the factors that affect PWG survival.

RÉSUMÉ

Récemment, la région sud-ouest de la Nouvelle-Écosse a été sévèrement touchée par l’arpenteuse à tâches (Iridopsis ephyraria), un lépidoptère s’attaquant principalement à la Pruche du Canada (Tsuga canadensis). L’objectif principal de ce mémoire était d’explorer les facteurs pouvant expliquer la préférence alimentaire de l’arpenteuse à tâches pour le feuillage retrouvé dans la couronne inférieure des pruches du Canada. En premier lieu, j’ai décrit la distribution des œufs et des larves de l’arpenteuse à tâches entre les différentes strates des pruches matures. J’ai ensuite vérifié différentes hypothèses pouvant expliquer la distribution verticale de l’espèce dans la canopée : (1) la variation du microclimat (i.e. température et humidité) ainsi que (2) les changements dans la qualité du feuillage.

Les œufs et les larves de l’espèce étaient principalement localisés dans la partie inférieure de la canopée. La défoliation plus importante au niveau de la couronne inférieure des arbres matures pourrait, en partie, être expliquée par cette préférence chez les larves.

Dans le chapitre 3, mes résultats ont démontré que le stress hydrique (stress causé par une température élevée et un faible taux d’humidité) ainsi que la qualité du feuillage ont influencé la préférence alimentaire de l’arpenteuse à tâches. Toutefois, les effets négatifs du stress hydrique ont davantage influencé la distribution finale de l’espèce dans les pruches du Canada comparativement aux effets reliés à la qualité du feuillage. De plus, mes données ont révélé que l’arpenteuse à tâches préfère se nourrir sur le feuillage de l’année courante qui comporte plus d’eau et d’azote. Cette préférence pour le feuillage de l’année courante pourrait favoriser la survie des larves retrouvées dans le haut de la canopée où le stress hydrique est supérieur.

Ce projet a permis d’ajouter à la littérature scientifique de l’information sur les relations entre plantes et insectes. Ce mémoire permet aussi de comprendre

xi davantage l’histoire naturelle de l’arpenteuse à tâches mais surtout, il nous informe sur les facteurs qui affectent la préférence alimentaire de l’espèce dans les pruches matures.

CHAPTER 1: INTRODUCTION, LITTERATURE REVIEW, AND OBJECTIVES

1.1 Introduction

Insects provide a range of benefits for humans including, for example, nutrient cycling, pollination, sericulture and dung decomposition (Losey and Vaughan 2006). Many species, however, are known for their detrimental effects, notably pests that cause major economical losses in agriculture and forestry. Pimental et al. (2005), for example, reported an annual loss of $14,400 million from crop pests and $2,100 million from forest pests in the United States.

In forest ecosystems, insects affect the growth, mortality and the susceptibility to disease of economically important trees (Comtois, 1998). In addition to economic damage, insects can cause ecological and social impacts. Ecological impacts can be of various types; for instance, insect populations can alter community dynamic (Styrsky and Eubanks, 2007) and the influence of abiotic factors (e.g., crown defoliation increases light incidence). Social impacts are often linked to insects causing aesthetic damage on ornamental trees or flowers and in gardens (e.g., the lily leaf beetle, Lilioceris lilii (Ernst et al. 2007)). In the past decade, two well-known species of forest pests emerged in Canada: the mountain pine beetle (Dendroctonus ponderosae, a native species) and the emerald ash borer (Agrilus planipennis, an invasive species). These two species are famous woodborers because of their significant economic impacts on pine and ash trees respectively (Colautti et al. 2006). In addition to woodborers, defoliators such as the gypsy moth (Lymantria dispar) and the hemlock looper (Lambdina fiscellaria) can be as detrimental in certain regions of Canada.

Integrated pest managements (IPM) programs use a range of approaches to manage pest insects. For examples, biological, physical, semiochemical and chemical techniques can be used together to manage insect pest populations (Comtois, 1998) and reduce damage they cause. Before developing an IPM

2 program for any pest, it is important to understand the whole system that surrounds the studied species (i.e., interactions between abiotic and biotic components) to prevent ecological catastrophes.

This thesis focuses on the pale-winged gray moth (PWG) (Iridopsis ephyraria (Walker)) a defoliator of eastern hemlock trees (Tsuga canadensis (L.) Carr.)) that consumes needles of mature trees and saplings and causes significant damage in parts of southwest Nova Scotia (Pinault et al. 2007). More specifically, this thesis attempts to understand the species’ feeding preference for the foliage located in the lower and middle crown of mature hemlock trees. My research approach includes both field and laboratory studies, using observational and experimental approaches on both mature trees and saplings. This fundamental knowledge on the ecology of the PWG may help develop management tactics for this defoliator. In order to achieve this goal, I tested three factors that could explain asymmetrical consumption of foliage within crowns of eastern hemlock trees: (1) egg and larvae distribution, (2) foliage quality and (3) hygrothermal stress.

1.2 Literature review

1.2.1 Natural history of the pale-winged gray moth

The pale-winged gray (PWG) moth (Iridopsis ephyraria), historically referred as Anacamptodes ephyraria, is an indigenous species that belongs to the family Geometridae (Order Lepidoptera) and is distributed across Canada from Alberta to Nova Scotia (Rindge 1966). In the United States, the PWG is found from North Dakota and is distributed as far south as Texas and east to Florida (Rindge 1966).

The small eggs (0.5-0.8mm long) of PWG are characterized as bright green with a flattened red end (Pinault et al. 2007). Eggs hatch at the end of May or early June. Pinault et al. (2007) reported that eggs are laid singly in deep bark

3 crevices and under pieces of lichen on hemlock trees. It is reported that PWG egg density increases linearly from the bottom of the trees to the top. Various hypotheses could explain this preference: eggs in the upper crown may obtain higher sun exposure (Fortin and Mauffette 2002, Ide 2006), reduced egg predation or parasitism or lower mortality from fungal pathogens (Hajek 2001). Larvae are distinguished by their rust-coloured head capsule with two prominent dorsal lobes and their green body. Pinault et al. (2007) established that the PWG moth goes through five different instars (based on head capsule width). Each instar lasts about 7 to 10 days except for the fourth instar, which lasts for more than two weeks. Before pupation occurs in mid-July, larvae change morphologically becoming brighter, shorter and fatter. They then drop to the soil where they bury themselves and start the pupation process. The reddish-brown pupae are found ˂10cm deep in the soil, where they stay for one to two weeks.

The light grey coloured adults emerge at the end of July and are active until mid-August. Because of their camouflaged colour they are difficult to see on tree bark. Adults’ peak activity usually occurs during one week in early August (Pinault pers. comm.). During that period of time, they reproduce and females lay the overwintering eggs. The species is univoltine.

The PWG moth is a generalist species known to feed on various tree species, such as red maple, red oak, balsam fir, white pine and eastern hemlock, and some deciduous shrubs (e.g., sweetfern, gooseberry) (Landry et al. 2002, Pinault et al. 2007). Although the species is able to feed on multiple hosts, it cannot always complete its larval development on some hosts (e.g., white pine) and its survival rate is low on other hosts (e.g., balsam fir) (Pinault et al. 2007). The PWG is known as a minor pest of cranberry (Landry et al. 2002). Regardless of its polyphagous behaviour, to my knowledge, PWG only cause significant defoliation on eastern hemlocks which suggests a preference for this evergreen tree.

The natural enemies of the PWG moth are poorly known but predation on PWG by birds, red squirrels, various hymenoptera (ants and wasps) and small arachnids have been reported (Pinault et al. 2007, personal observation). Natural enemies also include a fungus (Entomophaga sp.) and a parasitoid wasp (Ichneumonidae: Pimpla pedalis (Cresson)) that emerge from the PWG moth pupae (Pinault et al. 2007, personal observation).

1.2.2 Eastern Hemlock - Tsuga canadensis

Eastern hemlock is a native hardwood species found in North America. In Canada, T. canadensis is distributed from south-central Ontario to Nova Scotia. In the United States, hemlock trees extend eastward from Minnesota to Maine and southward into northern Georgia and Alabama (Burns and Honkala 1990).

Eastern hemlock is a shade-tolerant species restricted to cool and humid climates and are found on moist soil with good drainage (Burns and Honkala 1990). Stands of mature eastern hemlock tend to recreate a similar microclimate due to their dense canopy: dense shading, deep layer of partially or fully decomposed organic matter, high moisture and low temperature (Burns and Honkala 1990).

Historically, eastern hemlock trees were mainly exploited for lumber production, reaching a peak production from 1890 to 1910. The species is commercially mainly used for pulping, newsprint and wrapping paper and for ornamental and aesthetic purposes. Ecologically, hemlock stands provide critical habitat and bedding for white-tailed deer and provide a cover for ground dwelling birds (e.g., ruffed grouse) (Burns and Honkala 1990).

In addition to the PWG, other insect species commonly feeding on eastern hemlock include the hemlock looper (Lambdina fiscellaria (Guenée)), the

5 hemlock woolly adelgid (Adelges tsugae (Annand)) and the hemlock borer (Melanophila fulvoguttata (Harris)).

1.2.3 The PWG research context

The PWG has never been considered as a pest since no outbreak had been recorded prior to 2002 (Pinault et al. 2007). The first known outbreak occurred in the Kejimkujik National Park (2002) in Nova Scotia where the PWG defoliated eastern hemlock trees. During the first year of an outbreak, the species is known to primarily attack sapling trees; they can kill up to 90% of saplings. In the following years, the PWG will move upward to reach the canopy of mature hemlock trees; they are capable of killing up to 40% of the trees after two consecutive years of intensive feeding on the needles (Pinault and Quiring 2008). The species is known to feed on all age classes of foliage but defoliation is more severe on current year foliage (Pinault et al. 2009). In fact, early instar larvae only feed on current-year needles while late instar larvae are capable of feeding on foliage of all ages (Pinault et al. 2009).

A consistent and repeatable pattern of defoliation occurs within endemic areas; the low and mid crown of mature hemlock trees are highly defoliated while the upper crown is usually spared from defoliation. Pinault et al. (2008) studied three different methods that could allow for predicting defoliation levels caused by the PWG on eastern hemlock trees and found that sticky tape is the only method that allows for quantitatively predicting site-specific defoliation. Although this study is very useful to predict the ravages of the PWG for a certain year, tree level effects are unknown as are the factors explaining the feeding preference of the larvae for the mid-lower crown. Several factors could explain this feeding preference including variations in nutrients and allelochemicals directly linked to foliage quality, hygrothermal stress (heat and water stress), female preference for oviposition sites and possibly larval performance differing between crown levels

6 due to variation in natural enemies, and these will be discussed in the following section.

1.2.4 Factors affecting PWG feeding preference

Foliage characteristics can be altered depending on the position of the leaf/needles on the tree due to a difference in sun exposure. Foliage grown under high solar radiance normally has a smaller area, a greater thickness and higher nitrogen contents (White 1984, Oishi et al. 2006). As proteins are the basic structural material of insects, nitrogen is an important element necessary for the survival of insects (Schoonhoven et al. 2005). It is well documented that leaf nitrogen content increases when directly exposed to sun (Coley 1983, White 1984, Dudt and Shure 1994, Fortin and Mauffette 2002, Oishi et al. 2006, Osier and Jennings 2007) and thus represents superior food quality for caterpillars, since nitrogen is usually limiting to insect growth. Nitrogen and water contents play an important role in larval performance (relative growth rate of foliage-chewing insects is higher when water and nitrogen contents are higher) (Scriber and Slansky 1981). Fortin and Mauffette (2002) looked at the biological performance and the feeding preference of Malacosoma disstria Hübner, a Lepidoptera from the Lasiocampidae family, when feeding on sugar maple (Acer saccharum Marsh.) leaves from different crown levels. Overall, the species performed better (i.e., higher pupal mass and egg number) when fed with leaves from the upper crown. After foliage chemical analyses, they reported that the amount of nitrogen and soluble sugars was higher at this level due to higher light incidence. Sugars can be used by insects as a cue for the selection of a food plant (Schoonhoven and van Loon 2002). Osier and Jennings (2007) arrived at similar conclusions when working on a saturnid moth (Callosamia promethea); final mass and developmental rate were higher for larvae fed with foliage grown in the sun.

The asymmetrical vertical distribution of the PWG moth larvae might be explained by variation in foliage quality at different heights of the host trees

7 regardless of sun exposure. The nutritional quality of foliage can vary in space (crown level) and time (foliage age-class or seasonality) within tree crowns (Quiring 1993, Carroll and Quiring 1994, Murakami et al. 2005). For example, it is known that the percentage of nitrogen and water contents decreases over time (Le Corff and Marquis 1999) and with age (Scriber and Slansky 1981).

Different hypotheses can explain how insects can survive changes in foliage quality over time and space. Nutrient requirements can change over the season for the different developmental stages of the insect. The ontogeny hypothesis implies that different developmental stages will feed on different age- foliage classes to either satisfy dissimilar dietary needs or to reflect changes in tolerance to plant chemicals with age or because of morphological changes of their mouthparts normally allowing older larvae to feed on tougher foliage (Hochuli 2001, Johns et al. 2009, Pinault et al. 2009). The complementary diet hypothesis states that individuals feeding on a mix of foliage-age classes perform better because of better balance of needed nutrients and attenuation of effects of harmful secondary chemicals (Singer et al. 2002, Johns et al. 2009).

Plants produce secondary metabolites that can deter herbivores (e.g., allelochemicals such as monoterpenes, tannins and phenols). Wallin and Raffa (1998) showed that the jack pine budworm feeds unequally within needles due to variation in allelochemicals. It was also demonstrated that allelochemical concentrations can vary between crown levels and thus change food use by the spruce budworm (Choristoneura orae Freeman) (Lepidoptera) (Carisey and Bauce 1997). Plant feeding’ deterrents can vary between the different age-classes of foliage; although most insects prefer feeding on younger foliage because of its chemical and physical attributes (i.e., it contains more water and is more tender), the opposite trend is also possible since young leaves can contain more defensive chemicals (e.g., phenols, monoterpenes) (Meyer and Montgomery 1987).

Leaves directly exposed to sun experience higher temperature and water loss. In mature trees, this creates a mosaic of microhabitats between crown levels for insects to exploit, with the upper crown more likely to be exposed to sun and higher winds. It has been demonstrated that some moth activities differ during the day due to variations in temperature and humidity between crown levels (Quiring 1994, Bento et al. 2001). Quiring (1994) reported a daily binomial activity of the spruce bud moth, Zeiraphera Canadensis Mutuura and Freeman, when temperatures are lower (around dawn and dusk). When temperature was at its highest and humidity at its lowest, moths were mainly found in the lower shaded crown; a possible direct consequence of hygrothermal stress. Bento et al. (2001) obtained the same results when studying the daily behaviour of the citrus fruit borer (Lepidoptera).

Other factors such as an unequal abundance and/or diversity of natural enemies between crown levels could also explain the observed pattern of defoliation by the PWG. In fact, several studies in different systems demonstrate that parasitism level varies within tree heights (Eikenbary and Fox 1968, Weseloh 1972, Kemp and Simmons 1978, Jennings and Houseweart 1983). Predation pressure by birds and arthropods can also vary by heights (Aikens 2010). Thus, an unequal diversity/abundance of natural enemies within crown levels could explain the feeding preference of the PWG moth. However, this factor will not be discussed since it goes beyond the scope of this thesis.

The female preference-larval performance hypothesis (PPH) implies that females have a preference in their oviposition sites in order to maximize their larvae performance before and after hatching. A recent meta-analysis study supports the PPH (Gripenberg et al. 2010). In fact, studies with insect groups as diverse as Hemiptera (Craig and Ohgushi 2002), Lepidoptera (Pokkyo 2006, Mphosi and Foster 2010), Coleoptera (Bertheau et al. 2009), Diptera (Videla et al 2006) and Hymenoptera (Carr et al 1998) support this hypothesis. Ultimately, females should prefer to oviposit at sites that maximize juvenile performance. For

9 instance, Poykko (2006) studied a lichenivorous geometrid moth and noticed that females prefer hosts that will allow a shorter larval developmental period rather than a higher growth rate for young instars. Females’ preference for these oviposition sites can reduce larval predation, hence allowing a higher larval survival (vertebrate example: Rieger et al. 2004). Fry et al. (2009) provide a different hypothesis for female preference in oviposition sites; the phenology hypothesis. Overall, females lay their eggs where the phenology of the foliage is more suitable for the hatching larvae. In addition, females’ preference for oviposition sites can be sex-biased and consequently selects for a particular gender. Only female juvenile yellowheaded spruce sawflies usually complete development on upper crown branches and consequently only female eggs are oviposited in the upper crown of host trees (Johns et al. 2010).

1.3 Objectives and Hypotheses

The main objective of this thesis is to investigate the environmental factors that best explain the higher levels of defoliation by the pale-winged gray moth in the mid-lower crown of eastern hemlocks located close to the Kejimkujik National Park, Nova Scotia.

Chapter 2 will describe egg and larvae distribution between crown levels which will ultimately help us understand the PWG feeding preference. Egg density is highest in the upper crown but the total number of eggs might be highest in the lower crown if we consider tree area. My first hypothesis is that egg number will vary with tree height. I expect the number of eggs to be higher in the lower crown. If the number of eggs varies with tree height, I expect the number of larvae to also vary with tree height. My second hypothesis states that the number of PWG larvae will differ with tree height. Because defoliation by PWG mainly occurs in the lower crown of eastern hemlocks, I expect that larvae will principally be found in the lower crown.

Chapter 3 evaluate two hypotheses that could explain the feeding preference of the PWG moth in the mid-lower crown of hemlock trees: (1) spatial variations in foliage quality and (2) hygrothermal stress hypotheses. My first hypothesis is that larval survival can change between crown levels because there are spatial variations in foliage quality within a tree. This could be possible because of a change in sun exposure between the different crown levels. According to the sun/shade foliage hypothesis, sun foliage contains more nitrogen, one of the most important nutrients for caterpillar growth. However, spatial variations in foliage quality could also be due to changes in crown level itself (independent of sun exposure). My second hypothesis is that larval survival will vary between crown levels because hygrothermal stress differs with crown height. Because exposure to sun and winds increase at higher crown

11 levels, this could affect larval survival of the PWG, as all species can only tolerate a certain range of environmental conditions

1.4 Literature cited

Aikens, K.R. 2010. Heterogeneity in a temperate forest canopy describing patterns of distribution and depredation of arthropod assemblages. MSc thesis. McGill University. Montreal. 2009. ProQuest Dissertations and Theses. Web. 2011.

Bauce, E., Crépin, M. and N. Carisey. 1994. Spruce budworm growth, development and food utilization on young and old balsam fir trees. Oecologia 97:499-507.

Bento, J.M.S., Parra, J.R.P., Vilela, E.F., Walder, J.M. and W.S. Leal. 2001. Sexual behavior and diel activity of Citrus fruit borer Ecdytolopha aurantiana. Journal of Chemical Ecology 27:2053-2065.

Bertheau, C., Salle, A., Roux-Morabitol, G., Garcia, J., Certain, G. and F. Lieutier. 2009. Preference-performance relationship and influence of plant relatedness on host use by Pytyogenes chalcographus L. Agricultural and Forest Entomology 11:389-396.

Burns, R.M. and B.H. Honkala. 1990. Silvics of North America, Vol. 1, Conifers. Washington DC: U.S.D.A. Forest Service Agriculture Handbook 654. http://www.na.fs.fed.us/pubs/silvics_manual/table_of_contents.shtm, last accessed 2011.05.26.

Carisey, N. and E. Bauce. 1997. Balsam fir foliar chemistry in middle and lower crowns and Spruce budworm growth, development, food and nitrogen utilization. Journal of Chemical Ecology 23:1963-1978.

Carr, T.G., Roininen, H. and P.W. Price. 1998. Oviposition preference and larval performance of Nematus oligospilus (Hymenoptera: Tenthredinidae) in relation to host plant vigor. Environmental Enlomology 27:615-625.

Carroll, A.L. and D.T. Quiring. 1994. Intratree variation in foliage development influences the foraging strategy of a caterpillar. Ecology 75:1978-1990.

Colautti, R.I., Bailey, S.A., van Overdijk, C.D.A. Amundsen, K. and H.J. MacIsaac. 2006. Characterised and projected costs of nonindigenous species in Canada. Biological Invasions 8: 45–59

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Forister, M.L. 2004. Oviposition preference and larval performance within a diverging lineage of lycaenid butterflies. Ecological Entomology 29:264- 272.

Fortin, M. and Y. Mauffette. 2002. The suitability of leaves from different canopy layers for a generalist herbivore (Lepidoptera: Lasiocampidae) foraging on sugar maple. Canadian Journal of Forest Research 32:379-389.

Fry, H.R.R., Quiring, D.T., Ryall, K.L. and P.L. Dixon. 2009. Inﬂuence of intra- tree variation in phenology and oviposition site on the distribution and performance of Ennomos subsignaria on mature sycamore maple. Ecological Entomology 34: 394-405.

Gripenberg, S., Mayhew, P.J. and T. Roslin. 2010. A meta-analysis of preference– performance relationships in phytophagous insects. Ecology Letters 13:383–393.

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Jennings, D.T. and M.W. Houseweart. 1983. Parasitism of Spruce budworm (Lepidoptera: Tortricidae) eggs by Trichogramma minutum and absence of overwintering parasitoids. Environmental Entomology 12:535-540.

Johns, R., Quiring, D.T., Lapointe, R. and C.J. Lucarotti. 2009. Foliage-age mixing within balsam ﬁr increases the ﬁtness of a generalist caterpillar. Ecological Entomology 34: 624–631.

Johns, R., Quiring, D., Ostaff, D. and E. Bauce. 2010. Intra-tree variation in foliage quality drives the adaptive sex-biased foraging behaviors of a specialist herbivore. Oecologia 163: 935-947.

Kemp, W.P. and G.A. Simmons. 1978. The influence of stand factors on parasitism of Spruce budworm eggs by Trichogramma minutum. Environmental Entomology 7:685-688.

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Osier, T.L. and S.M. Jennings. 2007. Variability in host-plant quality for the larvae of a polyphagous insect folivore in midseason: the impact of light

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Pinault, L., Georgeson, E., Guscott, R., Jameson, R., LeBlanc, M., McCarthy, C., Lucarotti, C., Thurston, G., and D.Quiring. 2007. Life history of Iridopsis ephyraria, (Lepidoptera: Geometridae), a defoliator of eastern hemlock in eastern Canada. Journal of the Acadian Entomological Society 3:28-37.

Pinault, L., Thurston, G. and D. Quiring. 2009. Interaction of foliage and larval age influences preference and performance of a geometrid caterpillar. The Canadian Entomologist 141:136-144.

Pinault, L. and D.T. Quiring. 2008. Sampling strategies and density-defoliation relationships for the pale-winged gray moth, Iridopsis ephyraria, on mature eastern hemlock. Forest Ecology and Management 255:2829-2834.

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Quiring, D. 1994. Diel activity pattern of a nocturnal moth, Zeiraphera canadensis, in nature. Entomologia Experimentalis et Applicata 73:111- 120.

Quiring, D.T. 1993. Influence of intra-tree variationin time of budburst of white spruce on herbivory and the behavior and survivorship of Zeiraphera canadensis. Ecological Entomology 18:353-364.

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CONNECTING STATEMENT

In Chapter 1, I reviewed the literature on pale-winged gray moth, eastern hemlock trees, insect-plant interactions and factors that can affect insect feeding preference. Chapter 2 will describe the PWG egg and larval distribution in mature eastern hemlocks to better understand the PWG feeding preference for foliage located in the mid-lower crown.

CHAPTER 2: EGG AND LARVAL DISTIBUTION OF THE PALE- WINGED GRAY MOTH (IRIDOPSIS EPHYRARIA) ON EASTERN HEMLOCK TREES IN NOVA SCOTIA.

2.1 Introduction

Insects are capable of exploiting different plant parts (e.g., roots, foliage, buds) as well as selecting for microhabitats within plants that might provide additional benefits. For example, defoliators might prefer sun-exposed foliage that normally contains high nutritional value (e.g., White 1984). In addition, female insects can select oviposition sites that benefit their offspring. The preference- performance hypothesis (a.k.a the naive adaptationist or the "mother-knows-best" hypotheses, Gripenberg et al. 2010) states that females select oviposition sites to increase offspring performance measurable by fitness proxies such as survival, pupal mass and developmental rate (Forister 2004, Poykko 2006).

There are several advantages for larvae to stay on female preferred oviposition sites after hatching: (1) to avoid predation/parasitism by natural enemies (e.g.,Videla et al. 2006), (2) to feed on highest foliage quality (e.g., Heisswolf et al. 2005) and (3) to be in synchrony with tree phenology (e.g., Quiring 1993, Fry et al. 2009). Oviposition sites between crown levels can be sex- biased; for instance only female juvenile yellowheaded sawflies normally complete their development in the upper crown (Johns et al. 2010). Larval distribution can also vary between species and over time. For example, some insect species tend to stay close to egg site after hatching (especially species with low mobility) and others move acropetally over the developmental season (Quiring 1993, Carroll and Quiring 1994).

Females of the pale-winged gray moth (Iridopsis ephyraria (Walker)), a generalist defoliator of eastern hemlocks in southwest Nova Scotia, prefer laying eggs in the upper crown of mature trees (highest egg density at this height, Pinault et al. 2007). Eggs are laid individually in deep bark crevices of hemlock trees or

19 under pieces of lichen. However, the total number of eggs per height possibly varies from egg density if I take into account the bole area. In order to increase knowledge about PWG natural history and to better understand why the species mainly consumed foliage in the mid-lower crown, I studied the PWG absolute number of eggs and the larval distribution, both per crown locations, on mature eastern hemlocks. Based on field observations, I expected the number of eggs to be highest in the lower half of the tree trunk because of a greatest surface area available. I also expected larvae to be more numerous in the lower crown based on the defoliation pattern seen on infested sites. Finally, I predicted that larvae number would decrease over the developing season because of many factors that can affect larval survival over time (e.g., predation, natural mortality, unfavourable environmental conditions).

2.2 Material and Methods

Pinault and colleagues (2007) reported that egg density of the pale-winged gray (PWG) moth increases linearly from the bottom to the top of the bole (y = 1.82x+0.44, y = log egg density per area and x = proportion up the bole). Using the linear regression from Pinault et al. (2007), I tested whether egg absolute number varies significantly with tree height with the additional consideration of surface area of the tree bole. The percentage of eggs laid on the upper, mid and lower foliated crown and on the tree bole (i.e., below the branches with foliage) was estimated on 5 mature trees at each of two different sites (n = 10) with PWG populations. The sites were located at Maitland Bridge (44°29'4.45"N/65°13'4.91"W) and at South Brookfield (44°23'18.84"N/64°58'28.30"W) in southwest Nova Scotia. Lower bole was defined as approximately 1.3 to 5m height in most mature trees. The crown area was divided into three sections where the lower crown was approximately positioned between 5 to 9m in height, the middle crown was normally located around 9 to 14m height and finally, the upper crown was defined as approximately 14 to 17m height. Trunk area for the first 1.3m and for the last 1.5m of the tree height was not included in the area calculation since they were omitted in Pinault et al. (2007) linear regression. For each tree section (i.e., lower bole as well as lower, middle and upper crowns), I measured the circumference of each extremity, except for the upper crown segment where the tip was unreachable. A hypsometer (Laser technology inc., SN#i02090) was used to measure the length of each segment and the total tree height. The average circumference of each tree section was multiplied by its respective height to obtain an area approximation for each trunk section. The area measured for the last segment (i.e., upper crown) was slightly overestimated because only the circumference of the base was taken into consideration in the calculations. However, only small variations in trunk diameter could be seen at this height (pers. obs.). Calculated areas were used to estimate space available for female to lay their eggs. Area of each section was multiplied by the average egg density

(linear regression) of each section to obtain an estimate of the total egg number per section.

To determine where larvae are mainly found in mature trees, the beating technique was used on the Maitland Bridge and South Brookfield sites with PWG populations. On 8, 9 June 2010, when larvae were mainly in the first instar, I randomly picked 4 trees per site on which I beat the lower and upper crown; the beating sheet was 1m2 in size. Here, upper crown is defined as foliage located in the top half of the tree crown whereas lower crown is defined as foliage located in the bottom half. For each height, 3 branches were selected and shook 5 times. The number of PWG on the beating sheet was recorded for each level. On 19 June 2010, when larvae were mostly second and third instar, I randomly picked 6 trees on the two same sites and followed the same technique to count larvae. To test the influence of crown level and collection date, I used a generalized linear mixed model with the glmer function for poisson distribution using the lme4 package in the R statistical program. The best model had the smallest AIC and did not include date and crown level interactions. Sites were considered as a random effect in the model.

2.3 Results

Although egg density is higher in the upper crown (Pinault et al. 2007) the absolute number of eggs was highest in the mid-lower crown when considering the availability of bole area for oviposition sites (Figure 2.1). Just after hatching, larvae were found primarily in the lower crown (z-value = -3.582, p ˂ 0.001) (Figure 2.2a) and their number decreased over the larval development season (z- value = -2.825, p ˂ 0.01) (Figure 2.2b).

Figure 2.1 Mean (±SE) percentage of eggs (estimated absolute number of eggs per section / estimated total number of eggs per tree) laid by PWG female for each tree section during summer 2010.

2.4 Discussion and Conclusion

The objective of this chapter was to describe the egg and larval distribution of the PWG in mature eastern hemlocks to better understand their feeding preference for the mid-lower crown needles. Various factors combined together can help explain egg and larval distributions of certain species (e.g., female preference, predation, foliage quality). As mentioned, female PWG prefers to lay their eggs in the upper crown of mature hemlocks (Pinault et al. 2007). The PWG egg distribution (when accounting for the trunk area) and larval distribution, however, have yet to be described. The main interest was to understand if PWG larvae mainly feed in the upper crown level partly because this is where the larvae are when they hatch.

My results showed that absolute egg number decreased when going up the tree presumably because less trunk area was available for females to lay their eggs. After hatching first instar larvae were mainly found in the lower crown, either suggesting that their movements are limited after hatching and/or that larvae silk down from the upper canopy after hatching. Further beating during the developmental season, when larvae were in second and third instar, also showed that larvae were mostly distributed in the lower crown. In addition, their average number of larvae per beating decreased seasonally (Fig. 2.2b). This finding was expected since several abiotic and biotic factors might affect larval survival temporally (e.g., natural enemies and hygrothermal stress) in additional to the spatial patterns I uncovered.

2.5 Literature cited

Carroll, A.L. and D.T. Quiring. 1993. Interactions between size and temperature influence fecundity and longevity of a tortricid moth, Zeiraphera canadensis. Oecologia 93:233-24.

Forister, M.L. 2004. Oviposition preference and larval performance within a diverging lineage of lycaenid butterflies. Ecological Entomology 29:264- 272.

Gripenberg, S., Mayhew, P.J. and T. Roslin. 2010. A meta-analysis of preference– performance relationships in phytophagous insects. Ecology Letters 13:383–393.

Heisswolf, A., Hobermaier, E. and H.J. Poethke. 2005. Selection of large host plants for oviposition by a monophagous leaf beetle: nutritional quality or enemy-free space? Ecological Entomology 30: 299–306.

Johns, R., Quiring, D., Ostaff, D. and E. Bauce. 2010. Intra-tree variation in foliage quality drives the adaptive sex-biased foraging behaviors of a specialist herbivore. Oecologia 163: 935-947.

Pokkyo, H. 2006. Females and larvae of a geometrid moth, Cleorodes lichenaria, prefer a lichen host that assures shortest larval period. Environmental Entomology 35: 1669-1676.

Quiring, D.T. 1993. Influence of intra-tree variationin time of budburst of white spruce on herbivory and the behavior and survivorship of Zeiraphera canadensis. Ecological Entomology 18:353-364.

Videla, M., Valladares, G. and A. Salvo. 2006. A tritrophic analysis of host preference and performance in a polyphagous leafminer. Entomologia Experimentalis et Applicata 121:105–114.

White, T.C.R. 1984. The abundance of invertebrate herbivores in relation to the availability of nitrogen in stressed food plants. Oecologia 63:90-105.

CONNECTING STATEMENT

Chapter 2 focussed on egg and larval distribution of the pale-winged gray moth in mature eastern hemlocks. I found that eggs and larvae are mainly located in the mid-lower crown throughout the growing season. Chapter 3 will focus on understanding the feeding preference of the PWG by testing two complementary hypotheses: foliage quality and hygrothermal stress.

CHAPTER 3: THE EFFECTS OF HYGROTHERMAL STRESS AND FOLIAGE QUALITY ON THE PALE-WINGED GRAY MOTH (IRIDOPSIS EPHYRARIA) IN HEMLOCK STANDS OF NOVA SCOTIA

3.1 Abstract

The pale-winged gray moth (Iridopsis ephyraria) is an important lepidopteran defoliator of mature and sapling eastern hemlock trees (Tsuga canadensis) in south-west Nova Scotia Canada. In situ, the species shows a preference for foliage located in the mid-lower crown. The objective of this research was to test two factors affecting larval feeding preference of the pale- winged gray (PWG) moth: variation in temperature and humidity (hygrothermal stress) and variation in foliage quality between the crown locations. These hypotheses were tested using field experiments in 2009-2010. Performance results revealed that hygrothermal stress was the main factor affecting the distribution of individuals between crown locations but that foliage quality also played a partial role in the feeding preference. Larvae survived best in shaded lower crown but adults were larger when raised on sun-exposed foliage located in the upper crown suggesting that sun-exposed foliage had a highest nutritional quality. PWG preferred feeding on current-year needle and results from foliage chemistry analysis revealed that this age-class foliage had more water and nitrogen contents than older foliage.

3.2 Introduction

The pale-winged gray (PWG) moth Iridopsis ephyraria (Walker) (Lepidoptera: Geometridae) is widely distributed across Canada (Rindge 1966). The first known outbreak of PWG occurred in the Kejimkujik National Park (Nova Scotia) in 2002 (Pinault et al. 2007). Here, PWG defoliated eastern hemlock trees (Tsuga canadensis (L.) Carr.) and killed up to 90% of sapling trees the first year of this outbreak. The following year, PWG attacked mature eastern hemlocks and was responsible for up to 40% of hemlock mortality in this part of Nova Scotia (Pinault and Quiring 2008).

Although PWG is known to mainly defoliate hemlocks, it can also feed on other tree species including sugar maple (Acer saccharum Marshall) and balsam fir (Abies balsamea (L.) Mill.). A previous study by Pinault et al. (2009) reported that PWG feed on all age classes of hemlock foliage but defoliation is higher on current year shoots. In situ, defoliation on mature hemlocks by PWG larvae is characteristic and thus easy to diagnose; larvae feed mainly on foliage located in the mid-lower crown and only sparsely attack foliage in the upper crown. Insect- plant interactions have been well studied (Schoonhoven et al. 2005) and often insects feeding behaviour directly interacts with the plants’ efforts to avoid herbivory. Production of allelochemicals by plants, for instance, is a well know example of coevolution where plants produce toxins to deter insect herbivory (Cornell and Hawkins 2003, Schoonhoven et al. 2005).

Several factors could explain the asymmetrical consumption of eastern hemlock foliage by PWG larvae: variations in foliage quality between crown locations, hygrothermal stress (i.e., heat and water stress), natural enemies, female preference for oviposition sites. The sun/shade hypothesis states that sun-exposed leaves have a higher nutritional quality than shaded leaves because they tend to have more nitrogen content (e.g., White 1984, Dudt and Shure 1994, Fortin and

Mauffette 2002, Osier and Jennings 2007) which is a limiting factor in insects’ growth (Feeny 1970). Foliage with higher nitrogen content but also with higher water concentrations can directly affect insect larval performance (e.g., Scriber and Slansky 1981, Fortin and Mauffette 2002,)

To escape the negative effects of hygrothermal stress, some insect species change their daily activities to avoid feeding during high temperatures and low humidity (Quiring 1994, Bento et al. 2001). Insects might also select for a particular crown location to escape from natural enemies. Several studies shows that predation rate by natural enemies can vary between crown locations (Eikenbary and Fox 1968, Weseloh 1972, Kemp and Simmons 1978, Jennings and Houseweart 1983, Aikens 2010). Also, females may have a preference in oviposition sites that determine where their larvae will mainly be and feed after hatching and larvae tend to do better on these preferred sites (female preference- larval performance hypothesis, Gripenberg et al. 2010).

Studies suggest that feeding in the mid-lower crown could be adaptive. Yet, based on the sun/shade hypothesis, we know that sun-exposed foliage in the upper crown should be best nutritionally but should also be under higher heat constraints. To understand if the PWG feeding habit is adaptive, I tested two main hypotheses: hygrothermal stress and foliage quality hypotheses. According to the sun/shade hypothesis, sun foliage contains more nitrogen, one of the most important nutrients for caterpillar growth. As foliage in the upper crown receives more sun rays, it should contain more nitrogen and be better nutritionally than foliage lower in the crown. If variation in foliage quality due to differences in sun exposure affects larval feeding preference, it is expected that PWG performance will be higher in the upper crown. If concentration of secondary metabolites affects the feeding preference of PWG larvae, it is likely that allelochemical content will be highest in the upper crown since this crown level seems less preferred based on field observations. If spatial changes in foliage quality affects PWG fitness, variations in larval survival between crown levels of mature

32 hemlocks is expected. Finally, PWG should perform better in the lower crown of mature trees, where temperature is lower and humidity higher, if PWG is affected by hygrothermal stress.

3.3 Material and Methods

3.3.1 Description of study sites and insect

Studies were carried out at Moose Hill (2009-2010) (44° 7’25.17’’N/64°47’44.34’’W) and Milton (2010) (44°4’40.64’’N/64°49’10.44’’W) in south-west Nova Scotia (Figure 3.1). One mature and one nearby (< 1km) young eastern hemlock stand was selected at each site. The selected stands did not contain any noticeable defoliation and very few PWG larvae were observed during the two-year study. Mature trees were approximately 25m tall, with mean diameter at breast height of 162.06 ± 8.48cm and 162.00 ± 6.74cm on Moose Hill and Milton (n=9). Sapling trees were approximately 2.43 ± 0.07m tall and 15.50 ± 0.39 years old at Moose Hill whereas they were about 2.46 ± 0.07m tall and 16.63 ± 0.73 years old at the Milton site. First instar PWG larvae used in manipulative experiments (described below) were collected during cool mornings from other unmanaged hemlock stands in Queens County and transported to the study sites in vials placed in coolers with ice.

Figure 3.1 Map of Queens County in Southwest Nova Scotia with insert illustrating the location of the two study sites, Milton and Moose Hill.

3.3.2 Experimental design

Experiment on mature hemlock trees

To evaluate the potential influence of hygrothermal stress and foliage nutritional quality on the performance of PWG developing in different crown locations, a manipulative field study was carried out on 10 mature hemlock trees at Moose Hill in 2009 and on 9 mature trees at Moose Hill and at Milton in 2010. Two branches in each of three crown locations were selected on each study tree: (1) sun-exposed upper crown; (2) shaded upper crown; and (3) shaded lower crown. To minimize the chance that branches in the shaded upper crown and shaded lower crown were exposed to sunlight, I selected shaded branches in the upper and lower crown that never extended > 1m from the tree bole. Climbing

35 equipment and single rope techniques were used to access branches on the large trees. One open vial containing five first-instar larvae was attached to each selected branch with twist ties. Each branch was enclosed in a sleeve cage as described in Pinault et al. (2009). Previous studies have demonstrated that sleeve cages protect caterpillars from parasitoids and predators but do not influence branch or caterpillar development, or leaf chemistry (Rossiter et al. 1988, Quiring 1993, Carroll and Quiring 1994, Parsons et al. 2005). Each sleeve cage contained current-year to 7 ± 2 year-old foliage. Relatively low levels of defoliation by sleeve-caged larvae (see results) indicate that this allowed sufficient foliage for five larvae to complete development.

As soon as larvae started to pupate in August, branches were cut (with sleeve cages still attached) and transported to a laboratory at the University of New Brunswick in Fredericton. Juveniles (5th instar larvae or pupae) were reared up to adult emergence in plastic containers with moistened vermiculite at the bottom. Water and current-year to two-year-old foliage was provided on a daily basis to mature 5th instars until they pupated. Newly emerged adults were sexed based on antennal morphology (Rindge 1966). The length of the subcostal vein on the forewing of emerged adults was also measured as a correlate for fitness. Realized fecundity of PWG females was positively related to the length of the subcostal vein in laboratory studies (Quiring unpubl. data).

In 2010, one half of each branch was randomly selected and defoliation was visually assessed on all current-year to 3 year-old shoots using the following defoliation classes: 0, 1–5, 6–20, 21–40, 41–60, 61–80, 81–99, or 100%. To determine the foliar content of water, other nutrients (nitrogen and sugars) and secondary compounds (phenol, tannins and monoterpenes), shoots from branches adjacent to branches bearing sleeve cages were collected from each crown position on three trees per site in 2010. Foliage for biochemical analyses was collected at the beginning (early June) and end (late July) of larval development, immediately frozen and stored at -20ºC. Current-year foliage and one to three

36 year-old foliage (hereafter “old foliage”) were processed and analysed separately in Eric Bauce’s laboratory at Laval University. For a complete description of the techniques used for biochemical analysis of the foliage, refer to Bauce et al. 1994. Additional branches were collected in August to determine foliage biomass between upper and lower crown. Dry mass of cut branches included current-year to 7 years old foliage.

To determine variations in temperature and humidity between crown locations, one DS1923 temperature/humidity logger iButtons (Dallas semiconductor ©) was placed inside one sleeve-cage in each of the three crown positions on two trees at Moose Hill and on two trees at Milton. IButtons recorded temperature (°C) and humidity (%RH) once per hour. They remained in the sleeve-cages throughout the season. Temperature and humidity data were only collected in 2010.

Experiment on sapling hemlock trees

To further test the relative effects of hygrothermal stress and foliar nutritional quality of sun versus shade leaves, on PWG performance, another manipulative field study was carried out on eastern hemlock saplings at Moose Hill in 2009 and on saplings at Moose Hill and at Milton in 2010. This experiment was done to tease apart the independent effect of hygrothermal stress from the effects of feeding on sun versus shade foliage by placing larvae on sun foliage that was directly exposed to sunlight or experimentally shaded. Fifteen (2009) or 10 (2010) saplings per site were assigned to each of three different sun exposure treatments: (1) sun-exposed; (2) shaded; and (3) previously sun-exposed but experimentally shaded saplings. Sun-exposed as well as previously sun-exposed but experimentally shaded saplings were immature eastern hemlocks located in a large canopy opening or in a clearing < 1km from the mature stand. A white or light-gray fabric, held taunt by wires and supported by a wood frame, was placed approximately 1m above study branches on previously sun-exposed but

37 experimentally shaded saplings. I assumed that this treatment would provide sun foliage to larvae but protect them from exposure to direct sunlight. Shaded saplings were located in the understory of the mature hemlock stands.

The influence of the three treatments on PWG performance was assessed using the same techniques described above for mature trees. One open vial containing five first-instar larvae was attached to each of two mid-crown branches on each sapling. A sleeve cage was then placed over and attached to each study branch. As the saplings were younger than the mature trees, sleeve cages only contained current-year to 5 ± 2 year-old foliage. Sleeve-caged branches were cut and brought to the University of New Brunswick in Fredericton when larvae started to pupate in August. Last instar larvae and pupae were reared, emerged adults sexed and measured, and defoliation on branches visually assessed as described above for matures. Similarly, foliage was collected from branches adjacent to the ones bearing sleeve cages in early June and late July and the nutrients and secondary chemicals in these foliar samples were examined at Laval University as described above.

DS1923 temperature/humidity logger iButtons (Dallas semiconductor ©) were placed in one sleeve cage on each of two (Milton) or three (Milton) trees in each sun exposure treatment. As previously mentioned, iButtons were programmed to record temperature (°C) and humidity (%RH) hourly.

3.3.3 Statistical analyses

The influence of crown location (mature trees) or sun exposure (saplings), year and their interactions on larval (i.e., first instar to pupation) and total (i.e., first instar to adult emergence) survival was evaluated with a generalized linear mixed model (glmm) using the glmer function for binomial distribution in the lme4 package in the R statistical program. The best models for larval and total

38 survival were selected based on the smallest Akaike information criterion (AIC). Trees nested in sites were considered as a random effect. Because there were strong crown location or sun exposure and year interactions for larval survival, glmm models were also run for each year.

The effect of crown location (mature trees) or sun exposure (sapling trees) on the sex ratio of PWG adults was assessed using a contingency table followed by a Pearson Chi-squared test. A generalized linear mixed model, using the lmer function for a gaussian distribution, was also used to evaluate the influence of crown location (or sun exposure), sex and year on adult size (subcostal vein length). However, the smallest AIC model from the analysis excluded the influence of year for the mature tree model and no interactions were tested for either model. Trees nested in sites were included as a random variable.

To test the effects of crown location (or sun exposure), month and foliage age on the foliar monoterpene, phenol, sugar and nitrogen content, generalized linear mixed models with the lmer function were used. The best model for each foliar compound was selected using the smallest AIC. Interactions between fixed variables were or were not tested depending on the best model kept, and trees nested within sites or the main effect of site considered as a random effect. The influence of the same three independent variables on water content was evaluated using glmer for binomial distributions. Again, no interaction between variables was tested and sites were considered a random effect.

The effects of crown location (mature trees) or sun exposure (saplings), foliage age and their interactions on percent defoliation by PWG larvae were tested using the glmer function with glmm. Trees nested in sites were included in the model as a random variable. The influence of crown level on foliage biomass was evaluated using the lmer function. Sites were considered as a random effect and dry weight data were log transformed to meet normality.

Temperature and relative humidity were highly correlated (see results) and therefore statistical analyses were only carried out for temperature only. The influence of crown location (mature trees) or sun exposure (saplings), month (June or July) and time (am or pm) on temperature was assessed using the lmer function with glmm. Data were root transformed to meet normality and homogeneity assumptions. Trees nested within sites (sapling) or the main effect of site (mature trees) was considered a random effect.

3.4 Results

Mature tree experiment

Defoliation by the PWG was influenced by crown location (includes crown level and the effect of sun exposure) (Figure 3.2a) and foliage age (Figure 3.2b, Appendix 1). Defoliation was higher in the shaded lower crown than the sun-exposed upper crown (Figure 3.2a), whereas defoliation in the shaded upper crown was intermediate. Defoliation was highest on current-year and lowest on one-year old foliage (Figure 3.2b). Small variation in the amount of defoliation on different-aged foliage among crown location resulted in small but significant interactions between crown locations and foliage age (Figure 3.2b, Appendix 1). Foliage biomass was more than two times highest in the sun-exposed upper crown (7.29g ± 1.26) compared to the shaded lower crown (3.05g ± 0.57). The shaded upper crown was an intermediate (5.47g ± 1.88).

Temperatures in the shaded upper crown ( = 17.97°C ± 0.06) were similar to those in the sun-exposed upper crown ( = 17.89°C ± 0.06) but temperatures in the shaded lower crown were significantly cooler ( = 17.44°C ± 0.06, p ˂ 0.001). Temperatures were higher in July ( = 20.49°C ± 0.05) than in May ( = 14.01°C ± 0.14) and June ( = 16.34°C ± 0.04). Also, temperatures were higher during afternoons ( = 19.68°C ± 0.05) than mornings ( = 15.86°C ± 0.04). Temperatures were negatively correlated to humidity (H = -0.109489T + 26.837433, where H = Humidity and T = Temperature; R-squared = 0.2296, p ˂ 0.001) and thus larvae developing in the cooler lower crown were exposed to higher humidity. Neither larval survival nor survival until adult emergence (total survival) was significantly influenced by crown location in 2009 (Figure 3.3, Table 3.1). In contrast, in 2010, larval survival was highest in the shaded lower crown and lowest in the sun-exposed upper crown (Figure 3.3b, Table 3.1). Although the same trend (Figure 3.3b) was observed for total survival, the trend

41 was not significant. Sex ratios were more female-biased in the upper crown exposed to sun (Figure 3.4a, Chi squared = 6.01, p ˂ 0.05). Similarly, both male and female size, as indicated by subcostal vein length, was highest for adults that developed in the sun-exposed upper crown (Figure 3.4b). Females (8.66mm ± 0.05) were smaller than males (8.94mm ± 0.06, t-value = 4.18, p ˂ 0.001) but adult size was not influenced by year.

Larger adult size and a more female-biased sex ratio, despite lower larval survival (2010), in the sun-exposed upper crown suggests that foliage nutritional quality may be higher in sun-exposed leaves in the upper crown, supporting the sun/shade hypothesis. However, contrary to the prediction emanating from the sun/shade hypothesis, nitrogen content was slightly higher in the shaded lower crown (2.03% ± 0.09) compared to the shaded upper crown (1.84% ± 0.10) and the sun-exposed upper crown (1.88% ± 0.08) (Table 3.2, p ˂ 0.05). There were no differences between crown locations in foliar water content (p ˃ 0.05). Water content was higher in current-year than older foliage (Figure 3.5, z-value = -5.037, p ˂ 0.001). Foliar nitrogen content was similar among different-aged foliage in June but was highest in current-year foliage in July, resulting in an interaction between month and foliage age (Table 3.2). Sugar concentrations were two times higher in ≥ one-year old than current-year foliage (t-value = 3.510, p ˂ 0.001).

The foliar content of secondary compounds (monoterpenes, phenols and tannins) were not directly affected by crown location but were influenced by crown location, month or their interaction. The foliar content of a-phellandrene was higher in July (80.54 ± 4.92) than in June (35.58 ± 6.34, t-value = -5.043, p ˂ 0.001) (Appendix 2). In June, a-phellandrene and myrcene concentrations (ng/mg of dry foliage) were higher in the shaded upper crown (49.28 ± 14.53 and 162.89 ± 27.61) than the shaded lower crown (26.21 ± 8.50 and 151.22 ± 27.61) and sun- exposed upper crown (31.24 ± 8.60 and 139.66 ± 24.32) (Appendix 2), but in July concentration of a-phellandrene was similar in the three crown locations whereas

42 myrcene concentration was highest in shaded lower crown in July, resulting in an interaction between crown location and month (t-values = 2.199 and 1.672, p ˂ 0.05) (Appendix 2). A-phellandrene was also influenced by a three ways interaction between crown location, month and foliage age (Appendix 2). Some monoterpenes were influenced by foliage age or month or by their interaction. Myrcene content was highest in June (t-value = 5.028, p ˂ 0.001) and in current- year foliage (t-value = 2.319, p ˂ 0.05). In June, the myrcene concentration between the two months was more pronounced resulting in a month- age interaction (t-value = 4.865, p ˂ 0.001. In June, but not July, b-pinene content was highest in current-year foliage, resulting in a month-age interaction (t-value = 2.93, p ˂ 0.01) (Appendix 2). The foliar content of a-pinene was highest in current-year foliage (t-value = -2.311, p ˂ 0.05) whereas terpinolene and limonene contents were highest in older foliage (t-values = 2.974 and 2.282, p ˂ 0.01 and p ˂ 0.05) (Appendix 2). In June, limonene content in current-year foliage was higher than older foliage whereas an opposite trend was observed in July leading to an interaction between month and age (t-value = 2.195, p ˂ 0.05). Bornyl acetate and camphene were influenced by month (t-value = -3.173 and 3.390, p ˂0.01 and p ˂ 0.001) and foliar age (t-value = -2.144 and 2.901, p ˂ 0.05 and p ˂ 0.01). Foliar concentrations of bornyl acetate were influenced by an interaction between month and foliage age (t-value = -1.684, p ˂ 0.05) (Appendix 2).

Sapling tree experiment

Defoliation on eastern hemlock saplings was influenced by sun exposure and foliage age. Defoliation was highest on shaded trees and lowest on sun- exposed trees (Figure 3.5a). Generally, defoliation was highest on the two oldest age-classes (Figure 3.5b) however, the influence of foliar age-class on defoliation varied with sun exposure, resulting in an interaction between these factors (Figure 3.5b, Appendix 1). Although defoliation of 2 and 3 year-old foliage was highest on previously sun-exposed but experimentally shaded saplings, defoliation of

43 current-year and one year old foliage was highest on shaded saplings (Figure 3.5b).

Temperatures on previously sun-exposed but experimentally shaded saplings ( = 18.21°C ± 0.08) were similar to sun-exposed saplings ( = 18.29°C ± 0.08), whereas temperatures were lowest on shaded saplings ( = 17.06°C ± 0.06, p ˂ 0.01). Temperature was lower in June ( = 16.44°C ± 0.05) than in July ( = 20.25°C ± 0.07) and temperature was higher in afternoons ( = 19.79°C ± 0.06) compared to mornings ( = 15.92°C ± 0.05). Temperatures were negatively correlated to humidity (H = -0.166473T + 32.410234, where H = Humidity and T = Temperature; R-squared = 0.3539, p ˂ 0.001) and thus larvae feeding on shaded trees were exposed to the lowest temperatures and had the highest humidity. Survival of first instar PWG larvae placed on sapling trees varied between years (Table 3.3). In 2009, both larval and total survival was highest on artificially shaded trees that were previously sun-exposed but sun exposure of trees did not significantly influence survival in 2010 (Figure 3.6, Table 3.3). Sapling sun exposure did not influence the sex-ratio of survivors in either year (Chi-squared = 1.61, p ˃ 0.05). Similarly, sun exposure did not influence the size of adult survivors, estimated by the length of the subcostal vein. As for individuals that developed in mature trees, females that developed on saplings were smaller than males (8.66mm ± 0.05 versus 8.94mm ± 0.06, p ˂ 0.001). Adult size was also larger in 2009 than in 2010 (8.82mm ± 0.05 versus 8.79mm ± 0.06, p ˂ 0.01).

Foliar content of nutrients (nitrogen and sugar) was not influenced by leaf sun exposure. However, the concentrations of three secondary compounds varied with sun exposure (Appendix 2). The foliar contents of b-pinene and myrcene were higher in shaded saplings than in previously sun-exposed but experimentally shaded saplings (t-values = -1.910 and -1.924, p ˂ 0.05) while terpinolene content was highest in sun-exposed saplings (1.774 p ˂ 0.05) (Figure 3.8, Appendix 2). There were some interactions between sun exposure and foliage age or month. Foliar content in b-pinene was slightly higher in July on shaded and artificially

44 shaded saplings whereas sun-exposed saplings had higher b-pinene content in June (treatment and month interaction, t-value = 1.697, p ˂ 0.05) (Appendix 2). There was a more distinct difference in camphene concentration between months in shaded saplings compared to artificially shaded and sun-exposed saplings (Appendix 2), resulting in an interaction between sun exposure and month (t- value = 1.848, p ˂ 0.05). Limonene content was influenced by an interaction between treatment, month and age (t-value = 1.735, p ˂ 0.05). Foliar content of a- phellandrene was highest in July for shaded and artificially shaded saplings whereas it was highest in June for sun-exposed saplings, resulting in a sun exposure and month interaction (t-value = 1.735, p ˂ 0.05) (Appendix 2).

The foliar content of many nutrients and secondary compounds varied with foliage age and month. Just like in mature trees, nitrogen and water content of saplings was highest in current-year foliage (t-value = -6.706, pnitrogen ˂ 0.001, z-value = -4.357 pwater = ˂ 0.001) and sugar concentrations were highest in older foliage (t-value = 1.75, p ˂ 0.05) (Appendix 2). Nitrogen was also highest in June (t-value = 2.074, p ˂ 0.05). Different monoterpene concentrations differed between foliar age-classes. Myrcene content was highest in current-year foliage while terpinolene content was highest in older foliage (C+1 to C+3) (Appendix 2). Foliar concentrations of b-pinene were highest in current-year foliage in June, while in July concentrations did not vary between different-aged foliage, leading to a month and foliage age interaction (t-value = 1.963, p ˂ 0.05). Finally, some monoterpene concentrations differed over time (from June to July); camphene, a- phellandrene and bornyl acetate concentrations increased throughout the season while myrcene concentration was highest in June in current-year foliage but highest in July in older foliage (Appendix 2).

A AB B

Fitness Std. Source Estimate z-value p-value correlate Error % larval survival- Intercept -0.125 0.299 -0.416 0.677 2009 Treatment 0.399 0.295 1.352 0.176 Ushade Treatment 0.354 0.295 1.202 0.229 Usun % larval survival- Intercept -0.102 0.199 -0.512 0.609 2010 Treatment -0.409 0.220 -1.862 0.063 Ushade Treatment -0.635 0.230 -2.758 0.006 Usun *In analyses, effects of Ushade and Usun were compared to Lshade (dummy variable).

A AB B

b a a B A A

Nitrogen content (%) Source Estimate Std. t- value p-value Error (intercept) 1.94 0.09 21.66 0.00 TreatmentUshade -0.18 0.09 -2.04 0.02 TreatmentUsun -0.14 0.09 -1.59 0.06 MonthJune 0.65 0.10 6.30 1.54E-08 AgeOlder -0.11 0.10 -1.08 0.14 MonthJune:AgeOlder -0.72 0.15 -4.91 3.36E-06 *In analyses, effects of Ushade and Usun were compared to Lshade (dummy variable).

a b

Figure 3.57Mean (± SE) percent nitrogen (open bars) and water (closed bars) content of current- year (CY) and older (1 to 3 years old) foliage of eastern hemlock during summer 2010. Bars with different letters represent significantly different results.

A AB B

Fitness Source Estimate Std. z-value p-value correlate Error % larval Intercept -0.964 0.229 -4.221 2.44e-05 survival- 2009 Treatment 1.671 0.319 5.245 1.56e-07 SS Treatment 0.364 0.323 1.128 0.259 Sun % larval Intercept -1.422 0.319 -4.459 8.22e-06 survival- 2010 Treatment 0.216 0.303 0.715 0.474 SS Treatment 0.482 0.298 1.618 0.106 Sun *In analyses, effects of SS and Sun were compared to Shade (dummy variable).

B a a A A

A A

3.5 Discussion

PWG larvae cause higher levels of defoliation in the lower than upper crown of mature eastern hemlock trees. This could be partially explained by oviposition preference by adult females (Pinault et al. 2007) and/or the foraging behaviour of young larvae (Chapter 2). In the current study higher levels of defoliation were observed in the lower than upper crown when similar numbers of first instar larvae were forced to develop in one crown location. In 2010, higher defoliation in the lower crown was associated with higher larval survival, suggesting that this preferential feeding is adaptive, at least in the absence of natural enemies. My study tested two separate yet complementary hypotheses and I show that both are supported: the increased performance of PWG juveniles in the lower than upper crown was due to contrasting effects of crown level on hygrothermal stress and foliage nutritional quality.

3.5.1 Hygrothermal stress hypothesis

On mature hemlock trees, larval survival was highest in the shaded lower crown during one of the two years of a manipulative field experiment, and this partially supports the hygrothermal stress hypothesis. Temperatures were lower and humidity was higher in the shaded lower crown than the sun-exposed or shaded upper crown. Similar temperatures and humidity in the two upper crown locations (shaded vs sun-exposed) suggest that larvae in these two upper crown locations experienced similar environmental conditions. Crown location significantly influenced larval survival in 2010 but not in 2009. The difference between years may be explained because temperatures and direct solar insulation were lower in 2009 than 2010. The average temperatures for the month of June and July in 2009 (15.8 ± 0.46°C, 18.2 ± 0.41°C) were lower compared to 2010 (16.3°C ± 0.55, 20.3 ± 0.46°C) at the Kejimkujik 1 station in Nova Scotia (Environment Canada 2011). It also rained more in June 2009 (total of 160.4mm)

56 compared to June 2010 (total of 97.8mm) and the much lower rainfall in 2010 could explain why hygrothermal stress had an effect on PWG in 2010.

Results from the sapling trees experiment also supports, in part, the hygrothermal stress hypothesis. In 2009, both larval and total (i.e., first instar to adult emergence) survival was highest on sun-exposed but experimentally shaded foliage. It is surprising that shading only increased juvenile performance in 2009, when temperatures were lower than in 2010. However, temperatures and humidity were similar inside sleeve cages in 2010 (measurements were not made in 2009) for the two sun-exposed treatments. These data, along with the results from experiment on mature trees, suggests that increased mortality in sun-exposed larvae may be due primarily to their direct exposure to sun rays rather than differences in air temperature or relative humidity per se.

It is documented that higher ambient temperatures can accelerate insect developmental rates (e.g., Lindroth et al. 1997). However, insects can benefit from increased heat until the internal body temperature reaches a certain threshold (i.e., lethal temperature) where their survival can be compromised (Casey 1976). Feeding activities can stop if temperatures become too warm (Casey 1976). Some species are believed to change their daily foraging activities to avoid high temperatures. For instance, the spruce budworm (Choristoneura fumiferana) is known to forage daily during dawn and dusk presumably to avoid high temperatures (e.g., Quiring 1994). PWG larvae likely exhibit similar behavioural response during foraging to avoid the negative impacts of sun exposure.

3.5.2 Foliage quality hypothesis

Spatial variations in foliage quality

Although survival of PWG that developed in the sun-exposed upper crown (i.e., foliage in the upper crown located on branch extremities) of mature trees was lower, the sex ratio of adult survivors was female-biased and adults were bigger than adults that developed in the lower crown. Wing lengths of individuals that developed in the shaded upper crown (i.e., foliage in the upper crown close to the bole) had intermediate values for females. These contrasting influences of crown location on the survival and size of survivors may reflect the contrasting effects of sun exposure on hygrothermal stress and foliage quality. Wing lengths of males were longer than those of females, presumably to facilitate dispersal. Thus the higher female-biased sex ratio and larger size of adults that developed in the sun- exposed upper crown suggests that foliage quality was highest in this crown location.

Several studies show that sun-exposed foliage have a smaller area, a greater thickness and contains more nitrogen and sugars than shaded foliage (e.g., White 1984, Fortin and Mauffette 2002, Oishi et al. 2006 and Osier and Jennings 2007). Nitrogen and sugars play an important role in larval performance (e.g., Scriber and Slansky 1981, Fortin and Mauffette 2002). However, in opposition to the sun/shade hypothesis, nitrogen content in the lower crown was slightly higher than in the upper crown and both water and sugar contents were similar between crown locations. The increased size of adults that developed in the sun-exposed upper versus the shaded lower crown is, therefore, not related to higher levels of the nutrients as measured in this study. It is possible that feeding on foliage with marginally higher nitrogen content in the shaded lower crown might have been responsible for part of the increased survival rate of juveniles that developed in this location. Small variations in foliar nitrogen content can increase development

58 rate, growth rate and consumption rate as well as the food assimilation efficiency of insects (Lindroth et al. 1997).

Secondary chemicals can vary between crown locations (e.g., Carisey and Bauce 1997) and may be responsible for potentially higher nutritional quality in the sun-exposed upper crown. It is documented that allelochemicals (monoterpenes, tannins and phenols) can be toxic and act as feeding deterrents for many insects taxa (Carisey and Bauce 1997, Wallin and Rafa 1998). During larval development (in June), the foliar content of two monoterpenes (a-phellandrene and myrcene) was higher in shaded foliage in the lower crown than in sun- exposed foliage in the upper crown. Lagalante et al. (2006) identified myrcene concentrations in Tsuga canadensis as a potential chemical deterrent of hemlock wooly aldegid (Adelges tsugae Annand). Myrcene can be attractive for some species (e.g. Mountain pine beetle, Borden et al. 2008) but can also be an effective repellent that can negatively affect insect performance (e.g., Zou and Cates 1997). A-phellandrene was tested as a natural insecticide and was very efficient at killing two beetle pests (Park et al. 2003). Although, it is not exactly known how nutrients and secondary compounds act on PWG, it seems that performance of PWG might be affected by myrcene and a-phellandrene.

Sun exposure did not influence the sex ratio or size of adults that developed on saplings, regardless of treatment, and thus these results do not support the foliage quality hypothesis. However, in 2009, larval survival was highest on previously sun-exposed foliage but experimentally shaded foliage on saplings. This result partially supports my conclusion, based on the study in mature trees, that sun foliage is of higher nutritional quality than shade foliage. Unfortunately, the nutrient and secondary chemical content of foliage was not tested in 2009 and thus it is impossible to comment on whether they were correlated to survival. In 2010, when juvenile survival was similar in all three sun exposure treatments, foliar nutrient content (nitrogen and sugar) was not influenced by sun exposure treatment. Thus the prediction of the sun/shade hypothesis that sun leaves would

59 have more nutrients than shade leaves was not supported in either the sapling or mature tree study.

In contrast to results for nutrients, the secondary chemistry of sapling foliage was influenced by sun exposure. B-pinene and myrcene were highest in shaded saplings and terpinolene was highest in sun-exposed saplings. Cates et al. 1987 reported that high level of b-pinene mixed with nitrogen can improve the larval growth of western spruce budworm (Choristoneura occidentalis Freeman) while it was also concluded that b-pinene possibly reduced the developmental time of 6th instar larvae of the spruce budworm, Choristoneura fumiferana (Clem.) (Carisey and Bauce 1997). Although b-pinene can be a feeding deterrent for bark beetles, this monoterpene often acts as an insect attractant or a feeding stimulant (Leather 1996). Terpinolene can negatively affect larval growth rate and pupal weight of the western spruce budworm (Zou and Cates 1997) and is an efficient natural insecticide against two pest beetles (Park et al. 2003). Overall, secondary compounds may act positively or negatively on insect performance depending on the species. The foliage chemistry of sapling hemlocks suggests that myrcene and b-pinene might have a greater effect on PWG than terpinolene. Different sun exposure and month interactions also suggest that secondary compounds might explain why PWG survive better on previously sun-exposed foliage but experimentally shaded.

The highest defoliation in the lower crown could be explained by a variation in foliage biomass between crown locations. My results showed that foliage biomass was more than two times smaller in the lower crown compared to the upper crown. On sapling trees, defoliation was highest on shaded trees and lowest on sun-exposed trees supporting the foliage quality hypothesis. Shaded foliage might be poor nutritionally and therefore, larvae need to feed more to compensate for the nutritional lost.

Temporal variations in foliage quality

Similar to a previous study carried out in the same area (Pinault et al. 2009), defoliation by PWG larvae on mature trees was highest on current-year foliage and older larvae fed on all ages-classes of foliage, presumably to obtain a balanced diet. As expected (Feeny 1970, Scriber and Slansky 1981), current-year foliage contained more water and nitrogen (in July only) than older foliage. Sugar content was two times higher in older foliage than current-year foliage suggesting that it is not limited (Feeny 1970) for PWG larvae. Previously, I mentioned that nutrients did not seem to affect larval performance between crown locations. However, when looking at foliage age only, it appears that larvae select foliage age that is better nutritionally, a possible behavioural adaptation to increase their performance.

It is documented that monoterpenes concentration in eastern hemlocks can vary with foliage age and time of year (Lagalante et al. 2006). In this study, tannins and phenol content did not differ between foliage ages. Again, we looked at allelochemicals content on different age-class foliage to see if they had an effect on the PWG feeding preference. Overall, myrcene, a-pinene, camphene and bornyl acetate contents were highest in current-year foliage but terpinolene and limonene contents were highest in older foliage. These results suggest that the higher nutritient content in current year foliage outweighs the possible negative effects of monoterpenes.

As for mature trees, nitrogen and water were most abundant in current- year foliage, and sugar in older foliage of saplings. However, in contrast to results from the study on mature trees, defoliation on sapling trees was highest on the two oldest foliage age-classes and there was an interaction between sun exposure and foliage age. The highest levels of defoliation occurred on 2 and 3 year-old foliage of sun-exposed but experimentally shaded saplings whereas current-year and 1 year-old foliage experienced the highest levels of defoliation on shaded saplings.

We do not know if the lack of preference for current-year foliage in some saplings is due to small differences in secondary chemicals, or variations in other unmeasured factors such as leaf toughness (Feeny 1970).

3.6 Conclusion

The objective of this research was to unravel some of the feeding preferences of the pale-winged gray moth by testing two complementary hypotheses: foliage quality and hygrothermal stress. My results showed that PWG larvae preferred to feed on foliage located in the lower crown of mature hemlock trees because their survival was improved when feeding on this foliage. Results from the two experiments showed that larval survival of the PWG was highest in the shaded lower crown of mature hemlocks because of a more suitable weather (lower temperatures, higher humidity and less sunrays exposure) that outweighed the benefits of feeding on nutritionally better sun-exposed foliage in the upper crown. Yet, adults that survived in the sun-exposed upper crown foliage benefited from highest nutritional value foliage and were bigger than larvae grown on other crown locations. Larvae were able to survive at all crown locations possibly because they selected a specific kind of foliage: current year foliage was preferred to older foliage and contained more water and nitrogen contents. Feeding on foliage with higher water content might have help larvae to survive in conditions where heat was prevailing. In conclusion, both hygrothermal and foliage quality played a role in the feeding preference of the pale-winged gray moth. Knowing that the species is affected by heat and water stress, this knowledge could reinforce the development of strategies to control population outbreaks: selective cuts of mature hemlocks could increase light incidence on foliage located in the lower crown which would directly affect the PWG survival.

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CHAPTER 4: GENERAL CONCLUSIONS

The main objective of this thesis was to assess different factors that could explain the feeding preference of the pale-winged gray moth for foliage located in mid-lower crown of mature eastern hemlocks. Chapter 1 was a review of the published literature on the two species studied (PWG and eastern hemlock) and on the possible factors that can affect insects’ feeding preference.

In Chapter 2, I described the egg and larval distribution of PWG in mature eastern hemlocks. The objective was to understand the species distribution within mature trees and see if this could be correlated with the feeding pattern observed in infested hemlock stands. The results showed that eggs and larvae were mainly located in the lower crown, where the highest defoliation occurred.

In Chapter 3, I looked at the influence of two factors on the PWG feeding preference: hygrothermal stress and foliage quality. The results from my two manipulative field studies revealed that PWG is affected by sun exposure and as a result, the species consumes foliage in the lower crown to avoid heat and water stress. However, some of my performance results revealed that larvae are also affected by foliage quality; adults feeding on foliage from the sun-exposed upper crown were bigger than adults fed on shaded foliage in the lower crown. Overall, this chapter demonstrated that both hygrothermal stress and foliage quality affected the feeding preference of the PWG. However, it was clear that benefits from feeding on foliage with highest nutritional quality were overweighed by the disadvantages from heat and water stress and thus, larvae ended up consuming foliage in the lower crown that provide some protection against sun exposure.

The findings of this thesis allowed us to better understand the PWG feeding preference. Overall, female preference in oviposition site (Pinault et al. 2007), larval preference for lower crown (Chapter 2), highest foliage biomass (Chapter 3) in the lower crown, hygrothermal stress and foliage quality (Chapter 3) are all factors that partly explain the feeding preference of PWG for foliage in

67 the mid-lower crown of mature hemlocks. Although we now have a better understanding of the PWG feeding preference, additional factors could be tested such as the occurrence of natural enemies between crown levels.

APPENDICES

APPENDIX 1- Results of a generalized linear mixed model evaluating the effects of crown location (mature trees) or sun exposure (saplings), month (June or July) and foliage age (current year and older) on defoliation of a) mature hemlock trees and b) sapling hemlock trees by PWG during summer 2010. a)

Fitness Source Estimate Std. z value P value correlate Error % Intercept -0.242 0.165 -1.464 0.143 defoliation Treatment Ushade -0.381 0.068 -5.625 1.86e-08 Treatment Usun -0.768 0.071 -10.817 < 2e-16 Mature trees Age C+2 0.325 0.082 3.980 6.88e-05 Age C+3 0.182 0.099 1.843 0.065 AgeCY 1.169 0.081 14.373 < 2e-16 TreatmentUshade:AgeC+2 -0.005 0.105 -0.048 0.962 TreatmentUsun:AgeC+2 -0.139 0.111 -1.254 0.210 TreatmentUshade:AgeC+3 0.338 0.128 2.652 0.008 TreatmentUsun:AgeC+3 0.270 0.135 2.003 0.045 TreatmentUshade:AgeCY 0.321 0.106 3.023 0.003 TreatmentUsun:AgeCY -0.077 0.105 -0.735 0.462 *In analyses, effects of Ushade and Usun were compared to Lshade (dummy variable). b)

Fitness Source Estimate Std. z value P value correlate Error % Intercept -0.840 0.145 -5.786 7.22e-09 defoliation Treatment SS -0.201 0.206 -0.973 0.331 Treatment Sun -0.433 0.207 -2.088 0.037 Sapling trees Age C+2 0.503 0.072 6.969 3.20e-12 Age C+3 0.662 0.104 6.350 2.16e-10 AgeCY 0.099 0.063 1.565 0.118 TreatmentSS:AgeC+2 0.237 0.115 2.065 0.039 TreatmentSun:AgeC+2 0.206 0.118 1.750 0.080 TreatmentSS:AgeC+3 1.357 0.255 5.332 9.72e-08 TreatmentSun:AgeC+3 0.096 0.230 0.420 0.674 TreatmentSS:AgeCY -0.086 0.086 -0.997 0.319 TreatmentSun:AgeCY -0.328 0.091 -3.584 3.38e-04 *In analyses, effects of SS and Sun were compared to Shade (dummy variable).

APPENDIX 2- Influence of crown location (mature trees) or sun exposure (sapling trees), month and foliar age on a) monoterpenes (mean ± SE, in ng/mg of dry foliage) and b) nitrogen, water, sugar and phenol contents (mean ± SE, in %) of eastern hemlock foliage during summer 2010. a)Experiment Month Age Treatment a-pinene Camphene b-pinene Myrcene a-phellandrene limonene terpinolene bornyl acetate Mature trees June CY Lshade 359.9 ± 30.2 272.1 ± 15.8 106.4 ± 11.0 235.2 ± 22.5 0.0 ± 0.0 88.9 ± 9.4 0.0 ± 0.0 446.3 ± 20.5 Ushade 382.0 ± 36.5 276.6 ± 23.7 113.7 ± 16.8 254.8 ± 18.6 44.4 ± 29.8 95.3 ± 14.0 0.0 ± 0.0 441.1 ± 27.3 Usun 348.4 ± 20.0 209.0 ± 9.1 79.6 ± 2.6 213.4 ± 20.2 11.8 ± 11.8 63.4 ± 2.1 0.0 ± 0.0 365.2 ± 14.0 Older Lshade 211.7 ± 16.6 206.0 ± 15.3 57.1 ± 5.2 67.2 ± 5.2 52.4 ± 6.6 78.4 ± 5.3 27.7 ± 6.4 384.4 ± 31.9 Ushade 210.6 ± 14.1 202.3 ± 10.1 59.9 ± 4.6 71.0 ± 4.4 54.1 ± 5.6 79.3 ± 4.1 24.8 ± 8.2 375.3 ± 21.6 Usun 216.9 ± 20.6 199.8 ± 12.5 52.8 ± 3.0 65.9 ± 4.2 50.6 ± 5.9 73.6 ± 3.7 22.6 ± 4.6 375.3 ± 35.1 July CY Lshade 377.3 ± 18.6 364.2 ± 16.2 90.5 ± 6.7 143.4 ± 6.1 91.3 ± 9.6 88.9 ± 6.7 10.7 ± 10.7 600.6 ± 63.3 Ushade 353.3 ± 25.7 332.4 ± 21.6 80.2 ± 8.7 119.8 ± 10.6 79.4 ± 10.4 87.4 ± 9.8 23.9 ± 8.7 604.4 ± 39.8 Usun 377.5 ± 33.2 350.1 ± 25.7 90.6 ± 10.5 131.9 ± 10.8 92.4 ± 11.8 87.7 ± 10.3 6.3 ± 6.3 635.2 ± 51.7 Older Lshade 296.2 ± 19.8 285.4 ± 20.1 95.0 ± 11.6 101.0 ± 11.8 66.0 ± 15.3 118.3 ± 11.1 42.7 ± 10.6 496.4 ± 32.3 Ushade 286.2 ± 30.9 271.3 ± 30.7 80.5 ± 10.6 94.4 ± 12.7 78.0 ± 10.8 108.8 ± 13.0 39.1 ± 11.2 485.8 ± 55.9 Usun 297.9 ± 39.9 262.5 ± 28.4 79.1 ± 11.0 90.4 ± 10.3 76.1 ± 15.1 100.3 ± 15.1 25.8 ± 9.9 467.5 ± 48.9 Sapling trees June CY Shade 447.1 ± 18.5 345.2 ± 12.2 141.1 ± 12.0 359.8 ± 45.4 39.2 ± 25.6 118.1 ± 10.3 0.0 ± 0.0 488.1 ± 38.9 SS 482.2 ± 49.4 374.9 ± 35.0 122.6 ± 18.8 277.8 ± 24.6 0.0 ± 0.0 102.7 ± 15.6 0.0 ± 0.0 627.3 ± 42.9 Sun 512.6 ± 84.9 415.3 ± 76.1 150.2 ± 27.9 287.7 ± 39.5 71.0 ± 38.4 137.2 ± 29.1 0.0 ± 0.0 659.2 ± 156.7 Older Shade 262.0 ± 12.1 240.4 ± 12.0 72.4 ± 7.1 94.9 ± 8.1 69.5 ± 9.1 101.9 ± 8.4 31.4 ± 7.1 449.9 ± 30.1 SS 290.0 ± 3.9 254.8 ± 8.1 73.4 ± 4.3 91.7 ± 4.4 79.4 ± 3.6 101.1 ± 5.4 36.0 ± 5.0 474.6 ± 17.1 Sun 249.4 ± 32.6 231.5 ± 25.1 58.3 ± 7.4 77.2 ± 10.4 54.3 ± 7.0 83.1 ± 8.9 28.8 ± 4.7 447.6 ± 49.5 July CY Shade 454.2 ± 42.2 425.4 ± 40.8 123.9 ± 12.3 199.6 ± 15.2 134.2 ± 14.7 120.9 ± 10.7 12.7 ± 12.7 683.9 ± 75.3 SS 423.1 ± 42.7 359.5 ± 34.9 98.9 ± 13.2 155.2 ± 19.4 115.6 ± 18.0 95.4 ± 11.6 18.0 ± 8.7 675.2 ± 62.9 Sun 443.4 ± 48.2 376.7 ± 33.6 88.4 ± 6.5 139.6 ± 13.5 103.1 ± 17.7 95.0 ± 5.5 31.5 ± 7.5 713.2 ± 69.1 Older Shade 388.1 ± 39.6 352.0 ± 36.3 106.7 ± 11.4 132.9 ± 12.7 112.4 ± 14.9 142.8 ± 14.5 42.9 ± 9.1 650.7 ± 70.5 SS 425.5 ± 54.2 332.8 ± 39.7 108.1 ± 20.9 128.2 ± 21.1 118.0 ± 34.0 139.8 ± 24.7 48.5 ± 11.0 653.8 ± 66.6 Sun 432.7 ± 45.7 371.9 ± 36.5 108.0 ± 15.9 134.1 ± 18.2 108.9 ± 18.1 146.5 ± 19.8 52.8 ± 10.2 734.5 ± 80.5

Experiment Month Age Treatment Nitrogen Phenol Sugar Water Mature June CY Lshade 2.5 ± 0.1 0.5 ± 0.0 11.9 ± 1.3 79.8 ± 0.4 trees Ushade 2.5 ± 0.1 0.7 ± 0.1 13.5 ± 1.4 77.4 ± 0.3

Usun 2.7 ± 0.2 0.9 ± 0.4 18.6 ± 1.8 74.5 ± 0.5 Older Lshade 1.9 ± 0.2 1.2 ± 0.6 22.3 ± 2.4 45.8 ± 0.4 Ushade 1.2 ± 0.2 0.8 ± 0.3 27.1 ± 4.2 44.8 ± 0.7

Usun 1.8 ± 0.1 0.5 ± 0.2 24.6 ± 4.4 44.6 ± 0.7 July CY Lshade 2.1 ± 0.1 1.1 ± 0.5 14.1 ± 4.6 60.4 ± 0.8 Ushade 1.7 ± 0.2 0.8 ± 0.4 10.6 ± 0.8 60.0 ± 0.3

Usun 1.8 ± 0.0 0.3 ± 0.1 14.1 ± 0.7 58.7 ± 0.3 Older Lshade 1.9 ± 0.1 1.9 ± 1.3 28.4 ± 4.3 50.7 ± 1.0 Ushade 1.9 ± 0.1 0.5 ± 0.0 26.5 ± 2.3 48.0 ± 0.5

Usun 1.7 ± 0.2 0.8 ± 0.3 26.1 ± 2.1 49.7 ± 1.3 Sapling June CY Shade 2.4 ± 0.0 0.3 ± 0.1 5.1 ± 0.9 81.1 ± 0.3 trees SS 2.5 ± 0.2 0.8 ± 0.4 9.8 ± 0.9 77.7 ± 0.3

Sun 1.9 ± 0.1 0.5 ± 0.1 11.0 ± 1.5 76.8 ± 0.4 Older Shade 1.7 ± 0.2 1.2 ± 0.4 20.0 ± 1.8 45.5 ± 0.4 SS 1.7 ± 0.1 1.2 ± 0.5 18.9 ± 3.1 45.8 ± 0.6

Sun 1.7 ± 0.1 0.4 ± 0.2 20.8 ± 1.3 44.7 ± 1.0 July CY Shade 1.9 ± 0.2 0.7 ± 0.3 10.7 ± 3.5 64.2 ± 0.3 SS 2.0 ± 0.3 0.8 ± 0.4 8.0 ± 0.6 59.2 ± 1.5

Sun 1.9 ± 0.1 0.5 ± 0.1 16.6 ± 5.7 59.4 ± 0.6 Older Shade 1.6 ± 0.1 0.7 ± 0.4 18.0 ± 3.1 55.1 ± 0.9 SS 1.2 ± 0.1 0.6 ± 0.1 18.8 ± 4.4 55.0 ± 1.2

Sun 1.4 ± 0.1 0.5 ± 0.1 14.4 ± 3.4 55.0 ± 1.1