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ABSTRACT 

Effects of elevated greenhouse gasses (GG: O3, CO2, CO2+O3, and control) on aspen 

leafminer oviposition were studied. Most eggs were found on leaves from the control and 

fewest on those from elevated O3 but treatment effect on oviposition was not significant. 

Epicuticular wax components differed among aspen clones. Elevated O3 increased 

deposition of wax and raised the amounts of alkyl esters. Numbers of leafminer eggs 

declined with increasing amounts of fatty acids and primary alcohols in the leaf wax. 

Leaves from the treatments used in an oviposition-preference experiment did not differ in 

wax composition and no preference for any treatment was detected. Epicuticular wax on 

leaves from elevated O3 (and somewhat less from the other GGs) was dense and 

amorphous, lacking the crystalline configuration and plate-like structures seen in the 

control. The leafminer preferred to lay eggs in larger leaves and those with crystalline 

wax with low amounts of alkanes.

There have been many people and organizations that provided me with valuable 

assistance and advice throughout the completion of my thesis. I am grateful to my cosupervisors 

Drs. Kevin Percy, Canadian Forest Service (CFS), Atlantic Forestry Center 

and Marek Krasowski, University of New Brunswick Faculty of Forestry and 

Environmental Management. I am also grateful for Professor Dan Quiring’s support as 

member of my advisory committee. I also appreciate the advice and support of Dr. 

William Mattson, United States Forest Service (USFS). I sincerely thank the CFS for my 

stipend and facilities for laboratory analysis and office space. 

I thank Professor Miloslav Nosal, University of Calgary Division of Statistics and 

Actuarial Science and Dr. Peter Ott, Government of British Columbia, Ministry of 

Forests for their careful and thorough statistical guidance. 

I 

cannot express more sincerely, my appreciation towards Mr. Jaak Sober and Ms. 

Wendy Jones at the Aspen-FACE site in Harshaw, Wisconsin. Gratitude is also extended 

to Ms. Joanne Lund and all the staff at the USFS Research Station in Rhinelander, 

Wisconsin who offered support and services. I would also like to thank Mr. Gerry Bance 

at the UNB Microscopy Unit, and Mr. Gary Henderson, Ms. Gretta Goodine, Mr. John 

Malcolm and Dr. Gaetan Moreau at the CFS. 

Thanks to Mr. R.C. Johns for his earlier reviews and feedback. Dr. Caroline 

Awmack provided motivating energy in the early days of my research therefore, I am 

grateful for the time and enthusiasm she offered. 

And, of course, my warmest acknowledgements towards my family and all my 

friends.

ABSTRACT........................................................................................................................ ii 

ACKNOWLEDGEMENTS..............................................................................................iii 

TABLE OF CONTENTS.................................................................................................. iv 

LIST OF TABLES..............................................................................................................v 

LIST OF FIGURES............................................................................................................vi 

1.0 INTRODUCTION........................................................................................................ 1 

2.0 METHODS AND MATERIALS............................................................................... 10 

2.1 Study Overview................................................................................................10 

2.1.1 Site location and experimental design...........................................10 

2.1.2 Fumigation.........................................................................................12 

2.2 FACE Ring Population Survey......................................................................13 

2.2.1 Leafminer Density............................................................................ 13 

2.2.2 Leaf Epicuticular Wax Production and 

Chemical Composition.....................................................................14 

2.2.3 Statistical Analysis...........................................................................16 

2.3 Leafminer Preference..................................................................................... 17 

2.3.1 Leafminer Egg Density....................................................................17 

2.3.2 Leaf Epicuticular W ax....................................................................19 

2.3.3 Epicuticular Wax Structure........................................................... 19 

2.3.4 Statistical Analysis...........................................................................21 

3.0 RESULTS...................................................................................................................... 23 

3.1 FACE Ring Population Survey......................................................................23 

3.1.1 Leafminer Density............................................................................23 


3.2 Leafminer Preference Study..........................................................................33 

3.2.1 Leafminer Egg Density....................................................................33 


3.2.3 Leaf Morphology.............................................................................. 38 

3.2.4 Dependence of Egg Quantity on Leaf Area and Wax................ 40 

4.0 DISCUSSION................................................................................................................42 

5.0 LITERATURE CITIED..............................................................................................53 

APPENDIX A ...................................................................................................................... 60 

CURRICULUM VITAE

1. Summary of number of cages that contained eggs and number of wax samples 

collected from leaves with and without eggs in preference tests replicated four 

times........................................................................................................................19 

2. Results of analysis of variance on effects on egg density in the population 

survey. The REML method of the Mixed Procedure of SAS was used to 

test effects of the fixed factor. The random statement identified random 

factors of the design (Blk; Blk*Treatment) enabling the program to determine 

appropriate error terms for testing the effects of the fixed factor. Covariance 

parameter estimates are also included, showing percent of variance 

(bracketed) attributed to each random factor........................................................24 

3. Results of the analysis of variance on leaf epicuticular wax and its constituents 

in leaves of clone 271,216,259 used in the population survey........................ 26 

4. Results of analysis of variance on the proportions of eggs on leaves of aspen 

from the four greenhouse gas treatments in the preference study. The REML 

method of the Mixed procedure of SAS was used to test effects of fixed 

factors. All random factors were entered into the Random Statement of the 

Mixed Procedure enabling the program to use appropriate error terms for 

testing the effects of the fixed factor (treatment). Covariance parameter 

estimates for random factors are included, indicating percent of random 

variation explained by each.................................................................................. 34 

5. Results of the analysis of variance on leaf epicuticular wax and its constituents 

in leaves of clone 42E used in the leafminer preference experiment (leaves 

from test 3 only)..................................................................................................... 36 

6 . Best Subsets Regression: Egg quantity versus leaf surface area, total wax 

amount, alkanes, fatty acids, alcohols and alkyl esters....................................... 41

1. Temporal distribution of the ontogenic stages of Phyllonorycter apparella. 

Adapted from Martin (1956)..................................................................................... 6 

2. The Aspen - FACE site in northern Wisconsin, USA. Block (1,2, 3) and rings 

(1 = control; 2 = CO2; 3 = O3; 4 = CO2+O3) distribution....................................... 10 

3. A typical allocation of tree species in an experimental ring at the Aspen - 

FACE site.................................................................................................................11 

4. Typical cage set-up, which included three leaves per treatment and four 

treatments per cage...................................................................................................18 

5. Effect of treatment with elevated greenhouse gases on leaftniner oviposition 

preference (Clone 42E) from population survey. Bars marked with the same 

letter represent means not significantly different from each other at a = 0.05. 

Capped lines represent halves of standard errors...................................................24 

6 . Effect of greenhouse gas treatments on leaf epicuticular wax amount averaged 

across clones (259, 216, 271) in leaves from the population survey. Bars of 

the same color marked by the same letters are not significantly different from 

each other at a = 0.05. Capped lines represent halves of standard errors...........27 

7. Effect of greenhouse gas treatments on leaf epicuticular wax composition 

averaged across clones (259, 216, 271) in leaves from the population survey. 

Bars of the same color marked by the same letters are not significantly 

different from each other at a = 0.05. Capped lines represent halves of 

standard errors.......................................................................................................... 27 

8 . Effect of clone on total epicuticular wax amount averaged across treatments (4) 

in leaves from the population survey. Bars marked with the same letter 

represent means not significantly different from each other at a = 0.05. 


9. Effect of clone on leaf epicuticular wax alkyl ester amount averaged across 

treatments (4) in leaves from the population survey. Bars marked with the 

same letter represent means not significantly different from each other at a = 

0.05. Capped lines represent halves of standard errors........................................ 29 

10. Effect of clone on leaf epicuticular wax alkane amount averaged across 



0.05. Capped lines represent halves of standard errors......................................... 29

11. Effect of clone on leaf epicuticular wax fatty acid amount averaged across 



0.05. Capped lines represent halves of standard errors......................................... 30 

12. Effect of clone on leaf epicuticular wax primary alcohol amount averaged 

across treatments (4) in leaves from the population survey. Bars marked with 

the same letter represent means not significantly different from each other at a 

= 0.05. Capped lines represent halves of standard errors..................................... 30 

13. Comparison of epicuticular wax amount on unmined and mined leaves 

averaged across treatments (4) and clones (3) in leaves from the population 

survey. Bars marked with the same letter represent means not significantly 

different from each other at p=0.05. Capped lines represent halves of standard 

errors........................................................................................................................31 

14. Mean amounts of wax constituents (alkyl esters, alkanes, fatty acids and 

primary alcohols) on mined and unmined leaves from the population study 

averaged across all clones (259,216,271) and treatments. Letters pertain 

separately to each pair of bars representing the same compound. Pairs of bars 

marked with the same letter represent means not significantly different from 

each other at a = 0.05. Capped lines represent halves of standard errors............32 

15. Effect of greenhouse gas treatments on leafminer oviposition preference 

(Clone 42E was used). Bars marked with the same letter represent means not 

significantly different from each other at a = 0.05. Capped lines represent 

halves of standard errors..........................................................................................34 

16. Effect of greenhouse gas treatments on leafminer oviposition preference 

(Clone 42E was used) in preference Test 4. Bars marked with the same letter 



17. Effect of greenhouse gas treatments on epicuticular wax composition of 

leaves from clone 42E used in the leafminer preference experiment. Bars of 

the same color marked by the same letters are not significantly different from 

each other at a = 0.05. Capped lines represent halves of standard errors...........37 

18. Mean amounts of wax constituents (alkyl esters, alkanes, fatty acids and 

primary alcohols) on leaves (clone 42E used) with and without eggs from the 

preference study. Letters pertain separately to each pair of bars representing 

the same compound. Pairs of bars marked with the same letter represent 

means not significantly different from each other at a = 0.05. Capped lines 

represent halves of standard errors.........................................................................37

19. Impacts of elevated CO2 and O3 on abaxial leaf epicuticular wax structure 

shown in scanning electron micrographs from aspen clone 42E grown under 

a) control, b) elevated CO2, c) elevated O3 and d) elevated CO2 + O3. Arrows 

indicate wax features. “S” indicates location of stomata.................................... 40

1.0 - Introduction 

Since the mid 18 century, atmospheric concentrations of greenhouse gases 

(GHG’s) such as CO2 and O3 have increased by 30-35% (IPCC, 2001). Exposure to 

elevated O3 is detrimental to forest health (Percy et al., 2003). The increasing release of 

CO2 and O3 into the atmosphere affects both climate and the structure and functioning of 

ecological communities on local and/or global scales (IPCC, 2001). Many experiments 

evaluating the ecological impacts of climate change on forest ecosystems have focused 

on its effects on plant growth (eg. Chappelka and Samuelson, 1998; Isebrands et al., 

2001; Kamosky et al., 2003) and the preference and performance of individual 

phytophagous insects (eg. Awmack et al., 1996; Bezemer and Jones, 1998; Coviella and 

Trumble, 1999; Heagle et al., 1994; Lindroth et al., 1993; Percy et al., 2002; Watt et 

al.,1995; Kopper and Lindroth, 2003). Of relevance to this study, it has been reported 

that elevated CO2 reduces foliar nitrogen concentration and increases synthesis of carbonbased 

defensive metabolites (Wilsey, 1996). 

In trembling aspen (Populus tremuloides Michx.), CO2 increases height, diameter 

and volume growth, (Isebrands et al., 2001). As well, there are increases in root biomass, 

phenolic glycosides and in the carbon-nitrogen ratio (Lindroth et al., 2001; King et al., 

2001; Kopper and Lindroth, 2003). Elevated CO2 also increases foliar consumption by 

herbivorous insects, resulting in reduced performance and high mortality rates (Hughes 

and Bazzaz, 1997; Stiling et al., 1999). Some herbivore species, particularly aphids, show 

increased fecundity at elevated CO2 (Awmack et al., 1997; Goverde et al., 2002; and 

Stacey and Fellowes, 2002), although these benefits to insects may be offset by increases 

in vulnerability to natural enemies due to a decrease in aphid response to alarm 

pheromones (Percy et al., 2002; Stiling et al., 1999).

In general, increased concentrations of O3 have been associated with high 

frequency of insect attack (Dahlsten et al., 1997), increases in enzyme activity associated 

with general plant defence mechanisms (Kangasjarvi et al., 1994) and potentially with an 

increase in insect fecundity (Percy et al., 2002). Specifically, elevated levels of O3 have 

been shown to increase pupal weight of forest tent caterpillars (Percy et al., 2002). 

Our knowledge about the combined effects of CO2 and O3 on herbivore 

performance is limited (Lindroth et al., 1993; Bezmer and Jones, 1998; Isebrands et al., 

2001; Percy et al., 2002), yet we need to investigate combined effects of the two 

pollutants interacting with living organisms before we can reliably predict their future 

impacts on terrestrial ecosystems (Heagle et al., 1994; Coviella and Trumble, 1999; Watt 

et al., 1995; Kopper and Lindroth, 2003). Only one study (Kopper and Lindroth, 2003) 

has examined the combined effects of CO2 and O3 on herbivore oviposition preference 

and performance, investigating the colonization rates and survivorship of the herbivore. 

Although studies have shown individual effects of greenhouse gases, CO2 and O3, on 

plant tissue composition and plant-herbivore relationships (preference), no study has 

directly investigated the interactions of both gases on leaf surface quality and their 

resultant changes in behavioral and reproductive strategies of herbivorous insects. 

Insect preference and performance are determined by both plant quality and the 

insect’s vulnerability to natural enemies. Plant nutritional status and defensive 

mechanisms both affect the preference and performance of individual herbivores 

(Awmack and Leather, 2002a). They may be affected by changes in their nutrition, their 

defences, and susceptibility to their natural enemies. These factors may all be affected by 

both the biotic and abiotic environment (Awmack and Leather, 2002a,b). Although

changes in plant tissue quality caused by elevated CO2 and O3 have been investigated 

(Lindroth et al., 2001; Kangasjarvi et al., 1994; Kamosky et al., 2002), less is known 

about how these changes affect host preference by herbivorous insects (Awmack et al., 

1996). 

Leaf epicuticular waxes influence herbivory directly or indirectly through various 

cues including chemical, morphological and visual stimuli (Bemays and Chapman, 1994; 

Dutton et al., 2000; Eigenbrode and Espelie, 1995). However, these influences are only 

partly understood (Eigenbrode and Espelie, 1995). Leaf epicuticular waxes are located 

on the outermost layer of the plant cuticle and comprised of long-chain, saturated 

aliphatic molecules (Baker, 1982). The most common types of aliphatic compounds are 

alkanes, alkyl esters, primary alcohols, fatty acids and aldehydes (Barthlott et al., 1998). 

Chemical composition of epicuticular waxes determines their morphology (Baker, 1982; 

Barthlott et al., 1998; Holloway, 1982; Jeffree, 1994). Many plant species can be 

identified based on wax morphology and chemistry. Most epicuticular wax studies have 

involved plants such as wheat, cabbage and other crop species (Baker, 1974; Baker and 

Jeffree, 1981; Eigenbrode and Espelie, 1995). Some studies have investigated the 

characteristics of epicuticular waxes of trees such as Eucalyptus sp. (Neinhuis et al., 

2001) and Prunus sp. (Jetter et al., 2000; Jetter and Schaffer, 2001) and Populus sp. 

(Kamosky et al., 1999; Percy et al., 2002). 

The main function of epicuticular waxes is providing protection against insects, 

diseases, and pollutants. They also act as barriers against excessive water loss 

(Holloway, 1994). Chemical wax components are biosynthesized by the plant epidermal 

cells (Kolattukudy, 1996). Chemical components of epicuticular waxes move within the

cuticular water current to the site of their disposition in a process similar to steam 

distillation (Neinhuis et al., 2001). Leaf epicuticular wax morphology and chemistry 

vary with changing environmental conditions (Baker, 1982). Air pollutants, such as CO2 

and/or O3, alter epicuticular wax composition through direct modification of wax 

biosynthesis (Kamosky et al., 1999; Kamosky et al., 2002; Kamosky et al., 2003; Percy 

et al., 1994; Percy et al., 2002). Epidermal cells are sensitive to environmental factors; 

therefore, the biosynthetic process of wax formation also responds to environmental 

factors (Kolattukudy, 1996) such as changes in concentrations of CO2 and O3. Kamosky 

et al. (2 0 0 2 ) determined an increase of alkane homologue ratio, increased wax amount, 

and a change in wax structure from a crystallite to an amorphous form as a result of 

exposure of trembling aspen to elevated O3 and CO2. These alterations to leaf-surface 

chemistry and morphology may affect the preference and performance of phytophagous 

insects (Eigenbrode and Espelie, 1995) and those of their enemies. The chemical cues 

used by the parasitoid to locate its concealed host after landing on an infested area are 

plant-derived, as in other tri-trophic systems and not insect-derived (Dutton et al., 2000). 

Relevant to this study, Phyllonorycter spp. has been shown to rely on plant-derived 

semiochemicals (chemical signals) for host location and ovipositional preference (Dutton 

et al., 2 0 0 0 ). 

Herbivore population dynamics may be affected by food quality and quantity, by their 

natural enemies (Price, 1992), and by changes in the abiotic environment. Bottom-up and 

top-down population regulation may both be affected by changes in atmospheric 

composition (Awmack and Leather, 2002b). Food webs may be directly impacted via 

changes in the relative strength of this dynamic, determined by changes in specific

mechanisms in response to abiotic factors (eg. greenhouse gases) (Price, 1992). The 

relationship between oviposition preference and the subsequent performance of offspring 

is one of the most important aspects in the evolution of associations between herbivores 

and host plants (Thompson, 1988). Some studies have shown a positive relationship 

between preferred oviposition and larval performance (McClure et al., 1998). However, 

others have provided evidence that some insects show no relationship between 

oviposition preference and offspring fitness (Kopper and Lindroth, 2003). As elevated 

levels of greenhouse gases affect plant quality, herbivore oviposition preferences may 

change and natural selection should favor those females that lay on hosts with best 

nutritional status (Quiring and McNeil, 1987). This might be expected, providing they 

obtain the correct cues and are not choosing oviposition sites to avoid natural-enemy 

attack. Plant nutritional quality is not the only factor determining a female insect’s choice 

of oviposition sites since higher-trophic level interactions must also be considered. Plant 

quality may affect tri- trophic relationships either via prey/host food source modification 

or by the provision of refiigia allowing prey to avoid predators and parasitoids (Awmack 

and Leather, 2002a). In other words, herbivores may select poorer quality hosts, in order 

to decrease risk of attack by natural enemies. 

This study is a part of the world’s largest international FACE (Free Air Carbon 

Dioxide Enrichment) project examining the effects of increased levels of carbon dioxide 

and ozone on trembling aspen ecosystems (http://aspenface.rntu.edu). Independent and 

interacting effects of elevated CO2 and O3 on the preference and performance of aspen 

blotch leafminer (Phyllonorycter apparella Herrich-Schaffer (Gracillariidae)) will be 

examined.

Martin (1956) was the first to describe the life history of Phyllonorycter apparella. 

The distribution of ontogeny stages is shown in Fig. 1. Phyllonorycter apparella is a 

univoltine leaf-mining moth that has a wide native distribution in North America (Martin, 

1956). The adult moth prefers to oviposit on trembling aspen as oviposition sites, but 

also will lay eggs on Populus grandidentata Michx. and Populus balsamifera L. 

(Auerbach, 1991). The overwintering adult oviposits on the margins of the abaxial 

(underside) surface of leaves in late May until mid-June. Adult moth wings are small and 

slender with bronzy-brown forewings, which range from 3.6 to 4.8mm in length (Davis 

and Deschka, 2001). There are five larval instars, the timing of which is shown in Figure 

1 . 

Stage April May June July August September 

Egg 

1st Instar 

2nd Instar 

— 

3rd Instar 

4th Instar 

5th Instar 

Pupa 

Adult 

(overwintering) 

Figure 1. Temporal distribution of onto genic stages of Phyllonorycter apparella. 

Adapted from Martin (1956). 

The following description of the ontogenic stages of the aspen blotch leafminer is 

based on Martin (1956). The first instar larva feed on the spongy mesophyll by moving 

its head from side to side, in a fan shape, advancing towards the mid-vein of the leaf. 

The mine width is approximately 3mm after the initial instar. In the second instar, the 

larva continues to feed in the same way as the first (Martin, 1956). The larva begins to 

form the mine in a circular shape and the mine reaches a diameter of approximately 6 mm. 

The third instar larva continues to feed on the mesophyll forming a more oblong mine.

Larva frass, which is black and round, is quite apparent around the edges of the mine. In 

the fourth instar, the larva undergoes the most obvious changes. The body becomes 

cylindrical, opaque and yellowish, while the mouthparts are extending ventrally, allowing 

the larva to feed into the upper palisade mesophyll layer. During the latter stages of this 

instar, the larva begins to spin silk webbing in the mine, which attaches the upper mine 

surface with the lower surface. 

By the fifth instar, the mine is completely woven with silk. Some feeding continues 

with the larva avoiding the vascular bundles. Unique identifying feature of this stage is 

the appearance of the body, which is dark yellowish-brown, and of the frass, which has 

been piled neatly and attached to the upper epidermal wall. At this stage, the mine has 

attained an oval shape and reaches its maximum size, averaging 17 mm long and 1 2 mm 

wide. 

During late-July and early August, the larva pupates within the mine. The fifth and 

sixth abdominal segments remain exposed, which permits the pupa a great deal of 

movement within the mine. Immediately before adult emergence in mid-August, the 

pupa pierces a hole through the lower epidermis and the pupal case remains in this 

protruding position for a few weeks. 

Only a small percent of individuals reach the adult stage of development due to 

predators and parasites (Martin, 1956). From the period of emergence until 

overwintering, male and female moths mate. Overwintering sites are located in the 

cervices of bark of coniferous trees such as Pinus strobus L., Pinus resinosa Ait., and 

Pinus bankisana Lamb.

At high density, leafminers can cause premature browning of the foliage (Martin, 

1956). Information regarding economic impacts at times of severe infestations is not 

available. Since trembling aspen is the most important deciduous species in the North 

American boreal forest (Hogg, 2001), any reduction to its growth and yield may have a 

significant economic impact. The leafminer was chosen as a model species for this study 

for four reasons: 1) its presence on the research site at endemic population levels; 2 ) 

ability to identify this species based on mine size. Mine size has been shown to vary in 

other Phyllonorycter spp. under CO2 treatments (Stiling et al., 1999); 3) observed 

differences in oviposition behavior on different aspen clones reared at elevated CO2 

and/or O3 (Kopper and Lindroth, 2003); and 4) leafminers are ideal experimental subjects 

because the larvae spend their entire lives inside mines that they form within leaves 

(Hesman, 2000); therefore, one can accurately determine population behavior. There is 

evidence of larval presence whether the herbivore is living or dead (from parasitoid) or 

has emerged (leaving behind frass and the pupal case) (Awmack, unpublished data, 

2001). 

Natural enemies of P. apparella include parasitoids from the family Braconidae (eg. 

Apanteles sp. and Pholetesor salicifliella), Eulophidae (eg. Chrysocharis sp., Cirrospilus 

sp., Pediobius sp., Pnigalio sp., Sympiesis sp.), Ichneumonidae (eg. Alophosterum 

foliicola, Diadegma sp., Scambus decorus) and Pteromalidae (Davis and Deschka, 2001). 

The main objective of this study was to link the changes in aspen leaf epicuticular 

wax chemical composition and morphology, due to exposure to elevated levels of CO2 

and/or O3, with aspen blotch leafminer oviposition site selection. This was the first study

to use population and bioassay studies to examine this link for aspen blotch leafminer. 

This objective was addressed by developing the following hypotheses: 

1 - If ovipositing leafininers prefer leaves with higher amounts of alkanes and 

fatty acids in epicuticular wax (Eigenbrode and Espelie, 1995), and these amounts are 

increased by the individual and combined effects of elevated CO2 and O3 levels 

(Kamosky et al., 2002; Percy et al., 2002), then colonization should be highest on leaves 

exposed to elevated CO2 and O3. Furthermore, on clones more sensitive to O3, alkane 

and fatty-acid amounts should increase more than in less sensitive clones (Kamosky et 

al., 1999). 

2 - If ovipositing leafininers prefer leaves with amorphous epicuticular wax surface 

structure (Eigenbrode and Espelie, 1995), and structure type changes from crystallite to 

amorphous by the individual and combined effects of elevated CO2 and O3 levels 

(Kamosky et al., 2002), then colonization should be highest on leaves exposed to 

elevated CO2 and O3 treatments. 

Based on these two hypotheses, it is predicted that colonization will be higher on 

clones more sensitive to O3. Furthermore, a critical component of defining the linkage 

between leaf surface properties and aspen blotch leafminer oviposition is to verify that 

leaf surface chemistry and morphology of aspen change in response to additive and/or 

interacting effect of CO2 and O3, as shown in previous studies (Kamosky et al., 2002; 

Percy et al., 2002).

2.1 Study Overview 

2.1.1 Site location and experimental design 

The Aspen-FACE (Free Air Carbon Enrichment) site (Fig. 2) is located near 

Rhinelander in northern Wisconsin, USA (45 .06 N; 89 .071W), on the Harshaw 

Experimental Farm (32ha) of the USDA Forest Service. The experiment established in 

1997 on the site comprises three randomized blocks each containing four 30-m diameter 

FACE rings, each randomly assigned to one of four treatments (control, elevated CO2, 

elevated O3 and elevated CO2+ O3). Rings are spaced at least 100 m apart to exclude 

ring-to-ring air drift. 

Figure 2. The Aspen - FACE site in northern Wisconsin, USA. Block (1, 2, 3) 

and rings (1 = control; 2 = C 02; 3 = 0 3; 4 = C 0 2 + 0 3) distribution.

Spaced at 1 m x 1 m, 760 trees were planted in each ring. Half of each ring 

contains trembling aspen (Populus tremuloides Michx.; 5 genotypes). The other half 

consists of two quarters: one quarter has white birch (Betula papyrifera Marsh.) mixed 

with trembling aspen and the other has sugar maple (Acer saccharum Marsh.) with 

trembling aspen (Fig. 3). Samples for this study were taken exclusively from the pure 

aspen communities. 

Figure 3. A typical allocation of tree species in an experimental ring at the 

Aspen - FACE site.

2.1.2 Fumigation 

In 2003, ambient levels in the control rings for CO2 and O3 were 362 ppm and 

38.4 ppb, respectively. In 2004, they were 373 ppm and 34.9 ppb, respectively. Elevated 

levels of CO2 and O3 were based on concentrations predicted for the year 2050 in the 

Great Lakes region (Dickson et al., 2000). Trees inside the CO2 rings were fumigated 

from dawn until twilight (i.e. when the sun elevation angle exceeded 6 ° from the 

horizon). 

During 2003, elevated CO2 levels were 537 ppm (averaged across three rings) 

from May to October for 145 days. During 2004, elevated CO2 levels were 512 ppm 

(averaged across three rings) from May to October for 150 days. 

During 2003, the O3 elevated hourly average levels were 51.4 ppb, that is, 1.34 X 

ambient (averaged across three rings). During 2004, the O3 elevated hourly average 

levels were 41.1 ppb, that is, 1.18 X ambient (averaged across three rings). Elevated 

levels of O3 were aimed at 1.34 -1.4 times that of ambient levels. The intended 

concentrations were based on a daily-alternating exposure that followed a diurnal 

ambient profile based on actual O3 data (Kamosky et al., 1999). Depending on 

meteorological conditions and forecasts, during both years, O3 maximum hourly average 

concentrations ranged from 46 ppb (on cool, cloudy days) to 85 ppb (on hot, sunny days). 

Trees fumigated with CO2 + O3 were exposed to a combination of the elevated levels.

2.2 FACE Ring Population Survey 

2.2.1 Leafminer Density 

Aspen clone 216 was intended for use in the population survey of leafminer 

density and leafminer preference study in order to maintain consistency with the wax 

analysis in the population survey. However, in the spring of 2004, the FACE site 

suffered from an outbreak of the pathogen, Venturia sp. Aspen clone 216 was particularly 

susceptible to this fungus. Although it does not induce tree mortality, it significantly 

affected crown form and leaf surface quality. Hence, aspen clone 42E (moderate 

sensitivity to O3) was used in leafminer density assessment because it was relatively 

unaffected by the pathogen. Leaves affected by the fungus were not selected. Fiftycentimeter 

branch segments were sampled from the middle of the upper tree canopy 

(approx. 4-5 m above ground). Branch segments were excised from full-sun laterals 

using a pole pruner. In order to reach live-leaf crowns in the CO2 rings, which began at 

approximately three meters height, a ladder was used. One segment was sampled from 

each of five haphazardly selected trees in each of the twelve FACE rings. On average, 

there were 73 leaves per branch segment. In total, 4400 leaves were examined for 

presence of eggs or early mines. 

The total number of leaves per segment was tallied. The proportion of the 

leafminer was expressed as a number of eggs or mines per leaf. Parasitized eggs (i.e. 

collapsed-dome shaped and no mine present) and parasitized mines (i.e. light brownish 

and/or with a tear/hole) were included in the count because they included a chosen 

oviposition site. These criteria were used because the survey was carried out during the

second week of June 2004 when moth oviposition was already ending and egg hatch and 

mine initiation began. 

2.2.2 Leaf Epicuticular Wax Production and Chemical Composition 

Leaves for wax analysis were collected in spring 2003, before the Venturia sp. 

outbreak. Aspen clones 259,216 and 271 (in decreasing sensitivity to O3) were used in 

epicuticular wax analysis. Leaves were selected based on the leaf plastachron index (LPI) 

system. The LPI is a method used to identify the physiological age of a leaf on a plant 

shoot (Percy, pers. com.). Leaves from LPI 6 - 8 located on full-sun, first order laterals 

were removed. Trees were selected based on average height and diameter in a ring, 

within 5m of the ring center. There were 215 samples collected; three leaves per clone 

(one tree), three clones per ring, twelve rings, and two levels of leafminer damage (mined 

or unmined). Leaf epicuticular wax was collected on the site by rinsing the adaxial and 

abaxial leaf surfaces with 3ml of CHCL3 using a glass syringe. Mined leaves were cut 

with a razor blade into mined and unmined portions. Wax was collected only from the 

unmined portion. Leaf surface area was measured (± 1cm2) at the USDA Forest Service 

lab in Rhinelander, Wisconsin using a leaf surface area meter (Type LI-3050A/2, Lambda 

Instrument Corporation, Lincoln, NB, USA). 

The solvent/wax solution was filtered, solvent evaporated, and wax weighed to 

the nearest ±10 ng and expressed as p,g cm'2 leaf area. Wax samples were analyzed using 

a Varian 3410 (Varian Incorporated, Walnut Creek, CA, USA) gas chromatograph (GC). 

Retention times were determined on a DB1-HT capillary column (15 m; 0.32 mm inner 

diameter [i.d.]) with methyl silicone liquid phase (0.1 |xm film thickness). Carrier gas 

(He) flow was 7.5 mL/min. Column programming was 70°C to 120°C at 20°C m in 1;

121°C to 390°C at 6 °C min'1. The septum programmable injector was programmed at 

75°C to 125°C at 18°C min'1; 126-395°C at 12°C min'1; hold 15min. The flame 

ionization detector (FID) was operated at 400°C. Wax samples were silylated with n- 

trimethylsilylacetimide (TMSI) at 35°C for 30 min. The homologue assignments and 

peak integration were determined with Varían version 6.0 Workstar (Varían Incorporated, 

Walnut Creek, CA, USA) software and injection of pure reference homologues and 

reference wax mixtures. 

A Hewlett-Packard 5989 GC-MS (Hewlett Packard - Palo Alto, CA, USA mass 

spectrometer was used in final confirmation of homologue assignments. A DBH1-HT 

column (15m; 0.32mm i.d.; 0.1 jim film thickness) was temperature programmed from 

70°C to 345 at 12°C m in _1 (hold 30 min). Helium carrier gas flow rate was 1.0 mL'1. 

Injection was on-column at 250°C. The electron impact (El) mass spectra were searched 

against Wiley, NIST and CFS pure homologue libraries. Ion source and quadrupole 

temperatures were 275°C and 100°C, respectively. 

The number of counts, associated with each homologue was combined into 

classes (alkanes, fatty acids, primary alcohols, and alkyl esters; 98% of resolved peaks). 

Homologue class value was expressed as a percentage of the total amount of wax per unit 

leaf surface area (ng/cm2). The counts for each sample were then normalized to account 

for the FID efficiency response by multiplying all values by a previously determined ratio 

for each homologue class.

2.2.3 Statistical Analysis 

The analyses of variance (ANOVA) were done using the REML method of the 

Mixed Procedure (PROC MIX) of SAS© (Version 8.2 for Windows - The SAS Institute 

Inc., Cary, NC, USA). The ANOVA was performed according to the completely 

randomized block design. In all analyses of variance in the thesis, differences between 

means were compared using the Least Square Means option. 

The analysis of leafminer egg density was performed on the average number of 

eggs per sample. In the analysis, the random statement was used (here and in other 

analyses made in Mixed Procedure of SAS) listing the random factors (i.e. block and 

block*treatment) enabling SAS to use correct error terms for testing the fixed variable(s) 

(here, the treatment). Analysis of leaf wax was performed on the total amount of wax and 

on each of its constituents; alkyl esters, alkanes, fatty acids, and primary alcohols. The 

elements of experimental design included three blocks; four treatments (CO2, O3, CO2 + 

O3 and control); three clones (259,216,271); and the presence of damage from leafminer 

(mined or unmined). 

Residuals in the analyses of variance were tested for normality of distribution 

using the Univariate Procedure of SAS. Where the data were found not to be normally 

distributed, the Box-Cox option of SAS’ Transreg Procedure was used to determine the 

most appropriate transformation of the data for normalization of their distribution. Then, 

the Box-Cox transformation was applied to the data. A transformation was required for 

the leafminer egg density analysis but not for the wax data. All data shown in the thesis 

are untransformed, even when transformation was used for their analyses. This approach

to testing and transformation of continuous data requiring normalization of distribution is 

consistently applied throughout this thesis. 

2.3 Leafminer Preference 

2.3.1 Leafminer Egg Density 

Female moths were collected from the surface of trunks of mature softwood trees (i.e. 

white pine, red pine, Abies balsamea (L.) Mill., Picea glauca (Moench) Voss and Picea 

mariana (P. Mill.) B.S.P.). Moths were removed from trunks using an aspirator and 

placed into 30 ml clear plastic vials. Moths were refrigerated for no longer than 48 hours 

at 2°C before the beginning of the experiment. After refrigeration, one female moth was 

added to each vial. 

Leaves were collected in 2004 from aspen clone 42E, from all FACE rings. 

Leaves from L P I6 - 8 on full-sun, main laterals were removed with attention not to 

damage the petiole. To ensure maximum leaf turgidity, petioles of excised leaves were 

placed in water sprigs. Three leaves on one shoot of each ring were placed in each water 

sprig. Leaves were transported to the United States Forest Service (USFS) greenhouse 

located near the site, in Rhinelander, Wisconsin. Mean temperature within the 

greenhouse was 25.4°C daytime and 19.3°C night time. Mean relative humidity was 

40.8% daytime and 60.9% night time. One sprig per treatment (i.e. 3 leaves/treatment) 

was inserted into a stryofoam base and covered in a 0.5 mm white mesh cage held in 

place by a metal cage (Fig. 4).

Figure 4. Typical cage set-up, which included three leaves per treatment and four 

treatments per cage. 

The cage was a conical structure, approximately 50 cm in height. Moths were removed 

from cool storage to adjust to greenhouse temperature. One live female was added to 

each cage. After 24 hours, eggs laid on each leaf were counted. Egg position on the leaf 

and the status of the female moth (i.e. dead or alive) was also recorded because some 

moths were found dead. 

This experiment was carried out at four different times over a 20-day period. 

Cages without eggs were not used in preference analysis. Where the female moth was 

found dead, it was assumed it died before it could oviposit. When no eggs were found, 

but the female was alive, it was assumed she did not have any eggs to lay. The numbers 

of cages from which data were gathered are given in Table 1.

Table 1. Summary of the number of cages that contained eggs and number of 

wax samples collected from leaves with and without eggs in preference 

tests replicated four times. 

Replicate Number of Cages Wax Samples 

With Eggs Without Eggs With Eggs Without Eggs 

1 48 45 0 0 

2 25 11 0 0 

3 2 0 16 39 1 0 1 

4 24 1 2 37 6 6 

2.3.2 Leaf Epicuticular Wax 

Epicuticular wax was removed from leaves from replicates three and four (Table 

1). Leaf wax was collected from abaxial sides of leaves with or without eggs (Table 1) 

using 3 mL of CHCL3 dispensed with a syringe. The wax samples were labeled for 

identification (including the treatment from which the leaves were originally collected, 

the cage, and the replicate number). The solvent/wax solution was filtered, solvent 

evaporated, and wax weighed to the nearest ± 1 0 |ig and expressed as ^g cm' 2 leaf area. 

Wax was analyzed using GC-MS from replicate three only. Time constraints did 

not permit wax samples from replicate four to be analyzed. Thirty-two and thirty-six 

samples were analyzed from leaves with eggs and without eggs respectively. The 

procedure for epicuticular wax analysis was that used in the population survey described 

in section 2 .2 .2 . 

2.3.3 Epicuticular Wax Structure 

Leaves for scanning electron microscopy (SEM) analysis were collected from 

FACE and stored in coolers to maintain leaf turgidity. In order to maintain consistency 

with the preference experiment, leaves were removed from full-sun, main lateral

anches at L P I6 -8 . Leaves from the same LPI were presumed to be at the same stage of 

wax development, based on Baker (1982). One day after leaf excision, leaves were 

viewed and photographed under cryo-scanning electron microscopy. Only turgid leaves 

were viewed. Surfaces of twelve leaves were examined (i.e. one leaf/treatment/block). 

The University of Minnesota Hitachi S-3500N SEM with Quartz PCI digital 

imaging, photo CRT recording camera, environmental secondary electron detector 

(ESED) and Windows NT operating system was used. Optimum SEM operating 

conditions were: 5kV-accelerating voltage and 12mm detector working distance. The 

SEM was adapted cryogenically with the Emitech K-l 150 Cryogenic System, including a 

cryo-preparation unit, airlock interface to SEM and sputter coater. The liquid nitrogen 

reservoir for the cryo-SEM system was frequently replenished during specimen 

preparation and viewing to maintain low temperatures in the devices. 

Square sections approximately 1cm2 were removed from the mid-section of the 

leaves using a scalpel and razor blade. Care was taken not to abrade or damage the 

epicuticular wax structure by specimen handling. Each specimen was mounted onto a 

bronze stub with the abaxial surface exposed for viewing. Non-conductive, self-sticking 

adhesive tabs were used to mount the section to the stub. Conductive carbon paint was 

then applied to the edges of the leaf section. The stub was then placed on the transfer rod 

and slowly plunged into liquid nitrogen slush under vacuum. Upon freezing, the 

specimen stub was transferred under vacuum to the cryo-SEM preparation chamber. The 

preparation stage was pre-cooled with liquid nitrogen to -1 83°C. Any ice or frost that

was present was removed using sublimation by heating (to the maximum of -75°C) the 

stub from below and above. Stub temperature was lowered again to -183°C. 

The specimen was then transferred to the coating area of the cryo-SEM system 

preparation chamber. The specimen stub was sputter coated twice for two minutes at a 

sputtering current of 35mA. The gold-coated sample was then available for examination 

under the SEM. Leaf surface characteristics that were deemed to be representative of the 

viewed leaf surface were photographed over a consistent set of magnifications. 

2.3.4 Statistical Analysis 

The analysis of variance on leafminer egg density and leaf wax data were done 

using the REML method of the Mixed Procedure of SAS© (Version 8.2 for Windows - 

The SAS Institute Inc., Cary, NC, USA). Egg density was expressed as a proportion of 

eggs on leaves from the four treatments. The ANOVA on leafminer egg density was 

performed according to split-split plot with treatment being the main plot, test the splitplot, 

and the bench as the split-split plot. The analysis was performed on proportions of 

eggs whereby eggs in each treatment per cage were expressed as a proportion of the total 

number of eggs in that cage. The model random statement was used listing the random 

factors (i.e. bench*replicate, bench*treatment, replicate*treatment, bench*replicate* 

treatment). In terms of experimental design, “bench” is considered a replicate. 

The ANOVA on leaf wax data was performed according to the split-plot design with the 

main plot being the treatment, leaf-damage status as the split-plot. The bench was treated 

as a replicate and was the only random factor. The random statement was used again 

specifying the random factor and its interactions with the fixed factors. Leaf wax total

amount and its constituents; alkane, fatty acid, primary alcohol and alkyl ester were 

analyzed separately in this way. All data were found to be normally distributed and no 

transformations were required. 

Residuals for both analyses were tested for normality using the Univariate 

Procedure of SAS. Regression analysis in MINITAB® was used to examine the 

relationship between leafminer preference, as indicated by the number of leafminer eggs 

and epicuticular wax composition. In addition to total wax amount and composition, leaf 

surface area was included as a predictor variable. Best subsets regression is an efficient 

method used for building a best-fitting regression model with as few predictor variables 

as possible (MINITAB® Release 14.1). The strength of this model was then determined 

based on p- and R-squared values for different predictor variables. 

The predictors were selected based on Best Subsets Regression (BSR) analysis 

that results in the best model. The best model is determined by the minimum number of 

parameters (p) with the highest R-squared (R2) adjusted value, while taking standard 

deviation (S) and the C-p into account. The C-p is the measure of the difference of a 

fitted regression model from a true model, along with the random error. The objective 

was to select a model with a C-p of (p+1) or below.

3.0 - Results 

3.1 FACE Ring Population Survey 

Egg density was assessed in the spring of 2003, however data collected did not 

accurately reflect the status of the population due to extremely low numbers of eggs 

surveyed on leaves. Therefore, improved sampling techniques were employed in spring 

2004. Moths were observed flying at the FACE site May 25,2004. This date was later 

than in previous years (Kopper, pers. com.) due to spring meteorological conditions (i.e. 

cool, wet weather). 

The first flight of leafminer moths corresponded with leaf expansion. They were 

observed flying during twilight hours. The beginning of flight appeared to be 

synchronized among the majority of moths, as also perceived by others (Kopper, pers. 

com.; Auerbach, pers. com.). Upon landing on an aspen branch, leafminer moths were 

observed rapidly walking to leaves. When reaching the leaf, moths did not appear to 

encounter difficulty walking on either adaxial or abaxial surfaces. However, the act of 

oviposition was not observed. 

3.1.1 Leafminer Density 

Density was assessed in mid-June, 2004 and egg density was not significantly 

affected by treatment (Table 2). Differences in least squares means comparison between 

the control treatment and the O3 treatment revealed that density was marginally not 

significantly (p=0.055) greater on leaves from the control (Fig. 5). Egg density tended to 

be greater in the control and least in the O3 treatment (Fig. 5).

0.8 

co 

^ 0.6- 

(O 

o> 0.5 

a> 

* 0.4- 

X 

X 

X 

X 

« 

1 0 .3 - 

© 

Q 0.2- 

O) 

u? 0.1 

0 - 

Control C02 03 C02+03 

T reatment 

Figure 5. Effect of treatment with elevateci greenhouse gases on leafminer 

oviposition preference (Clone 42E) from population survey. Bars marked 

with the same letter represent means not significantly different from each 

other at a = 0.05. Capped lines represent halves of standard errors. 

Among the random factors, the least of variation was explained by block (Table 

2). The greater density of eggs was in block 3, followed in decreasing order by block 1 

and block 2. Block*treatment explained almost one fifth of the random variation (Table 

2 ), indicating inconsistent distribution of eggs in treatments in different blocks. 

Table 2. Results of analysis of variance on effects on egg density in the population 

survey. The REML method of the Mixed Procedure of SAS was used to test 

effects of the fixed factor. The random statement identified random factors of the 

design (Blk; Blk*Treatment) enabling the program to determine appropriate error 

terms for testing the effects of the fixed factor. Covariance parameter estimates 

are also included, showing percent of variance (bracketed) attributed to each 

random factor. 

Dependent 

Variable 

Sources of 

Variation 

Num 

df 

Den 

df 

F- 

value 

P- 

value 

Covariate 

Parameter 

Estimate 

Eggs Treatment 3 6 2.04 0.20 

Block 

Block*Trt 

Residual 

Total 

0.0028 (7.2%) 

0.0075 (19.4%) 

0.0284 (73.4%) 

0.0387 (100%) 

Block: 1,2, 3; Treatment: 1- control, 2-C02, 3-03, 4-C02+ 0 3; df=degrees of freedom, 

Num = numerator, Den = denominator


ANOVA are presented in Table 3. Results are described following the source of 

variation effects on different wax constituents. Averaged across clones 271,216, and 

259, total wax amount was significantly (p

Table 3. Results of the analysis of variance on leaf epicuticular wax and its 

constituents in leaves of clone 271,216,259 used in the population survey 

Dependent Sources o f Variation Num Den F P-value 

Variable df df 

Total Wax Treatment 3 6 7.52

Treatment 

Figure 6. Effect of greenhouse gas treatments on leaf epicuticular wax amount 

averaged across clones (259, 216, 271) in leaves from the population 


different from each other at p = 0.05. Capped lines represent halves of 

standard errors. 

Eo 

a> 

o 

E 

< 

Control 

Alkyl Ester 

Alkane 

Fatty Acid 

Primary Alcohol 

C 0 2 

Treatment 

C O 2 + O 3 

Figure 7. Effect of greenhouse gas treatments on leaf epicuticular wax 

composition averaged across clones (259, 216, 271) in leaves from the 

population survey. Bars of the same color marked by the same letters are 

not significantly different from each other at a = 0.05. Capped lines 

represent halves of standard errors.

Clones varied significantly in the total amount of wax and the differences were 

consistent across treatments (no interaction) (Table 3). Leaves from clone 259 had a 

significantly larger amount of total wax than leaves from clones 271 and 216 (Fig. 8). 

Clones varied significantly in alkyl ester amount and the differences were consistent 

across treatments (no interaction) (Table 3). Leaves from clone 259 had a significantly 

larger amount of alkyl esters than leaves from clones 271 and 216 (Fig. 9) corresponding 

with decreasing 0 3 -sensitivity in clones as reported by Kamosky et al. (1999). The effect 

of clone on alkyl esters was significant (Table 3). Wax from leaves of clone 271 had 

significantly more alkanes compared to clones 216 and 259 (Table 3, Fig. 10). The 

amounts of fatty acids on leaves from clone 271 were greater than in clones 216 and 259 

(Fig. 11), but the clone effect was not significant (Table 3). The amounts of primary 

alcohols were not significantly affected by clone (Table 3) even though the mean of clone 

259 was the smallest (Fig. 12). 

D) 

3 

C 

O 

3 

X 

CO 

CO 

•+-» 

o 

Figure 8. Effect of clone on total epicuticular wax amount averaged across 


same letter represent means not significantly different from each other at a 

0.05. Capped lines represent halves of standard errors.

E 

o 

~Q) 

3 

 

CD 

-*-» 

0) 

LU 

Figure 9. Effect of clone on leaf epicuticular wax alkyl ester amount averaged 

across treatments (4) in leaves from the population survey. Bars marked 



271 

e 

O 

O) 

D 

 

© C(0 

216 

259 

C lone 

Figure 10. Effect of clone on leaf epicuticular wax alkane amount averaged 



other at a - 0.05. Capped lines represent halves of standard errors.

271 216 259 

C lone 

Figure 11. Effect of clone on leaf epicuticular wax fatty acid amount averaged 



other at a - 0.05. Capped lines represent halves of standard errors. 

C lone 

Figure 12. Effect of clone on leaf epicuticular wax primary alcohol amount 

averaged across treatments (4) from the population survey. Bars marked 


other at a = 0.05. Capped lines represent halves of standard errors.

The total wax amount was not significantly different between leaves that were 

mined and those that were unmined (Table 3, Fig. 13). The amounts of alkyl esters and 

alkanes in mined and unmined leaves were not affected by leafminer presence (LP) 

(Table 3, Fig. 14). There was a significant effect of LP on amounts of fatty acids (Table 

3) that were smaller in mined than unmined leaves (Fig. 14). Primary alcohol amounts 

were also significantly (Table 3) larger in unmined than in mined leaves (Fig. 14). 

unmined 

mined 

Leafminer Presence 

Figure 13. Comparison of epicuticular wax amount on unmined and mined leaves 

averaged across treatments (4) and clones (3) in leaves from the population 



standard errors.

20 

E 

o 

15> =3C 

3 O 

E 

< 

15 

10 - 

5 - 

a a 

Il I. 

Ester 

Unm ined 

Mined 

A lkane Fatty Acid Alcohol 

Wax Constituent 

Figure 14. Mean amounts of wax constituents (alkyl esters, alkanes, fatty acids 

and primary alcohols) on mined and unmined leaves from the population 

study averaged across all clones (259, 216, 271) and treatments. Letters 

pertain separately to each pair of bars representing the same compound. 

Pairs of bars marked with the same letter represent means not significantly 


standard errors. 

The only significant (p=0.05) interaction in the analysis of wax constituents was 

between treatment and clone for alkyl ester amount. There are different responses in the 

amount of alkyl esters to treatments in different clones.

3.2 Leafminer Preference Study 

It was challenging to keep moths alive from the period of collection to initiation 

of the preference tests. Twice the amount of moths required was collected to ensure a 

sufficient number of moths for the tests. 

Moths were not observed ovipositing on leaf (clone 42E) surfaces. 

Approximately 50% of cages contained leaves with successfully ovipositing moths. Eggs 

may not have been laid because female moths either did not have eggs to lay or they died 

before laying eggs. 

3.2.1 Leafminer Egg Density 

Eggs were located on the abaxial surface of aspen leaves, usually towards leaf 

margins and were visible with a 10X hand lens (Appendix A, Fig. 1). Analysis was 

performed on proportions of eggs whereby eggs in each treatment per cage were 

expressed as a proportion of the total number of eggs in that cage. When all tests were 

combined for the analysis of variance, egg proportion was not significantly affected by 

treatment (Table 4). There was a trend for more eggs on leaves from the control and CO2 

(Fig. 15), similar to the population survey. Contribution of random elements of the 

experimental design to the overall random variation was very small. They jointly 

explained only 8.3% of variation in the distribution of eggs while the unexplained 

(residual) random variation accounted for the remaining 91.7% of variance (Table 4).

Table 4. Results of analysis of variance on the proportions of eggs on leaves of aspen 

from the four greenhouse gas treatments in the preference study. The REML 

method of the Mixed procedure of SAS was used to test effects of fixed factors. 

All random factors were entered into the Random Statement of the Mixed 

Procedure enabling the program to use appropriate error terms for testing the effects 

of the fixed factor (treatment). Covariance parameter estimates for random factors 

are included, indicating percent of random variation explained by each. 

Dependent 

Variable 

Sources of 

Variation 

Num 

df 

Den 

df 

F- 

value 

P- 

value 

Eggs Treatment 3 6 0.33 0.80 

Covariate 

Parameter 

test 

bench 

Test*bench 

Test*Trt 

Bench*Trt 

Test*bench*trt 

Residual 

Estimate 

0 

0 

0 

38.70 (3.5%) 

49.82 (4.5%) 

3.72 (0.3%) 

1011.67(91.7%) 

Total 1103.91 (100%) 

Test: 1,2, 3; Bench: 1,2, 3; Treatment: 1- control, 2-C02, 3-03, 4-C02+ 0 3; df=degrees of freedom, 

Num = numerator, Den = denominator 

0) 

B> 

RS 

.O 

IS o> o> 

d> 

"rë 

c 

o 

r oaos_ 

a. 

o> 

O) 

LU 

Control CO2 O3 

Treatment 

C02+03 

Figure 15. Effect of greenhouse gas treatments on leafminer oviposition 

preference (Clone 42E was used). Bars marked with the same letter 


Capped lines represent halves of standard errors.

When tests were analyzed individually, there were no significant effects of 

treatment in first three tests (the earliest tests). However, there was a significant (p=0.03) 

effect of treatment on egg proportion in test 4. The egg proportion in the control was 

greater than in the CO2, O3 and CO2 + O3 (Fig. 16). 

Control C 02 0 3 

Treatment 

CO2+O3 

Figure 16. Effect of greenhouse gas treatments on leafminer oviposition 

preference (Clone 42E was used) in preference replicate 4. Bars marked 




There were no statistically significant effects of treatment or leafminer 

presence on total wax amount and wax constituent classes (Table 5). Unlike the 

population survey, total wax amount was not significantly greater in the O3 treatment 

compared to the control, CO2 and CO2 + O3 treatments ( Table 5). As in the population 

survey, alkyl esters composed the largest proportion of leaf epicuticular wax deposit (Fig. 

17). However, there was not a significant effect of treatment on alkyl ester amount (Table

5). As a matter of fact, there were no significant effects of treatment or leafminer 

presence on any wax constitiuents (Table 5). The leaves with eggs tended to have 

smaller amounts of alkyl esters than leaves without eggs (Fig. 18), but this trend was not 

significant (Table 5). 

Table 5. Results of the analysis of variance on leaf epicuticular wax and its constituents 

in leaves of clone 42E used in the leafminer preference experiment (leaves from 

test 3 only). 

Dependent Sources of Num Den F P-value 

Variable Variation df df 

Total Wax Treatment 3 5 0.40 0.75 

LP 1 2 2.36 0.26 

Treatment x LP 3 5 2.45 0.17 

Error 68 

Alkyl Esters Treatment 3 5 0.17 0.91 

LP 1 2 3.51 0.20 


Error 16 

Alkanes Treatment 3 5 0.78 0.55 

LP 1 2 0.02 0.91 


Fatty Acids 

Error 16 

Treatment 3 5 0.20 0.89 

LP 1 2 0.31 0.63 


Error 16 

Primary Alcohols Treatment 3 5 0.35 0.79 

LP 1 2 1.42 0.35 


Error 16 

♦Treatment: 1- control, 2-C02, 3-03, 4-CO2+O3; LP: 1 - unmined, 2 - mined 

+ LP = Leafminer Presence

a 

25 

I 20 

là 

§ 15 - 

o 

E 

< 

c§ 10 - 

5 

0 

Treatment 

Alkyl Ester 

Alkane 

Fatty Acid 

Primary Alcohol 

Figure 17. Effect of greenhouse gas treatments on epicuticular wax composition 

of leaves from clone 42E used in the leafminer preference experiment. Bars 

of the same color marked by the same letters are not significantly different 

from each other at a = 0.05. Capped lines represent halves of standard errors. 

25 

20 - 

a 

No Eggs 

Eggs 

Ester Alkane Fatty Acid Alcohol 

Wax Constituent 

Figure 18. Mean amounts of wax constituents (alkyl esters, alkanes, fatty acids 

and primary alcohols) on leaves (clone 42E used) with and without eggs 

from the preference study. Letters pertain separately to each pair of bars 

representing the same compound. Pairs of bars marked with the same 

letter represent means not significantly different from each other at a = 

0.05. Capped lines represent halves of standard errors.

3.2.3 Leaf Morphology 

Wax production in the control treatment resulted in crystalline and irregular platelike 

structures projecting from the leaf surface (Fig. 19a). In the CO2 treatment, 

crystalline projections were prevalent, however amorphous surfaces were also present on 

the epicuticular ridges (Fig. 19b). Epicuticular wax appeared to be amorphous, with 

dense agglomerations lacking irregular plate-like structures projecting from the leaf 

surface (Fig. 19c). Leaves from the combined CO2 and O3 treatment had dense wax 

structures on the guard cells around the stomata, however the area surrounding the 

stomata was covered by a combination of amorphous and agglomerated wax formation 

(Fig. 19d). 

lOuitl 

a) Crystalline and irregular plate-like structures projecting from epicuticular ridges.

) Crystalline projections and amorphous surfaces present on the epicuticular ridges. 

lOum 

c) Amorphous, dense agglomerations of wax, without irregular plate-like structures.

d) Combination of amorphous and agglomerated wax formation. 

Figure 19. Impacts of elevated CO2 and O3 on abaxial leaf epicuticular wax structure 

shown in scanning electron micrographs from aspen clone 42E grown under a) 

control, b) elevated CO2, c) elevated O3 and d) elevated CO2 + 0 3. The entire leaf 

surface was examined before a representative sample area was selected. Arrows 

indicate wax features. “S” indicates location of stomata. 

3.2.4 Dependence of Egg Quantity on Leaf Area and Wax 

The model for predicting leafminer oviposition was optimized when leaf surface 

area (p

Table 6. Best Subsets Regression: Egg quantity versus leaf surface area, total wax 

amount, alkanes, fatty acids, primary alcohols and alkyl esters. 

Vars R2 R2 

(adj) 

Mallows 

C-p 

S 

Leaf 

Surface 

Area 

Wax 

Amount 

Alkanes 

Fatty 

Acids 

Primary 

Alcohols 

1 65.7 64.3 -1.3 0.25205 X 

1 25.2 22.1 23.3 0.37249 X 

2 68.1 65.4 -0.7 0.24836 X X 

2 66.3 63.4 0.4 0.25534 X X 

3 70.2 66.2 -0.0 0.24537 X X X 

3 69.4 65.1 0.0 0.24549 X X X 

4 72.3 67.1 0.7 0.24212 X X X X 

4 71.4 65.9 1.3 0.24630 X X X X 

5 73.1 66.3 2.3 0.24481 X X X X X 

5 73.0 66.3 2.3 0.24501 X X X X X 

6 74.1 65.9 3.7 0.24625 X X X X X X 

Alkyl 

Esters 

Each model appears on a different line in the output. “Vars” is the number of 

predictor variables used in the model. The R-sq (R2), Adj. R-sq (Adj. R2), C-p and S are 

then displayed. The list of possible predictors appears next. If the predictor is included 

in that particular model, then an 'X' is placed under the column header for that variable. 

For example, the first line tells us that the first model included in the output has a R-sq of 

65.7%, an adjusted R-sq of 64.3%, a C-p of-1.3 and s is 0.25205. The only predictor 

included in this model is leaf surface area. 

Best subset regression analysis determined that the 5th model was optimal. The 

results of this model were: R-sq of 70.2%, an adjusted R-sq of 66.2%, a C-p of 0.0 and S 

is 0.24537.

In the FACE Ring population survey, aspen blotch leafminer exhibited no 

preference for any treatment. Egg density was lowest on O3 leaves but did not differ 

significantly from leaves in the other treatments. The preference for leaves in control 

treatments may be attributable to specific host-plant characteristics. 

These data do not support previous investigations which determined leafminer 

egg densities at the FACE site were significantly larger in the control treatment compared 

to the CO2 ,0 3 and CO2 + O3 treatments (Kopper and Lindroth, 2003). A distinct 

difference between Kopper and Lindroth (2003) and this study at FACE was the overall 

leafminer population density. The former data were collected in 1999, when aspen blotch 

leafminer populations were more than double 2004 FACE leafminer egg density. Mean 

egg density was 0.25 eggs per leaf in 1999 (Kopper and Lindroth, 2003) and 0.12 eggs 

per leaf in 2004. 

Additionally, in this study, there were significant effects of block and 

block/treatment interaction. These effects have not been reported in other publications 

from research conducted at the FACE site (eg. Percy et al., 2002; Kamosky et al., 1999; 

Kopper and Lindroth, 2003). However, there were no effects of block or block/treatment 

interaction in the wax population survey analysis. It is important to point out that egg 

density in blocks 1 and 3 responded similarly to the treatment effects and that these two 

blocks contained the greatest densities of eggs compared to block 2. In fact, there 

appeared to be no effect of treatment on egg density in block 2. As a result, the lack of 

treatment effect in block 2, potentially due to lower egg densities, should be considered

as an explanation of not only the block effect but, the influence of treatment in the 

present and 1999 investigation. 

In the preference study, there was no significant effect of treatment. However, 

there did appear to be a trend similar to the population survey in that the control treated 

leaves tended to have higher densities of eggs than the O3 treated leaves. In fact, this 

trend was significant in the fourth and last test. This study was replicated four times 

during a 20-day period. Egg density was significantly greater in the control compared to 

the O3 treatment in the fourth test. Although there was subtle variation in the results, egg 

quantity on O3 treated leaves were smallest in each replicate. This variation may be due 

to the time difference when the replicates took place. Leaves in the last tests were 

exposed to O3 for a longer duration, and therefore any deterring qualities of the leaf may 

have been augmented by longer exposure to O3. Furthermore, moth age may have been a 

factor. Moths used in replicate one emerged earlier from overwintering sites than those 

used in replicate four and were therefore younger and potentially carrying more eggs than 

moths in replicate four. 

The population survey results suggest that O3 treated leaves may have been least 

preferred due to phenology of the aspen blotch leafminer. The initiation of aspen blotch 

leafminer moth oviposition takes place shortly after P. tremuloides bud burst, when 

leaves have flushed (Martin, 1956). O3 delays aspen bud break (Kamosky et al., 1999; 

Kamosky et al., 2005). Therefore, while control and CO2 treatments had leaves available 

for oviposition, leaves from O3 treatments may not have reached the development stage 

corresponding with the timing of leafminer oviposition site selection. However, the 

preference study offers evidence that oviposition cues for the aspen blotch leafminer are

plant-based despite the lack of significant results in all but one of the preference tests, 

trends indicate that control leaves had higher egg densities and O3 leaves lower densities. 

The effects on oviposition are apparently due to O3 and CO2 mediated changes in plant 

chemistry and not due to the direct effects of 0 3 and CO2 on the leafminer. This 

conclusion is based on the similar trends in the manipulated preference and the 

population studies. 

Plant epicuticular waxes are typically composed of at least four major chemical 

classes of aliphatic compounds; 1) alkyl esters, 2) alkanes, 3) primary alcohols, and 4) 

fatty acids (Baker, 1982). GC analysis and MS confirmation confirmed 99% of wax 

chemical composition. In both population and preference samples, the epicuticular 

waxes of trembling aspen were approximately 50% alkyl esters. Alkanes (24%), fatty 

acids (20%) and primary alcohols (5%) composed the remaining total wax amount. 

In the population survey, epicuticular wax was significantly affected by CO2, O3 

and CO2 + O3 treatments, clones and leafminer presence. Total wax amount was largest in 

the O3 treatment. Total wax amount from the O3 sensitive clone 259 was largest 

compared to clones 271 and 216 when averaged across all treatments. The effects of 

treatment and clone are supported by the findings of others (Kamosky et al., 2002). 

Leaves from the O3 treatment produced significantly larger amounts of alkyl esters. The 

effect of clone on wax amount may explain some of the variation in these results. Alkyl 

esters, which composed the largest proportion of total wax amount, were significantly 

larger on leaves from clone 259. Additionally, the interaction between clone and

treatment had a significant effect on alkyl ester amount. The effects of O3 result in an 

increased production of alkyl esters on clone 259. 

The interaction between clone and treatment was also significant (p=0.03) for 

fatty acids. In contrast to alkyl esters, production of fatty acids decreased in the 0 3 - 

sensitive clones 216 and 259. This interaction of the tolerant clone 271 and treatment, 

suggests that clone 271 may not be predisposed to decreases in fatty acid amount under 

elevated levels of O3. 

It has been proposed that aspen blotch leafminers least prefer clone 271 leaves 

(Kopper and Lindroth, 2003). Wax class amounts, other than alkyl esters and primary 

alcohol, were higher from 0 3 -tolerant clone 271. This suggests that greater alkane and 

fatty acid amounts would have a negative effect on leafminer oviposition preference 

while alkyl esters and primary alcohols would have a positive effect. In fact, mined 

leaves had significantly lesser amounts of fatty acids than leaves without eggs. 

In the preference study, wax trends corresponded with those from the population 

survey (clone 42E), despite the lack of significant effects. Wax production tended to 

increase in the O3 treated leaves, which supports Percy et al. (2002). Neither treatment 

nor leafminer presence significantly affected wax and its constituents. However, leaves 

selected for oviposition that were treated with O3 singly and in combination with CO2 

tended to have larger amounts of total wax amount than leaves without eggs. 

It is difficult to speculate based on non-significant results, however, leaves upon 

which eggs were laid either possessed stimulating oviposition cues or leaves without eggs 

possessed deterrent oviposition cues. Leaves without eggs in the O3 treatment had total

wax amounts similar to those in the other treatments (eg. control), which were preferred. 

Therefore, total wax amount may not be a critical cue affecting egg lay. 

Alkyl ester amounts tended to be larger on leaves with eggs. This suggests that 

alkyl esters stimulated oviposition. Although, there was not a significant effect of 

leafminer presence on alkyl esters in the population survey, it may suggest that alkyl 

esters had a role this plant-insect interaction. 

Air pollutants, such as CO2 and/or O3, alter epicuticular wax composition through 

direct modification of wax biosynthesis (Percy et al., 1994). Wax chemical composition 

influences the formation of epicuticular waxes on plant surfaces (Baker, 1982; Jeffree, 

1994). Alkanes and primary alcohols produce plate-like structures while alkyl esters and 

fatty acids form an amorphous wax surface. 

Here, leaves from the control have morphologically distinct crystalline wax 

structure. Amorphous surface features, characterized by agglomeration of waxes on 

leaves in the CO2 ,0 3 and CO2 + O3 treatments were prominent, which is supported by 

others (Kamosky et al., 1999; Kamosky et al., 2002). The formation of this amorphous 

surface was directly influenced by the increase in total wax production in those 

treatments. Moreover, it has been shown that morphological leaf characteristics are at 

least as important as chemical characteristics as oviposition signals (Eigenbrode and 

Espelie, 1995). Therefore, it is suggested that reduced crystalline wax structure 

correlates with reductions in aspen blotch leafminer egg density. 

Epicuticular waxes are considered crucial to oviposition site selection 

(Eigenbrode and Espelie, 1995). Leaf epicuticular waxes influence insect movement 

(Eigenbrode et al., 1991; Powell et al., 1999), feeding (Adati and Matsuda, 2000) and

oviposition (Cervantes et al., 2002; Ulmer et al., 2002; Kanno and Harris, 2000; 

Steinbauer et al., 2004; Muller and Hilker, 2001). Insect oviposition site selection could 

be either stimulated (Steinbauer et al., 2004) or deterred (Hagley et al., 1980, Uematsu 

and Sakanoshita, 1989, Justus et al., 2000) on plants with large amounts of wax. 

The regression analysis showed that increasing wax amount had a negative effect 

on egg density. Kamosky et al. (2002) suggested this increased production of 

epicuticular waxes may be “.. .an O3 - mediated stimulation of de novo synthesis, which 

maybe a protective adaptation.. 

This implication is supported by the reduction in 

leafminer preference for O3 treated leaves. Therefore, we would expect to observe wax 

amounts to be smaller from leaves without eggs or mines. However, total wax amount 

was larger from leaves with eggs or mines from both population and preference studies. 

This suggests the effects of epicuticular wax on the plant-insect interaction are complex 

and not well explained by the total amount of wax amount. 

One explanation may be that oviposition on O3 treated leaves was due to the 

moths maximizing stimulating cues of the leaf epicuticular waxes. These cues are 

specific wax classes, that this study suggests leafminers prefer, such as alkyl ester and 

primary alcohol, which are increased due to exposure to O3. However, at the population 

level, these stimulants are not strong enough to overcome the combined ovipositiondeterring 

characteristics of increased total wax amount, decreased leaf surface area and 

amorphous leaf surface morphology. In other words, the leafminers that choose O3 

treated leaves may make the best of a bad situation. 

The results from this study suggest the factors influencing aspen blotch leafminer 

oviposition site selection are, at least in part, isolated around leaf surface characteristics,

namely: surface area, epicuticular wax morphology and chemical composition. Of these 

characteristics, two factors, surface area and wax chemical composition, could be 

modeled on data collected. Modeling determined that leaf surface area, total wax amount 

and alkane proportions are strong predictors of egg density. Others have determined 

specific chemical wax compounds that affect oviposition (Udayagiri and Mason, 1997, 

Muller and Hilker, 2001, Steinbauer et al., 2004, Shepard et al., 1999). Fatty acids have 

been shown to positively influence oviposition in some beetles (Parr et al., 1998) and 

aphids (Eigenbrode, 1996). Also, alkanes are suggested to stimulate oviposition 

(Eigenbrode and Espelie, 1995). Fatty acids and alkanes are both considered to stimulate 

host recognition, however based on evidence from this study, we suggest the opposite is 

true for aspen blotch leafminer. 

These chemical characteristics of epicuticular wax are known to be indicators of 

plant quality (Baker, 1982). Plant quality changes (eg. phenolic glycoside, carbonnitrogen 

ratio, tannins, starch) when exposed to CO2 and O3 (Kamosky et al., 2003; Percy 

et al., 2002) therefore it is likely that moths detect these differences in leaf quality 

resulting from epicuticular wax chemical alterations. 

A higher number of eggs were observed on leaves with larger surface areas. Leaf 

surface area had a significant influence on leafminer egg density, however it is difficult to 

separate its effect from those of other factors that are critical to oviposition site selection. 

It has been shown that leaf surface area is largest in CO2 treated leaves (Kamosky et al., 

2003). Since leafminers did not prefer CO2 treated leaves, leaf surface area cannot be the 

single factor influencing leafminer oviposition preference.

Other studies have shown that larger surfaces areas have a lower amount of 

epicuticular wax per unit area (Hagley et al., 1980). Regression analysis also determined 

leafminers preferred leaves with lower wax amounts. Leaftniner density results, which 

were not statistically significant, exhibited a trend in the population and preference 

studies that leaves exposed to CO2 singly or in combination with O3 tended to be 

preferred over leaves exposed singly to O3. This suggests that the implications of leaf 

surface area on leaftniner oviposition site selection are not fully understood. 

This was the first investigation using bioassays to examine if microlepidoptera 

exhibit a preference for leaves exposed to elevated levels of CO2 and O3, both singly and 

in combination. As a result, there are many suggestions for improved methods to 

determine proximate cues for leaftniner oviposition. 

Future investigations would involve choice bioassays testing the direct effects of 

leaf epicuticular wax. Two experiments are suggested: 1) offer adult gravid female 

moths choices between leaves with and without wax. Crystalline waxes can be removed 

using cellulose acetate, which does not damage the plant tissue (Baker et al., 1983; Percy 

and Baker, 1988). Obtaining images from scanning electron microscopy and comparing 

the different treatments would be used in both tests to illustrate the surface morphological 

differences of leaves with and without waxes intact; 2) apply waxes to artificial substrate 

(eg. glass) from four different treatments. The results of this experiment would 

determine the specific effect of epicuticular waxes on aspen blotch leaftniner host-plant 

oviposition site selection while minimizing biological influences. Furthermore, specific

chemical compounds could be isolated, both singly and in combination that directly 

influence leafminer oviposition site selection. 

To further test the effect of leaf surface area on leafminer preference, female 

moths could be given a choice of a variation of leaf sizes and ages. Others have shown 

that leaf age affects insect behavior (Steinbauer et al., 2004). Generally, the amount of 

wax decreases as leaves become older. As amount and composition of epicuticular wax 

changes throughout leaf development, leaves of different development stages could be 

selected and tested for preference. To this end, as older leaves in this experiment were 

used in the fourth test of the preference experiment, and this was the only test in which 

leafminer preference was exhibited, then analysis of the wax samples from that test may 

have proven useful. 

An additional suggestion for future investigations would be to standardize the 

status of the number of eggs adult female moths possessed as in Hora and Roessingh 

(1999). The moths used in the preference study were selected randomly from pure aspen 

stands within a 10km radius of the population survey site. As a result, the complement of 

eggs females possessed was unknown. The number of eggs a female moth has often 

influences the selectiveness of oviposition site (Singer, 1982). This can be addressed by 

standardizing the complement of adult female moth eggs. Rearing female and male 

moths together from pupae achieves this, as there is a higher probability females will be 

gravid because they are provided the opportunity to copulate and they have not been 

exposed to oviposition substrate. Adult female aspen blotch leafminer moths have a 

complement of approximately 45 eggs to lay (Auerbach, 1992). In this study, the range 

of eggs laid was from 0 to 51, with an average of 10 eggs/female.

It could also be interesting to test the effect of clones on oviposition preference 

because clones of varying sensitivity to O3 had significant effects on epicuticular wax. 

Therefore, in order to elucidate characteristics, which have a marked influence on 

leafminer oviposition, clones (from the same treatment) should be tested in a choice 

bioassay. 

Eggs were located on the margins of leaves, which supported what others have 

reported (Martin, 1956; Auerbach, 1991). It would be useful to analyze waxes from leaf 

margins to determine if there are differences in their composition or morphology. Any 

differences in wax chemical composition and morphology on leaf margins may be further 

linked to oviposition. 

Quantifying epicuticular wax crystalline density from different treatments and 

clones would be another parameter that could be incorporated into models, which predict 

aspen blotch leafminer preference. Systematically measuring epicuticular wax density 

could be done with SEM modifications (eg. Cervantes et al., 2002). 

The main objective of this research was to link the changes in aspen leaf 

epicuticular wax chemical composition and morphology, due to exposure to elevated 

levels of CO2 and/or O3, with aspen blotch leafminer oviposition site selection. This was 

the first study to offer evidence, using population and bioassay studies, that aspen blotch 

leafminer moths assess the suitability of oviposition sites using epicuticular wax cues. 

Regression analysis using parameters of wax composition and leaf surface area to 

predict egg density, along with wax morphological interpretation, suggest that specific 

wax components are involved in plant/insect interactions. These data suggest that

amorphous epicuticular wax morphology, increased total wax amount and alkane amount 

had negative effects on leafininer oviposition, however they were not the only factor 

affecting leafininer oviposition. Rather, this behavior appears to involve a suite of cues, 

including leaf surface areas. 

Future investigations could reveal other factors which are involved in aspen 

blotch leafininer oviposition site selection. Nevertheless, the results of this study strongly 

suggest that epicuticular waxes influence aspen blotch leafininer oviposition. In addition, 

these results have improved the understanding of the role of epicuticular waxes in aspen 

blotch leafininer oviposition site selection. 

Further investigation is recommended on the general effects of leaf surface 

epicuticular waxes on aspen blotch leafininer oviposition site selection. These methods 

should be incorporated with further leafininer bioassays and leaf physiological responses 

to CO2 and/or O3. As a result, our knowledge of epicuticular waxes with respect to 

leafininer oviposition and elevated levels of greenhouse gases will be further improved.

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APPENDIX A

200om 

Figure 1. Scanning electron micrograph of Phyllonorycter apparella Herrich-Schaffer 

egg on abaxial surface of Populus tremuloides Michx.

CURRICULUM VITAE 

Candidate's full name: Robert Frank Partridge 

Universities attended: University of New Brunswick, 2002, Bachelor of Science in 

Forestry 

Publications: none 

Conference Presentations: University of New Brunswick Graduate Student Association 

Conference, February 2005

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