Terpene Composition of Lodgepole and Jack Pine and Its
Relationship to the Success of the Mountain Pine Beetle
Erin Clark
B.Sc, Denison University, 2002

Thesis Submitted In Partial Fulfillment of
The Requirements For The Degree Of
Master Of Science
In
Natural Resources and Environmental Studies
(Biology)

The University of Northern British Columbia
December 2008
© Erin Clark, 2008

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Abstract
Host tree defensive ability in new geographic regions and in different species could play
a role in the success of mountain pine beetle. Constitutive and induced terpene-based
defenses were tested in lodgepole and jack pine. Sampled lodgepole pines were assessed
for number of beetle attacks. Trees with higher historical exposure to the insect had
lower levels of defensive terpenes and lower attack densities, which may be explained by
reduced apparency. Significant differences existed in constitutive and induced levels of
terpenes between species; higher levels of most terpenes were found in lodgepole pine.
Jack pine had higher levels of a-pinene, which has implications for beetle success as this
terpene is involved in pheromone biosynthesis. This insect faces different terpene-based
chemical defenses in host species that it encounters in new geographic ranges and these
differences should be taken into account in management plans across large geographic
ranges and multiple host species.

ii

Table of Contents
Abstract

ii

Table of Contents

iii

List of Tables

v

List of Figures

vii

Acknowledgements

ix

Chapter One

Chapter Two

Literature review and overall objectives
1.1 Tree Defense
1.2 Mountain Pine Beetle Biology
1.3 Eruptive Populations
1.4 Factors Relating to Beetle Success
1.5 Geographic Ranges
1.6 Overall Objectives

1
3
5
6
7
8

Differences in lodgepole pine constitutive defenses across a
geographic range in British Columbia and the correlation to
mountain pine beetle attack
Abstract
2.1 Introduction
2.2 Methods
2.3 Results
2.4 Discussion
2.5 Conclusions

10
11
15
27
46
57

Chapter Three

Comparison of constitutive resin chemistry of lodgepole, hybrid,
and jack pine stands in British Columbia and Alberta
Abstract
59
3.1 Introduction
60
3.2 Methods
63
3.3 Results
68
3.4 Discussion
78
3.5 Conclusions
84

Chapter Four

Induced response in lodgepole and jack pine to mountain pine
beetle inoculation and wounding
Abstract

86

in

Chapter Five
References

4.1 Introduction
4.2 Methods
4.3 Results
4.4 Discussion
4.5 Conclusions

86
88
93
122
128

Conclusion

130
136

IV

List of Tables
Chapter 2
List of sample locations, number of trees sampled and the
climatic suitability classes of the sampled plots

17

List of chemical standards used to process all phloem
samples using GC-FID

24

Mean amount of terpenes present (ppm, ± 1 SE) for constitutive
defenses by location

28

Mean constitutive levels terpenes (ppm, + 1 SE) by climatic
suitability class and location

29

Effect of location, levels of borneol, limonene, and a-pinene,
and dbh on attack density (attacks/m )

37

Effect of location, attack density (attacks/m ), and dbh (cm)
on the pupal chamber density (chambers/m2)

38

Effect of location, levels of borneol, limonene and a - pinene,
dbh, and attack density on attack density and pupal chamber density

39

Effect of location and climatic suitability classes, borneol,
a - pinene, and dbh on attack density (attacks/m2)

43

Effect of location and climatic suitability class, levels of borneol,
and a - pinene, and dbh on attack density (attacks/m )

44

Effect of location, levels of limonene and total terpenes,
and dbh on the probability of tree survival

45

Table 3.1.

Coordinates for sample locations and number of trees sampled

66

Table 3.2.

Mean concentration of terpenes (ppm, + 1 SE) of the two species

72

Table 3.3.

Mean percent concentration of terpenes (+ 1 SE) of the two species

75

Table 2.1.

Table 2.2.

Table 2.3.

Table 2.4.

Table 2.5.

Table 2.6.

Table 2.7.

Table 2.8.

Table 2.9.

Table 2.10.

Chapter 3

Chapter 4
Table 4.1.

Sampling locations, number of trees sampled at each location,

v

Table 4.2.

and dates of sampling

89

Sources of variation in the levels of terepenes (ppm) to time,
location, dbh, treatment (Trt), and time and associated P-values

94

VI

List of Figures
Chapter 2
Figure 2.1.

Map of sampling locations

18

Figure 2.2.

Diagram of an example of a fixed-radius experimental plot

20

Figure 2.3.

Mean levels (ppm, + 1 SE) of the predominant seven terpenes
found at four locations

30

Figure 2.4.

Mean levels (ppm, ± 1 SE) of the predominant seven terpenes
found at two locations and two climatic suitability classes

33

Figure 2.5.

Box plots of attack density (attacks/m2) by sampling locations

35

Figure 2.6.

Box plots of attack density (attacks/m2) by sampling locations and
climatic suitability classes

41

Figure 3.1.

Map of sampling locations in British Columbia and Alberta, Canada

64

Figure 3.2.

Cluster analysis of samples to separate into two groups based
on percentages of a-pinene and p-phellandrene

69

Chapter 3

Figure 3.3.

Mean level of terpenes (ppm, + 1 SE) that showed significant difference
between lodgepole pine and jack pine
73

Figure 3.4.

Mean percent of terpenes comprising of 1 % of the total
terpenes (ppm, ± 1 SE) in lodgepole pine and jack pine

76

Mean levels of terpenes (ppm, +1 S.E.) in inoculated trees at
sampling time day 0, day 2, and day 14

96

Mean levels of A-3-carene (ppm, + 1 SE) at each location for
each treatment overtime

98

Mean level of limonene (ppm, + 1 SE,) at the sampling locations
overtime

100

Chapter 4
Figure 4.1.
Figure 4.2.
Figure 4.3.

vn

Figure 4.4.
Figure 4.5.
Figure 4.6.
Figure 4.7.
Figure 4.8.
Figure 4.9.

Mean level of myrcene (ppm, + 1 SE) at the sampling locations
overtime

103

Mean level of p-phellandrene (ppm, ± 1 SE) at each location
for each treatment over time

105

Mean level of a-pinene (ppm, + 1 SE) at the sampling locations
overtime

108

Mean level of terpinolene (ppm, ± 1 SE) at each location for
each treatment over time

110

Mean total levels of terpenes (ppm, + 1 SE,) at each location for
each treatment overtime

113

Mean levels of P-pinene (ppm, ± 1 SE) at each location for each
treatment over time

116

Figure 4.10.

Mean level of linalool (ppm, ± 1 SE) at each location each treatment
by day
118

Figure 4.11.

Mean levels of pulegone (ppm, + 1 SE) at each location for each
treatment by day

120

vni

Acknowledgements
I would like to thank Dr. Dezene Huber for all of his support and guidance on this
project. Also, Dr. Staffan Lindgren and Dr. Allan Carroll for all of their hard work and
help on this project. I am also grateful to Anna, Caitlin, Jordie, Dan, Jeff, and Sean for
help in both the field and the lab and to Nancy-Ann for her help creating the maps. The
chemical analysis would not have been possible without Clive Dawson and David Dunn
of the British Columbia Ministry of Forests and Range Forest Research Laboratory in
Victoria, BC. Thank you also to all the members of the Lindgren, Aukema, Poirier, and
Huber lab for help and feedback on this project.
Thank you to the Natural Resources Canada Mountain Pine Beetle Initiative research
grant (8.45), the Natural Sciences and Engineering Research Council of Canada, Canada
Research Chairs Program, Canada Foundation for Innovation, and the British Columbia
Knowledge Development Fund without which this work would not have been possible.
I am also grateful to the many people who have supported and helped me throughout this
project: Crystal, Kyla, Irene, my office mates, Sara (MPBTI), Julia, and Marcel for moral
support and friendship throughout the process. Finally, I wish to thank my family for
their unconditional love and support.

IX

Chapter 1. Literature review and overall objectives
1.1

Tree Defense
The ability of bark beetles to locate and colonize their hosts is essential to their

survival and reproductive success. In order to successfully colonize their host, bark
beetles must overcome its defenses and kill the tree (Atkins 1966, Reid and Robb 1999).
Chemical defenses, both constitutive and inducible, have been related to defense against
pathogens and herbivores (Gershenzon and Croteau 1991, Raffa and Smalley 1995,
Logan and Powell 2001), including bark beetles (Raffa and Smalley 1995, Berryman
1972, Seybold et al. 2006, Wallin and Raffa 1999, Raffa and Berryman 1982) and the
fungi that they vector (Berryman 1972, Reid et al. 1967, Hofstetter et al. 2005).
Constitutive defenses are defenses that are maintained by a tree at all times to
inhibit or prevent initial attack. Induced defenses, on the other hand, are initiated by the
tree when it faces a challenge (Franceschi et al. 2005). The terpene profiles (Smith 1966,
Rocchini et al. 2000, Pureswaran et al. 2004) and quantity (Raffa and Berryman 1982,
Reid et al. 1967, Hodges et al. 1979) of constitutive and induced resin differs in conifer
species (Trapp and Croteau 2001), and seems to be partially under phytohormonal
control.
Conifer resin is primarily composed of terpenes (Franceschi et al. 2005).
Terpenes are a large class of organic compounds that are composed of multiples of C-5
isoprene units (Gershenzon and Croteau 1991). Terpene synthases, which biosynthesize
terpenes, can be divided into three classes based upon the number of isoprene units in
their products: monoterpene synthases (two isoprene units), sesquiterpene synthase (three
isoprene units), and diterpene synthases (four isoprene units) (Keeling and Bohlmann

1

2006). Multiple terpene products can be produced from individual synthases, and conifer
resins contain a great diversity of terpenes due to this feature of their biosynthesis and the
concurrent expression of multiple terpene synthases (Keeling and Bohlmann 2006).
Terpenes are often studied in the context of their use by bark beetles for long- and
short-range host selection, both as attractants and repellants (Gershenzon and Croteau
1991, Keeling and Bohlmann 2006, Pureswaran and Borden 2003). Mountain pine beetle
(Dendroctonus ponderosae, Hopkins; MPB) has been shown to be attracted to bolts of
host material that emit kairomonal (host volatile) cues, even in the absence of visual and
pheromone cues (Moeck and Simmons 1991). Gas chromatographic electroantennographic detection analyses have shown that MPB is capable of detecting
both host and non-host volatiles (Huber et al. 2001).
The ability of bark beetles to detect volatiles underlines the fact that host volatiles
play a large role in bark beetle biology. For instance, chemical stimuli in the phloem of
ponderosa pine have been found to stimulate boring behavior in Ips paraconfusus
(Lanier) (Elkinton et al. 1981), which showed preference for their host phloem in the lab,
although final selection was not made by the insect until after boring through the bark
(Elkinton and Wood 1980). In addition to their role in host identification, terpenes found
in the resin of pines have been shown to be toxic to attacking insects (Raffa and Smalley
1995, Smith 1961, Smith 1963, Raffa and Berryman 1983b). Raffa et al. (1985) found
mortality of the fir engraver beetles (Scolytus centralis, Le Conte) within four hours of
being exposed to monoterpene vapors in the lab.
Resin terpenes are also precursors for aggregation and antiaggregation pheromone
biosynthesis by bark beetles (Gershenzon and Croteau 1991, Borden 1985, Raffa and

2

Berryman 1983a, Hunt et al. 1989) which enable coordination of "mass attacks" by the
insects and which allow them to overcome their hosts defenses (Borden 1985, Borden
1982, Wood 1982, Berryman et al. 1985). Erbilgin and Raffa (2000) found that certain
monoterpenes inhibited Ips pini (Say) attraction to their aggregation pheromone and nonhost volatiles have been shown to reduce bark beetle attraction to its pheromone (Huber
and Borden 2003). Other studies have found that monoterpenes enhance beetle response
to their aggregation pheromone (Borden 1985). The effect of host monoterpenes on
beetle response to aggregation pheromones has been found to vary with release rate
(Erbilgin and Raffa 2000). Thus, the role of terpenes in the lifecycle of coniferophagous
bark beetles is complex, as compounds may enable host identification, be toxic to
invaders, attract insects, or provide precursors to their pheromone production.
1.2

Mountain Pine Beetle Biology
Mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera:

Scolytidae), is a native bark beetle that normally survives at endemic levels throughout
western North America. MPB is the primary insect affecting lodgepole pine ecosystems
(Amman and Cole 1983). Lodgepole pine (Pinus contorta var. latifolia, Dougl. ex.
Loud.) is the most-utilized host of MPB in BC (Safranyik and Carroll 2006), but they are
capable of successfully utilizing other species of pines throughout their range (Furniss
and Carolin 2002), including jack pine {Pinus banksiana, Lamb.) (Safranyik and Linton
1982, Cerezke 1995). Stands of jack pine extend across northern Alberta and are
contiguous with the currently unprecedented infestation in neighboring British Columbia,
which affected 10.1 million ha in 2007 aerial surveys (Westfall and Ebata 2008).

3

Lodgepole pine and jack pine hybridize where the two species overlap in western Alberta
(Moss 1949).
Newly-emerged adult MPB disperse through the forest and search for hosts in
mid-summer. Upon encountering a host pine tree, the adult female bores through the
outer bark and commences gallery construction in the phloem. Males, also capable of
producing pheromones, joining the aggregation, follow the females into their galleries
and mate (Reid 1962a). Reid (1962b) found that under favorable laboratory conditions
females established galleries with over 200 eggs, although under natural conditions
galleries contained fewer than 75 eggs. The significant differences between the average
gallery length being attributed to differences in the rate at which attacked tree defenses
deteriorated. Larvae go through four instars, pupate, and then emerge as adults.
Unlike many other herbivorous insects, the reproductive success of MPB is
dependent on the beetles' ability to kill their host (Logan and Powell 2001, Raffa and
Berryman 1983a). Because one pair of insects cannot accomplish this alone, attacking
beetles produce a powerful aggregation pheromone that coordinates the attack of many
individuals on a target tree (reviewed by Borden 1985; reviewed by Seybold et al. 2000).
MPB utilizes its host's own terpene-based defenses to assist in the production of their
aggregation pheromones (Raffa and Berryman 1983a). In addition, MPB is a vector for
pathogenic fungi (Reid et al. 1967, Lee et al. 2006). The fungi and the beetle benefit
from this symbiotic relationship. In fact, the relationship is so closely co-evolved that the
beetle has specialized mycangia that help it to carry the fungal spores from its brood tree
to new hosts, and in return the fungi help the invading beetles to rapidly kill the host tree
(Berryman 1972, Safranyik et al. 1975) and may even provide nutritional benefits

4

(Bleiker and Six 2007). Tree defenses are induced against the fungi, even in the absence
of the beetle (Raffa and Smalley 1995, Klepzig et al. 1995).

1.3

Eruptive Populations
In the past century, there have been four or five significant outbreaks in British

Columbia (Taylor and Carroll 2004). In the progression to, and from, an outbreak
population level, there are four categories of MPB populations: endemic, incipientepidemic, epidemic, and post-epidemic (Safranyik and Carroll 2006). During the
endemic phase, MPB are present at low population levels and they typically colonize
stressed trees (Amman 1984). A population is considered incipient-epidemic when it
reaches a level in which large-diameter trees are successfully attacked. This is often
correlated to events that weaken tree resistance (e.g., drought) or favor beetle populations
(e.g., mild winters) (Berryman 1976). Mortality of vigorous trees from events such as
wind-throw can contribute to significant population increases for bark beetles (Reid and
Robb 1999) and allow endemic populations to become incipient-epdemic or fully
epidemic. When a population reaches the epidemic phase, the beetles are able to make
use of vigorous, larger-diameter trees, and the infestation is detectable at the landscape
level instead of in small, scattered clusters.
The availability and suitability of host material are also considered major factors
in bark beetle success (Raffa and Berryman 1987). The shift to a post-epidemic
population in a particular area may be the result of several factors such as poor abiotic
conditions (e.g., a particularly harsh winter) or a depletion of suitable hosts. When MPB
have killed most of the desirable hosts with thick phloem, they are forced to infest

5

smaller trees with thinner phloem. Reproductive success in thin-phloem trees is reduced,
compared to trees with thick phloem, which contributes to population decline (Amman
and Pace 1976). At this point, the population size of the MPB decreases, and because
fewer individuals are present to join aggregations, host resistance once again becomes a
strong factor in MPB survival by affecting reproductive success and survival during
colonization of hosts.

1.4

Factors Relating to Beetle Success
The Shore and Safranyik (1992) model of stand susceptibility takes into account

many stand characteristics such as stand age, composition, density, and location, in
addition to beetle pressure in the vicinity. However, it does not take into account any
differences in tree defenses present in the stand. Risk ratings, based upon Shore and
Safranyik's (1992) model, have been shown to be of practical use for forest managers
(Dymond et al. 2006). In general, utilization of models to predict the spread and likely
areas of MPB infestation can be a useful tool for directing management efforts if the
models take into account important parameters that affect MPB success.
A number of parameters have been found to be important and influence such
models. Phloem thickness has been correlated with beetle success, (Amman 1972,
Berryman 1976, Amman and Pace 1976), as has the diameter of trees (Cole and Amman
1969, Paine et al. 1997), because phloem thickness generally increases as bark thickness
increases with increasing tree diameter (Amman 1969). Smith (1975) suggested that the
resistance of ponderosa pine (Pinus ponderosa, Laws) against the western pine beetle,
Dendroctonus brevicomis (LeConte), is in part a function of the resin quantity and quality

6

(as measured by the xylem monoterpene composition). Total attacks and brood
production have also been positively correlated to tree age and diameter (Safranyik and
Carroll 2006). Cole et al. (1981) found that monoterpene concentration is higher in
thicker phloem, which may be a factor in MPB selection of larger trees (Amman and
Cole 1983), although visual cues, potentially relating to host size, play a role in host
selection as well (Shepherd 1966, Rasmussen 1972).
In addition to stand and host characteristics, climatic factors play a role in
determining beetle success. Because insects are ectotherms, their physiology and
progress through their lifecycle is highly dependent upon environmental temperature
(Carroll et al. 2004). There are minimum winter temperatures below which there is 100%
beetle mortality under the bark (Safranyik and Linton 1998). Warming associated with
climatic changes can result in changes in climatically suitable habitat (Carroll et al.
2004). For instance, MPB outbreaks have recently been reported in high elevation white
bark pine forests, an area that was previously considered too cold for successful brood
development (Logan and Powell 2005).

1.5

Geographic Ranges
Conifer defenses are known to vary across physical space. Bannister et al. (1962)

found that the mean level of a-pinene varied with geographic origin between three
populations of Pinus radiata (D. Don) in California. Hanover and Furniss (1966) also
found a degree of geographical variation in the quantities of specific monoterpenes in
Douglas-fir (Pseudotsuga menziesii, (Mirb.) Franco) although the mean level of total
monoterpenes was similar across all locations. Forrest (1980) grew seeds in England

7

taken from the North American range of P. contorta and found chemotypic variation
there. Matson and Hain (1985) hypothesized that different pine species allocate different
amounts of energy to constitutive or induced defenses based upon geographic location
and the frequency with which they face attacks. Another hypothesis, suggested by Raffa
and Berryman (1987), is that defenses may be related to life history, with long-lived
species needing complex systems of defense.
Various studies have also examined geographical differences in beetle population
development and other lifecycle parameters. Bentz et al. (2001) found that parent beetles
had a greater influence on brood development and time to maturity than did the host
material, suggesting that MPB populations have geographically distinct, heritable traits.
In particular, they found that the data were consistent with the expectation that adult size
and development time decrease as latitude increases. Bentz et al. (2001) suggested that,
because development rate is a highly heritable trait, population response to changes in
climatic regime could occur in a short evolutionary time scale. There may also be
geographic differences in behavioral response of Dendroctonus spp. to sterioisomers of
monoterpenes (Erbilgin and Raffa 2000). In addition, geographic variation has been
noted in the enantomeric composition of other bark beetle semiochemical systems
(Borden 1985).

1.6

Overall Objectives
The purpose of this study was to examine the constitutive and induced oleoresin

terpene chemistry of lodgepole and jack pine stands as they relate to MPB colonization
and reproductive success. In addition, I examined the variation of the terpene

8

composition of the resin within lodgepole pine, across the geographic range of the hosts,
particularly comparing populations that historically have had higher beetle pressure with
each other and with those that have had lower historical contact with the beetle.
Information about the chemical characteristics of pines as they relate to beetle
colonization and reproductive success in lodgepole pine will contribute to the risk
assessment of jack pine and to developing new or improved predictive models of beetle
impact in their potential eastward spread from the pure lodgepole pine stands of BC and
western Alberta and into pure jack pine stands. In addition, these data may assist tree
breeders and forest managers in the selection of more resistant cultivars for replanting,
which may help to reduce the impact of future beetle infestations.

9

Chapter 2. Differences in lodgepole pine constitutive defenses across a
geographic range in British Columbia and the correlation to mountain
pine beetle attack.
Abstract
Mountain pine beetle {Dendroctonus ponderosae Hopkins) is considered to be the most
destructive insect pest in western conifer forests. Currently, British Columbia, Canada is
experiencing the largest outbreak of this insect in recorded history. This outbreak
includes areas such as the northern portion of the province that historically have had low
climatic suitability for the insect. Populations of lodgepole pine across BC were sampled
for constitutive resin terpenes for comparison between trees that may exhibit differential
resistance to mountain pine beetle attack based upon the likelihood of previous exposure
of tree populations to mountain pine beetle. Phloem samples were analyzed by gas
chromatography for 26 terpenes. Trees were assessed for number of mountain pine
beetle attacks, number of pupal chambers, and survival the following spring. Data were
analyzed using ANOVA, linear mixed effects models, and generalized linear models.
Significant differences existed between the levels of terpenes found in populations that
had likely experienced substantial mountain pine beetle infestations in the past compared
to populations that likely had not. While I expected southern populations to contain more
terpenes than northern populations due to higher historical exposure to the insect, the
converse was, in fact, true. The northern population generally contained higher levels of
terpenes than the south. There were also significantly higher levels of attack in the
northern lodgepole pine populations compared with those in the south, which might be
partially explained by the southern populations having reduced apparency to the beetles.
That is, southern lodgepole pines have evolved to release fewer attractant terpene

10

kairomones than have northern trees. Specifically, level of attack was correlated to the
quantities of borneol, limonene, and a-pinene, and levels of limonene and total resin
terpenes explained tree survival. Coevolutionary implications are discussed.

2.1

Introduction
One of the primary means of defense utilized by conifers against insect attack is

resin, which is composed primarily of terpenes. Bark beetles may exhibit positive or
negative chemotactic responses to terpenes while selecting a host (Gershenzon and
Croteau 1991, Pureswaran et al. 2006, Keeling and Bohlmann 2006). Some terpenes are
also toxic to invaders (Smith 1965, Smith 1963). Despite that, bark beetles, including the
mountain pine beetle (Dendroctonus ponderosae, Hopkins, MPB), are able to use some
host phloem resin terpenes as precursors to aggregation pheromone components (Hughes
1973a, Raffa and Berryman 1983a, Seybold et al. 2006).
The MPB is a native bark beetle normally present at endemic levels throughout
western North America, ranging from Mexico to northern British Columbia. It is a
primary insect herbivore in lodgepole pine ecosystems (Amman and Cole 1983) and has
been described as the most destructive of the tree-killing Dendroctonous spp. (Craighead
et al. 1931). Newly-emerged adults search for hosts in mid-summer, and upon
encountering a host pine tree, the adult female bores through the outer bark and
commences gallery construction in the phloem. Reproductive success is dependent on its
ability to kill its host (Raffa and Berryman 1983a, Logan and Powell 2001), therefore in
order to overcome host tree defenses, the beetles produce aggregation pheromone to

11

attract conspecifics for a mass attack of the tree (Raffa and Berryman 1983a, Borden
1985).
Once pioneer females have entered the phloem, they begin to produce aggregation
pheromone components (Pitman et al. 1968). The pheromone is comprised partly of
modified host terpenes, and some unmodified terpenes emanating from the entry holes
made by the beetle are synergists of the aggregation pheromone (Borden et al. 1983,
Conn et al. 1983).
Male beetles - which also produce aggregation pheromone components - are
attracted to the tree and more males and females rapidly join the aggregation. The rapid
recruitment of conspecifics of both sexes to the aggregation serves to increase the
production of pheromone and release of host-derived kairomones, thus the attack
escalates at a rapid rate. Upon landing on the trees, males follow the females into their
galleries and mate (Reid 1962a). Mated females then lay eggs in niches along the walls
of galleries that they have constructed in the resin-saturated host phloem. Once the eggs
hatch, the larvae generally go through four instars while feeding on host phloem. They
normally overwinter as either 2 n or 3 r instars, complete larval development the
following spring, pupate, and then emerge as adults during the summer.
In addition to their tunneling activity in the phloem, which serves to partially
girdle the host, the beetles vector fungi into the tree (Reid et al. 1967, Lee et al. 2006a).
The fungi, some of which are capable of killing the host on their own (Klepzig et al.
1995), also trigger induced defenses in the host (Raffa and Smalley 1995).
While endemic populations tend to persist in physiologically weakened trees,
MPB populations occasionally reach epidemic sizes at which time they can attack and

12

kill apparently healthy trees at a landscape level (Rudinsky 1962, Safranyik and Carroll
2006). In the past century there have been four or five significant MPB outbreaks in
British Columbia, Canada (BC) (Taylor and Carroll 2004). Currently, BC is
experiencing the largest outbreak on record (Carroll et al. 2004), affecting -13 million ha
since 1999 (Westfall and Ebata 2008). In addition, the current infestation extends further
north in the province than past records indicate has occurred previously (Raffa et al.
2008).
Considerable variation in resin monoterpene composition within populations of
various conifers (Squillace 1971, Rockwood 1973, Hanover and Furniss 1966, Franklin
and Snyder 1971, Smith 1983) and, between locations in lodgepole pine (Pinus contorta
Dougl. var. latifolia) (Forrest 1980), the primary host of the mountain pine beetle in BC,
has been found. Terpene composition in conifers has been shown to be a heritable trait
(Squillace 1971, Hanover 1966) controlled by numerous terpene synthase genes (Keeling
and Bohlmann 2006).
It has been suggested that both constitutive and induced terpene-based defenses
evolved partly in response to selective pressure by bark beetles (Mitton and Sturgeon
1982, Raffa and Berryman 1987). Thus, lodgepole pine could show within and between
population-variation in chemical defenses that could translate into variable levels of
susceptibility to beetle attack. Raffa and Berryman (1982) found no differences in terms
of the percent composition of the monoterpenes present between lodgepole pine that
showed resistance to MPB attack and those that were attacked. They also found no
qualitative difference in the induced phloem composition between the resistant and
susceptible trees although there were compositional changes. They concluded that it was

13

not the monoterpene composition that was distinct between resistant and susceptible
lodgepole pines, but, instead, the quantitative resin response, which was greater after
attack in resistant trees. However, this study took place in one location, and thus the
authors may have missed variation that is present at a larger geographic scale. In
addition, Huber et al. (2004) suggested that plasticity of the multigene-based terpene
defense in conifers is an important adaptation for these long-lived, immobile tree species
in defending themselves against shorter-lived, highly mobile herbivores and pathogens.
This would imply that trees in areas with prior exposure to bark beetle outbreaks may be
better adapted to defending themselves against attack. I predict that pines in regions that
have historically been exposed to higher MPB pressure will have evolved more effective
terpene defenses and responses against beetle attacks than pines in regions that have
historically had lower exposure to beetle pressure.
A climate suitability model developed by Carroll et al. (2004) uses climatic
factors to infer probable historical beetle pressure based upon the suitability of historic
climatic factors to sustain MPB populations. The climatic suitability model is based upon
climatic factors that are known to be important to MPB success such as degree days,
minimum winter temperatures, and precipitation. However, it does not take into account
stand structure (Carroll et al. 2004). Particular regions are placed into climatic suitability
classes (CSCs) via a high to low ranking system. This model allows for selection of
sampling sites in areas hypothesized to have been exposed to different levels of historical
MPB pressure.
While Hanover and Furniss (1966) found no qualitative difference in
monoterpene composition between attacked and unattacked Douglas-fir trees

14

(Pseudotsuga menziesii (Mirb.) Franco), attacked by the Douglas-fir beetle, (D.
pseudotsugae Hopkins), they did find higher levels of A-3-carene in unattacked trees than
those that resisted attack, implying the role of particular terpenes in resistance in some
trees. Sturgeon (1979) found that ponderosa pines (Pinus ponderosa Laws ) from
geographic areas that had historically been exposed to western pine beetle (Dendroctonus
brevicomis LeConte) outbreaks had higher concentrations of limonene. Smith (1975)
found that ponderosa pine with a higher percentage of limonene and higher resin flow
were more resistant to attack and colonization by the western pine beetle than were trees
with lower limonene levels or resin flow.
To assess if there are differences in constitutive chemical defenses that potentially
relate to susceptibility to attack by MPB in lodgepole pine in regions that have likely had
historically high exposure to the insect compared to regions that have not been so
exposed, I sampled pines across a geographic range and among different CSCs in British
Columbia. I hypothesize that there will be differences in constitutive terpene
composition and tree survival between populations that will correlate with historic
climatic suitability for MPB attack and with subsequent beetle success.

2.2.

Methods

2.2.1. Sample Collection and Processing
Four locations were selected based on accessibility and geographic spread along a
north/south transect across the MPB infestation in British Columbia (Table 2.1, Figure
2.1). At both the northern-most, (Quesnel, Q), and southern-most (Princeton, P)
locations, two sites were selected using historic climatic suitability class maps to choose

15

one site in a historically climatically favorable area for MPB and one in a less favorable
area. The map locations and associated CSCs were determined based on data from 19411970 (Carroll et al. 2004). Sites in both high and low CSCs with unattacked trees were
found near Princeton but sites with severe and moderate CSCs and uninfested trees were
found near Quesnel. Near 100-Mile House (C) and near Kamloops (K) only one site was
selected in the low climatic suitability class due to the high infestation level in those area
- suitably-sized, unattacked trees in the high climatic suitability class could not be found.
Only uninfested lodgepole pine trees were baited and/or sampled. Uninfested status was
determined by the absence of pitch tubes and/or frass and green foliage. At each site,
several 7 m fixed-radius, baited (mountain pine beetle lure, Pherotech International, Inc.,
Delta, BC, Canada) plots were established (Figure 2.2, Table 2.1). A minimum distance
of 25 m was maintained between the centers of adjacent plots. The number of plots
varied at each site, but enough plots were established to obtain a minimum of 50
experimental trees at each site. Baiting and sampling was completed 6-12 July, 2006,
before the major beetle flight. In each plot, a central tree was selected for baiting such
that the number of lodgepole pine with a dbh of 15 cm or greater within the plot was
maximized. In two cases, it was necessary to hang the central tree bait on a lodgepole
pine with a dbh <15 cm to maximize the number of large-diameter, surrounding
lodgepole pines in the plot. Each tree in each plot was marked with paint (tree marking
paint, Aervoe Industries Inc., Gardnerville, NV 89410, U.S.A.) for identification
purposes and their distance from the central bait and their dbh were measured.
Immediately after baiting, a phloem sample was taken from each tree in each plot at 1.3
m from ground level using a 10 mm diameter punch (No. 149 Arch Punch 10 mm, C.S.

16

Table 2.1. List of sample locations, site (representing CSC) number of trees sampled and
the climatic suitability classes1 (CSC) of the sampled plots.
^__
Location

Site

Plots

Quesnel

Higher

7

Number of trees
sampled
66

Quesnel

Lower

10

56

100 Mile House

Lower

6

79

Kamloops

Lower

3

55

Princeton

Higher

8

59

Princeton

Lower

4

51

Coordinates

CSC

N53°00.555'
W122°11.941'
N 53°08.827'
W121°51.741'
N51°47.587'
W 120°45.372'
N50°31.900'
W 120°32.102'
N 49°48.339'
W 120°31.743'
N49°49.597'
W 120°27.534'

severe
moderate
moderate
very low
high
low

Carroll et al. 2004

17

Figure 2.1. Map of sampling locations designated by black circles. QH - Quesnel higher
climatic suitability class (CSC), QL - Quesnel lower CSC, CH - 100-Mile House higher
CSC, KH - Kamloops higher CSC, PH - Princeton higher CSC, PL - Princeton lower
CSC. Cities of Prince George and Vancouver added for orientation represented by black
triangles. Blue color represents major rivers and lakes. Coordinates for sampling
locations can be found in table 2.1. Map created in ArcMap v. 9.2.

18

19

Figure 2.2. Diagram of an example of a fixed-radius experimental plot. • represents the
central mountain pine beetle bait (supplied by Pherotech International, Inc., Delta, BC,
Canada) attached to a tree, • represents lodgepole pine trees with dbh greater than 15
cm. All lodgepole pine within the 7 m radius with a dbh greater than 15 cm were
sampled prior to beetle flight in July 2006. A minimum distance of 25 m between the
central bait trees separated the plots.

20

•

A

7 meters

21

Osborne & Co., Harrison, N.J. 07029, U.S.A.). Each phloem and bark disk was stored in
an individual envelope (#1 coin envelopes, 5.7 cm x 8.9 cm, Staples brand, #438346) and
immediately placed onto dry ice where it was kept until it could be transferred to a -80°C
freezer (700 Series Formula ULT Freezer, Thermo Electron Corporation) in the
laboratory. All discs were stored at -80°C until they were shipped to the British
Columbia Ministry of Forests and Range Forest Research Laboratory, Victoria, BC, for
processing and analysis (see below).
In August, at the end of the major beetle flight (20-24 August, 2006) entrance
holes in a 10 cm x 20 cm rectangle placed on the east and west side of each attacked tree
in each plot at dbh were counted to determine the MPB attack density. The following
spring (May, 2007) the trees in the plots were revisited to count pupal chamber density as
a measure of beetle reproductive success on the trees. A 10 cm x 20 cm rectangle was
placed on the east and west side of each attacked tree at dbh. The bark was peeled off
and the number of pupal chambers in the rectangle were counted. In between these
sampling periods, woodpeckers or other animals had removed the bark of several
experimental trees resulting in a reduced data set. Thus, it could not be determined in all
cases whether lower pupal chamber numbers were due to predation or larval failure due
to being unprotected because of removed bark. In addition, several replicates were no
longer available for assessment in the spring of 2007. Therefore, only a subset of the
entire sampling group was sampled which served to reduce the data set, particularly at the
northern-most site.
Phloem samples were processed using gas chromatographic-flame ionization
detection analyses (GC-FID) to identify compounds by matching their retention time with

22

synthetic standards (Table 2.2) at the British Columbia Ministry of Forests and Range
Forest Research Laboratory. Frozen (-80°C) phloem samples were ground in liquid
nitrogen and the sample was extracted using 4 ml of hexane (with 250 ppm pentadecane
as an internal standard) for 48 h. Samples were then inverted to mix, allowed to settle for
24 hours, after which 0.5 ml of solution was transferred to a 2 ml autosampler vial for GC
analysis using either a PerkinElmer Clarus500, or PerkinElmer AutoSystem with built in
autosampler, fitted with an INNOwax column (J&W, 25m x 0.2mm ID, 0.4u film). The
injection was split (35 ml/min, -39:1; injector temperature 200°C). Helium was utilized
as the carrier gas (flow rate 21 PS I, 0.90 ml/min at 60°C). The oven temperature was
held at 60°C for 1 min, temperature was increased at a rate of 3.0°C/min to 85°C, then
increased at a rate of 8.0°C/min to 170°C. Finally, the temperature was increased to
250°C (rate of 20.0°C/min) and held for 7.0 min. The remaining contents of the vials
containing the extracted phloem and bark were evaporated in a fume hood and then oven
dried at 70°C overnight to remove residual moisture. A dry weight was obtained to
determine a moisture correction, which was applied to the results.
All data were analyzed using R v.2.6.2 (R Development Core Team, 2008).
Values of <5 ppm were considered to be zero for analysis. For all tests in which
differences between locations were examined, only samples from the lower CSC sites at
all four locations were examined. Tests examining the difference between CSCs were
done using data derived from the higher CSC and lower CSC sites in the northern-most
and southern-most sampling locations. Graphs were created using R v.2.6.2. (R
Development Core Team, 2008) and GNUPLOT v. 4.2.3.

23

Table 2.2. List of chemical standards used to process all phloem samples using GC-FID.
Compound
Purity (%)
Manufacturer
Supplier
borneol
99.00
Aldrich
Sigma Aldrich
bornyl acetate
97.00
Aldrich
Sigma Aldrich
camphene
95.00
Aldrich
Sigma Aldrich
camphor
100.00
Aldrich
Sigma Aldrich
2-carene
98.00
Fluka
Sigma Aldrich
A-3-carene
99.00
Fluka
Sigma Aldrich
a-caryophyllene
99.00
Fluka
Sigma Aldrich
a-copaene
90.00
Fluka
Sigma Aldrich
a-cubebene
90.00
Aldrich
Sigma Aldrich
/7-cymene
100.00
Fluka
Sigma Aldrich
a-humulene
98.00
Fluka
Sigma Aldrich
limonene
100.00
Fluka
Sigma Aldrich
linalool
97.00
Fluka
Sigma Aldrich
myrcene
90.00
Fluka
Sigma Aldrich
ocimene
65.00
Fluka
Sigma Aldrich
a-phellandrene
95.00
Fluka
Sigma Aldrich
P-phellandrene (1,8-cineole)
95.00
Aldrich
Sigma Aldrich
a-pinene
100.00
Aldrich
Sigma Aldrich
P-pinene
99.00
Fluka
Sigma Aldrich
pulegone
99.00
Fluka
Sigma Aldrich
sabinene
99.00
Indofine
Indofine
a-terpinene
85.00
Aldrich
Sigma Aldrich
y-terpinene
97.00
Aldrich
Sigma Aldrich
a-terpineol
90.00
Aldrich
Sigma Aldrich
terpinolene
97.00
Fluka
Sigma Aldrich
a-thujone
100.00
Fluka
Sigma Aldrich
a
Sigma-Aldrich Corp., 3050 Spruce St., St. Louis, MO 63103, USA; INDOFINE Chemical Company, Inc.,
121 Stryker Lane, Bldg 30, Suite 1, Hillsborough, NJ 08844, USA.

24

2.2.2. Terpene variation by location and by CSC
Data were checked for homogeneity of variance using a Levene's test (Mickey et
al. 2004) and a Shapiro-Wilk normality test. Phloem resin terpene data that met both
assumptions were analyzed using ANOVA (a = 0.05) followed by a multiple comparison
of means with a Tukey's post-hoc (a = 0.05). Data that could not be transformed to meet
the assumptions of homoscedasticity and normality were analyzed using a Kruskal-Wallis
rank sum test followed by a pairwise Wilcoxon's rank-sum test (a = 0.05).

2.2.3. Terpene effects on attack density and beetle reproductive success
Attack density at and between locations and CSCs were analyzed using ANOVA
(a = 0.05) followed by a multiple comparison of means with a Tukey's contrast (a =
0.05) to determine differences between attack density at the locations. A square-root
transformation was necessary to meet assumptions for these tests based upon a visual
examination of the residual plot for the comparisons between locations and CSCs.
A linear mixed-effects model was used to examine the effects of specific terpenes
and geographic variation on attack density and pupal chambers. The model, designed to
examine effects on attack density, was created using a combination of backward
elimination and forward addition, with all of the tested terpenes (but did not include the
total amount of all tested terpenes) eliminating highest F-values until all variables
remaining in the model were significant (a = 0.05). The final model contained a number
of fixed effects: dbh, location, and concentration of limonene. The random effect was
plot. No transformation was required to meet the assumptions of normality and

25

homoscedasticity based upon a visual inspection of the residual plots. The model
examining the variation in attack density by CSC class in the northern-most and
southern-most sample site required a square root transformation of attack density to meet
assumptions of normality based upon a visual examination of histograms. Borneol, apinene, dbh, and site were fixed effects and plot was the random effect.
The model for examining effects on pupal chamber density was created using a
combination of backward elimination and forward addition, eliminating the highest Pvalues until all variables remaining in the model were significant (a = 0.05). No
transformation was necessary to meet the assumption of normality based upon a visual
examination of the data. In addition, there was a non-linear relationship between attack
density and the number of pupal chambers. This was expected because intraspecific
competition for resources should reduce larval success before they have a chance to
pupate, regardless of host suitability. This outcome was accounted for in the model. The
model contained fixed effects of location, dbh, and attack density with random effects of
replication and individual tree.
2.2.4. Terpene effects on tree survival
Trees were also reassessed in the spring for survival and to count pupal density. Each
tree was categorized into binomial survival classes (green = alive, red/fading = dead) and
a combination of backwards elimination and stepwise addition was used to create a
binomial generalized linear model with random effect of plot, in which all terms were
significant (a = 0.05).

26

2.3

Results

2.3.1. Variation of terpenes between locations and climatic suitability classes
There were significant differences between the amounts of almost all the terpenes
between locations (Table 2.3). The northern-most site, Quesnel, often contained
significantly higher amounts of many of the analyzed terpenes. Total terpenes were also
significantly greater at that site. Most individual terpenes comprised < 1% of the total
terpenes present in the tree, but, seven terpenes comprised, on average, > 1 % of total
terpenes. These terpenes, which will be referred to as the predominant terpenes, were: A3-carene, limonene, myrcene, p-phellandrene, a-pinene, P-pinene, and terpinolene. They
showed similar trends across the sampled geographic area, with their levels at the
Quesnel sites being significantly higher than elsewhere for each of the seven (Figure 2.3).
A comparison between the higher and lower CSCs at Quesnel and Princeton showed
significant differences within each location between the CSCs in the levels of terpene
compounds. There were also significant differences between the locations, although
these differences were not as consistent (Table 2.4). The higher CSC sites in Q and P
were often not significantly different from each other in terms of particular terpenes. The
lower CSC site in the northernmost site had consistently higher levels of the predominant
seven terpenes while the lower CSC site in the southernmost site generally contained
lower concentrations of those same compounds compared with the higher CSC site at that
location (Figure 2.4).

27

K
P

c

Q

Q
C
K
P

Q
C
K
P

28

Borneol
Bornyl Acetate
Camphene
Camphor
2-Carene
A-3-Carene
q-Caryophyllene
51.6±18.9 a
240.0±81.5
179.9±29.0a
15.0±3.6a
1946.6+285.7a
46.0±6.r
0
70.6+14.6
7.6±1.9 b
46.9+5. l b
31.1+2.7"
580.3±122.6 b
7.9+2.2"
0
60.6±4.5
3.1+0.9°
31.9+3.9b
9.9+1.9 a
223.6±48.6C
3.7+1.2b
0
59.5+5.2
5.6+1.l b °
42.3±3.6b
2.1+0.7°
517.6+146.4"
8.8+2.7b
0
2
2
2
/3=0.3,
X 3=28.8,
X 3=105.8,
/ 3 =70.8,
^3=77.5,
X23=90.2,
P=0.956
P<0.001
P<0.001
P<0.001
P<0.001
P<0.001
q-Copaene
a-Cubebene
p-Cymene
a-Humulene
Linalool
Myrcene
Limonene
827.0+62.2 a
51.7±6.8 a
4.6±1.7 a
31.5+4.73
93.9±12.1 a
1891.9+350.9a
130.8±13.6a
bc
ab
a
b
215.3±25.3"
4.5+1.3
3.0±1.2
26.1±5.7
31.3±6.5
502.3+90.0"
16.9+2.3"
177.5+17.8"
2.5±1.6 b
0.7+0.5"
ll.l±2.7 b
16.3+4.0"
565.5±75.7C
10.4+2.7°
211.4+16.9"
8.0+2.3°
0.7±0.4ab
10.6±2.1b
16.7+4.3"
335.2±53.l"
16.4±3.3"°
/3=108.4,
X23=91.5,
223=9.1,
^3=20.8,
^3=57.7,
X23=66.5,
X23=123.8,
P<0.001
P<0.001
P=0.028
P<0.001
P<0.001
P<0.001
P<0.001
Ocimene
a -Phellandrene
p-Phellandrene
a-Pinene
P-Pinene
Pulegone
Sabinene
123.8+33.1"
306.4±18.0a
17869.5±972.5a
1366.7±147.3 a
2598.7±307.1 a
30.3±5.4a
260.4±19.2 a
15.7±4.8b
91.7±8.6 b
6266.4+468.9"
395.8±31.6"
584.1±75.9"
9.4±2.1 b
88.8±7.9 b
b
b
b
22.4±5.8 °
73.1+7.4"
4320.8+397.0°
276.5+20.6°
410.8±53.4
6.1±1.7
55.1±6.7°
33.8±8.6C
92.6+8.7"
5538.9+457.4"°
363.2+28.5"°
623.1+78.0"
8.0+1.8b
78.9±8.0 b
2
2
F3,23(,=91.9,
/3=46.2,
X 3=106.3,
/V105.5,
X 3=75.7,
^3=27.6,
^3=104.1,
P<0.001
P<0.001
P<0.001
P<0.001
P<0.001
P<0.001
P<0.001
q-Terpinene
Y-Terpinene
Terpineol
q-Thujone
Terpinolene
Total
14.5+2.7a
27.1+3.7a
124.7±10.2a
0
28639.4±1551.4a
406.6+40. l a
Q
5.7±1.5"
3.3+1.4"
25.9+4.8"
0.3+0.3
131.2+17.5"
9162.3±689.0b
c
b
b
K
2.1±0.9
16.3±3.0"
0.1 ±0.1
1.9+1.l
130.3+20.1"
6432.4+530.8°
P
4.2±l.l b
3.9±1.6b
23.4±5.2b
0.3±0.3
138.3+19.7"
8143.5+667.8b°
X23=22.1,
X23=69.2,
F3,236=109,
^3=100.6,
^3=64.8,
P<0.001
P<0.001
P<0.001
P<0.001
P<0.001

Table 2.3. Mean amount of terpenes present (ppm, + 1 SE) for constitutive defenses by location (Q - Quesnel site [lower CSC], C 100 Mile House site, K - Kamloops site, P - Princeton site [lower CSC]). Different letters indicate a significant difference with in
each terpene (Tukey's contrast or Wilcox's rank-sum pairwise test, a = 0.05).

QA
QB
PA
PB

QA
QB
PA
PB

QA
QB
PA
PB

QA
QB
PA
PB

Borneol
35.1±7.4a
51.6±18.9a
20.8±11.7b
5.6±l.l b
X23=30.9,
/•<0.001
a-Cubebene
3.9±1.7
4.6±1.7
2.5±1.0
0.7+0.4
/3=4.8,
P=0.184
p-Phellandrene
9917.4+941.l a
17869.5±972.5b
7774.3±601.2a
5538.9+457.4°
X23=81.2,
P<0.001
Terpinolene
318.7±49.7a
406.6+40. l b
251.3±32.3 a
138.3±19.7C
223=33.5,
F<0.001

Bornyl Acetate
217.8±49.9 a
240.0±81.5 b
149.5±62.7b
59.5+5.2"
X23=21.4,
F<0.001
p-Cymene
30.4±4.3 a
31.5±4.7 a
20.9±4.7 ab
10.6±2.1 b
X23=14.27,
P=0.003
a-Pinene
1000.4±110.0a
1366.7±147.3 b
610.8±60.7C
363.2±28.5 d
X23=68.2,
P<0.001
a-Thujone
1.9±0.9
0.000
0.3+0.2
0.3+0.3
Camphene
128.4+19.6a
179.9+29.0b
91.7+24.4 a
42.3±3.6°
/3=70.2,
P<0.001
a-Humulene
36.7±6.5 a
93.9±12.1 b
29.3±46.1 a
16.7+4.3a
X23=46.9,
P<0.001
P-Pinene
1363.7+161.7a
2598.7+307.1"
977.9+131.l ac
623.1±78.0C
^3,227=17.59,
P<0.001
Total
15846.3±1361.1a
28639.4±1551.4b
11993.3+819.9a
8143.5±667.8C
X23=90.8,
P<0.001

Camphor
27.3+2.4a
15.0+3.6"
24.5±12.2b
2.1+0.7C
*23=66.7,
P<0.001
Limonene
620.6+128.5ac
1891.9+305.9b
675.2+122.8a
335.2±53.1 c
/3=53.0,
P<0.001
Pulegone
35.1±8.0ab
30.3±5.4a
21.6±5.1 ab
8.0+1.8b
X23=13.3,
P=0.004
Linalool
54.5+6. l a
130.8+13.6"
32.4±4.1 c
16.4±3.3 d
/3=91.3,
P<0.001
Sabinene
211.7+43.5 a
260.4+ 19.2b
121.1±13.1 a
78.9+8.0°
X23=71.3,
P<0.001

2-Carene
0.5+0.5
0.000
0.000
0.000
A-3-Carene
1121.9+240.4a
1946.6±285.7b
612.0±123.4ac
517.6±146.4°
^3=50.5,
P<0.001
Myrcene
379.3+33.9"
827.0+62.2b
338.3±28.6a
211.4±16.9C
X23=89.8,
P<0.001
a-Terpinene
12.8±3.1ab
14.5+2.7a
6.2+1.6ab
4.2+1.4b
X23=9.2,
P=0.027

aCaryophyllene
19.6±3.7a
46.0+6. l b
14.3+2.6a
8.8+2.7a
X23=53.2,
P<0.001
Ocimene
33.5+7.7 a
123.8±33.1 b
38.1±6.7 a
33.8±8.6 a
^3=21.2,
P<0.001
y-Terpinene
18.4+4.4"
27.1+3.7"
7.7±2.0a°
3.9+1.6°
X23=33.4,
P<0.001

29

a-Copaene
18.5±4.2a
51.7+6.8"
9.6+2.8a
8.0+2.3a
^3=49.7,
P<0.001
a-Phellandrene
164.9±16.9a
306.4+18.0"
136.2±10.9a
92.6±8.7°
X23=75.8,
P<0.001
Terpineol
73.3±9.4 a
124.7±10.2b
26.6±4.6C
23.4±5.2C
/3=89.0,
P<0.001

Table 2.4. Mean constitutive levels terpenes (ppm + 1 SE) by climatic suitability class (CSC) and location (QA - Quesnel site, higher
CSC, QB - Quesnel site, lower CSC, PA - Princeton site, higher CSC, PB - Princeton site, lower CSC). Different letters indicate a
significant difference between CSC and location (Tukey contrast or Wilcoxon rank sum test, a - 0.05).

Figure 2.3. Mean levels (ppm, + 1 SE) of the predominant seven terpenes found at four
locations. P-Phellandrene was found at much higher levels and is scaled to the right-hand
y-axis. All six other terpenes are scaled to left-hand axis. Different lower case letters
indicate significant difference within each terpene between locations (Tukey's contrast or
Wilcox's rank-sum pairwise test, a = 0.05).

30

(3S I + 'Uldd) 9U9jpUBH9lld"^l J ° I9A9J U139p\[

( a s T + 'mdd) 9U9di9j j o |9A9| ire9j/\[

2.3.2. Effects of terpenes on attack density and beetle reproductive success
There were significant differences in attack density between the four locations.
The mean attack density was highest in the northern-most sampled site and lowest in the
southern-most sampled site. There was no significant difference in mean attack density
per tree between Quesnel and 100-Mile House or between Kamloops and Princeton, but
there were significant differences between those two groups of locations (i.e., Quesnel
and 100-Mile House together differed from Kamloops and Princeton) (Figure 2.5).
Attack density was influenced by several of the measured parameters. Location,
dbh, and the concentration of borneol, limonene, and a-pinene were all significant (a =
0.05) (Table 2.5). This relationship held for lodgepole pine at these experimental
locations with a dbh in the range of 15-34 cm and concentrations of borneol ranging from
0-1000 ppm, limonene from 0-12530 ppm, and a-pinene from 0-7180 ppm. An increase
in dbh and an increase in the concentration of limonene and borneol was correlated with
an increase in attack density. An increase in the concentration of a-pinene was associated
with a decrease in attack density (Table 2.7).
Location, dbh, and attack density played a significant role in larval success as
measured by pupal chamber density (Table 2.6). The levels of individual, or combined,
constitutive terpenes did not have a significant effect on pupal chamber numbers (Table
2.7).

32

Figure 2.4. Mean levels (ppm, ± 1 SE) of the predominant seven terpenes found at two
locations and two climatic suitability classes (CSC). P-Phellandrene was found at much
higher levels and is scaled on right-hand y-axis. All six other terpenes are scaled to lefthand y-axis. Different lower case letters indicate significant difference within each
terpene between each location and CSC (Tukey's post-hoc or Wilcox's rank-sum
pairwise test, a = 0.05).

33

0

500

1000

1500

2000

2500

3000

c

iac

ac

A-3-Carene Limonene IV^rcene a-Pmene (3-Pinene Terpiriolene

iac ac a

= Princeton Lower

= Princeton Higher

= Quesnel Lower

= Quesnel Higher

(3-Fhellandraie

a

0

2000

4000

6000

8000

10000

12000

1 14000

, 16000

J

1 18000

] 20000

Figure 2.5. Box plots of attack density (attacks/m2) by sampling locations (Q-Quesnel,
C-100-Mile House, K-Kamloops, P-Princeton). Different lower case letters indicate
significant differences (ANOVA, Tukey's post-hoc, a = 0.05, F3,236=17.53, P < 0.001)
between locations.

35

a

250

200

150

100

50 J

Quesnel

T

T

100 Mile House

Kamloops

Princeton

36

Table 2.5. Effect of location, levels of borneol, limonene, and a-pinene, and dbh on
attack density (attacks/m ) with their associated marginal fit F-value and P-value (df =
223).
Variable

F-value

P-value

Intercept

42.72

<0.001

Location

16.44

<0.001

Borneol (ppm)

11.35

<0.001

Limonene (ppm)

9.14

0.003

a - Pinene (ppm)

6.10

0.01

dbh (cm)

19.27

<0.001

37

Table 2.6. Effect of location, attack density (attacks/m ), and dbh (cm) on the pupal
chamber density (chambers/m ) with their associated marginal fit F-values and P-values
(df=223).
Variable

F-value

P-value

Intercept

223.09

<0.001

Location

6.30

0.001

Attack Density (attacks/m2)

14.83

<0.001

Attack Density2 (attacks/m2)

11.98

0.001

dbh (cm)

21.72

<0.001

38

42.09

(11.94)

-504.90

(61.38)

Attack Density

(attacks/m2)

Pupal Chamber

Density

(105.96)

-415.30

(27.14)

84.19

House

100 Mile

(47.73)

-366.24

(8.31)

37.33

Kamloops

(46.54)

-316.18

(8.37)

61.75

Princeton

Location of interest is used as intercept in ANCVOA model.

(chambers/m2)

Quesnel

Response

Location3

(0.05)

(0.0021)

0.0065

(ppm)

(PPm)
0.15

Limonene

Borneol

(0.0056)

-0.014

(ppm)

a - Pinene

3.42
(1.10)

(4.65)

Density

Attack

23.52

(0.84)

3.68

(cm)

dbh

(0.006)

-0.02

Density

Attack

Table 2.7. Effect of location, levels of borneol, limonene and a - pinene, dbh, and attack density on both attack density and pupal
chamber density. All effects are significant at P < 0.05. Means (SE below) are presented.

39

There were significant differences in attack density between the sampling
locations but not at the same location between CSCs. Higher attack densities were in the
northern location (Figure 2.6).
There was a significant effect of diameter, concentration of borneol and a-pinene
and the CSC classes on attack density (Table 2.8). This relationship again held for these
locations, and the range of levels of terpenes found at these sites. Increases in borneol
appears to significantly but weakly correlate with increase in attack density as does
increase in dbh (Table 2.9). Increases in a-pinene levels appear to relate to decrease in
attack density.

2.3.3. Terpene effects on tree survival
The model used to calculate the probability of a tree surviving an outbreak was
found to be dependent on levels of limonene and total terpenes, the location of the trees,
and dbh (Table 2.10). It is valid for trees with a dbh in the range of 15 to 34 cm,
concentrations of limonene ranging from 0 to 12530 ppm, and total terpene
concentrations ranging from 600 to 69900 ppm. The probability of a tree surviving a
MPB outbreak (Y) in an area can be calculated by applying the canonical link function to
the values found in table 2.10.
p(Y) = exp (Bo + Bix)
1 + exp (3o + Pi-*)

Limonene concentration, total terpenes, and dbh had a negative effect on the probability
of survival.

40

Figure 2.6. Box plots of attack density (attacks/m ) by sampling locations (Q- Quesnel,
P- Princeton) and climatic suitability classes (high and low). Different letters indicate
significant differences (ANOVA, Tukey's post-hoc, a = 0.05, F3,226= 19.83, P < 0.001)
between locations.

41

a

Quesnel
High

Quesnel
Low

Princeton
High

Princeton
Low

42

Table 2.8. Effect of location and climatic suitability classes (Site), borneol, a - pinene,
and dbh on attack density (attacks/m2) with their associated marginal fit F-value and Pvalues(df=214).
Variable

F-value

F-value

Intercept

235.89

<0.001

Site

23.28

<0.001

Borneol (ppm)

4.68

0.03

a - Pinene (ppm)

6.40

0.01

dbh (cm)

16.91

<0.001

43

Table 2.9. Effect of location and climatic suitability class, levels of borneol and a pinene, and dbh on attack density (attacks/m2)a. All effects are significant at P < 0.05.
Means (SE below) are presented.
Location6

Quesnel
Response

a
b

High

Princeton

Low

High

Low

Borneol

a - Pinene

dbh

(ppm)

(ppm)

(cm)

Attack Density

4~87

37i9

22A

L04

0.004

-0.0006

0.24

(attacks/m2)

(1.19)

(0.49)

(0.46)

(0.48)

(0.002)

(0.0002)

(0.058)

Response is square root transformed
Location of interest is used as intercept in ANCOVA model.

44

(1.917)

(1.346)

Location of interest is used as intercept in model.

a

L267

4.584

Tree survival

House

100 Mile

Quesnel

Response

(1.H4)

4.859

Kamloops

Location"

(1.095)

5.075

Princeton

(1.757e-04)

-4.175e-04

Limonene(ppm)

(4.022e-05)

9.939e-05

(ppm)

Total terpenes

(-3.484e-01)

-3.484e-01

dbh (cm)

Table 2.10. Effect of location, levels of limonene and total terpenes, and dbh on the probability of tree survival. All effects are
significant at P < 0.05. Means (SE below) are presented. Values are used in the canonical link function to determine probability.

45

2.4

Discussion

2.4.1. Variation in terpenes and environmental conditions
Many studies have been conducted for the purpose of studying the relationship
between levels of resin terpenes and susceptibility of trees to attacking insects (Seybold
et al. 2006, Wallin and Raffa 1999, Klepzig et al. 1996, Christiansen et al. 1987).
However, lodgepole pine occupies a large and partially overlapping geographic ranges in
which they also interact with many other organisms including MPB. Thus, there is
potential for geographic differences in the interactions between the bark beetle and its
host trees in different regions due to complex co-evolutionary interactions (Thompson
1997). I found significant differences in the titer of various terpenes between the
locations that I intensively sampled. This was particularly true of the northern-most site
(Quesnel) where I found significantly higher concentrations of most of the terpenes
tested, including A-3-carene, limonene, myrcene a- and P-pinene, P-phellandrene,
terpinolene, and total amount of terpenes. This is a population that has not had
significant prior exposure to MPB outbreaks. Limonene, A-3-carene, and a-pinene, are
terpenes that have been found to be lethal to MPB eggs during continuous contact and
volatile exposure (Raffa and Berryman 1983b). These terpenes are also toxic to adult D.
brevicomis when exposed to the vapors (Smith 1965). These geographic differences in
the terpene profiles could be due in part to the level of historical interaction these
populations have had with MPB. These differences in terpene profiles between these
locations also indicates that there is potential for differences in the interaction between
the MPB and the host tree, which I did observe and is discussed in the following sections.

46

The lower CSC site near Quesnel generally had significantly higher
concentrations of most of the terpenes - and of all of the seven predominant lodgepole
pine phloem resin terpenes. On the other hand, the lower CSC site near Princeton
generally had significantly lower concentrations of most terpenes - and of all of the seven
predominant lodgepole pine phloem resin terpenes. It is likely that differences between
these sites may be due to environmental differences in addition to historical beetle
pressure. The environment across a large geographic range is variable. Since defenses
require an investment of resources (Herms and Mattson 1992), differences in defense
against bark beetles and their associated fungi to environmental stressors may be linked
to factors that affect photosynthetic efficiency (Christiansen et al. 1987) and other
nutrient limitations (Waring and Pitman 1985, Lewinsohn et al. 1993, Gilmore 1977).
For instance red pine seedlings stressed by low light were found to have lower induced
chemical changes when challenged by a bark beetle vectored fungus (Klepzig et al.
1995). Thus, terpene composition may vary across a geographic range not only because
of historical beetle pressure, but also because of environmental differences at different
locations. Environment and historical beetle pressure are also linked because ectothermic
insects are highly dependent on temperature and other climate variables (Carroll et al.
2004). In fact, the variables utilized to determine the historical likelihood of beetle
success, which in turn determined the CSCs that utilized for this analysis, are based upon
historical climatic data (Carroll et al. 2004).

47

2.4.2. Terpene effects on attack density and beetle reproductive success
While phloem resin terpene levels varied by location (Table 2.3), only the levels
of limonene, a-pinene, and borneol were found to significantly affect attack density
between locations (Table 2.6.). High levels of limonene explained an increase in attack
density, which was an unexpected result. Limonene has been found to be toxic to the
southern pine beetle (Dendroctonus frontalis Zimmermann) in a laboratory bioassay
(Coyne and Lott 1976). In addition, of the compounds tested by Smith (1965) it is the
most toxic monoterpene vapor to the western pine beetle. It would therefore be expected
that higher limonene levels would result in lower beetle attack. Between the locations
and higher versus lower climatic suitability classes, limonene was not a significant
variable in the linear-mixed-effects model, despite the significant difference in levels
present in the resin. Any toxic effects of limonene, may not be evident with this level of
beetle pressure on the host trees, but could potentially play a role in endemic or incipient
endemic MPB populations. At lower population levels, when there are fewer
conspecifics on the landscape to attract to the same tree, toxicity may be more of a barrier
to successful colonization.
Attack density increases with an increase in the concentration of borneol.
Borneol does not appear in large concentrations in these trees, thus it was a surprise that
this terpene was significant in the model. Raffa and Berryman (1983b) found evidence
that lodgepole pines more susceptible to attack may have higher levels of oxygenated
monoterpenes, such as borneol, than do less susceptible lodgepole pines. This could
explain why the trees with higher attack density also had higher concentrations of this

48

terpene. It is also possible that borneol is highly correlated with a factor, environmental
or chemical, that is significant to the MPB.
Increased a-pinene levels appear to be correlated with a decrease in attack density
(Table 2.5.)- This was also surprising as a-pinene serves as both a precursor to the
aggregation pheromone, ^rans-verbenol, (Hughes 1973b, Conn et al. 1984) and was
shown to have synergistic properties with trans-verbeno\ in the laboratory (Pitman 1971).
It can also, however, be auto-oxidized to verbenone, an anti-aggregation pheromone
(Borden et al. 1987, Hunt et al. 1989). It may be argued that higher levels of a-pinene
allow MPB to optimize attack on the tree (Raffa and Berryman 1983a) which, in areas of
high beetle population pressure, could reduce the attack density required to kill an
individual tree, reducing intraspecific competition on such trees. Levels of a-pinene were
not found to be significant for tree survival (further discussion in 2.4.3.). This indicates
that on its own, a-pinene did not lower attack to the point of tree survival. However, the
presence of enough a-pinene in the host provided adequate precursor material for autooxidization to verbenone, ultimately lowering the attack density, reducing intraspecific
competition, with excess beetles likely spilling-over to neighboring hosts (Geiszler and
Gara 1978). This complex relationship between MPB and a-pinene may explain why, in
the comparison between all locations, increased levels of a-pinene were correlated with
decreased attack density. This relationship between increased levels of a-pinene relating
to decreased attack density was also found in a comparison between higher and lower
CSCs in the northernmost and southernmost locations.
It may also be important to consider the chirality of the compounds in the tree.
Volatile emissions from the bole of lodgepole pine collected near Princeton, BC were

49

67.7% (-)-a-pinene (Pureswaran et al. 2004). Dendroctonus brevicomis convert the
enantiomers of a-pinene to the corresponding enantiomers of frans-verbenol (Byers
1983). The (+) enantiomer of ?rans-verbenol did not cause attraction of either sex of the
MPB while the (-) and racemic mixture did (Borden et al. 1987). If lodgepole pine
populations differ in the ratio of enantiomers of this terpene, as has been found in
Norway spruce (Picea abies Linnaeus) (Lindstrom et al. 1989), this difference could
translate into differences in the ability of MPB to draw in conspecifics for the mass
attack. In addition, Ryker and Yandell (1983) found that racemic and (-)-verbenone had
antiaggregation properties in both field and laboratory tests, while (+)-verbenone did not.
This could again translate into differences in the MPB ability to utilize the host
effectively.
It is possible that the differences in attack density could be partially attributed to
differences in beetle population pressure at the different locations, and that the
relationships observed between the terpene levels and attack density were circumstantial.
However, all of the sites were selected with fading and red MPB-infested trees nearby
and the central bait in the middle of each plot should have ensured that large populations
were attracted to all of the plots. Once the beetles were initially attracted to the areas by
the baits, the effects of beetle-produced aggregation pheromone in the area should also
have ensured that there was significant population pressure throughout the experimental
areas.
Individual terpenes did not appear to influence beetle reproductive success, as
measured by the density of pupal chambers and the number of adult galleries that were
counted. At high attack density, like I found in this study, the host does not have a

50

suppressive effect on brood development (Raffa and Berryman 1983a). However, dbh,
location, and original attack density did relate to pupal chamber density. Total attacks
and brood production have previously been positively correlated to diameter (Safranyik
and Carroll 2006) which is consistent with my findings. Attack density should strongly
correlate with the number of pupal chambers but not in a linear way as once the optimum
attack density has been exceeded, intraspecific competition would be the main factor that
would reduce larval success and, ultimately, total pupal chambers (Raffa and Berryman
1983a).
The importance of the flow of resin, rather than differences in individual terpenes,
has been shown to be important in tree defense against bark beetles (Smith 1975). Resin
flow was not measured in this study, but it may have contributed to the differences in
attack density and reproductive success that I observed. However, since beetles must
choose a host and enter its bark before resin flow occurs, constitutive concentrations of
the terpenes should be a more important cause of differences in host preference.

2.4.3. Variation in resin terpene levels and tree survival
Large dbh, high concentrations of limonene, or high total terpenes are correlated
with a reduction in the probability of a tree surviving an outbreak in all areas that I
sampled. Because the sampling pertaining to tree survival represents a reduced data set,
it is possible the analysis misrepresents some dynamics that may occur elsewhere,
particularly in the northern area where the sample size was the most reduced. However,
this analysis does serve as an indication of what may be happening in the stands across
the locations, as the trend was found at all four locations. A previous study has

51

demonstrated that large-diameter lodgepole pine are generally the first trees to be
attacked and killed in a stand (Cole and Amman 1969). Phloem thickness, which is
related to tree size, has been correlated with beetle success (Amman 1972, Amman and
Pace 1976, Berryman 1976). Cole et al. (1981) found that resin monoterpene
concentration is higher in thicker phloem, which also relates terpene levels to dbh
(Amman and Cole 1983). While terpenes are often viewed as defense compounds, bark
beetles may in fact use such compounds as kairomones to identify and locate suitable
hosts. Thus lodgepole pine trees with high levels of resin terpenes, which are also likely
to be bigger and more visible, may be more apparent to foraging MPBs.
There should be selective pressure on MPBs to efficiently locate suitable hosts.
They must find a susceptible host in stands that also contain non-hosts and resistant hosts
(Atkins 1966). In addition, they have a limited energy supply to expend (Bell 1990), and
the less time that they spend searching for a host, the less time they are exposed to other
hazards such as inclement weather or predators and parasitoids associated with the bark
of trees (Dahlsten 1982). Pioneering beetles may locate hosts using visual cues (Cole and
Amman 1969, Shepherd 1966) or at random (Hynum and Berryman 1980) and then test
the potential host for suitability after landing on it (Pureswaran and Borden 2003, Wallin
and Raffa 2000, Elkinton and Wood 1980). But they also likely utilize chemical cues for
host selection while in flight. Moeck and Simmons (1991) found that MPBs are attracted
to host odors, even in the absence of aggregation pheromone or visual cues. Like other
bark beetles, this species has the ability to detect and avoid both host and non-host
volatiles in flight (Huber et al. 2003). Pureswaran et al. (2004) found that there were
enough quantitative differences in the volatile monoterpene profiles of several conifer

52

species that they could be used for host identification by flying insects. It is possible that
the trees in the south, with more previous exposure to MPB outbreaks, have been selected
to reduce their apparency to the beetle by maintaining low concentrations of terpenes
compared to those in the northern-sampling area. The lower CSC in Quesnel, the
northernmost site which had higher levels of all of the predominant seven terpenes than
the higher CSC in the same location, may support this. If there had been historical MPB
outbreaks near Quesnel they would more likely have occurred in the areas that were
climatically more suitable for the beetle. However, the lower CSC in Princeton had
lower levels of the predominant seven terpenes which is not consistent with this theory.
This may be explained by Princeton's location (southern British Columbia) where most
historic beetle outbreaks in Canada have been restricted to (Carroll et al. 2004). It is
probable that beetles, during prior outbreaks, were pushed into climatically less-suitable
areas around Princeton and that high tree mortality has occurred even in areas classified
in the lower CSCs. Locations with lower historical climatic suitability closer to the edge
of the historical range of beetles may have been less exposed to prior beetle outbreaks
due to lower population levels.
In an analogous situation, Tomlin et al. (1997) found that four clones of Sitka
spruce (Picea sitchensis (Bong.) Carr.) classified as resistant to the white pine weevil
(Pissodes strobi Peck) had significantly lower levels of ten terpenes than did the clones
classified as susceptible. Their results indicated that there were multiple resistant
chemotypes based on the terpene profiles, but that the resistance could be classified as
either repellency or lack of apparency.

53

In addition, the presence of other suitable hosts for the MPB, e.g., ponderosa pine
(Wood 1982) found in that same region, could also create selective benefits for trees that
the beetles cannot find. In the sampled stands individual lodgepole pines that make it
more difficult for the beetles to locate them by having reduced levels of constitutive
terpenes may be better off since other species of potentially more apparent hosts are
available. This apparency strategy may be more effective than producing enough
constitutive defenses to prevent attack may be more energy efficient for those trees that
have other species present in the same region.
It is not only essential for beetles to locate a suitable host, they must compete with
other species such as the western pine beetle, also found in ponderosa pine and in the
same southern region in British Columbia (Wood 1982). These variations in the
ecosystems, alternative hosts, and a variety of other competitors for resources across this
geographic range could result in different selective pressures in different populations of
MPB and lodgepole pine (Thompson 1997), which could have resulted in this variation in
terpene profiles between locations.

2.4.4. Implications for coevolution
The results of this study demonstrate that there is a large variation in the phloem
resin terpene composition of lodgepole pine across their range in British Columbia and
that variable tree survival seems to be partly influenced by the presence of certain
terpenes. Dendroctonus spp. were more tolerant of resin vapor of their host than nonhost
(Smith 1961a), indicating that the beetles have had selective pressures to adapt to utilize
their particular hosts. But, in order for natural selective processes to work on lodgepole

54

pine in the context of MPB infestation there must be a reproductive advantage to a tree
that survives an outbreak. Lodgepole pine is a species that produces viable seed at an
early age, with cone production beginning at five to ten years of age (Burns and Honkala,
1990). Seed production in lodgepole pine includes some seeds contained in serotinous
cones. If such cones are retained on branches rather than shed to the ground, the seeds
can remain viable for many years (Burns and Honkala, 1990). The ratio of serotinous
cones to cones that open at maturity depends on the geographic region, but even in areas
with high fire frequency the non- serotinous cone phenotype is still maintained (Perry and
Lotan 1979). Trees that survive a MPB outbreak would be able to continue to produce
these cones and their increased contribution of seeds to the soil seed bank may increase
the chances of their progeny to grow in the gaps left by the killed mature lodgepole pine.
On the other hand, if fire immediately followed an outbreak, burning the recently killed
forest, then the likelihood of those individuals who survived the outbreak having such a
reproductive advantage seems minimal. However, Bebi et al. (2003) found that areas in
the White River National Forest in Colorado that had been affected by the spruce beetle
(Dendroctonus rufipennis Kirby) outbreak in the 1940s did not show a higher
susceptibility to forest fires immediately following the infestation. In addition, MPBkilled trees in central Oregon were found to begin falling 3-5 years after death (Mitchell
and Preisler 1998). If the trees that are still alive post-outbreak are able to reproduce for
several more years after an infestation but before a fire, and if cones from trees that fall
have a lower success rate than cones from live trees, then the MPB could exert a
significant selection pressure on lodgepole pine stands in terms of differential survival of
tree progeny.

55

2.4.5. Potential future studies to expand on this study
My experiments were performed in the midst of an outbreak population. Further
studies that are designed to examine similar defense variables at different stages in the
insect population cycle could expand on the relationship between this bark beetle and
lodgepole pine. Wallin and Raffa (2004) showed that the spruce beetle, another eruptive
bark beetle, showed a decreased avoidance of high concentrations of a-pinene with an
increase in conspecifics, and this behavior was more pronounced in epidemic
populations. In addition, an examination of the particular concentrations of terpenes
necessary to affect the success of MPBs in locating suitable hosts, in maintaining
optimum attack density, and in reproductive success could clarify the importance of the
levels of particular terpenes in the phloem resin. For example, it would be interesting to
determine what levels of a-pinene, a terpene utilized by this insect for production of both
aggregation and anti-aggregation pheromone and with potential to be toxic, are necessary
for the beetles to be able to maintain optimum attack density and still avoid toxic effects
of the compound. Further toxicity or behavioral bioassays, which employ synthetic,
commercially-available terpenes individually or in combination, would be valuable to
further elucidate the role of various secondary metabolites in toxic, and in other
interactions of the host with bark beetles. Such assays could include vapor toxicity tests
(Smith 1961b) and feeding bioassays. Feeding bioassays would provide evidence both
for the level of feeding deterrence evoked by the compounds and their toxic or sub-toxic
effects on the insects. In addition to the differences in amounts present in the phloem, it
may be beneficial to examine if there is geographic variation between the chirality of the

56

terpenes found across the geographic range. This could also have implications on the
success of the MPB utilizing its host in different geographic areas as discussed in section
2.4.2.

2.5

Conclusions
•

I found variation in phloem resin components between the sample sites
and between CSCs. There is evidence that this variation influences tree
survival and attack density by MPB. Specifically, I found that phloem
resin terpene levels of borneol, limonene, and a-pinene influenced the
attack density. Phloem resin terpene levels of limonene influenced tree
survival. There did not appear to be any influence of terpene levels on
pupal chamber density.

•

I found that a high level of total terpenes increased the likelihood of
surviving an attack, except for limonene which had a negative relationship
with the likelihood of survival. However, high levels of terpenes may not
always be beneficial to lodgepole pines. Lower levels of specific terpenes
and total terpenes in the south part of my sampling range indicates that
lodgepole pine populations that have had more prior exposure to MPB
outbreaks may have been selected for reduced apparency to the insects.

•

Models of MPB interactions with hosts on a landscape scale could
potentially be refined if the differences in the host defense characteristics,
as they relate to MPB success, were incorporated.

57

•

Coevolution between MPB and lodgepole pine, in terms of host
constitutive defenses, has likely occurred but is at least partially dependent
on the probability and timing of fire following an outbreak.

58

Chapter 3. Comparison of constitutive resin chemistry of lodgepole,
hybrid, and jack pine stands in British Columbia and Alberta
Abstract
The mountain pine beetle (Dendroctonus ponderosae Hopkins) is a significant pest of
lodgepole pine in British Columbia, where it currently has reached an unprecedented
outbreak level and has moved into jack pine forests in Alberta. The ability of jack pine
trees to defend themselves could play a major role in the success of this insect in a new
geographic range and host. Lodgepole pines and jack pines were sampled in central
British Columbia and north-central Alberta for constitutive phloem resin terpene levels of
26 terpenes. Phloem resin terpenes were identified and quantified using gas
chromatography, and data were compared between the two species. Significant
differences existed between levels of terpenes between the two species of pines, aPinene levels were significantly higher in jack pine. This terpene is a precursor in the
biosynthesis of components of the aggregation and antiaggreagation pheromones of
mountain pine beetle. In general, levels of terpenes were lower in jack pine which, based
upon a comparison of lodgepole pine populations in British Columbia, could mean that
jack pine is less apparent to mountain pine beetle. However, lower levels of compounds
that are considered to be toxic to attacking insects, such as A-3-carene, were found in jack
pine, which means that it could prove to be an easily overcome host for mountain pine
beetle. The mountain pine beetle will face a different phloem resin terpene environment
when locating and colonizing a jack pine host in its new geographic range.

59

3.1.

Introduction
The primary host of the mountain pine beetle (Dendroctonus ponderosae, Hopk.,

MPB) in British Columbia (BC) is lodgepole pine (Pinus contorta Dougl. var. latifolia
Engelm.), but this insect is also capable of utilizing other species of conifers including
jack pine (P. banksiana Lamb.) (Furniss and Schenk 1969). Lodgepole pine is found in
northwestern Alberta, throughout BC, and south to California. Its range in BC and
western Alberta is contiguous with stands of jack pine in northeastern Alberta which
extend across Canada and into the United States. In the area in north-central Alberta
where the two species' ranges meet, the two pines are capable of producing fertile
offspring, thus forming a "hybrid zone" (Moss 1949) which has recently been
successfully invaded by the beetle (Langor et al. 2007). The MPB is normally a native to
southern Alberta and BC, and has not had a major impact in jack pine stands in recorded
history, but its recent immigration into stands in north-central Alberta has raised concerns
that it may have the capacity to spread eastward through Canada's extensive jack pine
forest. The MPB is capable of successful reproduction in jack pine, and under laboratory
conditions brood production was comparable in jack and lodgepole pine (Safranyik and
Linton 1982).
The natural movement of a native species into a new habitat is similar to an
invasive species introduction (Logan and Powell 2001). Both scenarios result in a
species encountering a new environment, with potentially new food sources and other
host species, in which they must be able to reproduce. Release from competition and
predation that a species is subjected to in its native habitat is one factor that contributes to
the success of an invasive species (Keane and Crawley 2002), but encountering plants

60

that are not able to defend themselves against specific forms of herbivory may also
enable a species to do well in a new environment. For instance, the emerald ash borer
(Agrilus planipennis Fairmaire), an insect native to Asia, where it is not considered a
major pest, has proven to be very destructive to several species of ash (Fraxinus spp.)
when introduced to North America (Haack et al. 2002, Poland and McCullough 2006).
In a common garden study, tree mortality and emergence hole data indicated that an
Asian species of ash was more resistant to the emerald ash borer than two North
American species (Rebek et al. 2008). Eyles et al. (2007) found that there were
significant differences in constitutive phloem chemistry, including compounds that are
toxic or deterrent to other herbivores, between the Asian species F. manchurica
(Ruprecht) and North American ash species. Trees that share an evolutionary history
with certain pests appear to be more resistant to those pests than do naive trees.
The introduction of the red turpentine beetle, Dendroctonus valens LeConte, in
China is an example of a bark beetle that is able to successfully use previously
unencountered hosts in a new habitat. This bark beetle is, at worst, a minor pest that only
attacks severely weakened trees in its native North American range. In China it is capable
of killing, and reproducing in, mature, seemingly healthy Chinese red pine, P.
tabuliformis (Carr.), and it has reached population levels in that country greater than
anywhere in its native range (Yan et al. 2005). It is still not clear why D. valens has been
so successful in China compared to its native range, but there is a possibility that a lack of
host chemical defense due to no prior history with the pest plays a role. In a similar
fashion, the MPB invading northern Alberta may be successful in the new hybrid and
jack pine hosts, or it may behave like other invasive species of insects that do not reach

61

outbreak populations in their new environment. Factors such as climate and the
abundance and distribution of susceptible and suitable host trees will likely play key roles
in determining the ultimate outcome of the current spread of this insect into the jack pine
forests of North America, but tree chemistry is likely to play a large role in determining
the insect's success, particularly in the event of significant global warming.
One of the major defenses utilized by conifers against attackers is the terpenes
contained in their resin. Terpenes are a large class of chemical compounds that serve
many functions. Terpenes can serve as both attractants and repellants for bark beetles
(Gershenzon and Croteau 1991, Pureswaran and Borden 2003, Keeling and Bohlmann
2006) and have been implicated in host selection (Moeck and Simmons 1991). They
have been found to be toxic (Smith 1965, Smith 1963, Smith 1961a), precursors to
aggregation and anti-aggregation pheromones (Conn et al. 1984), and synergists with
pheromones (Miller and Borden 1990, Conn et al. 1983, Borden et al. 1983). They are
important to all stages of a beetle's successful colonization of a host and, therefore,
reproductive success. Given that the MPB is very successful in lodgepole pine, a
comparison of the constitutive resin chemistry between lodgepole pine in British
Columbia and Alberta with jack pine and their hybrids in the northern Alberta region
most likely to experience attacks may provide insight into the beetles' ability to locate
suitable hosts, attract conspecifics, and utilize the resource to successfully reproduce in
this new host.

62

3.2.

Methods
Uninfested jack, hybrid, and lodgepole pine trees, determined by the absence of

pitch tubes and frass, were sampled from the Alberta/Saskatchewan border to Prince
George, BC (Figure 3.1). I attempted to sample trees at even intervals along the transect,
but large gaps where suitable pine could not be found due to agricultural or oil extraction
activities or natural breaks in the stand could not be avoided. In 2006, twelve locations
were sampled. In 2007, an additional five sites were sampled to increase the total
number of samples. At each location, GPS coordinates were obtained for the stand. A
maximum of ten trees in each stand, each with a minimum dbh of 15 cm, were sampled.
At some sites, ten trees meeting the minimum size requirements could not be found so
fewer than ten trees were sampled (Table 3.1). Because visual cues have also been
suggested to play a role in MPB host selection (Cole and Amman 1969, Shepherd 1966),
I recorded the dbh of all trees in my sample population. A 10 mm diameter punch (No.
149 Arch Punch 10 mm, C.S. Osborne & Co., Harrison, N.J. 07029, U.S.A.) was used to
remove a disk of bark and phloem at breast height (1.3 m). Each disk was stored in
individually labeled envelopes (#1 coin envelopes, 5.7 cm x 8.9 cm, Staples, 438346) on
dry ice until transferred to a -80°C freezer (700 Series Formula ULT Freezer, Thermo
Electron Corporation), where samples were kept until being shipped on dry ice to the
British Columbia Ministry of Forests and Range Forest Research Laboratory, Victoria,
BC, for processing and analysis.

63

Figure 3.1. Map of sampling locations in British Columbia and Alberta, Canada. Table
3.1 contains information for each sampling location: the number of trees, coordinates,
year sampled, and the number of trees of each species sampled at each location. Map
created with ArcMap v9.2.

64

65

Table 3.1. Coordinates for sample locations and number of trees sampled.

1

Number of trees
sampled (lodgepole
pine, jack pine)
10(1,9)

2

8 (0, 8)

3

10 (0, 10)

4

10 (9, 1)

5

10(10,0)

6

10(10,0)

7

5 (3, 2)

8

10(10,0)

9

10 (10, 0)

10

10(10,0)

11

10(10,0)

12

10 (10, 0)

13

8 (0, 8)

14

5 (0, 5)

15

10(0, 10)

16

10 (0, 10)

17

10 (10, 0)

Sample location

Coordinates

Year sampled

N54°28.110'
WU1°11.025'
N 54°43.363'
W113°16.452'
N55°01.594'
W113°49.192'
N55°01.578'
WU5°16.308'
N54°47.172'
W115°18.491'
N 54°29.283'
W115°30.020'
N53°36.833'
W115°22.701'
N53°36.471'
W115°55.101'
N 53°34.268'
W116°37.942'
N 53°33.675
W117°l 1.938"
N53°01.11'
W119°16.531'
N 53°03.587'
W119°36.880'
N 56°20.959'
Wlll°34.155'
N 55°56.692'
W112°01.709'
N57°21.308'
Wlll°32.363'
N 59°00.636'
W 109°28.586'
N 55°47.358'
W 121°18.037'

2006
2006
2006
2006
2006
2006
2006
2006
2006
2006
2006
2006
2007
2007
2007
2007
2007

66

Phloem samples were processed using gas chromatographic-flame ionization
detection analyses (GC-FID) to identify compounds by matching their retention time with
synthetic standards (Table 2.2) at the British Columbia Ministry of Forests and Range
Forest Research Laboratory. Frozen (-80°C) phloem samples were ground in liquid
nitrogen and the sample was extracted using 4 ml of hexane (with 250 ppm pentadecane
as an internal standard) for 48 h. Samples were then inverted to mix, allowed to settle for
24 hours, after which 0.5 ml of solution was transferred to a 2 ml autosampler vial for GC
analysis using either a PerkinElmer Clarus500, or PerkinElmer AutoSystem with built in
autosampler, fitted with an INNOwax column (J&W, 25m x 0.2mm ID, 0.4u film). The
injection was split (35 ml/min, -39:1; injector temperature 200°C). Helium was utilized
as the carrier gas (flow rate 21 PSI, 0.90 ml/min at 60°C). The oven temperature was
held at 60°C for 1 min, temperature was increased at a rate of 3.0°C/min to 85°C, then
increased at a rate of 8.0°C/min to 170°C. Finally, the temperature was increased to
250°C (rate of 20.0°C/min) and held for 7.0 min. The remaining contents of the vials
containing the extracted phloem and bark were evaporated in a fume hood and then oven
dried at 70°C overnight to remove residual moisture. A dry weight was obtained to
determine a moisture correction, which was applied to the results.
All data were analyzed using R v.2.6.2 (R Development Core Team, 2008).
Values of <5 ppm were considered to be zero for analysis. Figures were created using R
v.2.6.2., GNUPLOT v. 4.2.3. and ArcMap v. 9.2.
Pollack and Dancik (1985) found that a-pinene and P-phellandrene were the most
important variables to differentiate between lodgepole pine and jack pine and the putative
hybrid populations in Alberta. Based on this, I performed a cluster analysis using only

67

the percentage of a-pinene and P-phellandrene to separate the samples into two
chemogroups, which are likely synonymous with the species lodgepole and jack/hybrid
pine. I will refer to my two groups as lodgepole and jack pine for the remainder of the
chapter.
Terpene concentrations (ppm) and the percent of each terpene were analyzed
using independent two-sample t-tests to examine differences between lodgepole and jack
pine. A two-sample non-parametric Wilcoxon test was used to determine if there were
differences between the two species (a = 0.05) as most of the terpene data could not be
transformed to meet the assumption of homoscedasticity based upon a Levene's test.

3.3.

Results
Based upon the separation of trees by their relative resin a-pinene and P-

phellandrene components, I sampled 93 lodgepole and 63 jack pine trees (Figure 3.2).
There was no significant difference in dbh between the two species in my sample
population. There were three sample locations where trees were separated into both
groups (Table 3.1, Figure 3.1). Based upon sample location, the chemical analysis was
successful in separating the tree species, except for one sample that was taken in a
location that is further east than lodgepole pine are supposed to be present (Table 3.1).
There is no reason so assume that this is due to sampling or processing error. It is not
inconceivable that there was a single lodgepole pine in that region due to seed dispersal
by an animal. This sample, despite its unusual location, appears to be chemotypically
lodgepole pine and was treated as such

68

Figure. 3.2. Cluster analysis of samples to separate into two groups based on percentages
of a-pinene and P-phellandrene: gray indicates lodgepole pine, black indicates jack pine
and likely any hybrids sampled.

69

o

80 -

60 -

40 -

20 -

o

o

o

°« °
_

Q>

0

O
O O

0

20

O
O

oo

®®
O
° X> 0 Or, CD
CGDOOD W C0BO «3DO Od8fe @ O

40

60

T
80

a-Pinene

70

There were significant differences between most of the constitutive
concentrations (ppm) in lodgepole and jack pine (species determined by the cluster
analysis) (Table 3.2). Borneol, camphene, camphor, A-3-carene, a-caryophyllene, acopaene, p-cymene, a-humulene, limonene, linalool, myrcene, ocimene, a- and pphellandrene, a- and (3-pinene, pulegone, a- and P-pinene, a- and y-terpinene, terpineol,
terpinolene, and total terpenes sampled were all significantly different between lodgepole
and jack pine (Wilcoxon rank sum test, a = 0.05). Lodgepole pine had higher levels of
each terpene and of total terpenes sampled with the exceptions of three terpenes: linalool,
pulegone, and a-pinene (one of the terpenes used to determine species) (Figure 3.3).
There were also significant differences between the percentage composition of the
phloem resin of a number of terpenes in lodgepole and jack pine (species determined by
cluster analysis) (Table 3.3, Figure 3.4). Bornyl acetate, camphor, A-3-carene, acaryophyllene, a-copaene, p-cymene, a-humulene, limonene, linalool, myrcene, ocimene,
a- and p-phellandrene, a-pinene, pulegone, sabinene, a- and y- terpinene, terpineol, and
terpinolene all differed significantly between lodgepole and jack pine (Wilcoxson rank
sum test, a = 0.05). These terpenes were the same ones that were different in absolute
level of terpenes (ppm) with the exceptions of borneol, camphene, and p-pinene. There
were significant differences between the absolute levels of these three terpenes between
the species, however there was no significant difference between percent composition of
each as a fraction of total measured terpenes. In addition, lodgepole pines had a

71

LP
JP

LP
JP

LP
JP

LP
JP

Borneol
11.731+2.317
4.556+1.619
P = 0.029
a-Cubebene
1.355±0.799
0.762+0.610
P = 0.717
(S-Phellandrene
6905.591+649.716
288.016+124.307
P < 0.001
Terpinolene
294.473+30.899
74.111+22.375
P < 0.001

Bornyl Acetate
102.366+31.207
49.079+5.801
P = 0.557
p.Cymene
41.237+8.270
7.429+4.245
P< 0.001
a-Pinene
1041.968+109.339
2656.349+334.773
P < 0.001
a-Thujone
1.624+0.615
1.794+1.162
P = 0.397

Camphene
90.355+10.010
37.365+6.149
P< 0.001
a-Humulene
32.763+5.004
6.365+3.504
P< 0.001
p-Pinene
784.172+85.465
305.508+44.139
P< 0.001
Total
12347.753+1022.355
4382.349+518.182
P < 0.001

Camphor
10.979+1.593
2.778+0.916
P < 0.001
Limonene
663.280±107.275
210.365+47.351
P < 0.001
Pulegone
23.871+3.994
105.651+22.157
P = 0.006

2-Carene
0+0
0+0
NA
Linalool
44.516+5.745
114.810+16.272
P = 0.030
Sabinene
109.688+10.922
10.508+2.847
P < 0.001

A-3-Carene
1569.860+217.032
355.714+97.474
P < 0.001
Myrcene
353.667+30.468
116.095+17.873
P < 0.001
a-Terpinene
14.581+3.458
0.857+0.612
P < 0.001

a-Caryophyllene
14.979±2.941
6.048+2.204
P < 0.001
Ocimene
22.538+5.143
1.333+1.333
P< 0.001
y-Terpinene
21.742+3.445
1.794± 1.285
P < 0.001

72

a-Copaene
8.451+1.737
3.905+2.180
P = 0.002
a-Phellandrene
140.581+14.580
3.968+2.723
P < 0.001
Terpineol
41.247+7.902
17.238+6.386
P< 0.001

Table 3.2. Mean concentration of terpenes (ppm) (+ 1 SE) of the two species (as determined by the cluster analysis). Differences
between species as determined by a Wilcoxson rank sum test (a = 0.05, significant differences in bold). JP - Jack pine, LP - lodgepole
cluster analysis of ot-pinene and |3-phellandrene content
pine

73

Figure 3.3. Mean level of terpenes (ppm, + 1 SE) that showed significant differences between lodgepole pine and jack pine. All nine
terpenes were significantly different based upon species based upon a Wilcoxon rank-sum test (a = 0.05). (3-Phellandrene and apinene are scaled to the right side scale, all others scaled to the left.

r-

( a S + 'UJdd) 9U9dl9J JO pA9J U129]/\[

in

oo

m

a
a

|
H

§
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t

OH

f

I

t

8

1
a
a

00

00

( 3 S + 'tttdd) 9U9dj9J JO J9A9J lI129]/\[

JP

LP

LP
JP

LP
JP

LP
JP

Borneol
0.09+0.02
0.09+0.03
P = 0.143
q-Cubebene
0.006+0.003
0.04+0.04
P = 0.757
P-Phellandrene
52.24+1.54
4.13+0.84
P < 0.001
Terpinolene
2.96+0.57
1.50+0.36
P < 0.001

Bornyl Acetate
1.85+0.54
1.54+0.25
P < 0.001
p-Cymene
1.01±0.57
0.12+0.07
P < 0.001
q-Pinene
10.37+0.95
57.72+2.31
P < 0.001
q-Thujone
0.01+0.01
0.20+0.14
P = 0.416

Camphene
0.82+0.08
0.67±0.09
P = 0.482
q-Humulene
0.36+0.07
0.22+0.13
P < 0.001
B-Pinene
7.02+0.57
6.48+0.71
P = 0.363

Camphor
0.13337+0.03
0.07619+0.03
P = 0.001
Limonene
5.10+0.69
4.75+0.96
P = 0.003
Pulegone
0.30+0.09
3.95+1.20
P < 0.001

2-Carene
0+0
0+0
NA
Linalool
0.56+0.21
5.75+1.33
P = 0.011
Sabinene
0.87+0.06
0.22+0.06
P < 0.001

A-3-Carene
11.95+1.11
9.25+1.5
P = 0.002
Myrcene
2.86+0.17
2.27+0.28
P = 0.002
q-Terpinene
0.08±0.02
0.01+0.01
P < 0.001

q-Caryophyllene
0.11+0.02
0.29+0.14
P = 0.003
Ocimene
0.13+0.03
0.02+0.02
P < 0.001
y-Terpinene
0.13+0.02
0.01+0.01
P < 0.001

75

q-Copaene
0.06+0.01
0.05+0.03
P = 0.003
q-Phellandrene
1.02+0.05
0.03+0.02
P < 0.001
Terpineol
0.28+0.04
0.30+0.11
P < 0.001

Table 3.3. Mean percent concentration of terpenes (+ 1 SE) of the two species (as determined by the cluster analysis). Differences
between species as determined by a two-sample Wilcoxon test (a = 0.05, significant differences in bold). JP - Jack pine, LP lodgepole pine. Tree species determined by cluster analysis of a-pinene andft-phellandrenecontent.

76

Figure 3.4. Mean percent of terpenes comprising over 1% of the total terpenes (+ 1 SE) in lodgepole pine (hatched bars) and jack pine
(white bars). Significant differences as determined by Wilcoxon rank test (a = 0.05) designated by an astrix. (3-Phellandrene and apinene are scaled to right side y-axis, all others scaled to left side.

4

2

S

Limonene

Linalool

Myrcene

p-Pinene

Pulegone

Terpinolene

P-Phellandrene

a-Pinene

0

10

20

30

40

60

8

*

70

50

A-3-Carene

= lodgepole pine
= jack pine

10

o
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W
co
41,
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12

14

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77

significantly higher percent composition of most of the terpenes tested that were
significantly different except for a-caryophyllene (0.11 vs 0.29), linalool (0.56 vs 5.75),
pulegone (0.3 vs 3.95), and a-pinene (10.37 vs 57.72).

3.4.

Discussion
The invasion into boreal stands of jack pine, and subsequent success of the MPB

is dependent upon many factors. The climatic suitability of the new area, availability of
host material, and competition with other organisms - including other insects - for
resources could all play key roles. However, beetle success is highly dependent upon
successful location and subsequent successful colonization of a suitable host. This
process includes both detecting host kairomones and overwhelming the host trees'
defenses.
Terpenes play a role in the ability of the MPB to successfully and quickly locate
suitable hosts - not only of the correct species but also the more susceptible hosts in the
population (Raffa and Berryman 1987). When colonizing a host, tree defenses must be
overcome by attracting an adequate number of conspecifics for a mass attack. Finally,
the attacking insects must successfully produce brood, which then must be able to
develop in the host tissues. Differences between lodgepole and jack pine, both in terms
of the amount present (ppm) and the percent composition of individual terpenes in the
phloem resin, could affect all aspects of this process. Quantitative amounts could
influence the insects' ability to detect the correct hosts. Significant quantitative
differences in monoterpene blends between conifer species may be enough for bark
beetle species to determine the correct host (Pureswaran et al. 2004) or result in more

78

insects being attracted by higher concentrations of synergistic terpenes (Miller and
Borden 2000). Higher levels of toxic or feeding deterrents for these insects may increase
their lethal effectiveness (Smith 1965). Proportional amounts of terpenes could also be
important in host selection, or there could be an additive or synergistic effect of toxic
compounds with each other on the physiology of the insect or, specifically, its
detoxification enzymes (Berenbaum and Neal 1985).
My comparison of bark resin terpene compositions of lodgepole pine and jack
pine showed that lodgepole pine has higher levels of most terpenes than does jack pine
(Table 3.2). High levels of terpenes serve to increase the attack density of MPB on
lodgepole pine (Chapter 2). Raffa and Berryman (1983b) found evidence that susceptible
lodgepole pines may have higher levels of oxygenated monoterpenes than do lesssusceptible trees. The lower levels of such terpenes (linalool, borneol, terpineol, etc.) in
jack pine may mean that it is not as apparent, which will result in the potential novel host
being more difficult for a pioneering beetles to locate on the landscape. A-3-Carene at a
high release rate enhanced trap catch of MPBs (Miller and Borden 2000) and myrcene
was the most effective synergist in a baited tree study (Borden et al. 1983). Myrcene and
terpinolene was found to be an even more effective synergist than myrcene alone in
pheromone baited traps (Borden et al. 2008). Lodgepole pine has a significantly higher
concentration of all three of these terpenes in its bark resin, suggesting that lodgepole
pine is potentially an easier host for foraging MPB to locate.
However, a-pinene, which is one terpene that is found in much higher
concentrations in jack pine bark resin is metabolized by female MPB to produce transverbenol, a component of the MPB aggregation pheromone (Conn et al. 1984). The

79

aggregation pheromone is essential to MPB ability to successfully mass attack the tree by
drawing in conspecifics in order to overwhelm the host's defenses (Rudinsky 1962). Ips
paraconfusus Lanier were found to have a positive linear relationship between the
production of components of their pheromone emissions (czs-verbenol, trans-\evbeno\,
and myrtenol) and the concentration of a-pinene (Byers 1981), so, in an analogous
fashion, a higher concentration of a-pinene may enable MPB to increase attraction to the
tree. Hunt et al. (1989) showed that a-pinene is auto-oxidized to verbenone, an antiaggregation pheromone of the MPB. Production of the anti-aggregation pheromone is
also key to beetle success, as there is an optimal attack density after which the
competition within the tree between conspecifics reduces the reproductive success of the
beetles (Raffa and Berryman 1983a). This complex interaction between the insect and
resin a-pinene could be very important especially since levels in jack pines are
significantly higher than in lodgepole pine. This relationship also could be dependent on
the beetle population levels in the area. In the epidemic stage, beetles may more easily
overcome the lower levels of terpenes found in jack pine, relative to lodgepole pine, and
may be able to utilize the relatively high levels of a-pinene in jack pine to produce the
highly effective aggregation pheromone to draw in enough conspecifics for successful
colonization and to terminate aggregation at the appropriate moment.
Brood has successfully been reared out of jack pine in a laboratory environment
and brood production in jack pine was comparable to that in lodgepole pine in the same
laboratory experiment (Safranyik and Linton 1982). This result is now supported by the
fact that I found lower concentrations of the terpenes that are generally considered to be
toxic, such as limonene and A-3-carene, in jack pine than in lodgepole pine. Both

80

compounds have been found to be ovicidal (Raffa and Berryman 1983b). However, apinene, which was also ovicidal to MPB eggs in the same study (Raffa and Berryman
1983b), is found at significantly higher levels in jack pine which could potentially reduce
brood success.
Terpenes can explain the difference between more and less susceptible trees. A-3Carene explained over 40% of the variation between lodgepole pine that was more and
less susceptible to the Douglas-fir pitch moth Synanthedon novaroensis (Hy. Edwards)
(Rocchini et al. 2000). The lower levels of this terpene in jack pine could result in it
being more susceptible to attack by MPB, particularly in consideration that - 4 5 % of the
trees classified as jack pine had undetectable amounts of A-3-carene.
There is evidence that D. valens can differentiate between the enantiomers of apinene, and trap catches differed based upon the enantiomers both for a- and P-pinene.
This may be in part because this insect does not appear to have a strong pheromone
(White and Hobson 1993). The chirality of a- and p-pinenes found in jack pine vs.
lodgepole pine could play a role in the attractiveness of the host to the MPB. Volatiles
collected from bolts of lodgepole pine collected near Princeton, BC showed 67.7% and
100% of (-)-a- and (-)-P-pinene respectively (Pureswaran et al. 2004). Differences in the
ratios of enantiomers of terpenes in jack pine or in lodgepole pine collected at other
locations could be significant to the MPB in host selection and recognition, because
electroantennograms have shown that this insect could differentiate between enantiomers
of verbenol and verbenone (Whitehead et al. 1989). There has been correlation between
the chirality of a-pinene and the ratio of pheromone enantiomers in Ips (reviewed by
Seybold 1993). However, I can find no evidence that the toxicity of these compounds

81

will be strongly influence by their chirality. Furthermore, other than its influence as a
precursor, it is unlikely that the ratio of the terpenes would have much effect as a
karimone in the presence of the powerful aggregation pheromone produced by the MPB.
The oxygenated terpenes linalool and pulegone comprise 5.75% and 3.95%,
respectively, of jack pine resin terpenes while each comprises < 1 % in lodgepole pine.
Thus, jack pine contains ~2.5x the level of linalool and ~4x the level of pulegone of
lodgepole pine in its constitutive phloem resin. However, the mean levels (ppm) of
linalool and pulegone in jack pine are much lower than the amounts of many other
compounds found in lodgepole pine. Linalool was identified as one of the volatiles in
female MPB frass, and is a host secondary metabolite that does not appear to be wellmetabolized by the insect (Pierce et al. 1987). Linalool is implicated in inhibition of the
pheromone response between /. paraconfusus (Lanier) and Ips pini (Say), although it did
not have an effect on attraction of /. paraconfusus to ponderosa pine bolts infested with
conspecifics (Birch and Light 1977). Linalool elicits antennal response in both male and
female D. brevicomis (Shepherd et al. 2008). Linalool has an alcohol functional group,
and there is potential that MPBs would recognize it as a compound not normally
associated with an acceptable host, at least at these levels. Therefore, it could act as a
deterrent, similar to some alcohol non-host green leaf volatiles that reduced trap catches
in field experiments to bark beetles (Wilson et al. 1996, Poland et al. 1998, Huber and
Borden 2003). However, a trapping experiment showed that linalool did not affect trap
catches at the release rates tested (Libbey et al. 1985). At higher release rates there could
be an effect or, linalool could have an additive effect with other compounds. Blends of
non-host volatiles have been shown to be effective deterrents to bark beetles in trapping

82

studies (Huber and Borden 2003). Thus, the behavioral activity of linalool at levels
similar to that found in jack pine may be a worthwhile avenue of study.
Pulegone, the other terpene to be found in relatively higher levels in jack pine,
was found to be toxic to five species of stored-products insects during fumigation trials
(Lee et al. 2003). Linalool and pulegone may ultimately be found to play no major role
in the interaction between the MPB and its host; the levels may be too low to have any
significant effect on aggregation behavior or colonization and larval success. However, I
have not been able to locate any studies that examine their effects on this or other bark
beetle species. Most studies on the behavioral and toxic effects of terpenes on D.
ponderosae are conducted using lodgepole pine, in which these two compounds are
minor components. It would be valuable to examine the physiological and toxic effects
of these two terpenes to determine if the comparably higher levels in jack pine could
result in different behavior or survival in the new host.
MPBs must efficiently and precisely locate suitable hosts. Initiating beetles must
attract conspecifics to mass attack the tree and overwhelm its defenses. Colonizing
beetles must be able to indicate to arriving conspecifics when an attacked tree's defenses
have been successfully breached. Colonizing adults and new larvae must survive in the
toxic environment of the host's phloem resin if new brood is to emerge the following
year. Lodgepole pine, with its generally higher concentrations of many phloem resin
terpenes, may be easier to locate for MPB than is jack pine. However, the higher
concentrations of a-pinene, precursors to aggregation and anti-aggregation pheromones,
found in jack pine may result in the trees being more successfully attacked once a
suitable host is found. While I found that attack density was reduced on lodgepole pine

83

with higher concentrations of a-pinene (section 2.3), the levels in lodgepole pine were
still far below what they are in jack pine.
Lodgepole pine has higher concentrations of several terpenes that act
synergistically with the aggregation pheromone, such as myrcene, but they are present in
jack pine as well and the minimum levels of resin terpenes necessary to provide an
adequate attractant plume from an attacked bole are unclear, although there could be an
additive effect between a number of host kairomones (Conn et al. 1983, Borden et al.
1983). This, in combination with the higher levels of total terpenes present in lodgepole
pine, could indicate that lodgepole pine is easier for MPBs to locate, and therefore attack
than jack pine. However, the combination of lower levels of some terpenes generally
considered to be toxic and an increased concentration of a-pinene, important in the
synthesis of components of the aggregation and antiaggregation pheromones, may result
in an increased success for the MPB in this new geographic range and host.

3.5.

Conclusions
•

There are significant differences between both the qualitative and
quantitative amounts of terpenes found in jack and lodgepole pine based
upon separation by levels of a-pinene (higher in jack pine) and p~
phellandrene (higher in lodgepole pine).

•

Jack pine may be less apparent to MPB due to lower levels of terpenes.
However, the jack pines contain significantly higher concentrations of apinene - the precursor to aggregation and anti-aggregation pheromones for

84

MPB - which could have a significant impact on the success of attacking
MPBs in jack pine stands.
•

Lodgepole pine contained higher levels of terpenes considered to be toxic
to MPB such as limonene and A-3-carene which could indicate that the
MPB will have less difficulty overcoming the levels in jack pine.

•

These conclusions were drawn based upon an analysis of constitutive
defenses. In chapter 4,1 will examine differences in lodgepole and jack
pine induced defenses.

85

Chapter 4. Induced response in lodgepole and jack pine to mountain
pine beetle inoculation and wounding
Abstract
The ability of a host tree to successfully defend itself against mountain pine beetle is
partially dependent on its induced defensive response to attack. Two populations of
lodgepole pine in British Columbia, and one population of jack pine in Alberta, Canada,
were sampled for levels of induced phloem terpenes after simple wounding or inoculating
with mountain pine beetle complex. Different induced responses in ten terpenes and in
total levels of all terpenes measured were found both between the populations of
lodgepole pine, as well as between the two species of pine. These differences in induced
response provide support for the hypothesis that tree populations with prior exposure to
mountain pine beetle outbreaks have different defense characteristics than naive
populations. These differences also indicate that mountain pine beetle will be facing
different terpene-based induced defenses as they move into jack pine-dominated regions,
and that this may play a role in the success of mountain pine beetle in this new host.

4.1

Introduction
In addition to the constitutive chemical defenses employed by conifer trees,

induced defenses are employed when subjected to attack by insects. Induced defenses are
triggered when a plant is under attack and are an important part of a tree's defense system
(Raffa 1991). As with constitutive defenses, induced defenses may be physical or
chemical in nature. Induced defenses have been studied in several different conifer
systems (Reviewed by Raffa 1991). Pines have less-developed induced defenses than

86

some other conifers such as Abies spp. (Raffa and Berryman 1987). In a survey of 10
conifer species, Lewinsohn et al. (1991) found that the increase in biosynthesis of
terpenes after wounding in Pinus spp. was not as substantial as was seen in Abies spp.
Although there was a detectable increase in defense levels in Pinus spp., constitutive
levels were already quite high, so there was no significant difference between the induced
(wounded) and constitutive (control) levels of monoterpene cyclase activity.
When mountain pine beetles, Dendroctonus ponderosae (Hopkins) (Coleoptera:
Curculionidae: Scolytinae), attack a host tree, an induced response in the host tree is
triggered (Shrimpton 1973, Miller et al. 1986, Raffa and Berryman 1983a, Raffa and
Berryman 1982). The aggregating beetles must successfully overcome the host's
defenses in order to successfully reproduce. When attacking a host, they also vector a
variety of fungi into the tree (Lee et al. 2006a), which are also capable of inducing host
defense reactions (Raffa and Smalley 1995, Lee et al. 2006b). If the growing aggregation
of beetles does not reach numbers sufficient to overcome the host tree's defenses (both
constitutive and induced), individual insects will not be able to successfully reproduce in
the trees (Raffa 1991, Raffa and Berryman 1983b).
Induced changes in concentrations of terpenes, some of them minor components
of the resin, have been documented after a simulated insect attack on lodgepole pine,
Pinus contorta (Dougl.) var. latifolia (Engelm). (Raffa and Berryman 1987). Lodgepole
pine resistant to inoculum of fungi associated with the MPB had higher rates of resin
flow (Raffa and Berryman 1982). Lodgepole pine has been found to respond to attack by
the MPB and its associated fungi with large increases of total terpenes in the sapwood
and heartwood (Shrimpton 1973). In jack pine, Pinus banksiana (Lamb.), total

87

monoterpene concentrations have been found to be elevated in induced tissue compared
to levels in constitutive tissue after attack by jack pine budworm (Wallin and Raffa
1999).
The ability of a tree to strongly and rapidly respond to attack should have a
significant impact on MPB success as they must kill their host to successfully reproduce
(Atkins 1966, Reid and Robb 1999). I hypothesized that there would be a difference
among populations of trees in the induced responses to a mechanical wounding and an
inoculation of crushed beetle material, potentially due to fungi and other factors (proteins,
chitin, etc) associated with the bark beetles. In addition, I hypothesized that there would
be differences in localized, induced chemical defenses in response to simulated MPB
attack between southern and northern stands of lodgepole pines in BC and also between
lodgepole pine and jack pine stands. A comparison of the response of lodgepole pine to
that of jack pine to the same treatments could provide information about how effective
jack pine defenses will be as they interact with an insect that is on the verge of expanding
into Canada's boreal forest.

4.2.

Methods
Uninfested lodgepole pine trees, as determined by an absence of frass and pitch

tubes, were selected in a stand near Chetwynd, BC (CH) and near Kelowna, BC (K). In
addition, a stand of jack pine, a population previously unexposed to MPB, were selected
near Fort McMurray, AB (FM). Specific location information is given in Table 4.1.
Selected trees were randomly assigned to one of two treatments, inoculated or control, by

88

Kelowna
Chetwynd
Fort McMurray

Location

# trees
sampled
30*
22
20
N 49°57.684'
N49°53.145'
N57°21.640'

Coordinates
W119°42.551'
W120°25.121'
Wlll°32.281'

01 Aug 07
05 Aug 07
9 Aug 07

03 Aug 07
07 Aug 07
11 Aug 07

Dates Sampled
Day 2
Initial

15 Aug 07
19 Aug 07
23 Aug 07

Day 14

89

Table 4.1. Sampling locations, number of trees sampled at each location, and dates of sampling. *Due to attack by MPB between the
time of initial sampling and the final sample, 12 of the 30 thirty trees were removed from the analysis.

coin toss. Once half of the trees had received one of the treatments, the remaining trees
were treated with the other treatment to ensure an equal number of inoculated and control
trees.
Two initial bark punch samples (10 mm diameter) were taken from both the east
and west side of the inoculated trees at 1.3 m above ground level. Live MPB adults,
which had been collected previously in baited (Pherotech International, Inc., Delta, BC,
Canada; MPB bait) Lindgren funnel traps (Phertotech International, Inc.) near Prince
George, BC were crushed in tubes (2.0 ml polypopylene microcentrifuge tubes, Fisher
Scientific) immediately prior to application on the tree to create a homogenous mixture
made from 30 beetles per tube with -10 drops of distilled water. A clean toothpick was
used to apply the beetle mixture to both punch holes (one-tenth of the mixture of one tube
per hole) to completely cover the exposed phloem. Mixing the beetles was an attempt to
homogenize the variety of fungal spores carried by this insect (Lee et al. 2006a) although
multiple tubes, and therefore multiple mixtures of beetles, were used. In addition, the
number of spores was expected to more accurately represent the amount vectored by
beetles as opposed to inoculations with a single species of fungus. Due to evidence that
some oral secretions of insects have an effect on the plant defense response (Alborn et al.
1997, Ralph et al. 2006), the use of crushed beetles was also expected to allow for any
effect of a variety of beetle parts on induced defenses. Following application of the
crushed beetles, silicone plugs (Mack's® Pillow Soft® silicone earplugs, McKeon
Products, Inc. 25460 Guenther, Warren, MI) were used to cover the wound and secure the

90

beetle paste to the tree. Control trees received the same punch treatment and plugs, but
were not exposed to the mashed beetles.
Prior to punching the bole of the tree, the area where the punch was to be made
was scraped and then sprayed with ethanol (95%). The tools used in the treatment and
sampling process were also sprayed with ethanol before use on each tree to reduce
potential contamination. The bark and phloem material removed from each punch site
was saved in individual, labeled envelopes and immediately buried in dry ice in a cooler,
where it was kept until it could be transferred to a -80°C freezer for longer-term storage.
Each pine was sampled three times in August 2007 (Table 4.1): 1) at the time of
treatment; 2) two days after inoculation on one side, randomly selected by a coin flip, 10
mm above the initial punch; and 3) fourteen days after inoculation on the side of the tree
opposite from the two-day sample, again 10 mm above the initial punch. The timing of
the second sample at two days post-inoculation was used because mass attack is normally
completed in one or two days following contact of the first MPB with the tree (Safranyik
and Carroll 2006). Therefore, it is the initial response by the tree during this time that is
most likely to have an effect on successful colonization.
After all sampling was completed, samples were shipped to the British Columbia
Ministry of Forests and Range Forest Research Laboratory, Victoria, B.C. for processing.
Only one of the punches from the initial sample from each tree was processed (either east
or west side of the tree); the processed sample was determined by a coin toss. Phloem
samples were processed using gas chromatographic-flame ionization detection analyses
(GC-FID) to identify compounds by matching their retention time with synthetic
standards (Table 2.2) at the British Columbia Ministry of Forests and Range Forest

91

Research Laboratory. Frozen (-80°C) phloem samples were ground in liquid nitrogen
and the sample was extracted using 4 ml of hexane (with 250 ppm pentadecane as an
internal standard) for 48 h. Samples were then inverted to mix, allowed to settle for 24
hours, after which 0.5 ml of solution was transferred to a 2 ml autosampler vial for GC
analysis using either a PerkinElmer Clarus500, or PerkinElmer AutoSystem with built in
autosampler, fitted with an INNOwax column (J&W, 25m x 0.2mm ID, 0.4u film). The
injection was split (35 ml/min, -39:1; injector temperature 200°C). Helium was utilized
as the carrier gas (flow rate 21 PSI, 0.90 ml/min at 60°C). The oven temperature was
held at 60°C for 1 min, temperature was increased at a rate of 3.0°C/min to 85°C, then
increased at a rate of 8.0°C/min to 170°C. Finally, the temperature was increased to
250°C (rate of 20.0°C/min) and held for 7.0 min. The remaining contents of the vials
containing the extracted phloem and bark were evaporated in a fume hood and then oven
dried at 70°C overnight to remove residual moisture. A dry weight was obtained to
determine a moisture correction, which was applied to the results.
Data were analyzed using R (R Development Core Team, 2008). Twelve trees at
Kelowna were attacked by MPB between the time of the initial punch and the time that
the final sample was taken 14 days later. All samples from those trees were removed
from analysis, as any measured response would likely have been due to the many attacks
on the tree. This resulted in ten control and eight inoculated trees remaining for data
analysis at this site. As in previous analyses, levels of individual terpenes <5 ppm were
considered to be zero for analysis.
To compare differences between locations, treatments, and time, backwards
selection, starting with the largest interaction effect, was used to create a linear mixed

92

effects model for each of the nine individual terpenes that comprised over 1% of the total
resin terpenes in either lodgepole or jack pine (Chapter 3), as well as for the total amount
of terpenes. The models contained fixed effects of location, dbh, treatment, and time
with random effect of the individual tree. When necessary, a square root or log (x+1)
transformation was used to meet the assumptions of normality and homoscedasticity
based upon a visual examination of the residual plots.
Based upon the model, the effect of treatment at different locations and over time
were tested by comparing the difference in the level of terpene at each time period using
ANOVA. Where significant treatment effects existed, means were compared using
Tukey's post-hoc test (a = 0.05). Prior to analysis data were transformed using square
root or log (x+1) as necessary to meet the assumptions of the analysis. If data could not
be transformed a Kruskal-Wallis rank sum test followed by a pairwise Wilcoxon's rank
sum test was used (a = 0.05).
Where treatment was a significant term, either independently or as an interaction
term, the differences in terpene level between locations and the three sampling times in
inoculated trees was compared using ANOVA (a = 0.05). Where significant differences
between the locations existed, means were compared using a Tukey's post-hoc test.

4.3.

Results

4.3.1. A-3-Carene
The levels of A-3-carene differed by sampling location and increased with
increases in dbh and over time. The effect of treatment was affected by the location, as

93

Response
A-3-Carene
Limonene
Linalool
Myrcene
|3-Phellandrene
a-Pinene
(3-Pinene
Pulegone
Terpinolene
Total terpene

Response
A-3-Carene
Limonene
Linalool
Myrcene
(3-Phellandrene
a-Pinene
P-Pinene
Pulegone
Terpinolene
Total Terpene
Location:Time

0.01
Location:dbh
0.01

Location:Treatment
<0.001

0.02

0.02

0.002
<0.001
0.03

0.02

<0.001
<0.001
<0.001
<0.001
0.003
0.003

0.001
0.01
<0.001
<0.001
0.05
<0.001
<0.001
0.04
<0.001

0.01
0.03
0.009

0.01
0.007

0.005

0.03

Time:Treatment

Time
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.001
0.001
<0.001
<0.001

dbh
0.01

Treatment

Location
<0.001

<0.001

Location:dbh:Treatment

0.01

0.001

94

Location:Treatment:Time

Table 4.2. Sources of variation in the levels of terpenes (ppm) to time, location, dbh, treatment (Trt), and time and associated Pvalues.

manifested by a significant interaction between location and treatment, specifically at
Kelowna and Fort McMurray (Table 4.2). The changes in levels of A-3-carene were not
significant between the two treatments within each location. However, lodgepole pine at
Kelowna had a higher increase in levels of A-3-carene between day 0 and day 2 in
inoculated trees compared with jack pine at Fort McMurray (7*2,26 = 3.19, P = 0.05)
(Figure 4.2). Two days post-inoculation, the inoculated lodgepole pine trees had higher
levels of this terpene than jack pine, although this trend was not present at day 0
(constitutive) or day 14 samples (Figure 4.1).

4.3.2. Limonene
The level of limonene increased with time. The effect of time on the level of
limonene differed by location (Table 4.2). There were significantly higher levels of
limonene in the lodgepole pine at Kelowna compared with the lodgepole pine at
Chetwynd at day 2 (F2,57 = 3.73, P < 0.05) and at day 14 (F2,57 = 4.02, P < 0.05). The
southern population of lodgepole pine (Kelowna) had a larger increase in levels of
limonene compared with the northern population of lodgepole pine (Chetwynd) between
day 0 and day 2 (F2,57 = 3.21, P < 0.05). The difference in the levels of limonene
between day 0 and day 14 was significantly higher in southern lodgepole pine and jack
pine (Fort McMurray) compared with the northern lodgepole pine (F2,si = 7.26, P < 0.01)
(Figure 4.3).

95

Figure 4.1. Mean levels of terpenes (ppm, ±1 S.E.) in inoculated pine (species in
parenthesis) at sampling time day 0, day 2, and day 14. Only terpenes where there was a
treatment effect are included. Differences between levels of terpenes at the three
locations with in a time period were determined using an ANOVA, with a Tukey's posthoc. Significant differences (a = 0.05) are represented by small letters. P-Phellandrene
and total scaled to right-side y-axis, all other terpenes scaled to left-side y-axis.

96

-J

~<

CD

o

5 £

-o
D E.

CD

n

Bo-

Level of terpene (ppm, + 1 SE)

Level of terpene (ppm, ± 1 SE)

SB

o

i

Level of terpene (ppm, ± 1 SE)

Level of terpene (ppm, + 1 SE)

H-

o

|,

n

n

D

o

O

Level of terpene (ppm, + 1 SE)

*r

<T>

^H

n

o

^ 0QQ.00o
^J»^T3 -a

~ Z o

ii

Level of terpene (ppm, ± 1 SE)

Figure 4.2. Mean levels of A-3-carene (ppm, ± 1 SE) at each location (pine species in
parenthesis) for each treatment over time. There were no significant differences between
the changes in level of A-3-carene within each location between treatments. There was a
significant difference between the change in level of A-3-carene between inoculated trees
in Kelowna and Fort McMurray between day 0 and day 2 [F2,26 = 3.19, p = 0.05, Tukey's
post-hoc (p< 0.05)].

98

Kelowna (Lodgepole)
•

Inocluated

• - - - Control

V5

9

10

11

12

13

14

9

10

11

12

13

14

Chetwynd (Lodgepole)

10000

T — <

i-carene (ppm,

+1

eni

8000
6000
4000
2000

0
0

1

2

3

4

5

6

7

Day

>
4}

i

4>

10000

Fort McMurray (Jack)

8000 6000
4000 A
2000 ]
0 !
6

7

10

11

12

13

14

Day

99

Figure 4.3. Mean level of limonene (ppm, ± 1 SE) at the sampling locations (pine species
in parenthesis) over time (days). There were significantly higher levels of limonene at
Kelowna compared with Chetwynd at day 2 (7*2,57 = 3.73, P < 0.05, Tukey's post hoc)
and day 14 (F2,57 = 4.02, P < 0.05, Tukey's post-hoc). The lodgepole pine trees at
Chetwynd had significantly lower RRI in levels of limonene compared with lodgepole
pine at Kelowna and jack pine at Fort McMurray between day 0 and day 2 (#22 = 21.42, P
< 0.001) (Figure 4.3, Table 4.3).

100

— -A — Kelowna (Lodgepole)
- - • # • - - Chetwynd (Lodgepole)
•
Fort McMurray (Jack)

100 H

6

7

10

12

13

Days

101

14

4.3.3. Myrcene
The levels of myrcene increased with tree diameter and time. The effect of time
on the level of myrcene was different between the locations (Table 4.2). Northern
lodgepole pine (Chetwynd) did not have as large an increase in their levels of myrcene
between day 0 and day 2 compared to jack pine (Fort McMurray) and southern lodgepole
pine (Kelowna) (F2,57 = 5.45, P < 0.01) and between day 0 and 14 (F2,57 = 7.40, P < 0.01)
(Figure 4.4). There were significantly lower levels of myrcene in jack pine (Fort
McMurray) compared with lodgepole pine at Chetwynd at day 0 (^2,57 = 4.50, P < 0.05).
However, at day 14 the jack pine and the southern lodgepole pine population had higher
myrcene levels than the northern lodgepole pine (Chetwynd) (^2,57 = 6.17, P < 0.01).

4.3.4. (3-Phellandrene
The level of p-phellandrene was related to an increase in tree diameter and over
time. It was also different by location and treatment. There was also a significant
interaction among the effect of treatment, time, and location on the level of Pphellandrene (Table 4.2). Both lodgepole pine locations had significantly higher levels of
P-phellandrene in the inoculated trees than the inoculated jack pine trees at all three
sampling times (Figure 4.1). Inoculated trees in the population of lodgepole pine in
Kelowna had a higher increase in levels of P-phellandrene compared with the control
trees between day 2 and day 14 (Fi;]6 = 4.49, P - 0.05) and between day 0 and day 14 (j22
= 8.08, P < 0.01). Control lodgepole pine trees in Kelowna had significantly higher
increases in levels of P-phellandrene compared with control jack pine trees in Fort
McMurray ixi = 10.82, P < 0.01). In inoculated trees, the lodgepole pine trees had

102

Figure 4.4. Mean level of myrcene (ppm, ± 1 SE) the sample locations (pine species in
parenthesis) over time (day). Mean levels significantly different by location: a) day 0
lodgepole pine at Chetwynd higher than jack pine at Fort McMurray [7*2,57 = 4.50, P <
0.05, Tukey's post-hoc (P < 0.05)]; b) and day 14 jack pine and lodgepole pine at
Kelowna are higher than lodgepole pine at Chetwynd [.^2,57 = 6.17, P < 0.01, Tukey's
post-hoc (P < 0.05)]. Lodgepole pine in Chetwynd had a significantly lower RRI than
either lodgepole pine at Kelowna or jack pine at Fort McMurray between days 0 and 2
(y 2 2 = 16.01, P< 0.001).

— TA — Kelowna (Lodgepole)
- - • # • - - Chetwynd (Lodgepole)
•

Fort McMurray (Jack)

-*

// ±

0 +-1 2

3

4

5

6

7

9

10

11

12

13

14

Day

104

Figure 4.5. Mean level of P-phellandrene (ppm, ± 1 SE) at each location (pine species in
parenthesis) and treatment over time (day). Inoculated trees in the population of
lodgepole pine in Kelowna had a higher increase in levels of P-phellandrene compared
with the control trees between day 2 and day 14 (Fi,i6 - 4.49, P = 0.05) and between day
0 and day 14 ixi = 8.08, P < 0.01). Control lodgepole pine trees in Kelowna had
significantly higher increases in levels of p-phellandrene compared with control jack pine
trees in Fort McMurray (xi — 10.82, P < 0.01). In inoculated trees, the lodgepole pine
trees had significantly higher changes in the levels of p-phellandrene between day 0 and
day 2 tfj = 15.79, P < 0.001) and between day 2 and day 14 (/2 = 12.16, P < 0.01)
compared to the inoculated jack pine trees.

105

Kelowna (Lodgepole) - -•—Inoculated

9

a

- - Control

10

11

12

13

14

Chetwynd (Lodgepole)

50000

on

(ppm, ±

•

40000 30000 -

p

20000

-phe'

a

10000
0

£

CS3L

9

10

11

12

13

14

10

11

12

13

14

O

Mean level

c*

Fort McMurray (Jack)

50000 -,
40000 30000
20000 10000 -

0

1 • •

1

2

I

1

3

!

4

5

7

8

9

Day

106

significantly higher changes in the levels of (J-phellandrene between day 0 and day 2 (j 2
= 15.79, P < 0.001) and between day 2 and day 14 (j 2 2 = 12.16, P < 0.01) compared to
the inoculated jack pine trees (Figure 4.5).

4.3.5. a-Pinene
The level of a-pinene was positively correlated with time and differed by location.
The relationship between a-pinene levels over time was affected by the location (Table
4.2). Initially, jack pine (Fort McMurray) had higher levels of a-pinene than both
lodgepole pine populations. The northern population of lodgepole pine (Chetwynd) had
higher levels of a-pinene than the southern population of lodgepole pine (Kelowna) (7*2,57
= 24.38, P < 0.001). Jack pine had higher levels of a-pinene than either population of
lodgepole pine after 2 days (F2>57 = 18.46, P < 0.001) and after 14 days (F2,57 = 8.83, P <
0.001). There were significant differences between the locations in levels of a-pinene
over time between locations. Chetwynd had a lower increase in levels of a-pinene
between day 0 and day 2 than Kelowna or Fort McMurray ixi = 9.72, P < 0.01) and jack
pine had a higher increase in levels of a-pinene than either lodgepole pine population
between day 2 and day 14 (j 2 2 = 7.86, P < 0.05). Between day 0 and day 14 all three
populations differed in their change in levels of a-pinene. ( j 2 = 18.52, P < 0.001) (Figure
4.6).

4.3.6. Terpinolene
The level of terpinolene increased with both time and dbh and differed by
location. The effect of the inoculation treatment was affected by time, and the effect of

107

Figure 4.6. Mean level of a-pinene (ppm, + 1 SE) at sampling locations (pine species in
parenthesis). The mean levels of a-pinene at each time period were significantly
different: a) day 0 all three locations significantly different from each other, highest in
jack pine (Fort McMurray), lowest in southern lodgepole pine (Kelowna) [^2,57 = 24.38,
P < 0.001, Tukey's post-hoc (P < 0.05)]; b) day 2 jack pine (Fort McMurray)
significantly different from lodgepole pine [^2,57 = 18.46, P < 0.001, Tukey's post-hoc (P
< 0.05)]; c) day 14 jack pine significantly different from lodgepole pine [F237 = 8.83, P <
0.001, Tukey's post-hoc (P < 0.05)], levels of a-pinene. The difference in levels of apinene between day 0 and day 2 were larger in jack pine (Fort McMurray) and than
lodgepole pine in Chetwynd \%22 = 9.72, P < 0.01, pairwise Wilcox test (P< 0.05)];
between day 3 and 14, jack pine had a large difference in levels of a-pinene compared
with both populations of lodgepole pine \x 2- 7.86, P < 0.05, pairwise Wilcox test (P <
0.05)]; and all locations were different from each other between day 0 and day 14 \x"i =
18.52, P < 0.001, pairwise Wilcox test (P < 0.05).

108

25000

•A— Kelowna (Lodgepole)
• - - Chetwynd (Lodgepole)
Hi— Fort McMurray (Jack)

20000
00
*—i

(ppm,

+1
15000

O

10000

5000

2

6

7

10

11

12

13

14

Days

109

Figure 4.7. Mean level of terpinolene (ppm, + 1 SE) at each location (pine species in
parenthesis) for each treatment over time (day). Inoculated lodgepole pine trees at
Chetwynd had a greater increase in level of terpinolene between day 0 and day 2 than the
control trees at the same location (Fij2o = 4.48, P < 0.05).

110

Kelowna (Lodge pole)

2500

- • — Inoculated
•••-- Control

2000
1500
1000
500
0
0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

10

11

12

13

14

11

12

13

14

Day

+1

Chetwynd (Lodgepole)

2500

a,
& 2000
a

1500
c
o

1000
500

<t>

>

0 +-

c

0

1 2

3

4

5

7

8

9

Day

2500

Fort McMurray (Jack)

2000
1500
1000
500
0
9

10

111

time varied by location (Table 4.2). Inoculated lodgepole pine in Chetwynd had a
marginally significant larger increase in the level of terpinolene between day 0 and 2 than
the control trees at the same location (Fi^o = 4.48, P < 0.05) (Figure 4.7). Only the
constitutive levels of terpinolene were significantly different in the inoculated trees
between locations. Levels of terpinolene on day 0 were higher in the lodgepole pine at
both locations compared to jack pine (Figure 4.1).

4.3.7. Total terpenes
Location and dbh were significant variables on the total level of terpenes and
there was a significant interaction between location and time, treatment and time, location
and dbh, and location and treatment (Table 4.2). There was a greater increase in level of
total terpenes in the inoculated trees compared to the control trees in jack pine (Fort
McMurray) between day 0 and day 14 (Fi,i8 = 13.79, P < 0.01) and in the southern
lodgepole pine trees (Kelowna) between day 2 and day 14 (j 2 2 = 6.19, P < 0.05) and
between day 0 and day 14 (x 2 = 5.76, P < 0.05). There were also significant differences
between the changes in total terpene levels between locations in the inoculated trees.
There was a larger increase in total levels of terpenes in southern lodgepole pine trees
compared with northern lodgepole pine trees between day 2 and day 1 4 ( j 2 = 6.51,P<
0.05). The increase in levels of total terpenes was significantly higher in southern
lodgepole pine trees and jack pine compared to the northern lodgepole pine between day
0 and 14 (£2 = 12.39, P < 0.01) (Figure 4.8). The levels of total terpene did not differ
significantly in the inoculated trees by location until day 14, when lodgepole pine trees at

112

Figure 4.8. Mean total levels of terpenes (ppm, + 1 SE) at each location (pine species in
parenthesis) for each treatment over time (day). There were significant differences
between Chetwynd and Kelowna in the difference in levels of total terpenes between day
2 and day 14 in the inoculated trees (x2i = 6.51, P < 0.05) and between Kelowna and Fort
McMurray between day 0 and day 14 (x\ = 5.76, P < 0.05). Inoculated jack pine trees
(Fort McMurray) had a higher increase in total terpenes compared with control jack pine
trees between day 0 and day 14 (Fi,i8 = 13.79, P < 0.01). There was also significant
differences between differences in levels of total terpenes between day 2 and day 14 (x22
= 6.19, P < 0.05) and between day 0 and day 14 (j 2 2 = 12.39, P < 0.01).

113

70000
60000
50000 H
40000
30000
20000
10000
0

-•

Inoculated

• - - control

Kelowna (Lodgepole)

10

W
on
+1

s

70000

11

12

13

14

11

12

13

14

Chetwynd (Lodgepole)

60000
50000

c

40000
30000 H

C3

o

20000
10000

«<«

• • • • • >

0
>

9

10

a
ca

70000 n
60000 i
50000
40000 -|
30000 H
20000 -

Fort McMurray (Jack)

10000 n
0 -

^^--

•'
1

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Day

114

Chetwynd had significantly lower levels of total terpenes than either of the other two
locations (Figure 4.1).

4.3.8. Other terpenes
The level of linalool and pulegone were found to decrease with an increase in
dbh. The levels of P-pinene and pulegone increased with time. Only linalool was found
to decrease with time. Both P-pinene and pulegone had significant time and location
interactions; linalool was not affected by location, but was the only one of the three to be
affected by treatment which had a significant interaction with time (Table 4.2). The
inoculated lodgepole pine trees at Kelowna decreased more in levels of linalool (Fi>i6 =
4.60, P = 0.05) and pulegone {F\^ = 5.94, P < 0.05) than the control trees. In general,
jack pine was more similar to the southern lodgepole pine trees regarding differences in
levels of p-pinene [e.g. Fort McMurray and Kelowna having higher differences than
Chetwynd between day 0 and day 14 (%22 = 14.8, P < 0.01) (Figure 4.9)]. The lodgepole
pine at Kelowna, generally had lower levels of linalool when compared with the other
lodgepole pine population and the jack pine (Figure 4.10). The levels of pulegone
generally were higher in lodgepole pine than jack pine in inoculated trees (Table 4.1),
however the level of pulegone decreased in southern lodgepole pine, separating the
inoculated trees from the other two locations (Figure 4.11).

115

Figure 4.9. Mean levels of P-pinene (ppm, ± 1 SE) at each location (pine species in
parenthesis) for each treatment by day (x-axis). The level of p-pinene increased more in
the inoculated jack pine at Fort McMurray (#22 = 7.0, P < 0.05) between day 0 and day
14. In the control trees, the southern lodgepole pine at Kelowna had a higher increase in
level of p-pinene than the control trees in the northern lodgepole pine at Chetwynd
between day 0 and day 2 (x2i = 9.88, P < 0.01). There were significant differences
between the changes in levels of P-pinene in the inoculated trees between jack pine and
southern lodgepole pine and the northern lodgepole pine population between day 0 and
day 2 (j22 = 9.11, P < 0.05) and between day 0 and day 14 (j22 = 14.8, P < 0.01).

116

12000

- • — Inoculated

10000

V • -Control

Kelowna (Lodgepole)

8000
6000
4000
2000 0
7

10

11

12

13

14

10

11

12

13

14

Day

Chetwynd (Lodgepole)

12000
10000
8000
+1

s

6000 I
i
4000

I

a 2000
'a,
CO.

1
>

2

3

4

5

6

7
Day

8

9

Fort McMurray (Jack)

117

Figure 4.10. Mean level of linalool (ppm, ± 1 SE) at each location (pine species in
parenthesis) each treatment by days (x-axis). The difference in the level of linalool
between lodgepole pine at Kelowna and the other lodgepole pine population (Chetwynd)
and jack pine (Fort McMurray) in inoculated trees between day 0 and day 14 was
significantly lower (F2,26= 4.82, P < 0.05, Tukey's post-hoc). Inoculated trees at
Kelowna decreased in level of linalool more than the control trees between day 2 and day
14 (F U 6 = 4.60, P = 0.05) and between day 0 and day 14 (F U 6 = 4.85, P < 0.05).

118

Kelowna (Lodgepole)

9

•
Inoculated
Wt - -Control

10

11

12

13

14

Chetwynd (Lodgepole)

350

Fort McMurray (Jack)

300
250
200
150
100
50

0 L-

0

1 2

3

4

5

6

7

10

11

12

13

14

Day

119

Figure 4.11. Mean levels of pulegone (ppm, + 1 SE) at each location (pine species in
parenthesis) for each treatment by day (x-axis). There was a significant difference
between the control and inoculated lodgepole pine at Kelowna between day 2 and day 14
(F U 6 = 5.94, P < 0.05) and location FM (x\= 17.75, P < 0.001). There were significant
differences in the changes in the level of pulegone in the controls between all three
locations between day 0 and day 2 0f22,= 17.75, P < 0.001) and between day 0 and day 14
(X22,= 10.87, P< 0.01). There were significant differences in the inoculation treatment
between the two lodgepole pine populations between day 0 and day 2 (F2,26 = 5.24, P <
0.05). There were significant differences in the effect of inoculation between day 2 and
day 14 (F2,26 = 11-08, P < 0.001, Tukey's post hoc, p < 0.05), and day 0 and day 14 (F2,26
= 14.22, P < 0.01, Tukey's post hoc, P < 0.05) between Kelowna and both Chetwynd and
Fort McMurray.

120

Kelowna

1 2

3

4

-•
Inoculated
V - -Control

5

Chetwynd

700

Fort McMurray

600
500 -;
400 -;
300 -:
200
100
0
6

7

10

11

12

13

14

Day

121

4.4.

Discussion
The success of the invasion of the MPB into the boreal forest will depend upon a

number of factors. These include the climatic suitability of the new area, availability of
suitable host material, and competition with other organisms, including other insects for
the resources. However, differences in the induced resin composition of these species
will likely be a major influence on the success of the progression of the outbreak into a
new host type. If induced defensive responses in pines are appropriate and adequate,
beetles will sometimes abandon their colonization attempts (Raffa 1991).
Lodgepole pine forests in the southern interior of BC are currently heavily
infested by the MPB. Finding uninfested lodgepole pine trees near Kelowna for this
experiment was challenging. It should be kept in mind that, while the sampled trees were
uninfested, potentially due to chance or geographic distance from infested stands, it is
also possible that they may have possessed some characteristic that made them relatively
unsuitable for colonization.

4.4.1. Comparison of terpene levels by location and species and potential implications
for host colonization success
There were significant differences in levels of individual and total induced
terpenes between the location and the two pine species. Lodgepole pine had significantly
higher levels of A-3-carene than did jack pine two days after treatment. A-3-Carene is
considered to be toxic to bark beetles (Raffa and Berryman 1983b, Smith 1965). There
was also an interaction between time, location, and the treatment indicating that the levels

122

of this terpene were affected by this interaction. Inoculated trees at Kelowna had a higher
rate of increase in levels of this toxic terpene between day 0 and day 2 than the jack pine
trees at Fort McMurray. The relatively rapid increase in lodgepole pine may be due to its
location in the southern part of BC, where this species has likely had previous historical
exposure to MPB outbreaks. The lower level of increase by jack pine may provide the
beetle with a less resistant host.
Limonene, another terpene toxic to bark beetles (Raffa and Berryman 1983b,
Smith 1965), was found in significantly higher levels in lodgepole pine in Kelowna than
lodgepole pine in Chetywnd both at day 2 and day 14. In addition to the higher levels in
the southern population of lodgepole pine, there was a greater increase the level of
limonene between day 0 and day 2 compared to the northern population of lodgepole.
This stronger response by southern lodgepole pine compared with northern lodgepole
pine could support the hypothesis that tree populations with prior exposure to MPB
outbreaks maintain a more appropriate response to attack by the insect/fungi complex i.e., rapid increase of a toxic terpene. However, there was no significant effect of the
beetle/fungus treatment on the levels found, indicating that this terpene response may
occur after wounding only, not necessarily in response to this specific insect/fungi
complex. For this terpene, the response by jack pine was similar to that of southern
lodgepole pine and could be an appropriate generalized response to any attack which
could prove to be successful against the MPB.
The success of MPBs is highly dependent on their ability to mass attack the host
tree. In order to attract conspecifics, they have a powerful aggregation pheromone.
Female beetles produce the aggregation pheromone trans-werbenol by metabolizing a-

123

pinene (Conn et al. 1984), making this a potentially important compound in determining
beetle success. At initial sampling, jack pine had higher levels of a-pinene than
lodgepole pine in the north, which in turn were higher than levels found in lodgepole pine
trees in the south. At day 2 and 14 there was no difference between levels found in the
lodgepole pine populations, but jack pine maintained significantly higher levels than
lodgepole pine at either location. This is an expected result as a-pinene is the main
component of jack pine resin. Northern lodgepole pine had a lower rate of change in
levels of a-pinene in response to wounding compared to southern lodgepole pine and jack
pine between day 0 and day 2. Assuming that the trees at the site with the most prior
exposure to beetle outbreaks (Kelowna) respond appropriately to attacks, this could be an
indication that a strong a-pinene response is effective against attack. However, 2 days
post-inoculation, jack pine trees had significantly higher levels of a-pinene than trees in
either lodgepole pine location. The absolute amount of the terpene, not merely the
difference over time, likely plays a vital role in the amount of aggregation pheromone the
females can produce.
a-Pinene is also auto-oxidized to verbenone, an anti-aggregation pheromone
(Borden et al. 1987, Hunt et al. 1989), so levels of a-pinene may be essential to
optimizing attack density on a tree to prevent intraspecific competition (Raffa and
Berryman 1983a). However, the role of a-pinene is likely more complex than simply
high levels equating directly to high levels of aggregation and anti-aggregation
pheromones. a-Pinene has been shown to be ovicidal to MPB eggs (Raffa and Berryman
1983b). This complex interaction and the differences in the induced levels of a-pinene

124

between jack pine and lodgepole pine could play a very important role in the population
dynamics of the MPB in jack pine.
There is evidence that, in order for ?rans-verbenol to be attractive, it must be
combined with other monoterpene host-derived kairomones, (Conn et al. 1983)
specifically myrcene, (Borden et al. 1983) which was shown to be even more effective at
increasing baited trap catches when paired with terpinolene (Borden et al. 2008).
Myrcene levels were found to be higher in the jack pine compared to northern lodgepole
pine at day 0 although there was no difference between locations after two days - i.e.
induced levels. There were higher constitutive levels of terpinolene in lodgepole but after
inoculation there was no difference between the two species of pine. There would likely
be similar levels of these synergistic terpenes in the jack pine following MPB attack as
there are in lodgepole pine, which would indicate a similar pheromone effectiveness in
this new host.
In lodgepole pine, higher levels of total terpenes increased the likelihood of
surviving attack (Chapter 2). Location and dbh were significant, with the level of total
terpenes increased with an increase in dbh at all locations. In FortMcMurray, there was a
relatively greater increase in total terpenes in inoculated jack pine trees compared with
the control between day 0 and day 14. A similar result was seen in the southern
lodgepole pine population (Kelowna). Thus, the inoculation treatment ultimately had a
strong effect on the change of the level of total terpenes in jack pine that was similar to
that in the population of lodgepole pine found in the south. This indicates that jack pine
trees, based upon the levels of total induced terpenes, might have similar survival rates to
their lodgepole pine counterparts of the same dbh, and that jack pines may react more

125

strongly to the inoculation by beetles than do lodgepole pine in other locations.
However, this measure of total terpenes does not take into account the proportions of
individual terpenes that make up total terpene levels. The relative amounts of terpenes
are likely to play a greater role in differentiation between more resistant and susceptible
host to MPB.
In general, there were very few instances of any of the locations decreasing in
level of terpenes between day 0 and day 2. An exception was levels of pulegone in the
lodgepole pine at Kelowna in both the control and inoculated trees. The southern
lodgepole pine trees were expected to have optimum response to inoculation with MPB
which could indicate that this terpene is not an important one in trees' defense. In
general, both species of trees at all the locations increased the levels of total terpenes
found in their phloem in response to attack, both to control wounding and to the crushed
beetle treatment. The response by the tree during the initial two days is likely the most
important response as mass attacks are usually completed in two days (Safranyik and
Carroll 2006). If a tree cannot successfully defend itself before being overwhelmed, it
will not survive.
It should be noted that several trees of both species had no detectable levels of A3-carene at the initial sampling period. With the exception of two lodgepole pine in
Chetwynd, the lodgepole pines with no detectable resin A-3-carene did produce
detectable levels in response to either wounding or wounding and inoculation. However,
out of thirteen jack pine with no detectable constitutive levels of A-3-carene, only three
had any induced levels of this compound following treatment. This is a small sample
size, so drawing conclusions may not be appropriate; however, this result suggests that at

126

least some jack pine do not produce induced resin A-3-carene under tested conditions.
This has potential implications, as this terpene is often found to be toxic to bark beetles
and is generally considered to be an important defensive compound. It has been shown to
be ovicidal to MPB (Raffa and Berryman 1983b) and toxic to Dendroctonus brevicomis
LeConte in vapor form (Smith 1965). While it is unlikely that one compound would
completely determine a tree's susceptibility to MPB attack, there is the potential that trees
with no induced A-3-carene would be substantially more susceptible to successful
colonization. Levels of A-3-carene in lodgepole pine clones explained over 40% of the
variation in the number of Douglas-fir pitch moth (Synathedon novaroensis Hy. Edwards)
attacks, resistant clones consistently having higher levels than susceptible clones
(Rocchini et al. 2000). Trees that lack detectable constitutive levels of A-3-carene and
that do not seem to produce the compound after attack, could be a source of new,
susceptible hosts, and would allow better colonization and ultimately higher reproductive
success. Such an effect would be magnified if, as seems possible, MPBs could exploit
large diameter trees lacking this potentially important resin component. Such a situation
could play a significant role in the population dynamics of this insect. Low host
resistance, in addition to favorable environmental conditions, is thought to be the main
factor that allows the development of incipient populations into epidemic populations
(Safranyik and Carroll 2006).

4.4.2. Potential future studies

The presence of the MPB in jack pine forests of Alberta will allow new field
studies to test the induced chemical indicators of tree success, similar to that described in

127

chapter 2. In addition, an examination of jack pine's abilities to produce A-3-carene
could also contribute to the understanding of the number of more susceptible jack pine
trees on the landscape (see 4.4.1). Further, a closer examination of the effect of the
induced a-pinene levels in jack pine compared to lodgepole pine, particularly with
respect to its complex relationship to MPB attack dynamics (see 4.4.1), could enhance
our understanding of the bark beetle's ability to utilize standing, live jack pine, as
opposed to cut bolts in a laboratory (Safranyik and Linton 1982).

4.5

Conclusions
•

The changes in terpenes are complex and there is not one overlying trend except
that the MPB will face a very different constitutive and induced terpene complex
in jack pine (e.g. A-3-carene, a-pinene, |3-phellandrene, terpinolene). This will
likely affect their successful location and utilization of these new hosts.

•

There were significant differences between the two lodgepole pine locations (e.g.
limonene, P - pinene, total terpenes) suggesting that the historical exposure to
MPB outbreaks in these regions may play a role on the constitutive levels and
induced responses of these trees. Regardless of cause, the pattern of induced
defense differed by location therefore an examination of trees in a single area may
or may not accurately represent populations across their entire geographic range.

•

There was some effect of the inoculation treatment on the response of the trees
but not as significant as was expected. This could be due, in part, to an
insufficient level of inoculum in relation to the damage caused by the wounding
of the tree.

•

The high rate of increase of a-pinene and the generally higher levels of a-pinene
found in jack pine compared with lodgepole pine, could play a significant role in
the success of MPB, given a-pinene's complex relationship with the beetles'
biosynthesis of aggregation and anti-aggregation pheromones.

•

Higher levels of A-3-carene were found in lodgepole pine at both locations
compared to jack pine. In addition, there were several jack pine trees that had no
detectable levels of A-3-carene, even after wounding and/or inoculation. This
could indicate that jack pine is a more susceptible host than lodgepole pine.

•

Similar levels of total amount of terpenes present could indicate that the MPB will
have similar success in jack pine as lodgepole pine. However, it could be the
composition of the total terpenes found, rather than total amount, that influences
the success of the beetle.

•

Given the importance of induced responses in host resistance to attack, these
differences could potentially translate into differential success of MPB between
these two species.

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Chapter 5. Conclusion
The mountain pine beetle (Dendroctonus ponderosae, Hopkins, MPB) is one of
the most important forest pests in British Columbia, Canada (BC), primarily utilizing
lodgepole pine (Pinus contorta Dougl. var. latifolia) (Safranyik and Carroll 2006). The
current eastern spread of the beetles means that jack pine (Pinus banksiana Lamb.) stands
in Alberta are now threatened, and that the insect may have an unimpeded route through
the boreal forests across the rest of Canada and even into the United States. Beetle
success depends upon the availability of suitable host tree material. In addition, the
insect must be able to kill host trees to reproduce. Understanding the complex
interactions between these insects and their hosts can contribute to management, and
ultimately to the mitigation of the effects of outbreak populations. One aspect of the
interaction between the insect and its host is the constitutive and induced terpene-based
defensive response of pines to invading beetles. Terpenes, a large group of chemicals
based on a five-carbon isoprene structure (Gershenzon and Croteau 1991), play a role in
host colonization dynamics of the insect because they are often toxic, they provide a
physical defense by flushing out gallery-building beetles, they can be part of attractant
host volatile mixes (kairomones), and they may be metabolic precursors to some
components of the beetle's pheromone communication system.
Variation in the levels of terpenes between populations of lodgepole pine could influence
the success of mountain pine beetle at a landscape level. I found differences in the
constitutive levels of various terpenes between lodgepole pine populations at four
locations in BC ranging from Princeton in the south to Quesnel in the north. The
variation in the levels of certain terpenes explained some of the variation in attack density

130

(borneol, limonene, and a-pinene) and tree survival (limonene, total resin terpene levels).
Consideration of the terpene profile of the host, in addition to other physical
characteristics, could allow for development of more accurate models for classifying
stand susceptibility (e.g. Shore and Safranyik 1992).
It also seems that some level of coevolution has occurred between the lodgepole
pine hosts in the southern part of BC and the beetle, which has had substantial historical
impact on pine populations in the area. The fact that several Dendroctonus spp. were
more tolerant of host resin vapors than nonhost resin vapors (Smith 1961a) indicates that
beetles face selective pressures to adapt to their particular host. Lodgepole pines in the
southern part of BC had lower attack densities than trees in the north suggesting that
those trees may have a composition of terpenes that provides a more comprehensive
defense against MPB than do trees in the northern populations. The likelihood of
coevolution, however, is partially dependent on the probability and timing of fire
following an outbreak, as the interplay between cone serotiny and length tree survival
time between an outbreak and a later fire would likely have an impact on the reproductive
advantage to trees that survive beetle outbreaks.
There was lower density of attack on lodgepole pine in the southern sampling
locations - near Princeton, BC. While, in general, higher levels of total terpenes
increased the probability of tree survival, the lower levels of total terpenes found in
southern populations of lodgepole pine - the population that has had more prior exposure
to MPB outbreak - may indicate selection for reduced apparency to the insects. Since
terpene emissions from trees play a role in attracting foraging beetles to the trees, it may
be that reduced emissions - observed in my study as reduced total resin terpenes -

131

provide trees with protection by making them less apparent to mountain pine beetle. The
correlation of reduced attack density on trees with lower levels of resin terpenes supports
this hypothesis.
There are other hypotheses that could also explain the variation found in these
samples, and they are not mutually exclusive with reduced apparency. Higher attack
densities in the northern populations of trees could be the result of larger beetle
populations in that area. However, all selected sites had red attacked trees at least
adjacent to the stand if not actually intermixed with the healthy trees, and the powerful
baits used to attract beetle to the sample plots were highly effective in inducing attacks.
Once the pioneering beetles, drawn in by the synthetic pheromone baits, had made their
host selection, pheromone attraction of conspecifics should have been powerful enough
to draw a heavy beetle population to all sampling areas.
The differences between locations in the level of terpenes are potentially not due
entirely to historical beetle pressure. Variation in environmental conditions can also
influence the defensive capabilities of trees. Resource limitations have been found to
influence defensive capabilities in plants including induced defense response
(Christiansen et al. 1987, Klepzig et al. 1995, Waring and Pitman 1985). Development
and maintenance of defenses requires energy resources, and therefore there can be a
tradeoff between the necessities of growth, reproduction, and defense depending upon the
pressures facing trees in particular regions (Herms and Mattson 1992).
Competition is the most common interaction between trees (Oliver and Larson
1990). It can therefore be advantageous for trees to allocate resources to growth,
however this can result in a tradeoff with a trees ability to handle stressors in their

132

environment such as resistance to cold temperatures (Loehle 1998). This type of tradeoff
could also be seen in the trees ability to handle attackers. Even within a plant, there can
be tradeoffs in the types of defenses produced. Evidence has been found in tomato plants
that pathways that induce resistance to insects can conflict with pathways that induce
resistance to pathogens (Thaler et al. 1999). Even the type of attack facing the tree can
result in a tradeoff in defensive capabilities.
The geographic range covered by even a single species will likely result in
different populations experiencing different, and potentially conflicting, pressures of
competition (both intra- and interspecific) and herbivory. These conflicting pressures
result in differences in the ability of a plant to defend itself (Herms and Mattson 1992).
In fact, Herms and Mattson (1992) predict that plant species with large geographic
distributions - such as lodgepole pine - could result in different established phenotypes
across the landscape. These differences would potentially be seen in variation in
constitutive and induced terpene defenses. Such variation would be the result of
combinations of differential competition, resource limitation, and herbivory. Because
MPB is considered to be the primary insect affecting lodgepole pine ecosytems (Amman
and Cole 1983), it would be a significant cause of such variation.
Jack pine was found to have very different constitutive and induced terpene
profiles than lodgepole pine, indicating that beetle foraging for, and utilization of, this
host may follow different dynamics as the insect invades eastern pine forests. In
addition, jack pine was found to contain lower total constitutive levels of terpenes in a
comparison between individuals in many populations of lodgepole and jack pine across
central BC and Alberta, Canada, although this difference was not observed when a

133

smaller sample of the two species was compared. Because jack pines, like southern
lodgepole pines, have lower levels of resin terpenes, they, too, may be less apparent to
foraging beetles.
However, this relationship between the host terpenes and MPB in jack pine is
complicated by the significantly higher levels of a-pinene found in both constitutive and
induced responses in jack pines, compared to levels found in lodgepole pines. a-Pinene
has been shown to be important as a precursor for components of the beetle's aggregation
and antiaggregation pheromones (Conn et al. 1984, Hunt et al. 1989). Thus, the
availability of a-pinene in host tissues should have a substantial effect on the insect's
ability to locate and successfully utilize a host.
A-3-Carene, usually considered toxic to bark beetles (Smith 1965, Raffa et al.
1985, Raffa and Berryman 1983), was found in higher constitutive levels, and higher
induced levels two days after inoculation with MPB in populations of lodgepole pines
compared to jack pines. Lodgepole pines from southern BC also had a larger relative
increase in levels of A-3-carene two days after being inoculated with crushed MPB,
compared with jack pine, also inoculated with crushed MPB. This result provides
evidence for a stronger response by lodgepole pines to MPB than seen in jack pines. The
lower amount of this important toxin in jack pine and the lower relative increase in
response to insect invasion could indicate that it would be a more susceptible host.
The success of MPB in a new geographic range and new host remains to be seen,
however, the beetle faces a very different constitutive and induced terpene-based
chemical defense in jack pines compared to in lodgepole pines. These differences should

134

be considered when making predications about the success of MPBs in the jack pine
stands of Canada and, perhaps, the United States.

135

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