Intra-Tree Variation in Foliage Quality Drives the Sex-Biased Foraging Behavior of a Specialist Herbivore, Pikonema Alaskensis, Within Juvenile Black Spruce

INTRA-TREE VARIATION IN FOLIAGE QUALITY DRIVES THE SEX-BIASED FORAGING BEHAVIOR OF A SPECIALIST HERBIVORE, PIKONEMA ALASKENSIS, WITHIN JUVENILE BLACK SPRUCE

Robert Carson Johns

Bachelor of Science (Honors), Biology, St. Francis Xavier University (1999)

A Thesis Submitted in Partial Fulfillment of

the Requirements for the Degree of

Doctor of Philosophy

In the Graduate Academic Unit of Biology

Supervisor: Dan T. Quiring, PhD, Biology, University of New Brunswick Examining Board: Abdehaq Hamza, PhD, Department of Physics, Chair Gilles Boiteau, PhD, Biology, U.N.B. Joseph Culp, PhD, Biology, U.N.B. Jon Sweeney, PhD, Forestry, U.N.B.

External Examiner: Michael R. Wagner, PhD, School of Forestry, Northern Arizona University

This thesis is accepted by the Dean of Graduate Studies.

THE UNIVERSITY OF NEW BRUNSWICK

April, 2007

395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada

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In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these.

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ii ABSTRACT

In this dissertation I investigate the influence of intra-tree variation in foliage quality on the adult and larval foraging behavior and associated performance of a specialist herbivore, the yellowheaded spruce sawfly {Pikonema alaskensis Roh.)

(Hymenoptera: Tenthredinidae) within the crown of juvenile black spruce (Picea mariana [Mills] B.S.P.).

I report in Chapter 2 that most eggs of P. alaskensis were laid in the mid to lower crown (i.e., whorls 4 and 7) of black spruce, however, late-instar larvae dispersed acropetally, from the mid and lower to upper crown (i.e., whorls 1 and 2), to complete larval development. The few eggs that were laid in the upper crown were predominantly female, due presumably to female-biased egg allocation by mothers. Manipulative field experiments indicated that female larvae were also more than twice as likely as males to disperse acropetally through the crown.

In Chapter 3,1 test the hypothesis that sex-biased oviposition-site selection and female-biased acropetal dispersal within the crown of black spruce represent adaptive behavioral responses to variation in foliage quality. Although survival of early-instar larvae was lower in the upper crown, survival of male larvae appeared to be more negatively influenced than that of females. As predicted based on the observed foraging strategy, survival was highest overall for larvae feeding during early instars in the lower crown and during late instars in the upper crown, particularly for females.

In Chapter 4,1 investigate the influence of intra-crown variation on the foliage- age preference and performance of larvae. Although larvae preferred and generally had higher survival when feeding on current-year (i.e., developing) foliage, late-instar larvae often consumed most current-year foliage in the upper crown, after which some

iii individuals fed on mature foliage. Larvae feeding at high population densities ate one- year-old foliage in the upper whorls in quantities comparable to the current-year foliage consumed in lower whorls. Larvae that fed on mature foliage in the upper crown generally had similar survival and sex ratios to groups of larvae that fed on current-year foliage in lower whorls.

Collectively, this dissertation emphasizes the important role of intra-plant heterogeneity in determining the preference and performance of herbivorous insects within their host-plants.

Key words: acropetal dispersal, sex-bias, foraging behavior, intra-tree, variation, foliage quality, foliage age, herbivory

IV PREFACE

The body of this thesis is comprised of three independent but related manuscripts that have been prepared for publication in scientific journals. Thus, each chapter possesses its own abstract, key words, introduction, methods, results, discussion, acknowledgements, and references. The main chapters of this dissertation have been prepared and formatted for submission to Ecology. For all publications the order of authorship will be as follows:

a a a,b Rob C. Johns , Dan T. Quiring , Don P. Ostaff

Population Ecology Group, Department of Biology, University of New Brunswick,

Fredericton, New Brunswick, E3B 6E1, Canada b

Natural Resources Canada, Canadian Forest Service, Atlantic Forestry Centre, PO

Box 4000, Regent Street, Fredericton, New Brunswick, E3B 5P7, Canada

For these papers, I was primarily responsible for preparing the initial research proposal, planning and designing experiments, collecting and analyzing the data, and writing the manuscripts. Discussions with Dan Quiring were essential for developing many of the ideas presented in these papers. Dan also provided essential advice and feedback at all stages of this research and was the primary supervisor for all the above manuscripts.

Similarly, Don Ostaff also provided important feedback on experimental design and analysis for all studies and provided valuable contributions during the preparation of these manuscripts. ACKNOWLEDGEMENTS

I am deeply indebted to Dan Quiring for his support and friendship throughout my graduate studies at the University of New Brunswick. Dan was always available for interesting discussion and provided me with exceptional support, guidance, and opportunities. Also, Don Ostaff and Harald Piene provided important advice and comments on experiments, as well as subsequent manuscripts that significantly improved both the quality of my work and the manner in which I approach research.

Thanks to my supervisory committee member Steve Heard who provided valuable feedback on several manuscripts and the opportunity to teach a portion of the

Biology Field Course. Other professors from Biology provided me teaching assistantships, including Myriam Barbeau, Carl Bursey, Sandy Craft, Les Cwynar, Mike

Duffy, and Richard Riding. Linda Allen, Jackie Seely, Bonny Morrison, and Carol

Johnston were helpful throughout my time at the University of New Brunswick.

My lab mates, including Drew Carleton, Leah Flaherty, Heidi Fry, Roger Graves,

Finbarr Horgan, Jonathan Leggo, Chris MacQuarrie, Gaetan Moreau, Andrew Morrison,

Lauren Pinault, and Kate VanRoonen, were helpful with editing and provided constructive comments on earlier versions of many manuscripts. My discussions of science and entomology, with Gaetan and Finbarr in particular, were always helpful and interesting.

Thanks to my field assistants who were always in good humor and a pleasure to work with in the depths of central Newfoundland. A special thanks to my brother Timm who was kind enough to work for free as a field assistant during my first year in

Newfoundland. Thanks to both the Griffin and Howell families who were always accommodating and generous with 'Jigs' dinners while I was in Newfoundland. This

vi work would not have been possible without the support of Jim Evans, Abitibi-

Consolidated Ltd., and George VanDusen, Comer Brook Pulp and Paper, who were always supportive of my work and who were instrumental in providing funding for my four years of scholarship.

I am grateful to my family, in particular my parents Rick and Carol, who supported and encouraged me throughout my studies. My son Carson, born during the first year of my graduate studies, has always provided a welcome reprieve from work with his contagious enthusiasm, youthful innocence, and budding appreciation for caterpillars and the world around him. I would also like to thank my dedicated assistant

Bailie-Ann who spent many summers chasing squirrels and the occasional caribou. My housemates as well as my friends from the University of New Brunswick, the Right

Spot, and the Fredericton Loyalists Rugby Club have provided needed diversion, close friends, and many laughs, which I have appreciated throughout my time in Fredericton.

vn TABLE OF CONTENTS

ABSTRACT iii PREFACE v ACKNOWLEDGEMENTS vi LIST OF TABLES x LIST OF FIGURES xii

CHAPTER 1: GENERAL INTRODUCTION 1 References 7

CHAPTER 2: JUVENILE SEX DRIVES THE INTRA-PLANT OVIPOSITION-SITE SELECTION AND LARVAL FORAGING BEHAVIOR OF A SPECIALIST HERBIVORE 15 Abstract 15 Introduction 16 Methods 19 Study Areas 19 Seasonal distribution of, and defoliation by, immature sawflies 20 Seasonal distribution of male and female larvae 22 Sex-biased larval foraging behavior 25 Results 27 Seasonal distribution of immature sawflies and of defoliation 27 Seasonal distribution of male and female larvae 28 Sex-biased larval foraging behavior 29 Discussion 30 Acknowledgments 33 References 33

CHAPTER 3: SEX-BIASED OVIPOSITION-SITE SELECTION BY ADULTS AND ACROPETAL DISPERSAL BY LARVAE ARE ADAPTIVE RESPONSES OF A SAWFLY TO INTRA-TREE VARIATION IN FOLIAGE QUALITY 52 Abstract 52 Introduction 53 Methods 57 Description of study insect 57 Study areas 57 Are sex-biased oviposition-site selection and acropetal dispersal adaptive? 57 Influence of phenology 60 Effect of bud phenology on oviposition 60 Effect of bud phenology on young larvae 62 Acropetal dispersal and shoot phenology 63 Results 64 Are sex-biased oviposition-site selection and acropetal dispersal adaptive? 64 Influence of Phenology 65 Effect of bud phenology on oviposition 65 Effect of bud phenology on young larvae 65

viii Acropetal dispersal and shoot phenology 65 Discussion 66 Acknowledgments 70 References 70

CHAPTER 4: INTRA-TREE VARIATION SHAPES THE FOLIAGE-AGE PREFERENCE AND PERFORMANCE OF LARVAE OF A SPECIALIST HERBIVORE 86 Abstract 86 Introduction 87 Methods 89 Study Areas 89 Branch-level feeding preference 89 Tree-level feeding preference 92 Larval performance 92 Results 94 Branch-level feeding preference 94 Tree-level feeding preference 94 Larval performance 95 Discussion 96 Acknowledgments 99 References 99

CHAPTER 5: GENERAL DISCUSSION 109 Significance of Studies to the Management of Pikonema alaskensis 113 References 114

IX LIST OF TABLES

Table 2.1. Results of a mixed-model two-way ANOVA evaluating the influence of

stand (random effect) and whorl (fixed effect) (described in Fig. la) on defoliation

associated with feeding by Pikonema alaskensis larvae in 2001 41

Table 2.2. Results of a mixed-model repeated-measures ANOVA evaluating the effects

of stand (random effect), whorl (fixed effect) (described in Fig. la), and

developmental stage (fixed effect) on the number of Pikonema alaskensis per

current-year shoot and per branch in 2001 42

Table 2.3. Results of a mixed-model repeated measures ANOVA evaluating the effects

of stand (random effect), branch section (fixed effect) (described in Fig. lb), and

developmental stage (fixed effect) on the number of Pikonema alaskensis per

current-year shoot in 2001 43

Table 2.4. Results of a mixed-model three-way ANOVA evaluating the effects of year

(random effect), whorl (fixed effect), and developmental stage (fixed effect) on the

percentage of Pikonema alaskensis larvae that were female from 2001 through

2003 44

Table 3.1. Pattern of transfers simulating acropetal dispersal (*) and other potential

dispersal strategies within the crown of juvenile black spruce from 2001 through

2004. Treatments employing the leader are represented by 'L' and those employing

the upper crown (i.e., whorls 1 and 2) are represented by 'U' 78

Table 3.2. Comparisons of variance components from one-way ANOVAs evaluating

the influence of transfer, described in Table 1, on larval survival and on the

percentage of surviving larvae that were female in experiments conducted from

2001 through 2004.

x 79

Table 4.1. Results of a mixed-model ANOVA evaluating the effects of stand (random

effect), whorl (fixed effect) (described in Fig. la), and foliage age (fixed effect) on

percent defoliation associated with feeding by Pikonema alashensis larvae in 2001.

103

Table 4.2. Results of ANOVAs evaluating the effects of whorl (described in Fig. la)

and foliage age on percent survival of early- and late-instar Pikonema alashensis

larvae in 2002 and 2003 (late-instar larvae only). Note that analyses for early and

late-instar larvae for 2002 were nested ANOVAs with foliage age treatment nested

in whorl, whereas a model-1 two-way ANOVA was used for 2003 104

XI LIST OF FIGURES

Figure 2.1. (a) Schematic representation of a juvenile black spruce tree with seven

whorls. For the purposes of this study, whorl 1 included the leader and whorl 1

branch, (b) Defoliation was visually estimated for all age classes of shoots along

the first- and second-order branch axes (solid shoots and buds). Whorl 4 branches

were subdivided into four sections, consisting of the terminal shoot (i.e., section i)

and sections distinguished by the position of second-order branches that included

all shoots from that branch up to the next second-order branch 45

Figure 2.2. Frequency distributions of: (a) head-capsule widths for an all-male group of

prepupae; (b) head widths of adult males (shaded bars) and females (white bars);

and (c) pooled distribution of head-capsule widths of all prepupae examined in

transfer experiments used in this study 46

Figure 2.3. Mean (±1 SE) percent defoliation of black spruce attributable to feeding by

P. alaskensis on the leader (L) and on one branch in each of whorls 1, 2,4 and 7.

The four shades of bars represent different stands with differing population

densities of P. alaskensis. Raw data are presented, however data were arcsine

square-root transformed prior to analysis 47

Figure 2.4. Mean (±1 SE) density of eggs, mid-, and late-instar larvae of P. alaskensis

per current-year shoot (a-c) and per branch (d-f) on the leader (L) and in whorls 1,

2, 4, and 7 of black spruce. The four shades of bars represent different stands with

differing population densities of P. alaskensis. Note the different scale used in c.

Figure 2.5. Mean (±1 SE) density of eggs (a), mid- (b), and late-instar larvae (c) of P.

alaskensis from the tip to the base (i.e., i to iv) of whorl 4 branches. The four

xii shades of bars represent different stands with differing population densities of P.

alaskensis 49

Figure 2.6. Mean (±1 SE) percentage of early- and late-instar P. alaskensis larvae

located on the leader (L), and in whorls 2 and 7 of black spruce that were female in

three different stands from 2001 through 2003 (a-c). Asterisks indicate that either

no larvae were present on (i.e., early instars in all years), or no larvae were

collected from, the leader (i.e., late instars in 2001). Raw data are presented,

however data were arcsine square-root transformed prior to analysis 50

Figure 3.1. (a) Schematic representation of a juvenile black spruce tree with seven

whorls, (b) Classification of terminal (t), distal-lateral (dL), and medial-lateral

(mL) vegetative buds developing on a one-year old shoot 80

Figure 3.2. Mean (±1 SE) percent survival of P. alaskensis: (a-d) from first to fourth

instar on the leader (L) or on one branch in each of whorls 2,4, or 7 and (e-h) from

fourth instar to cocoon for P. alaskensis transferred either within whorls (i.e., 7 to

7, 2 to 2, leader to leader (Lto L)) (white bars), from upper to lower whorls (i.e., 2

to 7) (white bars), or acropetally among whorls (i.e., 7 to 2,4 to leader (L), 4 to

upper (U)) (slashed bars) from 2001 through 2004. Raw data are presented,

however, data were arcsine square-root transformed prior to analysis. "*" indicates

a significant difference in percent survival when compared to the acropetal transfer

treatment (i.e., 7-2) at a = 0.05 (Dunnett's Test) 81

Figure 3.3. Mean (±1 SE) survival of P. alaskensis from first instar to cocoon (a-d) and

percentage of survivors that were female (±1 SE) (e-h) for larvae that were initially

placed on the leader (L) or on a branch in whorl 2,4, or 7 and then transferred

within whorls (i.e., 7 to 7,2 to 2, leader to leader (Lto L)) (white bars), from upper

xiii to lower whorls (i.e., 2 to 7) (white bars), or acropetally among whorls (i.e., 7 to 2,

4 to leader (L), 4 to upper (U)) (slashed bars) to complete development from 2001

through 2004. Raw data are presented, however data were arcsine square-root

transformed prior to analysis. "*" indicates a significant difference in sex ratio

when compared to the acropetal transfer treatment (i.e., 7-2) at a = 0.05 (Dunnett's

Test) 82

Figure 3.4. Relationship between budburst phenology and oviposition by P. alaskensis.

(a) Percentage of buds in stages 1-5 on which P. alaskensis females oviposited in a

manipulative study, (b) The mean percentage of buds on 4 whorls (L (- •-), whorl 2

(-•-), whorl 4 (-T-), and whorl 7 (-•-)) within the crown of black spruce in a

suitable stage (i.e., stage 3-5) for oviposition and the temporal distribution of eggs

laid (shaded area) in a field survey 83

Figure 3.5. Effect of bud type (terminal/distal-lateral (T/dL) versus medial-lateral

(mL)) on the performance of first-instar larvae. Raw data are presented, however

data were arcsine square-root transformed prior to analysis 84

Figure 3.6. Temporal relationship between the mean percentage of larvae in the upper

crown (i.e., whorls 1 and 2) of black spruce (shaded area) and mean length of

current-year shoots on the leader (-•-) and in whorls 2 (-•-) and 7 (-•-) 85

Figure 4.1. (a) Schematic representation of a juvenile black spruce crown with seven

whorls. For the purposes of this study, whorl 1 was separated into its leader and

whorl 1 branch, (b) Defoliation was visually estimated for all age-classes of shoots

along the first- and second-order branch axes (solid shoots and buds), c, c+1, c+2,

xiv c+3, and c+4 refer, respectively, to current-year, and one-, two-, three-, and four-

year old shoots 105

Figure 4.2. Relationships between densities of early-instar P. alaskensis larvae and

subsequent defoliation on current- (closed circles), one- (open circles), and two-

closed triangles) year old foliage on branches in whorls 4 or 5 from trees in an

experiment using naturally occurring densities of larvae already on branches (a),

and in an experiment where larval density was manipulated on branches (b).

Defoliation data were arcsine square-root transformed prior to analysis to meet

model assumptions 106

Figure 4.3. Mean (±1 SE) percent defoliation per shoot per branch attributable to

feeding by P. alaskensis on current- (black bars) or one-year-old (gray bars) foliage

on the leader or on whorl 1, 2,4, or 7 branches in each of two stands (a,b) of young

black spruce. Defoliation data were arcsine square-root transformed prior to

analysis to meet model assumptions 107

Figure 4.4. Mean (±1 SE) percent survival (a-c) and percentage of survivors that were

female (d-f) for P. alaskensis allowed to feed on all, current-, one-, two- (in 2003

only), or three-year old (only in whorl 7 in 2003) foliage in either whorls 2 (white

bars) or 7 (slashed bars). Larvae were placed on branches as early- (a,d) or late-

instars (b,e) in 2002, but only late-instar larvae (c,f) were placed on branches in

2003. In (d) * denotes that there was no data available for the analysis due to low

survival 108

xv SYMBOLS AND ABBREVIATIONS

ANO V A: Analysis of variance h: Hour mm: Millimeter

SE: Standard error of the mean

xvi CHAPTER 1: GENERAL INTRODUCTION

"Something in the insect seems to be alien to the habits, morals, and psychology of this world, as if it had come from some other planet: more monstrous, more energetic, more insensate, more atrocious, more infernal than our own." Maurice Maeterlinck (1862-1949)

Ecologists have long debated how forests remain 'green' in the presence of rapidly evolving insect herbivores that, with such short generation time and high potential for genetic recombination, should easily overcome the relatively slow-evolving defenses of plants (White, 2005). Some argue that natural enemies and a myriad of chemical and physical plant defenses are responsible for suppressing herbivore populations below levels that could completely devour plants (Hairston et ah, 1960; Murdoch, 1966).

Recent studies suggest that the persistence of plants is more likely attributable to the indirect influence of temporal and spatial variation in plant traits on the preference and associated performance of herbivores (Denno and McClure, 1983). Such variation is speculated to prevent herbivores from becoming completely adapted to the quality of all foliage simultaneously, thereby reducing herbivory by restricting feeding to the relatively few modules that are most nutritious (Denno and McClure, 1983). In this way, natural selection may favor herbivorous insects that adopt foraging strategies increasing the likelihood that the best foliage available is encountered and acquired.

Herbivorous insects are highly capable of modifying their distribution in response to variation among plants in primary- and secondary-plant chemistry (Crawley,

1983; Scriber and Slansky, 1981), age-related declines in foliage quality (Bassett, 1991,

1992; Hunter, 1992), leaf or shoot size (Price et ah, 1987; Senn et ah, 1992), induced defenses (Karban and Myers, 1989), and risk of attack by natural enemies (Williams et

1 ah, 2001). However, because immature insects cannot fly, any benefits derived seeking superior foliage by moving large distances, such as between plants, can be offset by the relatively high energetic costs of such dispersal (Kareiva, 1982; Cain, 1985; Ballabeni et ah, 2001; Behmer et ah, 2003; Bernays et ah, 2004) and the added risk of exposure to mortality agents (Hassell and Southwood, 1978).

Most studies have focused on the influence of variations among plants on herbivore preference and performance, while few studies have investigated the potentially important influence of high levels of variation present within plants, particularly in large plants such as trees (Denno and McClure, 1983). Variability within plants should be most important in determining the oviposition and juvenile feeding behavior of herbivores that obtain most or all of their nutrients as juveniles. For herbivores whose progeny are constrained to the module selected by the mother, such as gallers, fruit or seed eaters, and leaf miners, natural selection should favour mothers that select the best available feeding site for their offspring (e.g., Roitberg et ah, 1981;

Quiring and McNeil, 1987; McClure et ah, 1998; Ozaki et ah, 2006).

Seasonal dispersal by juvenile insects within hosts has been demonstrated for only a few systems and may reflect changes in the nutritional quality of foliage due to phenological development (Quiring, 1993; Kessler and Balwin, 2002), changes in individual nutritional needs of juveniles (Reavey, 1993; Hochuli, 2001), or changes in the tolerance of juveniles to adverse microenvironments or natural enemies (Kessler and

Baldwin, 2002). The head and mandible morphology of larvae, for example, change dramatically with ontogeny and may alter the manner in which nutrients are acquired.

Many early-instar juveniles can use their small mouthparts to selectively skeletonize foliage, avoiding the tough silica in some types of leaves (e.g., Barbehenn, 1992;

2 Hochuli and Roberts, 1996), or the toxic resin canals of needles in conifers (Gaston et al., 1991). Late-instar juveniles, in contrast, often have large, tough mouthparts capable of efficiently chewing high quantities of foliage (reviewed in Reavey, 1993).

Assimilation efficiency, however, may be reduced significantly during late-instars

(Scriber and Slansky, 1981) presumably from ingesting a higher proportion of poor quality food (Slansky, 1993). The defensive capabilities of larvae may also change with age, as late-instar juveniles tend to develop thick cuticles, strong physical responses, and/or toxic exudates to deter natural enemies (e.g., Boeve and Pasteels, 1985).

The fitness of many insects maybe increased by the adoption of complementary foraging strategies by ovipositing adults and feeding juveniles. For example, spruce bud moth (Zeiraphera canadensis (Mutt. & Free.)) caterpillars disperse acropetally from the mid-crown of white spruce, where most eggs are laid, to the upper crown (Quiring,

1993), to account for acropetal budburst and to feed almost continuously on foliage of newly burst buds (Carroll and Quiring, 1994). Caterpillars on tobacco plants possess a similar foraging strategy to account for phenological variation in foliage quality, but also to avoid predators and hygothermal stress as well as to gain higher quality foliage

(Kessler and Baldwin, 2002). Acropetal dispersal may also benefit insects, such as tent caterpillars, by enabling them to avoid hygrothermal stress during early instars and by basking during late instars (Porter, 1982; Alonso, 1997). In contrast, some juvenile insects disperse to poorer foliage on protected parts of their host to reduce exposure to natural enemies (Dammon, 1987; Stamp, 1989; Stamp and Bowers, 1990) or adverse thermal conditions (Bardoloi and Hazarika, 1994).

Males and females of some arthropods may employ different fitness maximization strategies to overcome intra-plant variation (e.g., Hendrichs et al., 1991;

3 Craig et al, 1992; Craig and Mopper, 1993; Barker and Maczka, 1996; Jormalainen et al, 2001; Rhainds et al, 2002) due in part to the contrasting reproductive responsibilities of each sex. The fitness of males is often contingent on the number of females inseminated, while fitness of females is more often dependent on the number of offspring produced (Arnold and Duval, 1994). Such differences, coupled with the high relative metabolic cost of producing eggs compared to sperm, can exert strong selective pressure on adult insects to allocate females to better quality resources (Craig et al,

1992; Craig and Mopper, 1993) or on juveniles to adopt foraging strategies that accommodate their respective needs as males or females (Rhainds et al, 2002).

Most studies evaluating sex-biased egg allocation by insects have focused on parasitoids of caterpillars, where female offspring are preferentially allocated to larger hosts (e.g., Vinson, 1976; Charnov etal, 1981; Herre, 1985; King, 1987, 1989) or in locations where brood competition is lowest (reviewed in Cook, 1993). In contrast, only a few studies have addressed how variation could select for sex-biased oviposition behavior of herbivorous insects within plants (Hendrichs et al, 1991; Craig et al, 1992;

Craig and Mopper, 1993; Barker and Maczka, 1996). Similarly, only one study has demonstrated that differences in the nutritional needs of juveniles may also lead to differential foraging between males and females (Rhainds et al, 2002), despite the fact that juveniles of many insects bear the sole responsibility of accumulating resources to fuel dispersal and reproduction during adulthood (Heinrich, 1979; Shultz, 1983).

Thesis Objectives

This dissertation investigates the role of intra-tree variation in determining the foraging behavior of a specialist herbivore, the yellowheaded spruce sawfly (Pikonema

4 alaskensis (Roh)) (Hymenoptera: Tenthredinidae), within the crown of juvenile black spruce (Picea mariana [Mills] B.S.P.). Although generally small in size (i.e., 1-2 m), juvenile black spruce are architecturally well hierarchized (Begin and Filion, 1999), providing a range of potential microhabitats for P. alaskensis to select from. Feeding by

P. alaskensis larvae often results in shoot and branch mortality in the upper crown of trees (Kulman, 1971; Lavigne, 1996), suggesting that not all parts of the plant are equally exploited. Pikonema alaskensis larvae are sexually dimorphic with females developing through a sixth instar compared to males that only develop through five

(Vanderwerker and Kulman, 1974).

Black spruce is the primary host of P. alaskensis in central Newfoundland where my studies were conducted; however, white spruce (P. glauca [Moench] Voss) and blue spruce (P. pungens Engelm.) are also susceptible (Kulman, 1971). Detailed descriptions of the life history of P. alaskensis are available in Pointing (1957), Houseweart and

Kulman (1976a), and Katovich et al. (1995). Briefly, adults eclose soon after budburst and lay eggs at the base of needles in new flushing shoots. Offspring of unmated females are all male, whereas those from mated females maybe either male or female

(Houseweart and Kulman, 1976b). Young larvae feed mainly on current-year foliage, then drop to the ground and spin a cocoon in the upper duff layer, where they overwinter as prepupae (Rau et al., 1979).

The following studies were undertaken to test the general hypothesis that P. alaskensis oviposition-site selection and larval foraging behaviors are determined, at least in part, by variability in foliage quality within the crown of black spruce, and that these foraging behaviors are adaptive (i.e., result in the highest individual fitness) (Perry and Pianca, 1997). The following three related chapters of this thesis sequentially

5 address aspects of this hypothesis. In Chapter 2, seasonal patterns of defoliation, oviposition-site selection, and larval feeding among crown levels in black spruce were determined using a combination of field and manipulative experiments. The relative distribution of eggs and immature larvae (both male and female) due, respectively, to oviposition-site selection and larval dispersal, were also surveyed. Based on the results of these surveys, a manipulative experiment was also conducted to determine whether male and female larvae employed different dispersal behaviors to overcome intra-crown variation.

In Chapter 3,1 test the hypothesis that oviposition and larval foraging behaviors of P. alaskensis are adaptive responses to intra-tree variation in foliage quality. Intra- plant variation in the phenologjcal development of vegetative buds can limit the availability of suitable foliage during the spring and can thus have a significant effect on the foraging behavior of ovipositing adults and feeding larvae of early season herbivores

(Quiring, 1993, Carroll and Quiring, 1994; Kessler and Baldwin, 2002). Thus this study also tests the hypothesis that foraging patterns from Chapter 2 are determined by intra- tree variation in budburst phenology within the crown of black spruce.

Foliage-age specialization represents an important adaptation by herbivores, allowing them to become highly attuned to the general chemical and physical defenses of one or a few specific age-classes of foliage within their host-plant (Cates, 1980).

However, where intra-plant variation is high, as it often is in trees, the quality of non- preferred age-classes of foliage could occasionally exceed that of preferred age-classes of foliage. In Chapter 4,1 present results from manipulative field studies and surveys investigating the feeding preference of P. alaskensis on different-aged foliage within branches and among whorls of black spruce. I also tested the hypothesis that the

6 foliage-age preference of P. alaskensis represents an adaptive response to foliage quality by forcing both young and old larvae to feed on selected age-classes of foliage using sleeve cages in the upper or lower crown of black spruce.

Collectively, this thesis provides strong evidence that variability in foliage quality has an important influence on the distribution and abundance of insect herbivores within heterogeneous host plants. Chapter 5 presents a broad synthesis of the preceding chapters and suggests future avenues of research that could complement these studies.

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14 CHAPTER 2: JUVENILE SEX DRIVES THE INTRA-PLANT OVIPOSITION-SITE SELECTION

AND LARVAL FORAGING BEHAVIOR OF A SPECIALIST HERBIVORE

Abstract

Field surveys and a manipulative study were conducted to evaluate the influence of intra-plant variation within the crown of black spruce (Picea mariana [Mills.] B.S.P.) on oviposition and larval feeding behavior of yellowheaded spruce sawfly (Pikonema alaskensis [Roh.]). Most eggs were laid in the mid to lower crown of 1.5 to 2m tall trees. Few eggs were laid on the most apical shoots within the crown (i.e., whorl 1), however, most of the relatively few eggs that were laid in the upper crown (i.e., whorl 2) were female. Fourth- and fifth-instar larvae dispersed acropetally, from the mid and lower to upper crown, causing high levels of defoliation in the upper crown. Late-instar female larvae were generally more abundant than male larvae on the leader, the most apical shoot on a tree, strongly suggesting that more females than males disperse acropetally. This hypothesis was supported in a manipulative experiment, where only

15-20% of larvae in all male broods, but almost three-quarters of larvae in mixed broods, dispersed to the upper crown. This study emphasizes the important role of intra- plant variation in shaping both oviposition-site selection and the dispersal behavior of juvenile phytophagous insects within their hosts, and indicates that sex-biased foraging behaviors maybe necessary for some insects to accommodate the respective needs of immature females and males within heterogeneous host plants.

Key words: acropetal dispersal, intra-crown variation, Hymenoptera, Pikonema alaskensis, Picea mariana, sex-bias, oviposition, haplodiploid

15 Introduction

Many studies have described how herbivore preference and performance are influenced by variation among plants (Quiring and Butterworth, 1994; Alonso and Herrera, 1996;

Clark and Messina, 1998; Bernays et al, 2004; Holland et al., 2004) or plant species

(Gratton and Welter, 1998; Scheirs et al, 2000; Schiers and De Bruyn, 2002; Gottard et al, 2005). However, variation within plants maybe almost as high as that among plants, particularly large plants such as trees (Denno and McClure, 1983). Highly predictable patterns in module size and development (Powell, 1988; Quiring, 1993), foliage age

(Moreau et al., 2003), enemy-free space (Williams et ah, 2001), and microclimatic conditions (Bardoloi and Hazarika, 1994) within trees may exert consistent directional selective pressure favoring specialization of oviposition or feeding behaviors on certain parts of the host.

If there is large variation in the quality of feeding sites within host-plants, and if foraging behavior is at least partially genetically determined, then natural selection should favour herbivores that maximize their fitness by acquiring the most nutritious modules within individual plants. For example, adult females of many herbivore species are subjected to strong selective pressure to select the best of available modules to maximize the survival and growth of their progeny, particularly for gallers, fruit or seed eaters, and leaf miners whose progeny are constrained to the module selected by the mother (e.g., Roitberg et al, 1981; Quiring and McNeil, 1987; McClure et al, 1998;

Ozaki et al, 2006). Juveniles free to disperse following egg hatch may also increase their fitness by accounting for poor maternal decisions (e.g., Thompson, 1988; Courtney and Kibota, 1989; Mayhew, 1997,2001), changing nutritional needs associated with ontogeny (Scriber and Slansky, 1981; Hochuli, 2001), or for temporal and spatial

16 variations in plant quality associated with plant development (Carroll and Quiring, 1994;

Kessler and Baldwin, 2002; Moreau et ah, 2003). The ability to acquire the best resources available within a heterogeneous host maybe adaptive if the alternative strategy of dispersing to an adjacent host incurs higher costs to fitness. Such costs could include higher energy expenditure (Kareiva, 1982; Cain, 1985; Ballabeni et ah, 2001;

Behmer et ah, 2003; Bernays et ah, 2004) and/or increased exposure to mortality agents

(Hassell and Southwood, 1978), particularly for larvae that cannot "balloon" or apterous adults that are unable to fly between plants.

Differences in the reproductive responsibilities and associated nutritional needs of females compared to males may also influence responses of phytophagous insects to variation in plant quality. For insects, female fitness is often most highly influenced by fecundity, which is often directly related to the quality and quantity of food ingested and the resulting size of adults (Hedrick and Temeles, 1989). Male fitness is usually most closely correlated to the number of mates inseminated and may not be directly influenced by the size of individuals (Hedrick and Temeles, 1989). Such differences may explain why females are often more sensitive to variation in plant quality, resulting in differential survival between the sexes on different quality hosts (e.g., Hendrichs et ah, 1991; Craig et ah, 1992; Craig and Mopper, 1993; Barker and Maczka, 1996;

Jormalainen et ah, 2001; Rhainds et ah, 2002) and why there is such a prevalence of biased sex ratios within populations in nature, particularly in hymenopterans (reviewed in Craig and Mopper, 1993).

Sex-biased foraging behavior may be particularly important in hymenopterous insects due to their unique reproductive biology. Hymenopterans employ a haplodiploid mating system where diploid females are produced through fertilization of eggs and

17 haploid males develop from unfertilized eggs. Ovipositing females, through selective fertilization of eggs, may thus manipulate the sex of their offspring in response to host quality. Although this has been shown previously for many hymenopteran parasitoids of caterpillars (e.g. Vinson, 1976; Charnov et al, 1981; Herre, 1985; King, 1987, 1989;

Craig and Mopper, 1993), it has been demonstrated explicitly for only one herbivore.

Craig et al. (1992) showed that more female than male eggs of Euura lasiolepis, a tenthredinid sawfly, were laid on fast growing willows that contained larger shoots, and suggested that this behavior was adaptive because female fitness increased more rapidly than that of males with increasing plant growth (Craig et al, 1989, 1992). Other studies have not been able to differentiate between biased sex ratios associated with oviposition preferences and those associated with differential mortality between immature males and females (e.g., Barker and Maczka, 1996). Sex-biased foraging by immature insects has also only been reported in one study (Rhainds et ah, 2002), despite the high selective pressure that is presumably exerted on juvenile insects to forage adaptively within and among hosts.

The yellowheaded spruce sawfly, Pikonema alaskensis (Roh.) (Hymenoptera:

Tenthredinidae) is a common defoliator of juvenile open-grown black spruce (Picea mariana [Mills] B.S.P.) throughout Atlantic Canada. This spruce - herbivore system provides a unique opportunity to evaluate how a herbivorous insect overcomes intra-tree variation in feeding-site quality through oviposition and juvenile foraging strategies.

Field surveys indicate that feeding by larvae of .P. alaskensis may kill the leader (i.e., most apical shoot of the tree) and upper crown branches of spruce (Kulman, 1971;

Lavigne, 1996), suggesting that oviposition and/or larval feeding is biased towards the upper crown. As with other hymenopterans, differences in larval development between

18 the sexes of P. alaskensis may generate sex-biased oviposition strategies. Larvae of .P. alaskensis are sexually dimorphic with haploid males developing through five instars and diploid females developing through six instars (Houseweart and Kulman, 1976).

Here I report the oviposition behavior of adult females and feeding preference of larvae of P. alaskensis within the crown of juvenile black spruce. Using field surveys I evaluated defoliation and seasonal variation in the distribution of P. alaskensis juveniles

(i.e., eggs, mid-, and late-instar larvae) among whorls within the crown of black spruce.

I also conducted field surveys and manipulative field experiments to test the hypothesis that variation in the relative distribution of female and male juveniles reflects sex-biased foraging decisions made by ovipositing females and larvae.

Methods

Study Areas

All studies were conducted in intensively managed black spruce stands located approximately 50km (N 48°40'11.3", W 55°30'27.5") and 100km (N 48°17'07.3", W

55 29'01.4") south of Grand Falls-Windsor, Newfoundland. Trees had been planted at densities of approximately 2500 stems per hectare in each stand and ranged in height from 1.5 to 2.5m with 10 to 14 whorls of branches. The lowest 3-4 whorls of study trees were often dead, due presumably to shading by surrounding ground vegetation, including sheep laurel (Kalmia angustifolia L.) and blueberry {Vaccinium sp.). Thus, the actual vegetative crown studied often consisted only of 6-10 whorls. The branches of adjacent trees did not overlap. Pikonema dimmockii (Cress.) (Hymenoptera:

Tenthredinidae), a solitary non-outbreaking herbivore (Pointing, 1957) and the only

19 other defoliator present, was only observed in a few trees not included in the study.

Thus, any needle loss was due to P. alaskensis feeding and/or natural needle fall. A few balsam fir {Abies balsamea L.), eastern larch (Larix laricina [Du Roi] K. Koch), and white birch (Betula papyrifera Marsh) trees were interspersed within each stand.

Seasonal distribution of, and defoliation by, immature sawflies

I conducted a field survey in 2001 to evaluate the seasonal distribution of immature P. alaskensis and associated patterns of defoliation within the crown of black spruce. Prior to budburst, in each of four stands that had sustained defoliation associated with feeding by P. alaskensis, 25 trees with defoliation on one-year-old shoots, which was indicative of defoliation in the previous year, but no branch mortality in the upper crown, were selected haphazardly. On each tree, I evaluated previous defoliation/needle fall on the leader and on one west-facing branch in whorls 1,2, 4, and 7 (Fig. 2.1a) using a sampling method described in Johns et al. (2006). Using this method, branch defoliation was estimated visually before and after larval feeding (i.e., in mid June and late August) using defoliation classes of 0, 1-5, 6-20, 21-40, 41-60, 61-80, 81-99, or

100% for shoots located along the first- and second-order branch axes. Differences in defoliation estimates obtained before versus after the season were attributed to feeding by P. alaskensis larvae.

To determine seasonal variations in the distribution of P. alaskensis among whorls, all shoots on branches where defoliation was evaluated were carefully examined and P. alaskensis counted periodically throughout the season. Each week, larvae in all stands were visually compared with preserved samples of larvae of each instar to estimate the mean developmental stage of sawflies. Based on this information, sampling

20 to estimate sawfly density was carried out when most individuals were eggs (< 3% egg hatch, n = 25 per stand), mid-instars (mean (±1SE) instar 3.0 ±0.18, where egg = 0 and sixth instar = 6, n = 25 per stand), and late-instars (mean (±1SE) instar 5.1 ± 0.05, n = 25 per stand). Determination of the instar of larvae was based on the average head-capsule widths (Vanderwerker and Kulman, 1974) of 25 larvae collected in each stand.

During each sampling period (i.e., eggs, mid-, and late-instar larvae), I also determined the distribution of larvae within branches by counting sawflies in each of four distal to proximal sections within a whorl 4 branch of each tree. Sections were generally categorized according to the position of second-order branches along the primary branch axis, except for 'section i' which was the first-order terminal shoot of the primary branch (Fig. 2.1b). For each sample, eggs and larvae were counted on all shoots located within each section. To ensure that all sawflies were counted, needles on current-year shoots were spread with a probe to expose the relatively inconspicuous eggs and early-instar larvae. Withered needles were usually indicative of oviposition and/or larval feeding and provided a useful search image when trying to locate eggs or early- instar larvae.

Oviposition occurred exclusively, and larvae fed predominantly, on current-year foliage and larvae were only observed feeding on older age-classes when current-year foliage became scarce. Thus, egg and larval densities are expressed as the number of P. alaskensis per current-year shoot per branch. To facilitate inferences concerning larval movement among whorls, egg and larval densities were also expressed as the number per branch. The mean number of current-year shoots on the leader and on a branch in each of whorls 1,2,4, and 7, respectively, were 23,15, 30, 58, and 80 for all stands combined.

21 A complementary study was conducted in 2003, based on results from field surveys evaluating the seasonal distribution of P. alaskensis, to determine the age of larvae dispersing within trees. In this study, 10 first- or second-instar larvae collected from a nearby stand were placed on a south-facing whorl 4 branch in each of 5 trees in part of a stand where no P. alaskensis were present. Trees were checked daily and larvae found in whorls 1 or 2 were assumed to have dispersed. Larvae that had dispersed were immediately collected and preserved in 70% ethanol for subsequent determination of instar based on head-capsule width (Vanderwerker and Kulman, 1974).

The effects of stand (random effect) and whorl (fixed effect) on percent defoliation per shoot per branch were evaluated using a mixed-model two-way ANOVA

(SAS Institute, 1999). Defoliation data were arcsine square-root transformed prior to analysis to correct problems with heterogeneity of variance and normality (Zar, 1984).

The effects of site (random), whorl or branch section (fixed), and juvenile development stage (repeated factor: i.e., eggs, mid-instars, or late-instars) on the number of P. alaskensis per current-year shoot and per branch (for among whorl analysis only) were evaluated using a mixed-model repeated-measures ANOVA.

Seasonal distribution of male and female larvae

From 2001 through 2003,1 investigated the influence of oviposition and larval foraging behavior on the seasonal distribution of male compared to female P. alaskensis larvae in the upper and lower crown of black spruce. However, because the sex of P. alaskensis cannot be determined visually during egg or larval stages (Houseweart and

Kulman, 1976), larvae had to be collected in the field and reared until eclosion. Larvae were collected in each of three stands (i.e., a different stand each >ear) hosting sawfly

22 populations ranging in density from0.1 1 to 0.21 eggs per current-year shoot per tree.

Each year after oviposition was completed, I haphazardly selected 20 trees in each stand and marked them with flagging tape. From this group, ten trees were selected randomly by picking numbers out of a hat and assigned to an early (i.e., mean (±1SE) instar 2.2 ±

0.19 to 2.4 ± 0.20, n = 25 larvae) and the other ten trees to a late instar (i.e., mean

(±1SE) instar 5.0 ± 0.11 to 5.3 ± 0.06, n = 25 larvae) collection period. During each collection period, 15 larvae were collected haphazardly from whorls 2 and 7 (2001), and from the leader and whorls 2 and 7 (2002 and 2003) of selected trees. Ten larvae were placed in a mesh cloth bag and five larvae were placed in a vial in 70 % ethanol and labeled by tree and whorl number. Early-instar larvae feed near the site of egg-lay

(unpubl. data) and thus their location was considered to represent that of eggs. Larvae were transported in a cooler to a laboratory in Badger, Newfoundland.

To determine the mean larval instar cited above, I measured the head capsules of the 5 larvae that had been preserved in 70% ethanol immediately after sampling. The 10 remaining larvae were reared in a 25 x 25cm foil tray on shoots cut from whorl 2 or 3 branches obtained from the same stand where larvae had been collected. To prevent rapid desiccation, the base of shoots were inserted into floral piks and replaced every three days with fresh shoots. "Tree Tanglefoot Pest Barrier" (™The Tanglefoot

Company) was applied around the upper edge of trays to prevent larvae from escaping.

This treatment appeared to repel larvae, as they were never found stuck in the

Tanglefoot. Sifted peat was added to the bottom of trays to facilitate cocoon formation.

In late August, tray contents were sifted and cocoons counted to obtain an estimate of larval survival. Cocoons from each tray were counted to estimate survival and placed in

23 plastic test-tubes partially filled with sifted peat, then placed in a cold room at ~ 4 C for three months to overwinter. In the spring of each year, cocoons were removed from the freezer and left at room temperature until adult eclosion. Average survival of larvae to pupation was 83%.

Most P. alaskensis did not eclose, probably because of extended diapause

(Bartelt et ah, 1981; Eller et ah, 1989), making it difficult to determine the sex of individuals. Consequently, cocoons from which adults did not issue were dissected and sex was identified based on the width of prepupal head-capsules, as males have five instars and females have six instars (Vanderwerker and Kulman, 1974). Three procedures were tested to verify that head-capsule widths accurately distinguished male and female prepupae. First, to obtain a range of head-capsule widths for male prepupae,

I reared broods of larvae from virgin females, which produce all-male clutches of progeny, to the cocoon stage and measured head-capsule widths of 30 randomly selected individuals (Fig. 2.2a). Also, I measured the head-capsule widths of male and female adults, with the assumption that their size would be similar to that of prepupal head capsules (Fig. 2.2b) and I measured head-capsules of surviving prepupae from all transfer experiments (Fig. 2.2c). Based on these measurements I determined that prepupae with head-capsule widths < 1.7mm were males and those with head-capsule widths > 1.8 mm were females.

The effect of year (random), whorl (fixed), and larval instar (fixed) on the percentage of P. alaskensis larvae that were female was evaluated using a three-way mixed-model ANOVA. Pikonema alaskensis were not generally present on the leader during early-instars (pers. obs.), thus late-instar larvae collected from the leaders of trees

24 presumably dispersed from mid and lower whorls. For 2002 and 2003, when late-instar larvae were collected from the leader, variation in the percentage of females on the leader compared to whorls 2 and 7 was evaluated using one-way ANOVAs. Prior to analyses, data were subjected to an arcsine square-root transformation to correct problems with normality and variance (Zar, 1984).

Sex-biased larval foraging behavior

In 2004 I conducted an experiment to test the prediction that intra-tree dispersal behavior of P. alaskensis larvae, observed in field surveys discussed above, differs between males and females. In this experiment I investigated the relative propensity of male compared to female larvae to disperse from the mid (i.e., whorl 4) to upper (i.e., whorls 1 and 2) crown. Mated and unmated female adults were used to obtain broods of larvae that were either a mix of males and females (i.e., 'mixed brood') or only male

(i.e., 'all male'), respectively (Houseweart and Kulman, 1976). Unmated adult females were obtained from 20 pyramid emergence traps (0.5 x 0.5m) that had been placed, in mid June, below trees that had sustained high levels of defoliation due to sawfly feeding in the previous year. To prevent adults from escaping below the base of the trap the lower sides of each trap were covered with moss and soil. A removable plastic collection jar was secured into the top of each trap to collect adults. Traps were inspected each morning and afternoon. Male and female adults were collected from traps and placed in fine mesh-cloth sleeve cages (0.5 x 0.75m) on branches in groups of

1 female and 2 males (for the 'mixed brood' treatment) or 1 female (for the 'all male' treatment). Females used to obtain 'all male' broods were collected from traps with no males present and, thus, could not have mated prior to placement in sleeve cages. To

25 determine the sex ratio of offspring from the 'mixed brood', larvae not used in the experiment were left in the sleeve cage and reared until the end of the season so that the sex of prepupae could be determined based on head-capsule width.

In an area of a stand with no previous defoliation or P. alaskensis present, 10 trees were selected haphazardly and randomly assigned a treatment of 5 'mixed brood' or 5 'all male' larvae. The mean instar of larvae placed on branches was determined for

10 larvae collected from sleeve cages containing each of the 'mixed brood' and 'all male' larvae. On 14 July (i.e., day 1), young larvae (mean instar 2.2 ± 0.03, n = 25) were collected from sleeve cages and placed in groups of 5 on one whorl 4 branch in each tree. There is no effect of cardinal direction on larval abundance (Johns et ah,

2006), however, for simplicity west-facing branches were always selected. All branches on study trees were carefully examined and larvae in each whorl counted every 3 to 4 days.

An independent means t-test (Zar, 1984) was conducted to determine whether the percentage of larvae that dispersed to the upper crown (i.e., whorls 1 and 2) differed between 'mixed brood' and 'all male' trees at the end of the season (i.e., at the peak of larval abundance in the upper crown). Larvae found in the upper crown, where no larvae were present at the beginning of the experiment, were assumed to have dispersed there. Percentage data were arcsine square-root transformed prior to analysis (Zar,

1984). Data from one tree used as a 'mixed brood' treatment were omitted from analysis as all larvae died or dispersed away from the tree prior to the date of peak larval abundance in the upper crown.

26 Results

Seasonal distribution of immature sawflies and of defoliation

Defoliation attributable to P. alaskensis varied significantly among stands and increased from the lower to upper crown of trees (Fig. 2.3, Table 2.1). Although mean defoliation on branches in whorls 4 and 7 was < 9%, defoliation was always > 20% on leaders, even in stands with low densities of P. alaskensis. Stands with high densities of

P. alaskensis appeared to have more defoliation in the upper crown (i.e., whorls 1 and 2) but similar levels of defoliation in the mid and lower crown (i.e., whorls 4 and 7) compared to stands with relatively lower population densities, resulting in a significant interaction between stand and whorl.

The number of sawflies per current-year shoot and per branch varied significantly among stands, whorls, and developmental stages, and there were significant interactions between almost all factors (Table 2.2). Egg and mid-instar larval densities were generally lowest on the leader and on branches in whorls 1 and 7 and highest in whorls 2 and 4, particularly in the stand with the highest population densities (Fig.

2.4a,d). However, when considering the overall number of P. alaskensis occurring within a branch in each whorl (i.e., Fig. 2.4d,e), the numbers of eggs and mid-instar larvae per branch in whorls 2 and 4 were similar in the three stands with the lowest sawfly densities. A dramatic increase in the density of larvae on the leader and in whorl

1 during late instars, and a corresponding decrease in the number of larvae on whorl 4 and 7 branches, resulted in a significant interaction between whorl and developmental stage. Larval density appeared to increase within trees from third to fifth instar (i.e., compare Fig. 2.4b,e to Fig. 2.4c,f). This trend is probably an artifact owing to the greater number of shoots in the mid and lower compared to upper crown, h the

27 complementary study conducted to determine the developmental stage of dispersing larvae, 31 and 65% (n = 142) of larvae that had dispersed to the upper crown were fourth and fifth instar, respectively.

The density of P. alaskensis was significantly influenced by branch section (see

Fig. 2.1b) and stand, although not by their interaction (Table 2.3). Densities of the developmental stages of P. alaskensis varied significantly, although this variation was not consistent between stands or branch sections, resulting in interactions between developmental stage and branch and between developmental stage and stand (Table 2.3).

Most eggs were laid near the distal to middle part of branches in one stand where population densities were highest. However, in the other three stands with lower population densities, eggs were laid either near the base of the branch or fairly evenly throughout the branch (Fig. 2.5a). Juvenile density decreased slightly in middle branch sections (i.e., ii to iii) from egg to late-instar larval stages but remained similar near branch tips (i.e., section i) (Fig. 2.5c).

Seasonal distribution of male and female larvae

Survival of larvae from the beginning to the end of rearing was ~ 83%. The percentage of early- and late-instar larvae that were female was influenced by whorl, year, and larval developmental stage (Fi.2,108 > 2.08, P < 0.01), as well as by an interaction between year and whorl (F2,io8 = 4.24, P - 0.03) but not by any other interaction (Fi-2,ios < 1.13, P > 0.33). The percentage of larvae collected from whorl 2 and 7 branches that were female varied from 38.0 ±3.5 % (2002) to 66.3 ± 3.1 %

(2003). On average, the percentage of early-instar larvae that were female was slightly lower than that for late-instar larvae (48.1 ± 3.9 vs. 56.8 ± 3.2). In all 3 years, sex ratios

28 of both early-instar and late-instar larvae were more female biased in whorl 2 than whorl

7 (Fig. 2.6). Larvae initiated feeding on the foliage of buds where eggs were laid and usually remained there through second and third instars (compare Fig. 2.4a,e with Fig.

2.4b,f). Thus, female-biased sex ratios of early-instar larvae in the upper vs. the lower crown strongly indicate that more female eggs were laid in the upper than lower crown.

Groups of late-instar larvae collected from the leader in 2002, which presumably had dispersed there from the mid and lower crown, were more female-biased than groups of larvae collected from lower whorls during the same time period (F2 27 = 10.19,

P < 0.01) (Fig. 2.6b). A similar but non-significant trend of relatively female-biased sex ratios in the upper crown was noted in 2003 (F2 27 = 1.62, P = 0.22) (Fig. 2.6c).

Sex-biased larval foraging behavior

Approximately 76% of larvae in the 'mixed brood' treatments were female.

Larvae dispersed gradually from the mid and lower to the upper crown beginning on 15

July 2004 (Fig. 2.7). As all larvae from the all-male treatment dropped from trees to form cocoons or died nearly six days before those in the mixed brood treatment, only data from when larvae were at the peak of upper crown occupancy in both treatments

(i.e., day 25) were analyzed. Only -20% of late-instar larvae in 'all male' broods were located in the upper crown compared to -74% of late-instar larvae from the 'mixed broods' (Fig. 2.7) (t6= 3.17, P = 0.02).

29 Discussion

The foraging behaviors of egg-laying females and feeding larvae had a pronounced influence on the relative distribution and abundance of immature males and females within the crown of black spruce, supporting the hypothesis that inherent differences among male and female conspecifics can lead to different foraging strategies within heterogeneous plants (Craig and Mopper, 1993). Although young larvae of both sexes were found throughout the crown, a greater proportion of female compared to male early-instar larvae fed higher in the crown (i.e., whorl 2) (see Fig. 2.6c). As early instars feed on the bud where the egg is laid (pers. obs.), this strongly suggests that adult females tend to lay more female than male eggs in the upper crown. Similarly, the highest proportions of late-instar larvae that were female occurred on the leader (Kg.

2.6b,c), where no eggs were laid, indicating that more female than male larvae disperse acropetally (sensu Quiring, 1993), from mid and lower to upper crown branches. This hypothesis was supported in manipulative studies, where only 15-20% of 'all-male' larvae but over three-quarters of mixed brood larvae dispersed, 75% of which were female, to the upper crown (Fig. 2.7).

In general, insect foraging strategies employed to overcome intra-plant variation remain poorly understood and have only been examined in a few systems. Oviposition patterns for some insects maybe determined by inherent variations in shoot growth within plants, resulting in a concentration of progeny in particular parts of the host where shoot size is optimal (e.g., McKinnon et al, 1999). Juvenile insects may also adopt foraging strategies to overcome intra-plant variation in shoot phenology (Quiring,

1993; Kessler and Baldwin, 2002), foliage age (Moreau et al, 2003), vulnerability to natural enemies (Stamp and Bowers, 1990; Williams et al, 2001), and/or microclimate

30 (Bardoloi and Hazarika, 1994). In most systems where juveniles are able to disperse from the module selected by the mother, including the one studied here, there is presumably a strong link between the foraging decisions made by adults and juveniles that should collectively maximize the overall fitness (but see Schiers and DeBruyn,

2002.

To my knowledge, this is the first study to demonstrate preferential allocation of progeny sex through oviposition-site selection for a herbivorous insect within a host.

Craig et ah (1992) previously reported that female sawflies allocated more female than male eggs to fast growing plants. Presumably either female larvae benefit more from feeding in the upper crown or juvenile males suffer reduced performance when feeding in the upper crown. Extensive studies on parasitoid oviposition behavior have demonstrated that adult females preferentially allocate female progeny to larger hosts

(Vinson, 1976; Charnov etai, 1981; Herre, 1985; King, 1987,1989; Craig and Mopper,

1993) or where there is lower brood competition (reviewed in Cook, 1993), presumably to accommodate the higher nutritional needs of female offspring that must acquire additional resources to produce eggs. As in Craig et al. (1992), there maybe similar selective pressure for P. alaskensis adult females to lay their female offspring in parts of the crown where their fitness is maximized, since young larvae (i.e., first to third instar) tend to feed on the branch where they are oviposited (Fig. 2.4).

Even though more female than male offspring were laid in the upper crown (i.e., whorl 2), ovipositing mothers and young feeding larvae of P. alaskensis generally avoided the most apical foliage on whorl 1 and the leader, instead preferring foliage located in mid and lower whorls. While on a per shoot and per branch basis the highest densities of eggs and mid-instar larvae occurred on whorl 2 and 4 branches, there are

31 many more branches in whorl 4 and 7 compared to whorl 2. Thus, most P. alaskensis eggs and mid-instar larvae are probably located in the mid-crown, as in this study, as well as the lower crown, as was shown in a previous independent study (Johns et al.,

2006).

While acropetal dispersal has been shown in several previous studies (i.e.,

Quiring, 1993; Alonso and Herrerra, 1996; Kessler and Balwin, 2002; Straw et al.,

2006), sex-biased dispersal by the juvenile stages of a herbivorous insect has only been previously demonstrated in flightless lepidopterans, where female larvae disperse more often from defoliated host plants than males (Rhainds et al., 1998, 2002). Female-biased dispersal by juveniles may aid in maximizing fitness by enabling females to pupate on better (i.e., less defoliated) hosts, thereby benefiting their offspring (Rhainds et al, 1998,

2002). This contrasts with the behavior of P. alaskensis larvae, which disperse acropetally even though population density and associated defoliation are often much higher in the upper compared to mid and lower crown. Thus the present study may be the first study to unambiguously demonstrate sex-biased larval dispersal by juvenile insects in response to the quality of their feeding sites.

Where dispersal among plants is costly, herbivores may adopt foraging behaviors that increase their ability to exploit various feeding sites within their host. Efficiently exploiting limited resources within a heterogeneous plant maybe aided somewhat by sex-biased foraging, as such partitioning may reduce competition between male and female juveniles (Slafkin, 1984). Presumably the foraging strategies employed by P. alaskensis are adaptive responses to intra-tree variations in bud phenology, foliage quality, natural enemies and/or microclimate. These hypotheses were tested concurrently with this study and will be discussed elsewhere. This study emphasizes the

32 importance of considering juvenile sex as a factor in determining the preference and performance of herbivorous insects, particularly in species where immature females and males may differ in their relative sensitivity to the quality of feeding sites within their host plant.

Acknowledgments

I thank J. Boone, H. Crummey, J. Evans, G. Fleming, B. Gregory, C. Griffin, V. Howell,

B. Johns, T. Johns, E. Kettela, J. Leggo, M. Luff, J. Marshall, J. Park, G. VanDusen for technical assistance, and S. Heard, F. Horgan, J. Leggo, G. Moreau, H. Piene, and M.

Rhainds for comments on an earlier version of the manuscript. Financial support was provided by an IPS NSERC scholarship with Abitibi-Consolidated and Corner Brook

Pulp and Paper Ltd., a NSERC Discovery grant, the Spray-Efficacy and Research

Group, and BIOCAP/NCE. The Canadian Forest Service contributed additional logistical support.

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40 Table 2.1. Results of a mixed-model two-way ANOVA evaluating the influence of stand (random effect) and whorl (fixed effect) (described in Fig. la) on defoliation associated with feeding by Pikonema alaskensis larvae in 2001.

Effect df MS F P

Stand 3 2.315 46.30 <0.01 Whorl 4 4.475 28.50 <0.01 Stand x Whorl 12 0.157 3.16 <0.01 Error 480 0.050

41 Table 2.2. Results of a mixed-model repeated-measures ANOVA evaluating the effects of stand (random effect), whorl (fixed effect) (described in Fig. la), and developmental stage (fixed effect) on the number of Pikonema alaskensis per current->ear shoot and per branch in 2001.

Unit Effect df MS F P

Per current-year shoot Stand 3 1.927 23.63 <0.01 Whorl 4 1.802 15.53 <0.01 Stand x Whorl 12 0.116 1.42 0.15 Error 480 0.082

Dev. Stage 2 3.54 12.08 <0.01 Dev. Stage x Stand 6 0.293 7.59 <0.01 Dev. Stage x Whorl 8 2.038 52.60 <0.01 Dev. Stage x Stand x Whorl 24 0.201 5.19 <0.01 Error (Dev. Stage) 960 0.039

Per branch Stand 3 4562.999 41.18 <0.01 Whorl 4 869.541 3.16 <0.01 Stand x Whorl 12 275.420 2.49 <0.01 Error 480 110.808

Dev. Stage 2 1070.691 6.38 <0.01 Dev. Stage x Stand 6 167.935 3.11 <0.01 Dev. Stage x Whorl 8 1188.026 36.11 <0.01 Dev. Stage x Stand x Whorl 24 172.032 5.23 <0.01 Error (Dev. Stage) 960 32.896

42 Table 2.3. Results of a mixed-model repeated measures ANOVA evaluating the effects of stand (random effect), branch section (fixed effect) (described in Fig. lb), and developmental stage (fixed effect) on the number of Pikonema alaskensis per current- year shoot in 2001.

Effect df MS F P

Stand 3 1.072 17.57 <0.01 Br. Section 3 0.210 7.78 <0.01 Stand x Br. Section 9 0.027 0.44 0.64 Error 348 0.061

Dev. Stage 2 0.095 2.02 0.02 Dev. Stage x Stand 6 0.047 2.84 <0.01 Dev. Stage x Br. Section 6 0.057 3.45 <0.01 Dev. Stage x Stand x Br. Section 24 0.024 1.45 0.11 Error (Dev. Stage) 960 0.017

43 Table 2.4. Results of a mixed-model three-way ANOVA evaluating the effects of year (random effect), whorl (fixed effect), and developmental stage (fixed effect) on the percentage of Pikonema alaskensis larvae that were female from 2001 through 2003.

Effect df MS F P

Year 2 1.841 15.74 <0.01 Whorl 1 2.227 4.50 <0.01 Dev. Stage 1 0.150 2.08 0.01 Year x Whorl 2 0.495 4.24 0.03 Year x Dev. Stage 2 0.072 0.62 0.54 Whorl x Dev. Stage 1 0.013 0.12 0.73 Year x Whorl x Dev. Stage 2 0.132 1.13 0.33 Error 108 0.117

44 (a) (b) whorl 4 braoch

Figure 2.1. (a) Schematic representation of a juvenile black spruce tree with seven whorls. For the purposes of this study, whorl 1 was divided into its leader and a whorl 1 branch, (b) Defoliation was visually estimated for all age classes of shoots along the first- and second-order branch axes (solid shoots and buds). Whorl 4 branches were subdivided into four sections, consisting of the terminal shoot (i.e., section i) and sections distinguished by the position of second-order branches that included all shoots from that branch up to the next second-order branch.

45 20 (a) male prepupae 15 VA 10

0 'AY4 1.2 1.4 1.6 1.8 2.0 2.2 Prepupal head-capsule width (mm)

20 (b) 15 male female adults adults 10 as" u

300 (C) 250

200 -I

150

100

0 1.2 1.4 1.6 1.8 2.0 2.2 Prepupal head-capsule width (mm)

Figure 2.2. Frequency distributions of: (a) head-capsule widths for an all-male group of

prepupae; (b) head widths of adult males (shaded bars) and females (white bars);

and (c) pooled distribution of head-capsule widths of all prepupae examined in

transfer experiments used in this study.

46 Whorl

Figure 2.3. Mean (±1 SE) percent defoliation of black spruce attributable to feeding by

P. alaskensis on the leader (L) and on one branch in each of whorls 1, 2,4 and 7. The four shades of bars represent different stands with differing population densities of P. alaskensis. Raw data are presented, however data were arcsine square-root transformed prior to analysis.

47 0.50 i 75 (a) eggs (d) eggs

0.25 i 25

0.00 L JL SUb Jfi. 31s lliii

I 0.50 75 (b) mid-instar larvae (e) mid-instar larvae & 1 -i s 50 i. a 3 0.25 •a

1.50 75 (c) late-instar larvae (t) late-instar larvae 1.25

1.00 50

0.75

0.50 25

0.25

1^0 ll EH •Mfr— 11 liL Ik liL k 0.00 u ) 1 2 Whorl Whorl

Figure 2.4. Mean (±1 SE) density of eggs, mid-, and late-instar larvae of P. alaskensis per current-year shoot (a-c) and per branch (d-f) on the leader (L) and in whorls 1,2,4, and 7 of black spruce. The four shades of bars represent different stands with differing population densities of P. alaskensis. Note the different scale used in c.

48 0.50 -i (a) eggs

0.25 H

o o ja 0.00 fc ii hi i- « 0.50 i J. (b) mid-instar larvae a

i. 3 W u 0.25 a*5 1

A; 0.00 l Ilia la lil

0.50 (c) late-instar larvae

0.25 -\

0.00 ,1 lip Ifo fa) in IV Section

Figure 2.5. Mean (±1 SE) density of eggs (a), mid- (b), and late-instar larvae (c) of P. alaskensis from the tip to the base (i.e., i to iv) of whorl 4 branches. The four shades of bars represent different stands with differing population densities of P. alaskensis.

49 100 (a) 2001

0 w. L 100 (b) 2002 I [ early-instar larvae 75 late-instar larvae

75 •

50-

25 •

2 Whorl

Figure 2.6. Mean (±1 SE) percentage of early- and late-instar P. alaskensis larvae located on the leader (L), and in whorls 2 and 7 of black spruce that were female in three different stands from 2001 through 2003 (a-c). Asterisks indicate that either no larvae were present on (i.e., early instars in all years), or no larvae were collected from (i.e., late instars in 2001), the leader. Raw data are presented, however data were arcsine square-root transformed prior to analysis.

50 100 -•— mixed brood -o— all male 1 75 I I

Figure 2.7. Mean (±1 SE) percentage of P. alaskensis larvae present in whorls 1 and 2,

with either 'mixed broods' (-•-) or 'all male' broods (-o-), from 25 July 2004 (i.e.,

day 15 of the study) to 4 August 2004 (i.e., day 25). Raw data are presented,

however data were arcsine square-root transformed prior to analysis.

51 CHAPTER 3: SEX-BIASED OVIPOSITION-SITE SELECTION BY ADULTS AND ACROPETAL

DISPERSAL BY LARVAE ARE ADAPTIVE RESPONSES OF A SAWFLY TO INTRA-TREE

VARIATION IN FOLIAGE QUALITY

Abstract

Some herbivorous insects may maximize their fitness by adopting foraging strategies to facilitate the acquisition of the best available resources within heterogeneous host plants. Adults of yellowheaded spruce sawfly, Pikonema alaskensis (Roh.)

(Hymenoptera: Tenthredinidae), lay most eggs in the mid to lower crown of black spruce (Picea mariana [Mills] B.S.P.), and late-instar larvae disperse acropetally, from the mid and lower crown to the upper crown, to complete juvenile development. While female and male eggs are both laid throughout the crown, most eggs laid in the upper crown are female, and more female than male late-instar larvae disperse acropetally through the crown. Four years of manipulative sleeve-cage experiments were carried out to evaluate the hypothesis that sex-biased oviposition-site selection and acropetal dispersal by P. alaskensis are adaptive responses to intra-tree variation in foliage quality. I also investigated whether intra-plant variation in foliage quality was attributable to acropetal budburst.

In general, survival was highest for larvae placed in the mid or lower crown during early instars, then transferred acropetally during late instars to complete development in the upper crown, compared to all alternative foraging strategies.

Females benefited most from acropetal dispersal, as indicated by the relatively higher female biased sex ratios of surviving larvae that were transferred acropetally. Although survival was generally lower for larvae feeding exclusively in the upper crown, males

52 appeared to be most negatively affected, resulting in female biased sex ratios for survivors. Survival of larvae feeding in the lower crown during both early and late instars was similarly poor, however, sex ratios of survivors were more male biased, suggesting that females were more negatively affected than males. Oviposition-site selection and acropetal dispersal were unrelated to intra-plant variation in budburst phenology. Budburst occurred weeks before peak egg-lay and bud phenology had no significant effects on early-instar larval survival. Also, late-instar larvae dispersed nearly a week after shoot elongation was completed in the mid crown, indicating that shoot phenology did not influence the foraging of late-instar larvae. Thus, sex-biased egg allocation and acropetal dispersal by larvae appears to increase the individual survival of P. alaskensis, particularly for females, by accounting for phenology- independent temporal and spatial variations in the quality of foliage within the heterogeneous crown of black spruce.

Key words: Phenology, budburst, Pikonema alaskensis, Picea mariana, hymenoptera, oviposition, foraging strategy, sex-biased behavior, intra-plant

Introduction

Variation in the abundance and distribution of suitable plant material can have a significant influence on foraging strategies of herbivorous insects (Hassell and

Southwood, 1978). Finding nutritious foliage can be problematic for some herbivores as only a small proportion of available foliage maybe suitable, due to a prevailing lack of accessible nutrients (e.g., nitrogen in the form of soluble amino acids), and a myriad of well-developed chemical and physical defenses (Murdoch, 1966; Slansky, 1993).

Natural enemies, adverse microclimates, and previous herbivorymay further limit the

53 number of suitable oviposition or feeding sites available (e.g., Hassell, 1980; Crawley,

1983; Jones and Hassell, 1988; Williams et al, 2001). Most studies have focused on how variation in foliage quality among plants can shape herbivore foraging (e.g.,

Quiring and Butterworth, 1994; Alonso and Herrera, 1996; Clark and Messina, 1998;

Bernays et al, 2004; Holland et al., 2004), while fewer have examined the important influence of variation on herbivory within plants, particularly in large plants such as trees (see Denno and McClure, 1983).

High levels of temporal and spatial variation in microhabitat quality within plants may determine the quality and quantity of potential oviposition and feeding sites available to herbivores. Numerous studies have shown that adult females frequently maximize the fitness of their offspring by laying eggs on the most nutritious of available modules, particularly in species whose progeny are restricted to the module selected by the mother, such as for many gallers, fruit or seed eaters, and leaf miners (e.g, Roitberg et al, 1981; Quiring and McNeil, 1987; McClure et al, 1998; Ozaki et al, 2006).

However, some juvenile insects are also well adapted to obtaining needed nutrients within heterogeneous host plants, either through increasing their feeding rate (Krause et al, 1999) or by dispersing to better feeding sites (Quiring, 1993; Carroll and Quiring,

1994; Kessler and Baldwin, 2002).

Complementary foraging strategies between adults and juveniles within plants maybe critical to maximize fitness of insects feeding on ephemeral resources (i.e.,

Carroll and Quiring, 1994; Kessler and Baldwin, 2002) or for insects whose nutritional needs or tolerances change dramatically with ontogeny (Hochuli, 2001). For example, spruce bud moth (Zeiraphera canadensis Mutt. & Free) caterpillars disperse acropetally from the mid-crown of white spruce, where most eggs are laid, to the upper crown

54 (Quiring, 1993). This is an adaptive behavior that enables juveniles to account for acropetal budburst and to feed almost continuously on foliage of newly burst buds

(Carroll and Quiring, 1994). Eggs of some lepidopterans are laid in protected parts of plants, and juveniles subsequently disperse as late instars to the plant apices to maximize sun-exposure for basking (Porter, 1982; Alonso, 1997). The oviposition and juvenile foraging strategies of other insects may favor poorer foliage on protected parts of their host if risks of mortality due to natural enemies (Stamp, 1989; Stamp and

Bowers, 1990) or desiccation (Bardoloi and Hazarika, 1994) are high. Avoidance of predators and hygrothermal stress, as well as obtaining higher quality foliage, appears to influence the oviposition and larval foraging behaviors of a caterpillar on tobacco

(Kessler and Baldwin, 2002). In some systems, inter-plant variation in host quality may also lead to sex-biased oviposition-site selection (Craig et al, 1992; Chapter 2) and/or larval dispersal (Rhainds et al, 2002; Chapter 2), presumably reflecting the differences in relative nutritional needs and associated reproductive responsibilities of females compared to males (Hendrichs et al, 1991; Craig et al, 1992; Craig and Mopper, 1993;

Barker and Maczka, 1996; Jormalainen et al, 2001; Rhainds et al, 2002).

Previously, I demonstrated that adult females of yellowheaded spruce sawfly,

Pikonema alaskensis (Roh.) (Hymenoptera: Tenthredinidae), lay eggs, and young larvae feed predominantly, in the mid to lower crown (i.e., whorls 4 to 7) of black spruce

(Picea mariana [Mills] B.S.P.), while late-instar larvae disperse acropetally, from the mid and lower to the upper crown (i.e., whorls 1 and 2), to complete feeding in the upper crown of the tree (Chapter 2). Oviposition-site selection and acropetal dispersal by P. alaskensis both appeared to increase the relative density of female compared to male juveniles in the upper crown. For example, although most eggs off. alaskensis

55 were laid in the mid and lower crown, during a three-year study 57% of the relatively few eggs laid in the upper whorls (i.e., mainly in whorl 2, Fig. 3.1) were female, compared to only 39% in the lower crown (Chapter 2). Acropetal dispersal by late- instar larvae was also sex biased with most females, compared to only about 25% of males, dispersing through the crown (Chapter 2).

In this study, I conducted a series of manipulative field experiments to test the hypothesis that biased allocation of female eggs to the upper crown by adults and acropetal dispersal by female larvae of P. alaskensis are adaptive responses to intra-tree variations in foliage quality. I predicted that survival should be lower for larvae feeding for their entire development in the upper crown. However, if sex-biased egg allocation is an adaptive response to variation in plant quality, then the costs of feeding in the upper crown should be lower for female compared to male larvae, resulting in a more female-biased sex ratio for survivors. I also predicted that larvae dispersing acropetally would have higher fitness, estimated by survival, than those that develop in only one crown level, or that dispersed basipetally, and that females would benefit more (as indicated by changes in the sex ratio of survivors) than males from acropetal dispersal.

I also conducted field surveys and manipulative experiments to determine whether temporal and spatial variations in sawfly foraging behavior associated with foliage quality were attributable to intra-tree variation in phenology, such as acropetal budburst. If P. alaskensis foraging behavior is an adaptive response to intra-tree variation in budburst phenology, then oviposition or early larval development should be constrained to a narrow phenologjcal window of development and acropetal dispersal should enable young larvae to feed for a second time on still-developing foliage in upper whorls. Other hypotheses that could also explain acropetal dispersal by P.

56 alaskensis, such as intra-tree variation in microclimate or parasitism, were tested concurrently with this study and will be discussed elsewhere (i.e., Johns et al., 2007b;

Boone, 2006).

Methods

Description of study insect

Pikonema alaskensis defoliates juvenile, open-grown spruce throughout central and northeastern North America (Katovich et al., 1995). Black spruce is the primary host of P. alaskensis in central Newfoundland, however white spruce (P. glauca

[Moench] Voss) and blue spruce (P. pungens Engelm.) are also susceptible (Kulman,

1971). Detailed descriptions of the life history of P. alaskensis are available in Pointing

(1957), Houseweart and Kulman (1976a), and Katovich et al. (1995). Briefly, adults eclose soon after budburst and lay eggs at the base of needles in new flushing shoots.

Progeny of virgin females are all male, whereas those from mated females maybe either male or female (Houseweart and Kulman, 1976b). Young larvae feed mainly on current-year foliage through five (male) or six (female) instars, then drop to the ground and spin a cocoon in the upper duff layer, where they overwinter as prepupae (Rau et al.,

1979).

Study areas

This study was conducted in intensively-managed black spruce stands that are described in Chapter 2.

Are sex-biased oviposition-site selection and acropetal dispersal adaptive?

57 In 2001 through 2004,1 used sleeve-cage experiments to test the predictions that larval survival is highest for individuals developing first in the mid to lower crown and dispersing acropetally to the upper crown of black spruce (represented by a whorl 2 branch), but that female early-instar larvae of P. alaskensis develop better than males in the upper crown. Each year I haphazardly selected 15 trees in a stand without P. alaskensis or previous defoliation and marked one west-facing branch in each tree with flagging tape in whorls 2 and 7 (2001 and 2002), or in whorls 2, 4, and 7 (2003,2004)

(Fig. 3.1a). In 2002 and 2004 the leader of each tree was also marked. Twentyfirst- and/or second-instar larvae (i.e., mean instar 1.8 ±0.12 to 2.1 ± 0.24, where egg = 0 and sixth instar = 6) obtained from a nearby stand, were placed on selected branches by gently removing needles on which larvae were feeding and placing the larval-bearing needles between needles of current-year shoots on the study branches. To prevent larvae from dispersing from the branch and to protect them from natural enemies, a fine-mesh cloth sleeve cage (100 x 75 cm) was placed over each study branch and a twist-tie used to constrict and attach the cage to the base of the branch, as in Johns et al. (2006a).

Each cage enclosed a minimum of fifteen current-year shoots and foliage was never limiting due to the relatively low density of larvae used.

To test the prediction that larval fitness, particularly for females, will be highest for groups transferred acropetally through the crown of black spruce, larvae from sleeve cages in the above experiments were transferred within trees to simulate a variety of potential dispersal strategies, including acropetal dispersal. Once larvae began dispersing acropetally in an adjacent stand where P. alaskensis were feeding naturally, sleeve cages were removed individually from trees and surviving larvae (mean instar 4.5

± 0.34 to 4.9 ± 0.52, n = 25) were counted. Larvae were then placed into a tray and

58 transferred in groups of three to other branches within the same tree to simulate a variety of potential foraging behaviors (Table 3.1). Depending on the availability of larvae, all transfers in 2001 through 2003 were executed by transferring larvae to previously unused branches within the same tree, either within whorls (i.e., 2 to 2, 4 to 4, and/or 7 to 7), acropetally from mid or lower to upper whorls (i.e., 4 to 2, 7 to 2) or from upper to lower whorls (i.e., 2 to 7). After larvae were transferred, a sleeve cage was placed over treatment branches and secured at the base of the branch with a twist-tie. To facilitate cocoon formation, approximately 500 ml of sifted peat was placed in the bottom of each sleeve cage. To determine survival, in late August sleeve cages were removed and the contents sifted for cocoons, which were then placed in vials labeled by transfer treatment. Cocoons were reared in a freezer (-4 °C) for 3 months and then at room temperature (~23°C) until eclosion. Adult eclosion after the overwintering period was poor (< 23%), due in part, perhaps, to extended diapause (Bartelt et al., 1981; Eller et ah, 1989). Thus, head-capsule width was measured to determine the sex of uneclosed prepupae (Vanderwerker and Kulman, 1974; Chapter 2).

Transferring larvae within the same tree did not allow for treatments on the leader or on multiple whorls of the upper crown (i.e., whorls 1 and 2), where most foliage is consumed in natural field conditions (Chapter 2). Thus, I conducted a similar experiment in 2004 where larvae were transferred to new trees, with only one treatment per tree, to allow treatments that were not possible with the previous protocol (i.e., 4- leader (L), 4-upper crown (U), L-L, L-7), as well as transfers used in previous years (i.e.,

7-7, 4-4, 2-2, 7-2).

The effect of whorl during the pre-transfer period (i.e., first to fourth instar) on survival was analyzed for each year using a one-way analysis of variance (ANOVA).

59 The effect of transfer treatment on post-transfer survival and sex ratio was analyzed for each year using one-way ANOVAs, followed by a Dunnett's test (Zar, 1984), where the mean survival or sex ratio of groups of larvae transferred acropetally (e.g., from whorl 7 to 2) were designated the control and compared to each of the other transfers. To meet model assumptions of normality and homogeneity of variance, all survival data was arcsine square-root transformed prior to analyses (Zar, 1984).

Influence of phenology

Effect of bud phenology on oviposition

To test the hypothesis that oviposition by P. alaskensis is restricted within a relatively narrow phenological window of bud development, a sleeve-cage experiment was conducted in 2001 where females were isolated on shoots of different phenology.

Females were placed on branches with buds classified into stages similar to those used previously for white spruce (Turgeon, 1986). Briefly, shoots were classified visually into developmental stages based on the following characteristics: (1) bud cap is tight and no needles are visible; (2) needles are < 40% visible but the bud cap is still attached; (3) needles are > 75% exposed (bud cap is mostly or completely shed) but are still tightly aligned together; (4) bud cap has been shed and needles are beginning to flare at the tips;

(5) all needles are flaring at the tips.

To obtain adults for experiments, soil from beneath heavily defoliated trees in one stand was sifted, yielding 196 uneclosed cocoons. Sifting occurred approximately two weeks before budburst and three weeks before the first females were captured in sticky traps in the same stand (Johns et ah, 2006a). Cocoons were placed individually in plastic test tubes half filled with moistened peat. Initially, -50 cocoons were left at

60 room temperature to accelerate pupation and synchronize the eclosion of some adults with earlier stages of budburst than would be encountered under natural conditions. The remaining cocoons were placed in a fridge and every 5 days, ~50 cocoons were removed and reared at room temperature. Because of the extended period of development resulting from the different rearing conditions, eclosion from reared cocoons occurred over a two-week period, from before budburst (i.e., stage 1, 20 June) until all buds were flaring (i.e., bud stage 4,4 July). Females used for each phenological stage were less than 24 h old. However, because replication for each bud stage was dependant on the abundance of females available at a given stage of bud phenology and eclosion over time was variable, sample-sizes between treatments were unequal (i.e., bud stage (1) seven females; (2) six females; (3) eight females; (4) four females). No females were available from rearings to examine oviposition on shoots in stage 5. Instead females and males were collected in the field and cooled in the fridge to extend their reproductive period so that they could be placed on buds in stage 5.

To evaluate the suitability for oviposition of each of the five phenological stages of bud development, 5 trees with no burst buds were selected and, in each tree, five west-facing branches in whorls 4 or 5 were selected haphazardly and marked with flagging tape. During each of the five stages of bud development, one female and two males were placed on one branch in each of the 5 trees and enclosed in a sleeve cage (20 x 30 cm). The ends of sleeve cages were constricted so that only medial-lateral buds of the terminal one-year old shoot (Fig. 3.1b) were available for oviposition. One week after females were placed in sleeve cages the branches were cut and transported to the lab so that the buds could be more carefully examined under a dissecting scope.

Oviposition success was classified based on the percentage of available shoots in each

61 sleeve cage that contained eggs because I was only interested in whether females were able to lay eggs in shoots and was unable to ensure that females collected in the field had not already laid eggs before being placed in sleeve cages.

In 2001 a field survey was carried out to evaluate the prediction that egg lay occurs during a relatively narrow window in which buds are suitable for oviposition.

Prior to budburst, I haphazardly selected 15 trees in an area of a stand with < 11% mean previous defoliation. In each tree, the leader and one west-facing branch in each of whorls 2,4, and 7 (Fig. 3.1a) were marked with flagging tape. On selected branches the terminal (T), one distal-lateral (dL), and one medial-lateral (mL) shoot (Fig. 3.1b) were marked with a small piece of thread tied loosely at the base of the shoot. The stage of each of the marked shoots was evaluated every two days until after egg lay ceased for 2 consecutive samples (i.e., 4 days). Each day that bud phenology was evaluated, needles were also examined for eggs. Needles were carefully spread using a probe to expose the base of the needles where P. alaskensis lay most eggs (Pointing, 1957).

Effect of bud phenology on young larvae

To test the hypothesis that early-instar larvae of P. alaskensis are influenced by declines in foliage quality associated with bud development, which could limit how late after budburst eggs could be laid without jeopardizing the success of offspring, I evaluated the survival of larvae feeding on buds in stage 3 or stage 4 in 2001. In one stand, 15 trees were selected haphazardly and two west-facing branches in whorl 4 were identified with flagging tape. Within 24 h of emergence, first-instar larvae were collected from sleeve cages and placed in groups of three on a terminal and on a medial- lateral bud of one of two selected branches in each tree immediately after buds burst and

62 the bud caps were shed (i.e., stage 3) and enclosed in a sleeve cage. This protocol was repeated on an adjacent branch one week later when needles of buds were begnning to flare (i.e., stage 4). First-instar larvae for the second treatment were collected from sleeve cages containing adult females and males whose eclosion had been delayed for a week by cooling cocoons in a cold room (~4°C). After one week, sleeve cages were removed from branches and surviving larvae were counted. The influence of bud stage

(i.e., stage 3 or 4) and bud type (i.e., terminal/distal-lateral or medial-lateral) on early- instar larval survival was evaluated using a two-way mixed-model ANOVA (SAS

Institute, 1999). Survival data were arcsine square-root transformed prior to analysis to correct problems with normality and homogeneity of variance.

Acropetal dispersal and shoot phenology

In 2002,1 investigated the timing of acropetal dispersal relative to the end of shoot elongation in the upper and lower crown of black spruce to test the prediction that acropetal dispersal enables late-instar larvae of P. alaskensis to feed for a second time on developing foliage. Prior to budburst, 15 trees were selected in an area of one stand where no P. alaskensis were present. In each tree, the leader and one west-facing branch in each of whorls 2 and 4 were marked with flagging tape. On each branch, a terminal, distal-lateral, and medial-lateral bud on the terminal one-year old shoot was marked by loosely tying a piece of thread around the base. After budburst, the length of each selected shoot was measured using a caliper every three days until the end of shoot elongation. Mid-instar larvae (i.e., second and third instars) were collected from an adjacent stand and placed as a group of 5 on the marked whorl 4 in each tree. After larvae were placed in the tree, the leader and all branches of trees were examined for

63 larvae every three days. Larvae found on branches above whorl 4, where larvae were placed initially, were assumed to have dispersed acropetally. No larvae were found in lower whorls.

Results

Are sex-biased oviposition-site selection and acropetal dispersal adaptive?

Survival of larvae from first- to fourth-instar decreased from the lower to upper crown in all years (F^, 25-49 > 3.40, P < 0.02), except 2003 where the trend was similar but differences were not significant (F2,4i = 1.98, P = 0.15) (Fig. 3.2).

In two of the four years studied, groups of late-instar larvae transferred acropetally had higher survival than those transferred within whorls or from upper to lower whorls (Dunnett's Test, P < 0.05) (Fig. 3.2e,h) (Table 3.2). In 2004, there were no differences in survival among larvae in three acropetal transfers (i.e., 7-2 vs. 4-Lor 4-U)

(Fig. 3.2h). Survival of larvae transferred acropetallyin 2002 did not differ significantly from those transferred in other patterns within the crown (Fig. 3.2f). In 2003, survival of late-instar larvae was highest for individuals transferred acropetally, except when compared to larvae transferred from whorl 4 to 4, which had similar survival (Kg. 3.2g).

Multiplying pre-dispersal survival by post-dispersal survival to calculate overall survival for each year yielded similar or slightly amplified differences in survival among foraging treatments (Fig. 3.3a-d).

In both 2001 and 2004, sex ratios of survivors were more female-biased for groups of larvae transferred acropetally compared to groups that were transferred within whorls (i.e., 4 to 4 and 4 to 7 in 2004 and 7 to 7 in 2001), or from whorl 2 to 7 (Fig.

3.3e,h) (Table 3.2). Although overall survival was relatively low, sex ratios of larvae

64 transferred within whorl 2 (all years) or from leader to leader (2004) were similar to those of larvae transferred acropetally (Fig. 3.3e-h). In both 2002 and 2003, there were few significant differences between the sex ratios of larvae transferred acropetally and those transferred in other patterns within the crown (Fig. 3.3f,g).

Influence of Phenology

Effect of bud phenology on oviposition

Eggs were only found in buds that had already shed most of the bud cap (i.e., stage 3-5) (Fig. 3.4a). There was no stage evaluated that was too old for egg-lay as females oviposited on almost all stage 3,4, and 5 buds available (Fig. 3.4a).

Budburst did not occur acropetally within the crowns of black spruce trees examined in this study. Although there were slight differences among whorls, most shoots were in a stage suitable for oviposition nearly a week before female P. alaskensis eclosed and laid their eggs (Fig. 3.4b).

Effect of bud phenology on young larvae

Although survival was slightly lower on medial-lateral and stage 4 buds, there was no significant difference in survival for larvae feeding on buds in stage 3 compared to stage 4 (Fi>59 = 0.76, P = 0.39) or on terminal/distal-lateral compared to medial-lateral buds (Fi,59= 0.27, P = 0.60) (Fig. 3.5).

Acropetal dispersal and shoot phenology

Most larvae dispersed acropetally between 29 July, when < 25% were in the upper crown, and 12 August, when approximately 80% of larvae were in the upper

65 crown (Fig. 3.6). Shoots in whorls 2 and 7 completed elongation by 29 July, whereas shoot elongation of the leader was 96% complete by the same date.

Discussion

This study supports the hypothesis that sex-biased oviposition-site selection and acropetal dispersal by larvae of P. alaskensis are adaptive responses to variation in foliage quality within black spruce. As predicted, survival was generally highest for larvae dispersing acropetally compared to all alternative foraging strategies (Fig.

3.2a,c,d). Females benefited most from this foraging behavior, as indicated by the relatively female-biased sex ratios for groups of larvae transferred acropetally (Fig.

3.3e,h). Although survival was generally lower for larvae feeding exclusively in the upper crown (i.e., compared to those dispersing acropetally), males appeared to be the most negatively affected, resulting in female-biased sex ratios for survivors (Fig.

3.3e,g,h, whorl 2-2). Survival for larvae feeding only in the lower crown was similarly poor, however, sex ratios of survivors were more male biased, suggesting that females were more negatively affected than males (Fig. 3.3e-h, whorl 7-7).

Foliage quality has previously been shown to influence patterns of distribution and abundance of immature insects within plants (e.g, McKinnon et ah, 1999;

Williams et ah, 2001; Fortin and Mauffette, 2002; Kessler and Balwin, 2002). To my knowledge, adaptive sex-biased oviposition-site selection in response to foliage quality has only been demonstrated in one other study. Craig et al. (1992) showed that adult females ofEuura lasiolepis preferentially allocate female eggs to the larger shoots of fast growing willows and concluded that differences in the mass of male compared to mixed-brood larvae among plants were attributable to mated females laying more

66 female than male eggs on faster growing shoots. For P. alaskensis, survival of early- instar larvae is best in the mid to lower crown (Fig. 3.2a-d), where most eggs are laid

(Chapter 2). However, the majority of the eggs laid in the upper crown were female, presumably because the survival of early-instar females developing in the upper crown is higher than that of males.

Sex-biased dispersal by the juvenile stages of a herbivorous insect has only been previously demonstrated in flightless lepidopterans, where female larvae disperse more often than males from defoliated host plants (Rhainds et ah, 1998, 2002). Because flightless adult lepidopterans have relatively low mobility, this foraging behavior was speculated to maximize fitness by enabling females to find better (i.e., less defoliated) hosts for their offspring (Rhainds et ah, 1998, 2002). In contrast, sex-biased acropetal dispersal by late-instar P. alaskensis larvae appears to be, at least partly, an adaptive response to variability in foliage quality within plants, independent of previous defoliation (Chapter 2). Both survival and the percentage of survivors that were female was highest for larvae that dispersed acropetaHy, strongly suggesting that females, which disperse acropetaHy more frequently than males (Chapter 2), benefit more than males from this behavior.

Several studies have investigated the adaptive advantage of acropetal dispersal by juvenile insects, identifying intra-plant variation in phenology exclusively (Quiring,

1993, Carroll and Quiring, 1994) or in combination with thermal environment and natural enemies (Kessler and Baldwin, 2002) as primary factors driving this foraging strategy. Acropetal dispersal by P. alaskensis, however, is unrelated to intra-tree variation in budburst phenology. Timing of budburst did not significantly influence oviposition as peak egg-lay occurred nearly a week after buds were suitable for

67 oviposition. Similarly, host-plant phenology did not influence first-instar larval survival and did not appear to play a significant role in acropetal dispersal. Late-instar larvae dispersed nearly a week after shoot elongation was completed in the mid crown and thus did not benefit from feeding a second time on still-developing foliage of apical shoots.

Acropetal dispersal may instead be a response to seasonal changes in the quality of similarly aged foliage within the crown of black spruce, driven by inherent source- sink competition among modules (Honkanen and Haukioja, 1994) and/or shading by overtopping branches (Henriksson, 2001; Fortin and Mauffette, 2002; Yamasaki and

Kikuzawa, 2003). Alternatively, age-related changes in the nutritional needs or feeding habits of larvae, such as those associated with changes in larval size and morphology

(Hochuli, 2001), may compel larvae to disperse within the crown to find more suitable food. Female-biased allocation of offspring to better quality plant material has often been attributed to higher relative nutritional needs of females compared to males, associated with the differing reproductive responsibilities between the sexes (Hendrichs et al, 1991; Craig et al, 1992; Craig and Mopper, 1993; Barker and Maczka, 1996;

Jormalainen etal., 2001; Rhainds etal., 2002). The sex ratio of many of these herbivores is generally more male-biased when developing on poorer quality hosts

(Craig et al., 1992; Mopper and Whitham, 1992; Barker and Maczka, 1996).

Intra-tree variability in foliage quality influencing the fitness of P. alaskensis was only clearly observed in 2 of the 4 years during this study but was, nevertheless, consistent from year to year as the fitness of larvae transferred acropetally was either the same or better than that of larvae forced to forage in other ways. In two different years in which the intra-tree distribution of P. alaskensis was evaluated, most eggs and early instar larvae were in the mid crown (Chapter 2) or in the mid and lower crown (Chapter

68 3), suggesting that the influence of crown level on foliage quality may vary somewhat among seasons. Thus, while it may not always be advantageous to disperse acropetally, foliage quality in the upper crown is presumably always at least equal to that of foliage in the mid and lower crown.

Survival and sex ratio were the only fitness correlates evaluated for P. alaskensis in this study due, in part, to my inability to estimate fecundity either through measuring prepupal weight, which involved destroying cocoons and killing prepupae, or by examining adult females, which rarely eclosed. Examining other fitness correlates might have facilitated further inferences regarding the benefits of acropetal dispersal to P. alaskensis. For example, in two of the four years where I found no effects of foliage quality on survival or sex ratio of P. alaskensis, there may have been sub-lethal effects that were not detected.

This study did not directly address the potential costs of acropetal dispersal. For example, feeding in high densities could significantly increase competition for limited foliage in the upper crown, forcing some larvae to feed on less suitable foliage.

Aggregation by larval insects can also increase rate of the spread of pathogens through populations (i.e., Beisner and Myers, 1999), although none have been reported for P. alaskensis. Studies of parasitism off. alaskensis larvae conducted in the same area as the present study indicate that parasitism of both early- and late-instar larvae is highest for larvae feeding in the mid to lower crown (Boone, 2006). This suggests that acropetal dispersal has a cost and a benefit in terms of increased parasitism for early- and late- instar larvae, respectively. Also, microclimatic conditions such as temperature and solar insolation did not differ significantly between upper and lower whorls (Johns et al,

2007). As larvae tend to feed with most of their body on the shaded bottom of shoots, it

69 is unlikely that microclimate drives acropetal dispersal by P. alaskensis. Consequently,

I conclude that sex-biased egg allocation by adults and acropetal dispersal by larvae of

P. alaskensis is, at least in part, an adaptive response to intra-tree heterogeneity in foliar quality. As shown in this study, such effects of intra-plant variation in foliage quality on herbivore fitness can vary considerably among years, and might not be detected in short- term studies.

Acknowledgments

I thank J. Boone, H. Crummey, J. Evans, G. Fleming, B. Gregory, C. Griffin, V. Howell,

B. Johns, T. Johns, E. Kettela, J. Leggo, M. Luff, J. Marshall, J. Park, and G. VanDusen for technical assistance, and S. Heard for comments on an earlier version of the manuscript. Financial support was provided by an IPS NSERC scholarship with Abitibi

Consolidated and Corner Brook Pulp and Paper Ltd., a NSERC Discovery grant, the

Spray-Efficacy and Research Group, and BIOCAP/NCE. The Canadian Forest Service contributed additional logistical support.

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77 Table 3.1. Pattern of transfers simulating acropetal dispersal (*) and other potential dispersal strategies within the crown of juvenile black spruce from 2001 through 2004. Treatments employing the leader are represented by 'L' and those employing the upper crown (i.e., whorls 1 and 2) are represented by 'U'.

Year Initial whorl Final whorl'

200 lb 7 • 7

7 ^ 7* 7 ^ 7

2002b 7 • 7

7 k. T*

b 2003 7 • 7 4 P1 4 I ^ z 1 P Z* 2 7

2004° 7 • 7 4 ^ 4 I F" Z L W L 7 w 2* 4 p. j^m 4 p> UT L F 7

a Pikonema alaskensis larvae were reared for 4 instars on 15 or more shoots and then transferred to a similar number of shoots on a branch in the final whorl. b Transfer treatments were all applied within each tree. c One transfer applied within each tree.

78 Table 3.2. Comparisons of variance components from one-way ANOVAs evaluating the influence of transfer, described in Table 1, on larval survival and on the percentage of surviving larvae that were female in experiments conducted from 2001 through 2004.

Fitness correlate Year ^transfer •> "terror ^transfer terror F P

% survival 2001 3,52 0.395/0.078 5.84 <0.01 2002 2,39 0.059/0.056 1.07 0.35 2003 4,70 0.465/0.119 3.91 <0.01 2004 7,110 1.329/0.226 5.89 <0.01

% female 2001 3,48 1.150/0.118 9.72 <0.01 2002 2,26 1.104/0.331 3.33 0.05 2003 4,67 0.609/0.236 2.58 0.05 2004 7,110 0.999/0.239 4.17 <0.01

79 (a) (b)

t shoot Leader tbud

1 dLbud

2 mL bud

Figure 3.1. (a) Schematic representation of a juvenile black spruce tree with seven whorls. For the purposes of this study, whorl 1 was divided into its leader and a whorl 1 branch, (b) Classification of terminal (t), distal-lateral (dL), and medial-lateral (mL) vegetative buds developing on a one-year old shoot.

80 100 100 (e) 2001 (a) 2001 75 75

50 50

25 25

0 0 2 7 7-7 2-2 7-2 2-7 100 100 (b)2002 (1) 2002 75 75

T 50 • 50 T 25 25

0 ' r"i 0 7-7 2-2 7-2

50 50

25 • 25

0 0 7.7 4-4 2-2 7-2 2-7

100 100 (d) 2004 (h)2004 75 75 A

50 50

25 25

0 0 ULtx 2 4 7 7.7 4-4 2-2 LrL 7-2 4-L 4-U h-7

Whorl Transfer

Figure 3.2. Mean (±1 SE) percent survival of P. alaskensis: (a-d) from first to fourth instar on the leader (L) or on one branch in each of whorls 2, 4, or 7 and (e-h) from fourth instar to cocoon for P. alaskensis transferred either within whorls (i.e., 7 to 7, 2 to

2, leader to leader (L to L)) (white bars), from upper to lower whorls (i.e., 2 to 7) (white bars), or acropetally among whorls (i.e., 7 to 2,4 to leader (L), 4 to upper (U)) (slashed bars) from 2001 through 2004. Raw data are presented, however, data were arcsine square-root transformed prior to analysis. "*" indicates a significant difference in percent survival when compared to the acropetal transfer treatment (i.e., 7-2) at a = 0.05

(Dunnett's Test).

81 100 100 (a) 2001 (e) 2001 75 75

50 50

25 25

0 0 7-7 2-2 7-2 2-7 7-7 2-2 7-2 2-7

100 100 (b) 2002 (f)2002 75 75

50 50

25 25

0 S o 7-7 2-2 7-2 7-7 2-2 7-2

50 50

25 25

0 0 7-7 4-4 2-2 7-2 2-7 7-7 4-4 2-2 7-2 2-7 100 (d) 2004 100 (h) 2004 75 75 50 CT 50 25 25 i 0 nn 0 .4. 7-7 4-4 2-2 L-L 7-2 4-L 4-U L-7 7-7 4-4 2-2 LL 7-2 4-L 4-U L-7 Transfer Transfer

Figure 3.3. Mean (±1 SE) survival of P. alaskensis from first instar to cocoon (a-d) and percentage of survivors that were female (±1 SE) (e-h) for larvae that were initially placed on the leader (L) or on a branch in whorl 2,4, or 7 and then transferred within whorls (i.e., 7 to 7,2 to 2, leader to leader (Lto L)) (white bars), from upper to lower whorls (i.e., 2 to 7) (white bars), or acropetally among whorls (i.e., 7 to 2, 4 to leader

(L), 4 to upper (U)) (slashed bars) to complete development from 2001 through 2004.

Raw data are presented, however data were arcsine square-root transformed prior to analysis. "*" indicates a significant difference in sex ratio when compared to the acropetal transfer treatment (i.e., 7-2) at a = 0.05 (Dunnett's Test).

82 100 (a) 75 I 1 50 25

12 3 4 5 Bud phenological stage

100 §

o '> o

0) 1 •3 o o

Jun-14 Jun-16 Jun-18 Jun-20 Jun-22 Jun-24 Jun-26 Jun-28 Jra-30 Jul-02 Jul-04 Date

Figure 3.4. Relationship between budburst phenology and oviposition by P. alaskensis.

(a) Percentage of buds in stages 1-5 on which P. alaskensis females oviposited in a manipulative study, (b) The mean percentage of buds on 4 whorls (L (- •-), whorl 2 (-

•-), whorl 4 (-T-), and whorl 7 (-•-)) within the crown of black spruce in a suitable stage (i.e., stage 3-5) for oviposition and the temporal distribution of eggs laid (shaded area) in a field survey.

83 1W

80 •I 60 B so 40

20 n ILIL T/dL mL Bud type

Figure 3.5. Effect of bud type (terminal/distal-lateral (T/dL) versus medial-lateral

(mL)) and shoot phenology (black-stage 3, white=stage 4) on the performance of first- instar larvae. Raw data are presented, however, data were arcsine square-root transformed prior to analysis.

84 July 14 Aug 12

Figure 3.6. Temporal relationship between the mean percentage of larvae in the upper crown (i.e., whorls 1 and 2) of black spruce (shaded area) and mean lengh of current- year shoots on the leader (-•-) and in whorls 2 (-•-) and 7 (-T-).

85 CHAPTER 4: INTRA-TREE VARIATION SHAPES THE FOLIAGE-AGE PREFERENCE AND

PERFORMANCE OF LARVAE OF A SPECIALIST HERBIVORE

Abstract

Foliage-age specialization is an important adaptation that enables some herbivorous insects to more efficiently acquire nutrients within their host-plant.

However, where intra-plant variation in foliage quality is extremely high, as it often is in trees, the quality of non-preferred age-classes of foliage could occasionally exceed that of preferred age-classes of foliage. To better understand how such variability influences herbivore foraging, I investigated the foliage-age preference and associated performance of larvae of yellowheaded spruce sawfly, Pikonema alaskensis (Roh.), within the crown of black spruce (Picea mariana [Mills.] B.S.P.) using a combination of field surveys and manipulative field experiments. In general, current-year foliage in the upper crown was more defoliated by P. alaskensis larvae than any other age-class, and larvae developing on this foliage had the highest survival and most female-biased sex ratios. Generally, defoliation of one-year-old foliage (i.e., a non-preferred age-class of foliage) in the upper crown was similar to that on current-year foliage (i.e., the preferred age-class of foliage) in the lower crown. Survival and sex ratios of late-instar larvae that fed on one- year-old foliage in the upper crown were generally similar to that of larvae that fed on current-year foliage in lower whorls. Thus, results supported the hypothesis that the foliage-age preference of P. alaskensis larvae is adaptive and that in some jears this preference maybe as strongly influenced by variation among crown levels as by variation among foliage age-classes. This study demonstrates an interesting dynamic in spatial compared to age-related variation in plant quality that can shape patterns of

86 herbivore preference and performance, particularly in large architecturally complex plants such as trees.

Key words: Pikonema alaskensis, Picea mariana, sex bias, acropetal dispersal, foliage quality

Introduction

Herbivorous insects that feed on plants with persistent foliage, such as conifers, generally specialize on either developing or mature needles (All and Benjamin, 1975;

Geri et al, 1993; Dodds et al, 1996; Carisey and Bauce, 1997; Moreau et al, 2003), although some insects feed on both (e.g., Wilson, 1971; Carroll, 1999). Pressure to specialize relates, presumably, to the tremendous contrast in quality between developing foliage, which is soft and nutritious with high quantities of toxins (Cates, 1980; Quiring,

1992; Carisey and Bauce, 1997), and mature foliage, which is often less toxic but is tough and contains large amounts of digestibility reducing compounds (Cates, 1980;

Geri et al., 1993; Carisey and Bauce, 1997). Feeding on foliage that is too young or old and presumably lacking an optimal complement of nutrients often results in reduced prepupal or pupal weight, slower development, and/or lower survival (Geri et al, 1993;

Dodds et al, 1996; Carisey and Bauce, 1997; Moreau et al, 2003). Thus, being able to efficiently evaluate the suitability of foliage and respond appropriately is critical for folivorous insects seeking to successfully exploit hosts possessing high levels of variability in foliage quality.

Spatial and temporal variation within either developing or mature foliage is also an important determinant of insect foraging behavior, both within and among plants.

For example, some herbivores that specialize on developing foliage disperse within their

87 host tree to feed continuously on the youngest foliage available (Quiring, 1993; Carroll and Quiring, 1994). Alternatively, juvenile insects that prefer mature conifer foliage may eat needles from a variety of age-classes of shoots to obtain their needed nutrients

(Carroll, 1999; Moreau et al., 2003). Where variation in foliage quality is extremely high within a plant with persistent foliage, as it often is in large plants such as conifer trees (see Denno and McClure, 1983), it is plausible that the quality of non-preferred age-classes of foliage will occasionally exceed that of preferred age-classes of foliage.

The direct influence of foliage age in shaping the preference and associated performance of herbivorous insects within conifer branches has been demonstrated in several laboratory studies (Geri et al., 1993; Carisey and Bauce, 1997; Carroll, 1999) and in the field (Moreau et al., 2003). However, such studies have not considered the potential influence of high levels of spatial variation within large conifers in shaping relationships between foliage-age preference and performance of herbivores.

In this study, I investigated the foliage-age preference and performance of a univoltine defoliator, the yellowheaded spruce sawfly (Pikonema alaskensis Roh.)

(Hymenoptera: Tenthredinidae), within the crown of juvenile black spruce (Picea mariana [Mills] B.S.P.). While larvae of P. alaskensis generally prefer current-year foliage (i.e., developing foliage) (Pointing, 1957) of black spruce, some late-instar larvae may also feed on older age-classes of foliage (Wilson, 1971) with unknown consequences to fitness. Adult females lay eggs and young larvae of P. alaskensis feed predominantly in the mid and lower crown, while late-instar larvae disperse acropetally

(sensu Quiring, 1993), from the mid and lower crown to upper apical crown positions to complete feeding (Chapter 2). In years where population densities are high, defoliation can significantly reduce the availability of current-year foliage in the upper crown,

88 posing a dilemma for larvae that have not yet completed development. These larvae can either feed in the upper crown on older age-classes of foliage, or they can feed on the abundant, but less nutritious (Chapter 3), current-year foliage in lower whorls.

Here I present results from field surveys and manipulative field studies investigating the feeding preference of P. alaskensis on different ages of foliage in the upper versus lower crown of black spruce. I also conducted a manipulative field experiment to test the hypothesis that the observed foliage-age preference of P. alaskensis within the crown of black spruce is adaptive, with respect to food quality (i.e.,

Perry and Pianca, 1997), by forcing both young and old larvae to feed on selected age- classes of foliage in the upper or lower crown of black spruce.

Methods

Study Areas

These studies were conducted in intensively-managed black spruce stands, described in Chapter 2.

Branch-level feeding preference

In 2000,1 investigated the foliage-age preference of P. alaskensis within branches of black spruce using two sleeve-cage experiments, one where naturally occurring densities of larvae were caged on mid-crown branches ('natural density' experiment) and one where the number of larvae on branches was manipulated

('manipulated density' experiment). In each of two stands, prior to budburst, 15 or 20 trees were selected haphazardly and five or six east- or west-facing mid-crown branches

(i.e., whorls 4 or 5) were selected and marked with flagging tape. Neither the densities

89 of eggs and mid-instar larvae of P. alaskensis, nor defoliation attributable to sawfiy larvae, are influenced by cardinal direction in black spruce (Johns et ah, 2006a). Branch defoliation was estimated visually before (i.e., mid-June) and after (i.e., late-August) larval feeding using defoliation classes of 0, 1-5, 6-20, 21-40, 41-60, 61-80, 81-99, or

100% for shoots located along the first- and second-order branch axes, as described by

Johns et ah (2006a). Five age-classes of foliage (i.e., current-year, one-, two-, three-, and four-year-old) were evaluated on each branch. For each age-class of foliage, mean percent defoliation attributable to P. alaskensis was calculated by subtracting defoliation estimates obtained prior to larval feeding from those obtained after larval feeding was completed.

In the 'natural density' experiment, I selected 15 trees and marked 5 branches per whorl with flagging tape in a stand with 0.03 to 0.86 P. alaskensis larvae per current- year shoot and with 1 to 39% defoliation per shoot per branch (i.e., based on initial counts and assessments of defoliation described below). After egg hatch, the mean instar of 25 larvae was determined, based on head-capsule width (Vanderwerker and

Kulman, 1974). Early-instar larvae (i.e., mean instar 1.8 ± 0.45, where egg = 0 and sixth instar = 6) were counted on marked branches in each tree and branches were enclosed in fine-mesh cloth sleeve cages (0.50 x 0.75m) filled with approximately 500ml of sifted peat to facilitate cocoon formation, as in Johns et ah (2006b). To ensure that all P. alaskensis were counted, I used a dissecting needle to carefully spread the needles of current-year shoots to expose the cryptic early-instar larvae. Needles often withered when P. alaskensis adults inserted eggs or when larvae fed on them and, thus, provided a useful search image when trying to locate early-instar larvae. The base of each sleeve cages was secured to the branch using a twist tie. After larval feeding was completed,

90 sleeve cages were removed, branches examined, and cocoons counted to provide estimates of larval survival and the density of late-instar larvae per branch.

The 'manipulated density' experiment was carried out on 20 trees in a stand with no P. alaskensis present and no previous defoliation. First- and second-instar larvae

(i.e., mean instar 2.3 ±0.17) were collected from a nearby stand (N 48°40'11.3", W

55°30'27.5") with relatively high densities of P. alaskensis (0.22 ± 0.02 larvae per current-year shoot). First-instar larvae (verified using head-capsule widths

(Vanderwerker and Kulman, 1974)) were collected by clipping individual shoots occupied by larvae and placing them in an open tub for transport to the study site, as in

Johns et al. (2006b). Six mid-crown branches were selected in each tree and randomly assigned a density treatment ranging from 0 to 0.53 larvae per current-year shoot

(originally applied as 0, 2, 4, 8, 12, or 18 larvae per branch). To minimize injury to larvae during transfer, forceps were used to gently remove the needles occupied by larvae from shoots and to move them onto the study branches or leaders. Needles with larvae were placed between the flaring needles of current-year shoots and enclosed with sleeve cages as described above. After placement, larvae moved almost immediately from the transferred needle onto adjacent shoots (pers. obs.).

Relationships between the density of early-instar P. alaskensis larvae and subsequent defoliation of each age-class of foliage for each experiment were evaluated using linear regression (SAS Institute, 1999). Differences between relationships for each foliage age-class were not evaluated directly due to a violation of the assumption of independence (Roa, 1992). A log-log transformation was used to straighten lines prior to analysis and to satisfy model assumptions of homogeneity of variance and normality

91 (Zar, 1984). Where values of zero were present a small value of 0.10 was added (Zar,

1984).

Tree-level feeding preference

To determine the foliage-age preference of P. alaskensis larvae throughout the crown of host plants, field surveys were conducted in 2001 in each of 2 stands that had previously sustained feeding associated with P. alaskensis. In each stand, 25 trees with one to two years of previous defoliation and no top-kill were selected prior to budburst.

On each tree, the leader and one west-facing branch in each of whorls 1,2, 4, and 7 was selected and marked with flagging tape (Fig. 4.1a). To estimate feeding caused by P. alaskensis, branches were evaluated for defoliation before and after larval feedingusing the method described above. The total number of age-classes, including current-year, examined for branches in each whorl was 2, 2, 3, 5, and 8, respectively, for the leader and whorls 1,2, 4 and 7.

The effects of whorl (fixed) and foliage age (fixed) on percent defoliation within tree crowns in each site were evaluated using a two-way model-1 ANOVA (Zar, 1984).

Data were transformed with an arcsine square-root function to meet model assumptions.

Larval performance

To test the prediction that the foliage-age preference of P. alaskensis larvae is adaptive, a manipulative study was carried out in 2002 and 2003 to determine the influence of foliage-age in upper and lower whorls on the survival and sex ratio of early- and late-instar larvae. Each year, 30 (2002) or 15 (2003) trees were selected in two different stands with no previous defoliation. In each tree, three or four west-facing

92 branches in both the upper crown (whorls 2 or 3) and the lower crown (whorls 7 or 8) were marked with flagging tape. Each branch was randomly assigned to one of the following treatments of foliage availability within a branch. In 2002 and 2003, treatments consisted of: (1) all age-classes; (2) only current-year; or (3) only one-year- old. In 2002 an additional treatment of three-year-old foliage in the lower crown was also used, whereas in 2003 a treatment of two-year-old foliage was used in both the upper and lower crown. To confine larvae to the current-year foliage treatment, one- year-old shoots were wrapped with mesh-cloth strips made from the same material used to make sleeve cages, and secured at the base of the shoots with twist ties. Shoots were wrapped tight enough to exclude larvae while still allowing the needles to sit normally.

To isolate larvae on mature foliage, current-year shoots were enclosed individually in small sleeve-cages (3 x 15cm) secured to the base of the shoot and to the adjoining one- year old shoot using twist ties. In these treatments mesh cloth was used to cover all mature age-classes of foliage other than that selected for a treatment (e.g, if only one- year-old foliage was specified in the treatment, current-year foliage was enclosed in small sleeve cages and two-year-old foliage was wrapped in cloth).

In both years, early- and late-instar sawfly larvae were collected from an adjacent stand < 1 km away and transported to the study site using the methods described in the

"Branch-level feeding preference" section. In 2002, first- to third-instar larvae (mean instar 2.3 ± 0.22, n = 25) collected in early July were placed in groups of 5 on selected branches of 15 trees and covered with a sleeve cage. This method was repeated using fourth-instar and fifth-instar larvae (mean instar 4.9 ± 0.35; n = 25) on the remaining 15 trees in 2002, and all 15 trees in 2003. After cocoon formation was completed, sleeve cages were collected and contents sifted for cocoons. Cocoons were counted to estimate

93 percent survival and placed in a freezer (-4 °C) for three months, then removed and allowed to complete pupation at room temperature. Cocoons that did not eclose were dissected and head capsules were measured to determine sex (Vanderwerker and

Kulman, 1974; Chapter 2).

The effects of foliage age and whorl on survival and sex ratio were evaluated using a nested ANOVA (i.e., with foliage age treatment nested in whorl) for early-instar larvae and late-instar larvae in 2002 and using model-1 two-way ANOVAs for late-instar larvae in 2003. For 2003 data this analysis was followed by Tukey's HSD post-hoc contrasts (Zar, 1984). Percent survival and percent female data were transformed using an arcsine square-root function prior to analysis to meet model assumptions of homogeneous variance and normality (Zar, 1984).

Results

Branch-level feeding preference

In both the 'natural density' and 'manipulated density' experiment, there was a significant positive relationship between early-instar larval density and subsequent

2 defoliation of current-year foliage (Fli73-ii7 > 254.17, P < 0.01, r > 0.73) (Fig. 4.2).

Defoliation also increased with larval density on one-year-old foliage in both experiments (Fi^-in > 19.58, P < 0.01, r2 > 0.14), and for two-year-old foliage in the

2 'natural density' (F1;73 = 44.05, P < 0.01, r = 0.38) (Fig. 4.2a) but not in the

2 'manipulated density' experiment (FMi7 = 0.38, P = 0.54, r < 0.01) (Fig. 4.2b).

Tree-level feeding preference

94 In both stands, defoliation associated with feeding by P. alaskensis larvae occurred only on current- and one-year-old foliage, and thus only these age-classes were included in analyses. Defoliation varied significantly among stands, whorls, and the two age-classes of foliage (Table 4.1). Generally, defoliation was highest on current-year foliage in the upper crown (Fig. 4.3). There was also a significant whorl x foliage age interaction (Table 4.1), due presumably to the relatively low defoliation on one-year-old foliage of leaders (Fig. 4.3). In general, defoliation of one-year-old foliage was highest on branches in whorl 2 and was similar to that of current-year foliage in whorls 4 and 7

(Tukey's HSD: P > 0.05). Small differences in the relative levels of defoliation on current- versus one-year-old foliage in the two stands (Fig. 4.3) resulted in a significant interaction between stand and foliage age (Table 4.1).

Larval performance

Survival of early-instar larvae was highest when they had access to current-year foliage (i.e., all age-classes or current-year foliage only), regardless of whorl (Table 4.2,

Fig. 4.4a). However, survival on the current-year foliage treatment was higher in whorl

7 than in whorl 2 but similar in both whorls of the "all" foliage-age treatment, resulting in a significant whorl x age interaction.

Survival of late-instar larvae in both 2002 and 2003 decreased significantly with increasing foliage age and was generally higher in whorl 2 compared to whorl 7 (Fig.

4.4b,c). There were no significant foliage age x whorl interactions in 2003. Based on post-hoc comparisons for late instars in 2002 and 2003, there were generally no significant differences in survival between larvae feeding with all foliage age-classes available or just current-year foliage within a whorl (Tukey's HSD: P > 0.07).

95 Furthermore, survival did not differ significantly between larvae feeding on one- to three-year old foliage in whorl 2 and larvae feeding on current-year foliage in whorl 7 in either year (Tukey's HSD: P > 0.25).

In 2003, the percentage of P. alaskensis that were female decreased significantly with increasing foliage age (F3>72= 4.77, P < 0.01) but was not influenced significantly by whorl (Fi;72 = 0.69, P = 0.41) (Fig. 4.4f), nor was there any significant interaction

(F3J2 = 0.14, P = 0.93). Although trends were qualitatively similar in 2002, there were no significant effects of whorl or foliage age on the percentage of surviving early-instar larvae (F4,4o < 1-94, P > 0.12) or late-instar larvae (F6>80 < 1.51, P > 0.19) that were female (Fig. 4.4d,e). In 2003 but not 2002, sex ratios were similar for larvae that fed on one-year-old foliage in the upper crown compared to those that fed on current-year foliage in the lower crown (Tukey's HSD: P > 0.05).

Discussion

This study demonstrates that P. alaskensis larvae prefer developing current-year, black spruce foliage, that this preference is adaptive with respect to foliage quality and that in some years this preference maybe influenced as strongly by variation among crown levels as by variation among foliage age-classes. Overall, current-year foliage in the upper crown suffered the most defoliation by larvae, and development on this foliage yielded the highest survival and percentage females. In natural conditions, however, the depletion of current-year foliage in upper whorls (i.e., whorl 1 to 2) resulted in a significant increase in defoliation of one-year-old foliage in the upper crown, in some cases to a level similar to that of current-year foliage in the lower crown. Survival and sex ratios of late-instar larvae that fed on one-year-old foliage in the upper crown were

96 generally similar to that of late-instar larvae that fed on current-year foliage in the lower crown (Fig. 4.4b,c). To the best of my knowledge, this is the first study to report that intra-plant variation can be so high that eating relatively high quality mature foliage in one part of the crown is equivalent to eating relatively low quality current-year foliage in other parts of the crown.

The foliage-age preference of herbivores may need to be somewhat flexible for species facing a variety of selective pressures. For example, caterpillars that perform best on new foliage may still select older foliage that is more proximally located within a plant to reduce exposure to natural enemies (e.g., Jefferies and Lawton, 1984; Stamp and

Bowers, 1990; Hawkins etal., 1993; Hopkins and Dixon, 1997). Juveniles of both hymenopteran and lepidopteran herbivores may increase their fitness by feeding on multiple age-classes of foliage to obtain a more balanced diet, rather than specializing on only one age-class (Carroll, 1999; Moreau et ah, 2003). For P. alaskensis, there was a consistent trend of foliage-age preference within branches, even among older age-classes of foliage where one-year-old shoots sustained higher levels of defoliation than two- year-old shoots (Fig. 4.2). When considering the entire tree crown, however, spatial variation in foliage quality was as important as age-related differences in foliage quality.

Overall, remaining in the upper crown to feed on one-year-old foliage may be less costly to P. alaskensis larvae than dispersing to lower whorls when current-year foliage in the upper crown becomes scarce. Such costs, which were not evaluated in this study, may include the energetic costs of dispersing or increased mortality due to higher levels of parasitism in lower whorls (unpubl. data, Boone, 2006).

The age of juveniles is also an important determinant of a herbivore's ability to exploit age-classes of foliage other than that selected by the mother during oviposition,

97 due in part to differences in the size, morphology, and feeding habits of young compared to old larvae (Hochuli, 2001). Early-instars tend to be far more sensitive than older instars to variation in foliage quality associated with foliage age (e.g., Geri et ah, 1993;

Dodds et ah, 1996; Carisey and Bauce, 1997; Moreau et ah, 2003). Similarly, most early-instar P. alaskensis larvae died when forced to feed on foliage > one-year old.

Late-instar P. alaskensis larvae, in contrast, were able to complete development on mature age-classes of foliage and presumably were responsible for the observed defoliation of one-year-old (field survey) and older (sleeve-cage study) foliage in black spruce.

Female P. alaskensis are generally more abundant in upper compared to lower whorls due to greater allocation of female eggs, female-biased acropetal dispersal

(Chapter 2), and higher survival of female larvae in the upper versus lower crown

(Chapter 3). Instances where female sex ratios were higher in whorl 2 compared to whorl 7 (Fig. 4.4f) support the latter hypothesis. The present study emphasizes the relatively high sensitivity to foliage quality of females compared to males. Overall, lower survival of late-instar larvae feeding on mature versus current-year foliage (Fig.

4.4b,c) was associated with decreases in the percentage of survivors that were female

(Fig. 4.4e,f). This indicates that increased mortality on older foliage was disproportionately due to the death of female larvae. In the few other studies reporting sex-biased mortality due to feeding on sub-optimally aged foliage, females also were more sensitive than males (e.g., Moreau et ah, 2003).

Foliage-age specialization represents a significant adaptation by herbivores, allowing them to become highly attuned to the general chemical and constitutive defenses of one or a few specific age-classes of foliage within their host-plant (Cates,

98 1980). This study demonstrates an interesting dynamic in the interacting effects of spatial compared to age-related variation in plant quality that can shape patterns of herbivore distribution and abundance, particularly in large architecturally complex plants such as trees.

Acknowledgments

I thank J. Boone, H. Crummey, J. Evans, G. Fleming, B. Gregory, C. Griffin, V. Howell,

B. Johns, T. Johns, E. Kettela, J. Leggo, M. Luff, J. Marshall, J. Park, and G. VanDusen for technical assistance, and G. Moreau and L Pinault for comments on an earlier version of the manuscript. Financial support was provided by an IPS NSERC scholarship with Abitibi-Consolidated and Corner Brook Pulp and Paper Ltd., a NSERC

Discovery grant, the Spray-Efficacy and Research Group, and BIOCAP/NCE. The

Canadian Forest Service contributed additional logistical support.

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102 Table 4.1. Results of a mixed-model ANOVA evaluating the effects of stand (random effect), whorl (fixed effect) (described in Fig. la), and foliage age (fixed effect) on percent defoliation associated with feeding by Pikonema alaskensis larvae in 2001. Effect df MS F P

Stand 1 3.851 51.67 <0.01 Whorl 4 3.091 25.13 <0.01 Age 1 12.758 22.82 <0.01 Stand x Whorl 4 0.123 1.65 0.16 Stand x Age 1 0.559 7.51 <0.01 Whorl x Age 4 0.452 6.07 <0.01 Stand x Whorl x Age 4 0.0341 0.45 0.77 Error 480 0.075

103 Table 4.2. Results of ANOVAs evaluating the effects of whorl (described in Fig. la) and foliage age on percent survival of early- and late-instar Pikonema alaskensis larvae in 2002 and 2003 (late-instar larvae only). Note that analyses for early and late-instar larvae for 2002 were nested ANOVAs with foliage age nested within whorl, whereas a model-1, two-way ANOVA was used for 2003. Experiment Effect df MS F P

Early instars Whorl 1 0.0001 <0.01 0.97 2002 Whorl (Age) 5 2.590 26.07 <0.01 Error 98 0.099

Late instars Whorl 1 0.673 4.08 0.05 2002 Whorl (Age) 5 0.925 5.61 <0.01 Error 98 0.165

Late instars Whorl 1 1.777 15.46 <0.01 2003 Age 3 0.639 5.56 <0.01 Whorl x Age 3 0.14 1.22 0.31 Error 112 0.115

104 (a) (b) whorl 4 branch

Figure 4.1. (a) Schematic representation of a juvenile black spruce crown with seven whorls. For the purposes of this study, whorl 1 was divided into its leader and a whorl 1 branch, (b) Defoliation was visually estimated for all age-classes of shoots along the first- and second-order branch axes (solid shoots and buds), c, c+1, c+2, c+3, and c+4 refer, respectively, to current-year, and one-, two-, three-, and four-year old shoots.

105 100 (a) • • T 75 • t • » -E • •• • • 0 a • • • '• 50 • ••» • • ° 0 •fi % +j • 0 U. •• * * 9i 25 a • „ 0 O C? O © •£ 0°

sh o 0' ^ 1 4» CL 5 100 r t•*i-

M a (b) .0 4> 75 • TJ

©^ ^ a 50 a • « •• • v • § \ * 25 jjfcdW 0 ^ft*2£&QeQX ^•OQ 0.0 0.2 0.4 0.6 0.8 1. Early-instar larvae per current-year shoot

Figure 4.2. Relationships between densities of early-instar P. alaskensis larvae and subsequent defoliation on current- (closed circles), one- (open circles), and two- (closed triangles) year-old foliage on branches in whorls 4 or 5 from trees in an experiment using naturally occurring densities of larvae already on branches (a), and in an experiment where larval density was manipulated on branches (b). Defoliation data were arcsine square-root transformed prior to analysis to meet model assumptions.

106 lOO.i

* 100

0s a

Figure 4.3. Mean (±1 SE) percent defoliation per shoot per branch attributable to feeding by P. alaskensis on current- (black bars) or one-year-old (gray bars) foliage on the leader or on whorl 1, 2, 4, or 7 branches in each of two stands (a,b) of joung black spruce. Defoliation data were arcsine square-root transformed prior to analysis to meet model assumptions.

107 early-instar larvae (2002) late-instar larvae (2002) late-instar larvae (2003) 100 (a) (b) (c) 75 50 I U X 25 0 L i All C 1 All C 1 3 All C 1 2 100 (d) (e) (f) 75 50 fi i X X x. X 25 0 * * fi _6lL All C 1 3 All C 1 3 All C 1 2

Foliage Age-class

Figure 4.4. Mean (±1 SE) percent survival (a-c) and percentage of survivors that were female (d-f) for P. alaskensis allowed to feed on all, current-, one-, two- (in 2003 only), or three-year-old (only in whorl 7 in 2003) foliage in either whorls 2 (white bars) or 7

(slashed bars). Larvae were placed on branches as early- (a,d) or late-instars (b,e) in

2002, but only late-instar larvae (c,f) were placed on branches in 2003. h (d) * denotes that there was no data available for the analysis due to low survival.

108 CHAPTER 5: GENERAL DISCUSSION

Results of the chapters presented in this thesis support the hypothesis that

Pikonema alaskensis forage adaptively to overcome variation in foliage quality within the crown of juvenile black spruce. Results in Chapter 2 support those from an independent study (Johns et ah, 2006a) that reported that most sawfly eggs are laid in the mid or lower crown. In addition, Chapter 2 showed that the relatively few eggs that were laid in upper whorls were mostly female. Both male and female late-instar larvae dispersed acropetally, although females were more than twice as likely to disperse.

Chapter 3 demonstrated that the female-biased oviposition-site selection and acropetal dispersal behaviors are adaptive responses to intra-tree variation in foliage quality, based on estimates of survival and sex ratio. Also, in contrast to past studies evaluating the adaptive value of acropetal dispersal for caterpillars (e.g, Carroll and Quiring, 1994;

Kessler and Baldwin, 2002), the adaptive value of this behavior for P. alaskensis was not associated with intra-crown variation in budburst. Chapter 4 demonstrated that larvae prefer and have the highest survival on current-year foliage in the upper crown of black spruce, but that when current-year foliage becomes scarce, may opt to feed on older age-classes in the upper crown, yielding the same survival as current-year foliage in the lower crown.

A myriad of studies have demonstrated the capacity of insects to utilize physiological mechanisms to overcome the chemical defenses of their host plants (e.g,

Brattsten, 1988; Dudt and Shure, 1994; Krause et ah, 1999; McKinnon et ah, 1999).

Similarly, behavioral adaptations, such as oviposition or larval feeding preferences, may enable herbivores to avoid unsuitable foliage (e.g., Hassell and Southwood, 1978;

109 Shultz, 1983), or obtain a balanced diet from a variety of modules of varying composition (e.g., Hagele and Rowell-Rahier, 1999; Moreau et ah, 2003; Held and

Potter, 2004). Pikonema alaskensis appears to have adapted to variability within the crown of black spruce by adopting sex-biased oviposition and larval foraging strategies.

Acropetal dispersal by P. alaskensis is a similar foraging strategy to that employed by some caterpillars to account for intra-plant variation in needle/leaf phenology exclusively (Quiring, 1993, Carroll and Quiring, 1994) or in combination with thermal environment and natural enemies (Kessler and Baldwin, 2002). For P. alaskensis, however, benefits derived from feeding as early instars in the lower crown, or as late instars in the upper crown, are unrelated to patterns of budburst phenology within the plant.

Intra-tree variation in the quality of foliage for P. alaskensis larvae maybe due to inherent source-sink competition among modules (Honkanen and Haukioja, 1994) and/or, to a lesser extent shading by overtopping branches (Henriksson, 2001; Fortin and

Mauffette, 2002). These variations could influence foraging in two possible ways.

Shading by overtopping branches could reduce the quality of foliage on branches in the lower crown (Henriksson, 2001), forcing individuals to disperse acropetally to obtain better quality (i.e., unshaded) foliage in the upper crown. This would explain why in

Chapter 4, unshaded one-year-old foliage in the upper crown yielded the same survival of larvae as shaded current-year foliage in the lower crown. However, the trees in this study were open grown and shading effects were probably not overly significant.

Alternatively, age-related changes in the nutritional needs or feeding habits of larvae, such as those associated with changes in larval size and morphology (Hochuli, 2001), may compel larvae to disperse within the crown to find more suitable food. Analysis of

110 foliage chemistry in upper and lower whorls throughout the season may aid in making further inferences as to the quality of foliage among whorls.

Chapter 2 of this dissertation is the first study to demonstrate preferential allocation of progeny sex through oviposition-site selection for a herbivorous insect within a host, and is only the second to demonstrate this strategy for any herbivorous insect (see Craig et ah, 1992). Presumably either female larvae benefit more from feeding in the upper crown, or juvenile males suffer greater reductions in performance when feeding in the upper crown. As in one previous study that investigated sex- allocation by an insect among plants in response to foliage quality (Craig et ah, 1992), there maybe similar selective pressure for P. alaskensis adult females to lay their female offspring, which are larger than males, in parts of the crown where their fitness is maximized, as young larvae (i.e., first to third instar) tend to feed on the branch where they are oviposited. Craig et ah (1992) showed that more female than male eggs of

Euura lasiolepis, a tenthredinid sawfly, were laid on fast growing willows that contained larger shoots, and that this behavior was adaptive because female size increased more rapidly than that of males with increasing plant growth (Craig et ah, 1989, 1992).

While acropetal dispersal has been shown in several previous studies (i.e.,

Quiring, 1993; Kessler and Balwin, 2002), sex-biased dispersal by the juvenile stages of a herbivorous insect has only been previously demonstrated in flightless lepidopterans, where female larvae disperse more often from defoliated host plants than males

(Rhainds et ah, 1998; Rhainds et ah, 2002). As the mobility of flightless adult lepidopterans is relatively limited, this foraging behavior was speculated to maximize fitness by enabling females to pupate on better (i.e., less defoliated) hosts for laying their progeny (Rhainds et ah, 1998; Rhainds et ah, 2002). In contrast, sex-biased acropetal

111 dispersal by late-instar P. alaskensis larvae appears to be an adaptive response to variability in foliage quality within plants, independent of previous defoliation (Chapter

2). Both survival and the percentage of survivors that were female was highest for larvae that dispersed acropetally (Chapter 3), strongly suggesting that females, which disperse acropetally more frequently than males (Chapter 2), benefit more than males from this behavior. Chapters 2 and 3 maybe the first studies to unambiguously demonstrate sex-biased oviposition-site selection by adults and sex-biased larval dispersal by juvenile insects foraging for food within their heterogeneous hosts.

The adaptive value of foraging behaviors has been the center of much debate (see

Endler, 1986; Pierce and Ollason, 1987; Sterns and Schmid-Hempel, 1987). The reductionist approach of only evaluating single parameters of fitness has been criticized in the past due to its limited value when analyzing field data (Roitberg et al., 1982;

Mangel and Clark, 1986; Cezilly et al., 1991). For insects such as P. alaskensis, the major function of the larval stage is to accumulate resources as fast as possible for subsequent adult activities, while avoiding potential mortality factors (Heinrich, 1979;

Shultz, 1983). Thus, survival, prepupal weight, and developmental time are all ideal correlates to evaluate fitness. I sacrificed evaluating prepupal weight in favor of obtaining sex ratio data. Survival and sex ratio were the only fitness correlates evaluated for P. alaskensis due, in part, to the challenge of measuring prepupal weight without destroying the cocoon and the difficulty of determining when larvae had spun their cocoon. Examining other fitness correlates, such as prepupal weight or development time, might have facilitated further inferences regarding the benefits of acropetal dispersal to P. alaskensis. For example, in years where we found no effects on larvae

112 that did not disperse acropetally, there may have been sub-lethal effects that were not detected.

Future studies might include evaluating the influence of previous defoliation on the foraging behavior of P. alaskensis. Several years of defoliation may reduce the quantity and quality of foliage in the upper crown making it less advantageous for larvae to feed there. This question was partially addressed in Chapter 4, where high levels of competition for the favored current-year foliage were found to influence the foliage-age preference. However, after several years of defoliation, current-year shoot quality may also decline, potentially forcing P. alaskensis to select alternative oviposition and feeding sites.

Significance of Studies to the Management of Pikonema alaskensis

Sampling insects in integrated pest management can be problematic in systems where the seasonal patterns of distribution and abundance of an insect pest varies unpredictably within the host (e.g., Straw et al., 2006). For P. alaskensis, acropetal dispersal may increase the difficulty of sampling because the life stage sampled (i.e., eggs or early-instar larvae in the mid-crown) does not occur in the same part of the plant where most damage occurs (i.e., the upper crown). I used our improved knowledge of the foraging behavior of this insect, described in Chapter 2, to establish important components of a monitoring program being developed for P. alaskensis in central

Newfoundland (i.e., Johns et al., 2006a, 2006b). Briefly, in Johns et al. (2006a) I established sampling units for P. alaskensis eggs and mid-instar larvae to precisely estimate the density of late-instar larvae in the upper crown of trees using only a portion of branches in whorls 2 and 4. I also established a simple and precise methodology for

113 evaluating defoliation within trees. These methods for estimating larval density and black spruce defoliation were used in Johns et al. (2006b) to establish robust relationships between P. alaskensis density and defoliation. Ultimately the results of these publications will be combined with relationships between defoliation and damage

(i.e., leader growth, shoot production, biomass, and top-kill) to help decision-makers determine if the application of suppression tactics is warranted in stands of black spruce subjected to defoliation by P. alaskensis.

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117 CURRICULUM VITAE

Candidate's full name: Robert Carson Johns

Education

University of New Brunswick (2000-2007) Doctor of Philosophy in Biology

St. Francis Xavier University (1995-1999) Bachelor of Science in Biology (Honors)

Teaching Experience

Lecturer, University of New Brunswick (2006) Forest Ecology: Communities, Populations, and Ecosystems

Lecturer/Demonstrator, University of New Brunswick (2004-2005) Biology Field Course, Insect Ecology and Sampling Procedures

Teaching Assistant, University of New Brunswick Introductory Zoology (2006) Introductory Biology (2001-2007) Introductory Botany (2003) Botany for Non-majors (2003-2004) Population Ecology (2001-2002)

Teaching Assistant, St. Francis Xavier University Introductory Botany (1999) Introductory Ecology (1998)

Refereed Publications

Johns, R.C., D.P. Ostaff, D.T. Quiring (2006) Relationships between yellowheaded spruce sawfly density and defoliation on juvenile black spruce. Forest Ecology and Management 22%: 51-60.

Johns, R.C., D.P. Ostaff, D.T. Quiring (2006) Sampling procedures for evaluating yellowheaded spruce sawfly density and defoliation in juvenile black spruce stands. Journal of the Acadian Entomological Society 2: 1-12.

Quiring, D., L. Flaherty, R.C. Johns, A. Morrison (2006) Variable effects of plant module size on abundance and performance of galling insects. Pg. 189-198 in Galling Arthropods and Their Associates: Ecology and Evolution, Springer, Sapporo, Japan. Selected Conference Presentations

Johns, R.C. (November 2006) Ecological processes shaping the foraging behaviors and associated performance of herbivorous insects within tree crowns. The Joint Meeting of the Entomological Society of Canada and the Societe d'entomologie du Quebec. Montreal, Quebec. Invited Symposium Speaker.

Johns, R.C. (November 2006) Intra-tree variation shapes the foliage-age preference and performance of a specialist herbivore larva. The Joint Meeting of the Entomological Society of Canada and the Societe d'entomologie du Quebec. Montreal, Quebec.

Johns, R.C., and D.T. Quiring (May 2006) Influence of intra-crown variation in foliage quality on foraging behavior and performance of caterpillars. The North American Forest Insect Work Conference. Asheville, North Carolina, USA. Invited Symposium Speaker.

Johns, R.C. (March 2006) Establishment of a sampling system and density-damage relationships for the yellowheaded spruce sawfly, Pikonema alaskensis (Roh.), in juvenile black spruce stands. Northeastern Forest Pest Council Meeting. Newport, Rhode Island, USA. Received "John Simeone "Award (1st place).

Johns, R.C. (February 2006) Intra-plant variation drives the sex-biased foraging behavior of a specialist herbivore. 14th Annual Graduate Student Association Conference. Fredericton, New Brunswick. Awarded Oral Presentation Prize (3rdplace).

Johns, R.C. (October 2004) Proximate and ultimate factors influencing the intra-plant foraging behavior of a sawfly. Joint Annual Meeting of the Entomological Society of Canada and the Acadian Entomological Society. Charlottetown, Prince Edward Island. Invited Symposium Speaker.

Johns, R.C. (November 2003) Proximate and ultimate factors influencing the intra-plant foraging behavior of a sawfly. Joint Annual Meeting of the Entomological Society of Canada and British Columbia. Kelowna, British Columbia.

Johns, R.C. (October 2002) Temporal and spatial variation in resource quality influences the foraging strategy of a sawfly larva. Entomological Society of Canada. Winnipeg, Manitoba.

Johns, R.C., D.P. Ostaff, D.T. Quiring (October 2001) Establishment of a sampling system and density-damage relationships for the yellowheaded spruce sawfly (Pikonema alaskensis Roh.) in central Newfoundland. Spray Efficacy and Research Group. Wheeling, West Virginia, U.S.A.

Johns, R.C., D.P. Ostaff, D.T. Quiring (October 2000) Density-damage relationship for the yellowheaded spruce sawfly {Pikonema alaskensis Roh.) in central Newfoundland. Joint Meeting of the Eastern Spruce Budworm Research Group Conference and the Spray Efficacy Research Group Workshop. Fredericton, New Brunswick.