University of Joensuu, PhD Dissertations in Biology
No:19
Global atmospheric change and herbivory:
Effects of elevated levels of UV-B radiation,
atmospheric CO2 and temperature
on boreal woody plants and their herbivores
by
Timo Veteli
Joensuu
2003
Veteli, Timo O.
Global atmospheric change and herbivory: effects of elevated levels of UV-B radiation,
atmospheric CO2 and temperature on boreal woody plants and their herbivores.
- University of Joensuu, 2003, 107 pp.
University of Joensuu, PhD Dissertations in Biology, No: 19. ISSN 1457-2486
ISBN 952-458-351-8
Keywords: Atmospheric change, UV-B, CO2, temperature, plant growth, secondary
compounds, herbivory, Betula, Salix, plant-insect interaction
The aim of this study was to assess the effects of elevated ultraviolet-B radiation (UV-B, 280320 nm), atmospheric CO2, temperature and soil nitrogen level on the growth and chemical
quality of boreal deciduous woody plants and on performance of the herbivorous insects
feeding on them. Eggs and larvae of Operophtera brumata (L.) (Lepidoptera, Geometridae)
were subjected to elevated UV-B radiation in the laboratory. Two willow species, Salix
phylicifolia L. (Salicaceae) and S. myrsinifolia Salisb., were grown in an UV-B irradiation field
where the responses of both plants and their herbivorous insects were monitored. S.
myrsinifolia, Betula pendula Ehrh. (Betulaceae) and B. pubescens Roth. were subjected to
elevated CO2 and temperature and different fertilisation levels in closed-top climatic chambers.
To assess the indirect effects of the different treatments, the leaves of experimental willows
and birches were fed to larvae of Phratora vitellinae (L.) (Coleoptera, Chrysomelidae) and
adults of Agellastica alni L. in the laboratory.
Elevated UV-B radiation significantly decreased the survival and performance of eggs and
larvae of O. brumata. It also increased concentrations of some flavonoids and phenolic acids in
S. myrsinifolia and S. phylicifolia, while the low-UV-B-absorbing phenolics, e. g. condensed
tannins, gallic acid derivatives and salicylates, either decreased or remained unaffected. Both
the height growth and biomass of one S. phylicifolia clone was sensitive to elevated levels of
UV-B radiation. Abundance of adults and larvae of a willow-feeding leaf beetle, P. vitellinae,
was increased under elevated UV-B; but this did not lead to increased leaf damage on the host
plants. There were no significant differences in performance of the larvae feeding on
differentially treated willow leaves, but adult A. alni preferred UV-B-treated leaves to ambient
control leaves.
Elevated CO2 and temperature significantly increased the height growth of S. myrsinifolia, B.
pendula and B. pubescens and the biomass accumulation of S. myrsinifolia. In the leaves, the
content of individual phenolic compounds and the total phenolic allocation of the plants were
affected by the treatments. Elevated CO2 reduced the levels of some phenolic compounds and
the level of nitrogen, while temperature elevation reduced the levels of many of the measured
compounds in the leaves of all the plant species studied. Increased nitrogen supply reduced the
levels of some of the individual compounds in birches. Performance of P. vitellinae fed with
willow leaves grown under elevated CO2 was reduced, while elevated temperature treatment
compensated for this effect. Feeding of A. alni on birches was not affected by the treatments.
These results show that the predicted atmospheric change will have various differential
effects on boreal deciduous woody plants and on their herbivores both directly and indirectly
via other trophic levels. These effects seem to be highly dependent on the particular species
and even on the genotype within the species as well as on the type of chemical compound or
plant growth parameter. Therefore, none of the existing hypotheses for predicting plant growth
and chemical responses to environmental changes can satisfactorily explain the observed
patterns of plant quality and herbivore performance.
Timo Veteli, Department of Biology, University of Joensuu, P.O.Box 111, 80101 JOENSUU,
Finland
3
CONTENTS
1. INTRODUCTION.............................................................................................7
1.1. OZONE DESTRUCTION AND INCREASING ULTRAVIOLET-B RADIATION ................................ 7
1.2. GREENHOUSE GASES AND CLIMATE CHANGE ...................................................................... 8
1.3. EFFECTS OF ATMOSPHERIC CHANGE ON PLANTS AND THEIR HERBIVORES........................... 9
1.3.1. Plants and phenolic allocation................................................................................... 9
1.3.2. UV-B........................................................................................................................... 9
1.3.3. CO2 and temperature ............................................................................................... 10
1.4. STUDY QUESTIONS ........................................................................................................... 11
2. MATERIAL AND METHODS......................................................................12
2.1. EXPERIMENTAL DESIGN .................................................................................................... 12
2.1.1. UV-B radiation......................................................................................................... 12
2.1.2. CO2 and Temperature .............................................................................................. 13
2.2. ORGANISMS...................................................................................................................... 13
2.2.1. Plants........................................................................................................................ 13
2.2.2. Herbivorous insects.................................................................................................. 15
2.3. GENERAL METHODOLOGY ................................................................................................ 17
2.3.1. Plant studies ............................................................................................................. 17
2.3.2. Animal bioassays...................................................................................................... 18
3. RESULTS.........................................................................................................18
3.1. UV-B ............................................................................................................................... 18
3.1.1. Direct effects on insect development and survival (I) .............................................. 18
3.1.2. Growth and quality of willows (II, III)..................................................................... 19
3.1.3. Insect herbivores and their damage on willows (III) ............................................... 19
3.2. CO2, TEMPERATURE AND NUTRIENT STATUS .................................................................... 19
3.2.1. Growth and leaf composition of plants (IV, V) ........................................................ 19
3.2.2. Effects of treated plants on herbivores (IV, V)......................................................... 20
4. DISCUSSION ..................................................................................................20
4.1. EFFECTS OF UV-B RADIATION ON PLANTS AND INSECT HERBIVORES ............................... 20
4.1.1. Plants........................................................................................................................ 20
4.1.2. Herbivorous insects.................................................................................................. 21
4.2. EFFECTS OF CO2 AND TEMPERATURE ............................................................................... 22
5. CONCLUSIONS .............................................................................................25
5.1. UV-B ............................................................................................................................... 25
5.2. CO2 AND TEMPERATURE .................................................................................................. 26
ACKNOWLEDGEMENTS................................................................................26
REFERENCES ....................................................................................................26
4
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following articles and previously unpublished results. The articles
are referred to in the text by their Roman numerals I-V.
I
Veteli, T.O. Direct effects of UV-B radiation on early development of Operophtera
brumata (L.). Submitted for publication
II
Tegelberg, R., Veteli, T., Aphalo P.J. and Julkunen-Tiitto, R. 2003. Clonal differences
in growth and phenolics of willows exposed to elevated ultraviolet-B radiation. Basic
and Applied Ecology 4: 219-228.
III
Veteli, T.O., Tegelberg, R., Pusenius, J., Sipura, M., Julkunen-Tiitto, R., Aphalo, P.J.
and Tahvanainen J. 2003. Interactions between willows and insect herbivores under
enhanced ultraviolet-B radiation. Oecologia 137: 312-320.
IV
Veteli, T.O., Kuokkanen, K., Julkunen-Tiitto, R., Roininen, H., and Tahvanainen J.
2002. Effects of elevated CO2 and temperature on plant growth and herbivore defensive
chemistry. Global Change Biology 8: 1240-1252.
V
Veteli, T.O., Tahvanainen, J., Julkunen-Tiitto, R., Kuokkanen K., Niemelä, P. and
Kellomäki, S. Effects of elevated CO2, temperature and nutrient level on growth and
herbivore defensive phytochemistry of two closely related birch species. Submitted for
publication
Publications are reprinted with permission from the publishers. Copyrights for publication II by
Urban & Fischer Verlag, III by Springer-Verlag Heidelberg and IV by Blacwell Science
5
ABBREVIATIONS
CBSC
carbon based secondary compounds
CNBH
carbon nutrient balance hypothesis
GDBH
growth differentiation balance hypothesis
CO2
carbon dioxide
HPLC
high performance liquid chromatography
IR
infrared radiation, λ = 700 - 3000 nm
PCM
protein competition model
RGR
relative growth rate
SLW
specific leaf weight
UV-A
ultraviolet-A radiation, λ = 320-400 nm
UV-B
ultraviolet-B radiation, λ = 280-320 nm
UV-BCIE
erythemally effective ultraviolet-B radiation
UV-C
ultraviolet-C radiation, λ =200-280 nm
6
1. Introduction
The world is changing – in more than one
sense. The ever-increasing human activity
within our biosphere is causing problems
for us, and for other living organisms, at an
unseen and accelerating rate. The rate of
change is the most rapid ever recorded –
only collisions with comets have more
pronounced effects on ecosystems than
does the present activity of man. Since the
1970’s, when concern over human-induced
environmental changes began, there have
been observations of extinctions of
thousands of species due to extensive
logging and destruction of both aquatic and
terrestrial habitats. In addition to direct
destruction of habitats and direct poisoning
of species with pesticides and herbicides or
chemical pollutants, we also change the
composition of the basis of life on the earth
- the atmosphere. The atmosphere consists
of gases, which are held near the surface of
the earth by gravity. Relatively small
changes in the concentrations of some of
these gases can substantially affect the
conditions on the earth’s surface, in
particular, ozone and carbon dioxide. The
changes in the atmospheric concentrations
of these gases as well as consequent
changes in the temperature on the surface
of the earth may have profound effects on
plant growth and chemical composition and
on the performance of plant-feeding
animals.
earth. In addition, this so-called ozone layer
absorbs part of the longer wave UV-B and
UV-A (λ = 320-400 nm) radiation. Thus,
only 2% of the photons reaching the surface
of the earth are in the ultraviolet range.
Photons travelling at these wavelengths
contain large amounts of energy. Therefore,
of the total solar energy reaching the
surface, UV-B and UV-A make up to 1.5%
and 6.4%, respectively (Frederick et al.,
1989; Chapman et al., 1995).
The thickness of the ozone layer controls
the intensity of UV-B radiation reaching
the earth’s surface. Natural variation in the
thickness of the ozone layer depends on
seasons, winds and solar cycles. In
addition, latitude, elevation above sea level,
time of year and time of day determine how
long a photon has to travel within the ozone
layer before entering the lower atmosphere
(Caldwell et al., 1980). Before reaching the
ground, the photon also encounters other
molecules and particles such as water in
clouds and in the air, aerosols and
tropospheric ozone molecules. These in
turn cause variation in irradiation
(Johansson 1997). Consequently, due to
gases and particles in the atmosphere and
also due to the surface albedo (scattering
from e.g. snow and leaves), almost half of
the UV radiation is scattered. Therefore, of
the UV-B radiation reaching the surface of
the earth, only half is direct radiation.
The concentration of ozone in the
atmosphere is low, only about ten ozone
molecules per million molecules in the air
(10 ppm, Graedel and Crutzen, 1993). It is
a highly unstable gas, and the ozone
molecules are continuously built up and
destroyed. Since the 1970’s the natural
balance between ozone formation and
destruction has been disturbed due to
anthropogenic emissions of nitrogen oxides
(NOx) and chlorofluorocarbons (CFC’s or
Freons) (Molina and Rowland 1974;
Prather et al., 1996). After reaching the
stratosphere, these compounds catalyse the
destruction of ozone (Molina and Rowland,
1974). This process is favoured by very low
temperatures, which may explain why
ozone depletion was first discovered and is
1.1.
Ozone
destruction
and
increasing ultraviolet-B radiation
The sun emits radiation, part of which can
be perceived by us as visible light (λ = 400700 nm, 28% of the photons reaching the
atmosphere). In addition to these
wavelengths, the sun also emits ultraviolet
(UV) radiation (λ = 200-400 nm, 5%) and
infrared (IR) radiation (λ = 700-3000 nm,
67%). The stratospheric ozone (O3) layer
and other oxygen molecules completely
absorb UV radiation below 290 nm (UV-C,
λ = 200-280 nm) and part of the UV-B (λ =
280-320 nm) preventing this part of the
radiation from reaching the surface of the
7
the IR active gases responsible for this
effect are popularly referred to as
“greenhouse gases”. Concentrations of
these gases in the atmosphere have
increased rapidly since the beginning of the
industrial period, which has given rise to
concern over potential changes in the
global atmosphere.
The principal greenhouse gases whose
concentrations have increased during the
industrial period are: carbon dioxide,
methane (CH4), nitrous oxide (N2O), and
CFC’s (Hansen et al., 1998; Schimel et al.,
1996). Mainly fossil fuel combustion and
cement production have caused the
observed increase of CO2 in the atmosphere
from about 280 ppm in the preindustrial era
to about 364 ppm in 1997 (Friedli et al.,
1986; Hansen et al., 1998). Changes in land
use produce a non-negligible but more
uncertain contribution to concentrations of
CO2 in the atmosphere (Schimel et al.,
1996). These anthropogenic sources of CO2
exceed the estimated uptake of CO2 by the
atmosphere and oceans.
Prediction of the future persistence of
anthropogenic greenhouse gases in the
atmosphere is based on mathematical
models that simulate future additions and
removals.
The
concentrations
of
greenhouse gases predicted by these models
are subject to large uncertainties as to the
effects of both natural processes and human
activities. Elevated concentrations are
predicted to persist in the atmosphere for
periods up to thousands of years (Houghton
et al., 1996; Schimel et al., 1996).
During the past 150 years, global mean
temperatures have increased by 0.3 to
0.6°C (Houghton et al., 1996). This change
is unusual in the context of the last few
centuries, even though on a timescale of
several thousand years there have been
larger climatic variations even during times
when variations in CO2 have been
relatively small. In general, however, there
have been large natural variations in CO2 in
the geologic past, which are correlated with
general features of climate change. There is
no known geologic precedent for large
increases in atmospheric CO2 without
most severe in the stratosphere over the
Antarctic (Molina and Rowland, 1974;
Farman et al., 1985). Recently, in the
1990’s, there were frequent occurrences of
springtime ozone depletion over the Arctic
as well (von der Gathen et al., 1995; Taalas
et al., 1996). Furthermore, it has been
found that the so-called “greenhouse gases”
cause stratospheric cooling, which further
favours the breakdown of ozone (Schindell
et al., 1998). Production of CFC’s has been
dramatically reduced in response to the
Montreal
Protocol
and
subsequent
international agreements, and atmospheric
concentrations of these compounds are
expected to diminish substantially during
this century (Prather et al., 1996)
Near the poles the ozone layer is thick.
Combined with low solar angles, this
means that historically, the polar and
subpolar regions have experienced far less
ambient UV-B radiation than the equatorial
regions have. According to the latest
predictions based on the stratospheric
chemistry and climate change models, in
the northern areas (60-90° N), compared
with the long-term means, the maximum
springtime UV-B radiation is expected to
increase up to 50-60% in 2010-2020
(Schindell et al., 1998; Taalas et al., 2000).
1.2. Greenhouse gases and climate
change
Radiation emitted by the sun warms the
atmosphere and the surface of the earth. Socalled IR active gases, mainly water vapour
(H2O), carbon dioxide (CO2) and also
ozone, which occur naturally in the
atmosphere, absorb the thermal IR radiation
emitted by the sun and by the earth’s
warmed surface and atmosphere. A
significant portion of this emitted energy
warms the surface and the lower
atmosphere even further. Therefore, the
average temperature of the surface air of
the earth is about 33°C higher than it would
be without atmospheric absorption of IR
radiation (Henderson-Sellers and Robinson,
1986; Kellogg, 1996; Peixoto and Oort,
1992). This phenomenon is commonly
referred to as the “greenhouse effect” and
8
simultaneous changes in other components
of the carbon cycle and climate system.
During this century alone, global
temperature has been predicted to rise by 25°C and the concentration of CO2 to double
(Houghton et al., 1996).
nitrogen on concentrations of CBSC are
essentially the same.
The protein competition model (PCM) of
Jones and Harley (1999) predicts very
variable outcome for CBSC under raised
CO2, depending, for example, on the
inherent growth rate of plant species, the
degree of photosynthetic acclimation of the
plant, nutrient availability and the state of
abiotic conditions, such as temperature and
shading. PCM is based on competition
between the pathways of phenolic
allocation and protein production over
phenylalanine. Phenylalanine is an amino
acid that is essential in both processes.
According to PCM, in non-acclimated
plants, at elevated CO2 CBSC are expected
to remain unaltered or to decrease slightly,
mainly via passive dilution due to increased
carbon content in leaves. In acclimated
plants, CO2 elevation is expected to cause
highly variable responses: no change, small
increase or small decrease in CBSC. PCM
also predicts that nitrogen enrichment of
the soil will result in a decrease in CBSC,
while in most plant species the moderate
increase in temperature expected with
global warming scenarios should have no
effects on phenolic concentrations. Overall,
the predictions of PCM with regard to the
effects of nitrogen fertilisation agree with
those given by CNBH and GBDH.
However, the predictions of PCM
concerning CBSC responses to elevated
CO2 are totally different from those of the
other two hypotheses. The PCM also makes
predictions about the effects of elevated
UV-B radiation: in general, in many
species the levels of CBSC are expected to
increase with increased UV radiation.
1.3. Effects of atmospheric change
on plants and their herbivores
1.3.1. Plants and phenolic allocation
Phenolic compounds, which are found in
all terrestrial plants, serve important
functions in these plants: lignin supports
plants and controls the decomposition rate
of plants, phenolics are, e. g., involved in
sealing of wounds following injury,
protection
against
herbivores
and
pathogens, and mediate interactions among
plants and between herbivores and soil
biota (Jones and Hartley 1998).
Elevated levels of CO2 are expected to
increase
photosynthesis
and
the
accumulation of carbohydrates in plants
over growth and maintenance. As predicted
by the carbon nutrient balance hypothesis
(CNBH) of Bryant et al. (1983), excess
carbon may then be directed to production
of carbon-based secondary compounds
(CBSC), such as phenolic compounds and
terpenoids. However, it is unclear how
excess carbon is distributed among
different
synthetic
pathways
and
compounds (e. g. Tuomi et al. 1988;
Hartley et al. 2000).
The growth differentiation balance
hypothesis (GDBH) of Herms and Mattson
(1992) predicts that resources and abiotic
factors that limit growth more than
photosynthesis increase the carbon
available for synthesis of CBSC. These
limiting factors may be, e. g. CO2,
nutrients, soil moisture and temperature.
According to GDBH, CO2 elevation is
expected to increase CBSC, while
temperature elevation and improvement in
nutrient concentration are expected to
decrease CBSC in boreal woody plants.
The predictions of the CNBH and GDBH
concerning the effects of elevated CO2 and
1.3.2. UV-B
Little is known about the effects of UV-B
on plant-herbivore interactions or the
repercussions they may cause in the food
chain. Plants and animals respond very
differently to environmental changes plants are sedentary and their responses are
mainly
restricted
to
changes
in
phytochemistry or morphology. Recently,
however, some field and laboratory studies
9
been a potent selective force in animal
evolution.
Insect herbivores, when allowed to feed
on leaves exposed to UV-B radiation, have
shown altered patterns of growth,
survivorship and feeding compared to
insects feeding on leaves without exposure
to UV-B (McCloud and Berenbaum, 1999;
Lindroth et al., 2000). However, few
studies have been conducted in natural
conditions. Most studies have approached
the problem by filtering out UV-B radiation
to prevent it penetrating the experimental
plots. Compared to control systems with
ambient levels of UV-B radiation, reduced
UV-B radiation has been found to increase
herbivore damage to plants (e. g. Ballaré et
al., 1996; Rousseaux et al., 1998; Ballare et
al., 2001; Rousseaux et al., 2001). In some
deciduous woody species, feeding on
leaves treated with increased UV-B
radiation has been shown to reduce insect
performance (Warren et al. 2002). Lindroth
et al. (2000) also showed that there are,
indeed, indirect effects on the growth of
herbivores induced by elevated UV-B.
These effects seem to be dependent on the
species and genotypes studied.
The larval and adult behaviour of some
insects seems to be affected by UV-B
radiation (e.g. Buck and Callaghan, 1999;
Antignus et al., 2001); but in general, there
is very little information about the direct
effects of UV-B on the performance and
survival of terrestrial arthropods. The
number of studies concerning the indirect
effects of enhanced UV-B on insect
herbivores (or other terrestrial arthropods)
is also extremely low. Work with natural
animal populations has been even more
rare. Herbivorous insects are major
contributors to functioning of the terrestrial
ecosystem, and effects on these insects
have ramifications for other trophic levels.
have reported the responses of terrestrial
plants and their interactions with herbivores
in the changing UV-environment (e. g.
Björn et al., 1997; Lindroth et al., 2000;
Warren et al. 2002; Ballaré et al., 2001;
Rousseaux
et
al.,
2001).
UV-B
enhancement has been shown to increase
the concentration of UV-B-absorbing
compounds in plants. These compounds, e.
g. flavonoids, protect plants against UV-Binduced DNA damage to leaves (Kootstra,
1994; Gehrke, 1999). The leaves can also
be altered structurally by producing hairs,
waxes and thicker palisade parenchyma
with greater amounts of UV-B absorbing
compounds.
Lesions
and
growth
abnormalities have also been reported in
plants under high UV-B radiation (e. g.
Wulff, 1999). These changes are thought to
be results of UV-B creating free oxygen
radicals that affect DNA regulation or
direct DNA or protein damage by UV-B.
It is a widely accepted view that the
effects of UV-B on ecosystem functioning
are mainly mediated by UV-B-induced
effects on primary producers. However,
little is known about the effects of direct
exposure to increasing UV-B radiation on
heterotrophic organisms other than
mammals and aquatic animals. UV-B
radiation has been shown to cause clear
detrimental effects on organisms; e. g. it
causes tumours in mammal skin; it may
increase the mortality of eggs and larvae,
cause reduction in growth rate of larvae and
finally, decrease the fecundity of adults
(Buck and Callaghan 1999, Berneburg and
Kruttmann 2000, McFadzen et al. 2000,
Roberts 2001). These changes may all be
related to DNA molecules, which respond
chemically to UV-B exposure, forming
double bonds between thymine molecules
and causing inactivation of DNA
(Berneburg and Kruttmann 2000). In most
of the organisms studied there are built-in
repair mechanisms for DNA damage.
Damage only occurs when these repair
mechanisms are disabled or saturated
(Berneburg and Kruttmann 2000). The
existence of repair systems for UV-induced
damage indicates that UV radiation has
1.3.3. CO2 and temperature
It is usually assumed that under enriched
CO2 atmospheres, carbon accumulation in
plants increases (e. g. Herms and Mattson
1992). This increase may influence patterns
of plant growth and carbon allocation,
10
dependent on them. The nutritive and
physical properties of plants and their
secondary metabolism have been shown to
influence the performance of herbivores as
well as the nutrient cycling through
variable decomposition of dead plant
material and herbivore faeces. The nutritive
value of the leaves may be lowered in an
elevated CO2 atmosphere due to
accumulation of carbohydrate structures in
the leaves. Fibre is hard to digest and
accumulated carbohydrates may also dilute
water and nitrogen levels in leaves. This
has been shown with various species of
plants (review by Bezemer and Jones,
1998). The effects of elevated atmospheric
CO2 on ecosystem functioning have been
studied in a number of studies, but
investigations of the effects of temperature
or the interactions between these two
factors are extremely rare. Few studies have
examined these two factors together.
which in turn determines the quality of
plants as food for herbivores. In general,
these quality factors include the
concentrations
of
water,
nitrogen,
allelochemicals, starch and other storage
carbohydrates as well as the fiber content
and toughness of the foliage (Lincoln et al.,
1993). Higher CO2 levels have been shown
to increase photosynthesis and biomass
production in certain species (e. g. Spunda
et al., 1998; Pospisilova and Catsky, 1999;
Kuokkanen et al., 2001). In most of the
species studied respiration decreases, water
use becomes more efficient and water
uptake is reduced (Pospisilova and Catsky,
1999). Because shortage of water is often a
major source of plant stress, under elevated
CO2 the overall stress tolerance of plants
improves. Depending on their differential
responses to environmental change,
increased CO2 may alter the competitive
balance among plant species, thus affecting
composition of the plant community. In
addition, the composition of plant
populations in various areas probably will
change. With greater growth, plants need
more nitrogen. One could expect that plants
successful in using more CO2 might also
need more nitrogen.
Increasing temperature is thought to
intensify the effects of elevated CO2 (Farrar
and Williams, 1991; Long, 1991; Stitt,
1991). There have been suggestions that,
especially in the boreal zone, lengthening
of the growing season and higher CO2
concentration and temperature will increase
plant growth (Kellomäki and Väisänen,
1997; Myneni et al., 1997; Kellomäki and
Wang, 1998). Levels of secondary
metabolites in plant react readily to
environmental variation. Allocation of
increased carbon supply to growth, storage
and secondary metabolites depend on the
demands of the whole plant sink, which can
vary considerably under increased CO2,
temperature and nutrient availability (Farrar
and Williams, 1991; Herms and Mattson,
1992; Jones and Hartley, 1999).
The variable allocation of the resources
within plants in a changing environment
may affect other organisms that are
1.4. Study questions
The main purpose of this thesis work was
to investigate the atmospheric impacts of
elevated UV-B, CO2 and temperature on
growth and secondary metabolites of
birches and willows and how possible
changes in plants may affect insect
performance and populations. More
specifically: Can UV-B radiation (i) affect
insect development directly (I)? Can
increased UV-B radiation affect (ii) the
quality of willows as food for insect
herbivores (II, III), (iii) the abundance and
distribution of insect herbivores on
different willow species (III), and (iv) the
amount of leaf damage caused by insect
herbivores on willows? (v) Does the
performance of insect larvae feeding on
plant tissues exposed to UV-B reflect
changes in the radiation regime (III)? Can
elevated CO2 and temperature and altered
nutrient status (vi) affect growth and the
amounts of secondary compounds in
willows and birches (IV, V); and (vii) do
possible plant responses affect herbivores
living on these species (IV, V)?
11
2. Material and methods
2.1. Experimental design
2.1.1. UV-B radiation
The effects of UV-B radiation on plants and
their herbivores were studied in two
different experimental set-ups: 1) the direct
effects of UV-B on insect eggs and larvae
in the laboratory (I), and 2) the direct effect
of supplemental UV-B radiation on S.
phylicifolia and S. myrsinifolia and their
interaction with their herbivores in the field
(II and III). In both experimental systems,
fluorescent tubes emitting UV-B radiation
(1.20 m; UVB-313, Q-Panel Co, Cleveland,
Ohio, USA) were used with combinations
of two special filters: (1) polyester filter
(PE-filter; 0.125 mm, FilmSales Ltd,
London, UK), which absorbed UV-B
radiation below 313 nm but transmitted
UV-A (0.56 – 1.12 % increase in
unweighted UV-A compared to sunlight;
see Tegelberg et al. 2001), and
(2)
cellulose-diacetate filter (CA-filter; 0.115
mm, FilmSales Ltd), which transmitted
both UV-B and UV-A radiation (0.75 –
1.54 % increase in UV-A compared to
sunlight) but absorbed radiation below 290
nm.
treatment was achieved by switching the
PE to a CA filter in the middle of the
exposure time (i.e. after 1h 40min at
+17oC). UV-B- and UV-B+ treatments were
achieved by covering the dishes with PE
and CA filters , respectively, during the
whole exposure. O. brumata was used as a
model insect. The survival rate for eggs and
larvae were determined as well as the
growth rates for larvae eating artificial
food.
Modulated UV-B irradiation field
This experiment was conducted in a UV-B
irradiation field in the Botanical Gardens of
the University of Joensuu (62°35’N,
29°46’E) from 5 June to 29 August 2000.
The UV-B irradiation system (Aphalo et al.
1999) consisted of 24 lamp frames (3.0 m x
1.5 m, Fig. 1), arranged in a randomised
block design with an ambient control, UVB treatment and UV-A control within each
of the eight blocks. Frames with
unenergized lamps provided ambient
radiation. The UV-B treatment was
obtained by covering the UV-B lamps with
CA filters. The lamps were adjusted once a
minute to keep a constant 50 % increase in
UV-BCIE (UV-BCIE based on the erythemal
action spectrum (McKinley & Diffey
1987)), which corresponds to a 20-25 %
reduction in ozone above central Finland
(Björn, 1990). The control for UV-A was
obtained by covering the UV-B lamps with
PE-filters. A more detailed description of
the UV-B field and performance of the
irradiation system is presented in Aphalo et
al. (1999) and in Tegelberg et al. (2001).
Laboratory experiment
In the laboratory the direct effects of UV-B
radiation on the development of O.
brumata were determined (I). An
irradiation-lamp system was built, and the
level of irradiance inside growth chambers
was controlled by using the special filters
described above. Three treatments were
created by switching the filters over the
petri-dishes: (1) near ambient level of UVB radiation (UV-BA) at our latitude in midJune, which corresponds to 3.3 kJ m-2 d-1
(Caldwell et al. 1983, plant damage action
spectrum), (2) UV-B exclusion (UV-B-)
and (3) supplemental UV-B radiation (UVB+), in which the level of irradiance was
about 50% higher than the ambient levels
today, representing an equivalent of the
dose that would be received in Finland
under conditions of 25% ozone depletion
under a clear sky in mid-June. The UV-BA 12
Figure 1. Pot arrangement under one
frame in the experimental UV-B irradiation
field. Above are the lamps covered by
plastic filter. Photo by TV.
2.1.2. CO2 and Temperature
Closed-Top Chamber System
This study, which is based on a factorial
design of elevated temperature and CO2,
was conducted at Mekrijärvi Research
Station, University of Joensuu (62° 47´N,
30° 58´E, 145 m a.s.l.), in eastern Finland.
Sixteen (16) closed-top climate chambers
(Fig. 2) were randomly assigned to four (4)
CO2 and temperature treatments, with four
(4) replicates in each treatment: (i) control
CO2 and temperature, (ii) elevated CO2 and
control temperature, (iii) control CO2 and
elevated temperature, and (iv) elevated CO2
and temperature. The mean control and
elevated concentrations of CO2 were 360
and 720 ppm, respectively. Elevated
temperature was obtained by raising the
temperature by 2°C, on average, from that
of the local ambient temperature to
correspond to the climate warming
scenarios predicted after doubling of
atmospheric concentrations of CO2
(Houghton et al. 1996). This system is
described in more detail by Kellomäki and
Wang (1998).
Figure2. Closed-top climate chamber in
Mekrijärvi study field. Photo by TV.
2.2. Organisms
2.2.1. Plants
Tea-leaved willow (Salix. phylicifolia L.)
The tea-leaved willow, S. phylicifolia L., is
a common and widespread shrub in
Scandinavia, found often on wet meadows,
wastelands, along lakesides, rivers and
drainage ditches (Jalas and Suominen 1976,
Hämet-Ahti et al. 1998, Skvortsov 1999).
S. phylicifolia is a typical early
successional shrub, which readily colonises
bare ground and uncultivated arable land. It
grows rapidly, and often forms dense but
relatively low vegetation together with
other willows. The leaves of S. phylicifolia
contain only small amounts of salicylates,
phenolic glucosides, which are thought to
be typical components of herbivore
resistance for willow species (Tahvanainen
et al. 1985, Julkunen-Tiitto 1989, Denno et
al. 1990, Matsuki and MacLean 1994,
Kolehmainen et al. 1995, Rank et al. 1998,
13
The salicylates, salicin and salicortin, are
the main secondary compounds found in
the leaves (Fig. 3.); and it almost
completely lacks condensed tannins and
flavonoids (Julkunen-Tiitto 1986, 1989,
Julkunen-Tiitto and Meier 1992, I).
Salicylates make the leaves very bittertasting compared to those of S. phylicifolia,
which are very mild-tasting. In contrast to
S. phylicifolia, S. myrsinifolia has fewer
generalist insect herbivores, apparently due
to the high concentrations of salicylates,
which are thought to protect the leaves
from non-adapted herbivores (Sipura and
Tahvanainen 2000; Sipura, 2000, 2002).
However, these phenolic glucosides can act
as feeding cues for specialist herbivores
(Tahvanainen et al. 1985, Pasteels et al.
1988, Rowell-Rahier and Pasteels 1990). S.
myrsinifolia may occasionally suffer severe
leaf damage caused by a specialist
herbivore,
Phratora
vitellinae
L.
(Coleoptera, Chrysomelidae).
II). On the other hand, its leaves contain
large amounts of other phenolics, including
ampelopsin and other flavonoids as well as
condensed tannins (Rank et al. 1998, II).
Due to the relatively low concentrations of
phenolic glucosides, S. phylicifolia serves
as host for a rich community of herbivorous
insects (Seppänen 1970, Liikanen 1997,
III).
Dark-leaved willow (Salix myrsinifolia
Salisb.)
The dark-leaved willow, Salix myrsinifolia
Salisb., typically grows in mixed stands
with S. phylicifolia, but usually is not as
abundant and is commonly found in drier
areas. Although S. myrsinifolia seems to be
morphologically and ecologically similar to
S. phylicifolia, the secondary chemistry of
its leaves is very different. The phenolic
glucoside concentrations of the leaves of S.
myrsinifolia are about fifty times higher
than those of S. myrsinifolia (Tahvanainen
et al. 1985, Julkunen-Tiitto 1989, Rank et
al. 1998, I).
Figure 3. A HPLC-run of a Salix myrsinifolia leaf extract detected at 220 nm
.
14
including propiophenone, cinnamic acid
and
chlorogenic
acid
derivatives,
flavonoids such as myricetin, quercetin and
kaempherol glycosides, and tannins
(Lavola
and
Julkunen-Tiitto
1994,
Keinänen, 1998, V, Fig. 4). B. pendula
serves as host for many insect herbivores
including lepidopteran, coleopteran and
hymenopteran herbivores, and also for
mammalian herbivores such as moose,
mountain hare and voles.
Silver birch (Betula pendula Ehrh.)
The silver birch, Betula pendula Ehrh., is
an early successional tree that usually
grows in moderately dry places with an
abundance of light. Ecologically it has
many features in common with Scots Pine
(Pinus sylvestris). B. pendula readily
colonises burned areas and forest openings
that have been cleared by logging or
storms.
The leaves of B. pendula contain various
carbon-based
secondary
compounds,
Figure 4. A HPLC-run of a Betula pendula leaf extract detected at 320 nm
chlorogenic acid and its ecological niche
and requirements (Veteli pers. obs.).
White birch (Betula pubescens Roth.)
The white birch, Betula pubescens Roth., is
morphologically a very variant species. It
resembles B. pendula but has a shorter life
span, grows more slowly and does not
usually reach the same height as B.
pendula. It also grows in a more shadowed
and moist environment than B. pendula
does and is usually found in peat lands and
along forest ditches as an understory.
The chemical composition of the leaves
of B. pubescens is similar to that of B.
pendula but contains about ten times more
chlorogenic acid derivatives, half the
myricetin glycosides and slightly more
tannins (V). B. pubescens is usually not
favoured as a host plant by most of the
insect herbivores that feeds on species of
Betula, probably due to its high content of
2.2.2. Herbivorous insects
All of the insect species mentioned below
are univoltine insects. The adult choice of
host plant for egg laying is crucial for the
larvae since they are very poor dispersers.
This is a common feature for herbivorous
lepidopterans and coleopterans. There are,
however, some exceptions for this rule,
especially among larger lepidopteran
generalists, whose host plants are
commonly found in mixed stands where
food is available.
15
the weather; and the larvae go through three
instars, eating for two to four weeks mainly on the lower surface of the leaves.
After that they pupate in the soil. Adults
emerge in August and September and eat
the leaves of various Salix species to gain
enough fat tissue for overwintering.
Operophtera brumata (L.) (Lepidoptera,
Geometridae)
The winter moth, Operophtera brumata
(L.), is a common species that feeds on
many different deciduous plants. The adults
emerge in the late fall when the flightless
females climb tree trunks to attract and
copulate with flying males. Eggs (Fig. 3),
which overwinter, are laid in crevices or
lichen epiphytes with minimal exposure to
UV-B. In spring, the eggs hatch and, if
suitable foliage is not found, the neonate
larvae (Fig. 4) start to disperse with the
wind over the canopy by ballooning on a
silk thread (Holliday 1977). Larvae develop
during the spring in leaf-rolls, going
through five (5) instars. Pupae occur in the
soil throughout the summer. The larvae are
extremely polyphagous feeding on almost
every species of deciduous tree found in
Finland (Seppänen 1970). This species may
also occur in outbreak densities, thus
causing serious damage to forest trees
(Tikkanen et al. 1998).
Figure 5. Phratora vitellinae L. in copula.
Photo by TV.
The larvae produce a defensive secretion,
salicylaldehyde, derived from salicylates,
salicin and salicortine, ingested from the
leaves of their host plant. Salicylaldehyde
has been shown to repel many natural
enemies of the larvae (Pasteels et al. 1988,
Rowell-Rahier and Pasteels 1990, Rank et
al. 1996). This may be the reason why
adults prefer salicylate-rich willows over
salicylate-poor ones (Tahvanainen et al.
1985, Pasteels et al. 1988, Denno et al.
1990, Rank et al. 1998, Sipura 2000), and
it has been suggested that feeding on
salicylate-rich willows provides an enemyfree space for P. vitellinae larvae (Pasteels
et al., 1988; Denno et al., 1990). However,
when larvae were grown on different
willow species, Rank et al. (1998) found no
differences in the mortality rates of the
larvae as a result of predation, although
they grew better on S. myrsinifolia than on
S. phylicifolia. This may be due to extra
energy gained from glucose detached from
salicin in the process of turning it into
salicylaldehyde. In addition, it has been
shown by Köpf et al. (1997) and Rank et
al. (1996) that larval secretion attracts
specialist predators, such as the larvae of a
syrphid fly (Parasyrphus nigritarsis Zett.).
Figure 4. Eggs and neonate larvae of the
winter moth Operophtera brumata L. Photo
by TV.
Phratora vitellinae L. (Coleoptera,
Chrysomelidae)
Phratora vitellinae L. is a chrysomelid
beetle that overwinters in litter or bark
crevices as an adult. In spring after bud
burst the adults colonise their host plants
and mate (Fig. 5). After mating they eat for
a few weeks and prepare to lay eggs in June
and July. The eggs, laid in batches of 1025, hatch after a week or two, depending on
16
climatic conditions, length of the shoots
was measured once a week for each plant
species. The leaf areas, dry weights and
specific leaf weights (SLW) for willows
were also determined at elevated UV-B and
in different CO2 and temperature treatments
after the growing season. By analysing the
nitrogen and water content of the leaves
and correlating them with relative growth
rate (RGR) and leaf palatability, the
nutritional value of leaves was determined.
Consequently, these specialist predators
and those generalist predators that can
avoid contact with larval secretion while
attacking can cause considerable mortality
in P. vitellinae populations (Rank et al.
1998).
Agellastica alni L. (Coleoptera,
Chrysomelidae)
The life cycle of Agellastica alni L. closely
resembles that of P. vitellinae, with the
exception that the adults are most
commonly found on alders (Alnus sp.) and
occasionally on birches (Betula spp.) (Fig.
6). The larvae are found in groups feeding
on alders. Occasionally, during outbreaks,
adults and larvae occur on S. phylicifolia
and Epilobium angustifolium growing in
sunny places (Veteli, pers. obs). There
seem to be very few natural enemies for
this species or else its populations are
extremely tolerant to predation and
diseases, since the numbers of individuals
found on suitable habitats are huge and the
leaf damage caused by the larvae is
considerable, often leading to total
defoliation of the host plants (Veteli, pers.
obs.). This may be related to the fact that
chrysomelid beetles have glands that
produce ill-tasting secretions.
High performance liquid
chromatography (HPLC) on secondary
phenolics
To analyse the leaves for water-soluble
phenolics, samples from the air-dried
leaves were excised with a cork-borer. The
leaf disks obtained (25 mm2, about 10 mg
DW) were then homogenized and extracted
with methanol (HPLC grade). The samples
were placed on ice and allowed to remain
there for 20 min before they were
centrifuged. The residues were extracted
four times with methanol. The solvent of
the combined extracts was dried under
nitrogen and dissolved in methanol. The
samples were then divided into three
aliquots and dried under nitrogen. One of
the aliquots was then dissolved in
methanol: water (1:1) and analysed with
high performance liquid chromatography
(HPLC) according to the procedure of
Julkunen-Tiitto et al. (1996). Salicylates,
flavonoids and phenolic acids were
identified based on their retention times and
UV-spectrum (Figs. 3 and 4). Phenolics
were quantified at 220, 270, 280, 320 and
360 nm as described in II and IV.
Tannin analysis
The concentrations of condensed tannins
were determined, both from the dissolved
methanol extract and from the residue,
using a butanol-HCL test according to the
procedure of Hagerman (1995). Tannin
content was the sum of the extract and
residue tannins. Quantification was based
on purified tannins from Salix purpurea L.
(purple willow) leaves.
Figure 6. Agellastica alni L. adult feeding
on grey alder (Alnus incana). Photo by TV.
2.3. General methodology
2.3.1. Plant studies
Growth and physical characteristics
To estimate the change in growth
characteristics of the plants in changed
17
2.3.2. Animal bioassays
Food-Choice
and
palatability
experiments
A few additional leaves were taken from
the same individual plants on the same
occasion as the leaves were collected for
chemical and RGR analysis. These
additional leaves were placed into an arena
in which an adult beetle could choose
between four (study IV) or two leaves
(study III) originating from different
treatments. The leaves were placed on filter
paper, and a plastic plate with evenly
distributed round holes was put on top of
them – one hole over each leaf, but
avoiding the main vein when possible. The
holes in the plastic plate had an area of 200
mm2. An adult beetle was then placed on
the plate, which was covered with the
bottom of a petri dish (9 cm in diameter).
The beetle was allowed to feed for 48 h.
Photoperiod was set at 17h:7h (L:D), the
humidity was 80% and the temperature was
20oC. After the experiments, the eaten areas
were measured using an ADC Area Meter
AM100 (Fig. 7).
Relative Growth Rate (RGR)
Relative Growth Rate (RGR) is a measure
of the short-term performance of an
organism. It is usually calculated as
follows:
RGR = (ln(Final mass)-ln(initial mass)) x
Time-1 (mg (mg x d)-1)
The
RGR-experiments
were
48h
experiments with second instar larvae,
which were weighed individually before
and after the experiment (Fig. 8). One larva
was put on a leaf excised from an
experimental plant in a 90 x 20 mm petri
dish, with a moistened filter paper on the
bottom. To prevent drying the dish was
covered with a lid and sealed with Parafilm.
In feeding experiments, the larvae were left
to eat for 48 h at 21oC with a photoperiod
of 17:7 (L:D). If the larva molted or died, it
was not included in the final data.
Figure 8. A full grown 2nd and a freshly
molted 3rd instar larva of Phratora vitellinae
skeletonizing a leaf of Salix fragilis. The 2nd
instar larvae were used in RGR
experiments. Photo by TV.
Figure 7. A food-Choice plate after the
experiment with Salix myrsinifolia and
Phratora vitellinae. Each leaf originated
from different treatments. Photo by TV.
3. Results
3.1. UV-B
3.1.1. Direct effects on insect
development and survival (I)
A high level of ultraviolet-B radiation
decreased egg and larval survival (by 15%
and 80%, respectively), reduced larval
growth (by 87%), slightly prolonged the
development of eggs and prevented the
development of larvae of O. brumata
compared to the situation without
ultraviolet radiation . In different irradiation
A no-choice test for palatability of B.
pendula and B. pubescens to A. alni was
also conducted (V) by inserting an intact
leaf into a petri dish with an adult beetle.
Afterwards the eaten leaf area and dry mass
were determined.
18
treatments, siblings also differed in terms
of egg and larval survival and in larval
performance, indicating genetic variation in
tolerance to ultraviolet-B radiation in this
species.
not suffer more herbivore damage than the
willows exposed to ambient solar radiation
(shade-control). As indicated by the UVtreatment block interactions, the observed
effects of UV-B on herbivore abundance,
feeding and growth showed significant
spatial variation in the field experiment.
3.1.2. Growth and quality of willows
(II, III)
UV-treatments clearly reduced the biomass
and height growth of the shoots of one
clone of tea-leaved willow. In contrast, the
growth of the other three tea-leaved willow
clones showed no significant reaction to
UV-radiation. Under elevated UV-B
radiation, the leaves of the latter clones
contained increased amounts of some of the
UV-B-absorbing quercetins, myricetins or
luteolins. In dark-leaved willows, biomass
production and growth were not affected by
UV-exposure.
In
general,
the
concentrations of leaf flavonoids were
clearly lower in dark-leaved willow than in
tea-leaved willow. In all clones of darkleaved willow, elevated UV-radiation
increased the concentrations of certain
quercetins, dihydromyricetin and phenolic
acids. Other willow leaf phenolics, i.e.
salicylates, condensed tannins and gallic
acid derivatives, were either decreased or
were unaffected by the UV-treatments.
Figure
9.
Feeding
preference
of
Agellastica alni on Salix phylicifolia leaves
excised from an experimental UV-B
irradiation field. Paired Sample T-test: N =
71, P = 0.017 (Unpublished result).
3.2. CO2, temperature and nutrient
status
3.2.1. Growth and leaf composition
of plants (IV, V)
Elevated
temperature
and
CO2
concentration increased the stem biomass,
and elevated CO2 increased the leaf
biomass and the total aerial biomass of S.
myrsinifolia. Patterns of biomass allocation
differed in different temperature treatments.
At elevated temperature there was less
branch and leaf material in relation to stems
than at the control temperature. Moreover,
the patterns of biomass allocation differed
among clones of S. myrsinifolia. CO2
enhancement increased the specific leaf
weight (SLW) and reduced both the water
and nitrogen content of the leaves.
However, leaf area was not affected by the
treatments.
The birch species, B. pendula and B.
pubescens, differed from each other in
nearly all of the variables measured.
Elevated temperature and elevated CO2
increased the growth of the plants, but more
so in B. pubescens. Leaf area and specific
leaf weight remained unaffected.
3.1.3. Insect herbivores and their
damage on willows (III)
The numbers of a leaf beetle, P. vitellinae,
on S. myrsinifolia were higher under UV-B
treatment compared with UV-A and shadecontrols. In laboratory tests, growth of the
2nd instar larva of P. vitellinae was not
affected by UV-B treatment on S.
myrsinifolia, but was retarded on UV-B
treated leaves of S. phylicifolia. A. alni
preferred willow leaves exposed to
enhanced UV-B over the ambient control
leaves (Fig. 9).
Naturally occurring insect herbivores
were more abundant on willows exposed to
elevated UV-B radiation than on those
grown under control conditions. Despite of
increased abundance of insect herbivores,
the willows treated with elevated UV-B did
19
Teramura, 1992; Sullivan et al., 1994;
Newsham et al., 1999; Tegelberg et al.,
2001). Since S. phylicifolia is an early
successional fast-growing species, in early
successional deciduous thickets the
intraspecific genetic variation in growth
responses may alter the competitive balance
in favour of more tolerant individuals.
However, this change in competitive
balance does not necessarily affect the
herbivore populations living on S.
phylicifolia since there were no clonal
differences in herbivore densities (III).
S. phylicifolia relies on flavonoid-based
protection against UV-B radiation, while S.
myrsinifolia seems to have a different but
effective method of protection against
elevated UV-B levels (II). A reactive
group, together with flavonoids, which
were present in small amounts in S.
myrsinifolia, was phenolic acids, which
have been shown to absorb UV
wavelengths (Landry et al. 1995; Lavola et
al. 1997). These compounds have been
found to protect plants against UV-Binduced DNA damage to leaves (Kootstra
1994, Gehrke 1999).
CO2 and T enhancement reduced the
concentrations
of
several
phenolic
compounds in the leaves, but the effect of
elevated CO2 on total CBSC was not
statistically different. In some cases, plant
clones showed specific responses to the
treatments. CO2 elevation reduced the
nitrogen and water content of the leaves of
birches, as well as the amounts of cinnamic
acid
derivatives
and
kaempherol
glycosides.
Elevated
temperature
significantly reduced the levels of most of
the CBSC. Fertilisation affected CBSC and
nitrogen content only moderately.
3.2.2. Effects of treated plants on
herbivores (IV, V)
CO2 enhancement for S. myrsinifolia
resulted in reduced RGR of the P. vitellinae
larvae. In contrast, the larvae of P.
vitellinae fed on willow leaves from
elevated temperature treatment had higher
RGR than larvae fed on leaves from the
ambient temperature treatment. Adult
beetles did not clearly discriminate between
willow leaves grown in different T and CO2
environments, but tended to eat more leaf
material from chambers with doubled CO2
concentration. There were no apparent
changes in the behaviour of A. alni feeding
on birch leaves.
4. Discussion
4.1. Effects of UV-B radiation on
plants and insect herbivores
4.1.1. Plants
In general, in this study UV-radiation had
no effects on growth of willows, despite the
decreased height and biomass growth of
one S. phylicifolia clone (II, Table 1). In
many other studies tree growth has also
been shown to be insensitive to elevated
UV-B radiation (Table 1; Petropoulou et
al., 1995; Newsham et al., 1996, Weih et
al., 1998; Liakoura et al., 1999; Newsham
et al., 1999). However, some long-term
studies have indicated that height growth,
biomass accumulation and carbon fixation
of plants may be susceptible to elevated
UV-B (Table 1; e. g. Sullivan and
20
Table 1. Summary of the predictions and results concerning the effects of elevated UV-B on
plants and herbivorous insects (UV-B literature: Sullivan et al. 1994; Weih et al., 1998; Buck
and Callaghan, 1999; Tegelberg and Julkunen-Tiitto 2001; Warren et al. 2002). Abbreviations:
+ = increase, - = decrease, 0 = no change, blank = does not exist.
Plants
Growth
Total phenolics
Leaf nitrogen content
Leaf water content
Herbivores
Performance
Survival
Predictions by
PCM
Literature
on UV-B
0/+/0/-
0/0/+
+
0/-
I
-
II
III
0/0/+
0/0/+
0/(+)
0
+
0
UV-B radiation affects the larval and
adult behaviour of at least some insects (III;
e. g. Buck and Callaghan 1999; Antignus et
al. 2001). For example, if enhanced UV-B
does have detrimental effects on larvae and
if adults or larvae can perceive this, some
kind of avoidance behaviour may evolve.
Avoidance behaviours could include
foraging and ovipositing under the leaves
and on other more shaded locations. The
diurnal rhythm of larvae might also change
to avoid increased UV-B radiation in the
middle of the day. These types of temporal
and spatial avoidance adaptations are
common in most cryptic herbivorous
insects. It is therefore unlikely that these
insects will normally be exposed to high
doses of UV-B at early developmental
stages, and in these insect species the
sensitivity to UV-B radiation tends to
persist.
Willows treated with elevated UV-B in
the field harboured more insect herbivores
(III), which may be due to the attraction of
UV-B radiation for insects. Probably the
best-known example of this attractive effect
of UV-B is the use of UV light, along with
other wave bands of light, by the bee (Apis
mellifera) in recognizing the source of
nectar in certain types of flowers (e. g.
Jones et al., 1986; Townson et al., 1998;
Joost et al., 1995). This is also the case for
many birds, which locate their prey by UVB vision (e.g. Viitala et al. 1995, Siitari et
al. 1999). Some butterflies may recognize
their con-specifics by UV markings on the
Flavonoids are produced by nearly all
higher plant species, and almost every
species contains its own distinctive
flavonoid profile, which some mono- and
oligophagous insects use to recognize their
host plant (Harborne and Grayer 1993).
Flavonoids can stimulate insect feeding but
can also inhibit insect growth. Some of
them even act as antibiotic and antiviral
agents in insects (Harborne and Grayer
1993). At least in some plants the flavonoid
profile varies even within species. For
example, in willows, there is genetic
variation in the content and composition of
the flavonoid profile (II). Thus genotypespecific changes in leaf chemistry due to
elevated UV-B radiation could affect the
choice of host plant, amount of feeding and
performance of herbivores.
4.1.2. Herbivorous insects
Juvenile forms of terrestrial insects
apparently are vulnerable to UV-B (Table
1; I; Buck and Callaghan, 1999). Reduction
in the egg development and larval
performance of O. brumata may be a result
of direct or oxidative damage to DNA or
proteins. In insects there may be some form
of morphological and/or physiological
protection mechanisms against increasing
UV-B radiation, especially if there is no
evidence of behavioural protection. The
number, size or thickness of body hairs,
development of pigmentation or thickening
of cuticle may change in response to UV-B
exposure (e. g. Buck and Callaghan 1999).
21
directly, which is suggested by the
laboratory experiment with O. brumata (I).
wings (e. g. Knuttel and Fiedler, 2001;
Imafuku et al., 2002). Flies and moths have
been shown to change their flight behaviour
according to UV-B radiation (e. g. Ozlov et
al., 1982; Antignus et al. 2001), which is
also widely used by insect collectors.
Increasing UV-B radiation may make
plants more obvious to their herbivores,
especially if the amount of waxes that
reflect UV wavebands in the leaf surface
increases. The amount of reflecting UV
light may also indicate the location of a
potential warm and sunny habitat. Higher
temperatures have been shown to enhance
insect performance (e. g. Lindroth et al.
1997).
In willows in the field, the increased
abundance of herbivorous insects under
elevated UV-B radiation, compared to the
ambient control, did not lead to increased
leaf consumption on those plants (III).
Nitrogen content in the leaves of willows
treated with UV-B seemed to be higher
than that in control leaves (III: Fig. 2).
Previously, nitrogen content has been
shown to increase in elevated UV-B and to
reduce leaf consumption by herbivores
(Hatcher and Paul 1994, Ballaré et al. 1996,
Rousseaux et al. 1998, Lindroth et al.
2000). Interestingly, in my laboratory
preference test, A. alni leaf beetles
preferred the UV-B-treated willow leaves
over control ones (Fig. 2). Increased
feeding may have been due to a simple
preference for leaves with higher nitrogen
content, since A. alni lives mainly on alder,
which is high in nitrogen.
The lack of increased consumption of
leaves on the plants exposed to UV-B in the
field (III: Fig. 6) could be due to structural
changes in the leaf material and/or limited
feeding capacity of the insects. Although
there were no differences between
treatments in SLW (a crude measure of leaf
toughness), there might have been
physiological structures such as hairs and
trichomes in the UV-B-treated plants that
made the leaves a poorer diet for
herbivores. Elevated UV-B may also have
reduced the food utilization of the insects
4.2. Effects of CO2 and temperature
Growth of the experimental plants was
significantly affected by both CO2 and
temperature, but these effects are clearly
dependent on the plant species or even
plant genotype (IV, V). Elevated CO2
raised the biomass gain of plants, whereas
temperature affected the height growth. For
elevated CO2, the same kinds of results
have been described earlier (Table 2;
Bezemer and Jones, 1998; Lincoln et al.,
1993; Kuokkanen et al., 2001). The
growth-stimulating effect of elevated
temperature is particularly interesting. With
elevated temperature, willows gained more
height and reduced lateral growth.
Fertilisation also increased the growth of
plants (V). At elevated temperature there
was less branch and leaf material in relation
to stems than at the control temperature.
Moreover, patterns of biomass allocation
differed among clones. All these and earlier
findings support the boreal-zone growthenhancement theory, which predicts that
lengthening of the growing season as a
result of rise in temperature and the
“fertilisation” effect of CO2 enhance the
growth of boreal trees (Kellomäki and
Väisänen, 1997; Myneni et al., 1997;
Kellomäki and Wang, 1998).
22
23
Herbivores
Performance
Leaf water
content
Plants
Growth
Total phenolics
Leaf nitrogen
content
0/+
+
+
-
+
-/0/+
+
-/0/+
T
CO2
CO2
T
Predictions by
PCM
Predictions
by
GDBH and
CNBH
-
-
+
-/0/+
-
Literature
on
CO2
0/(-)
-
+
0/+
-
CO2
+
0
+
0/0
T
Yes (?)
No
No
No
No
CO2 x T
Earlier
Mekrijärvi
studies
-
-
+
-
CO2
+
0
+
0
T
IV
No
No
No
No
No
CO2 x T
0
-
+
0
-
CO2
0
0
+
+
T
V
Yes
Yes
No
No (Yes)
No
CO2 x T
Table 2. Summary of the predictions and results concerning the effects of elevated CO2 and temperature on plants and insect herbivores
(CO2 literature: Lincoln et al., 1993; Bezemer and Jones, 1998. GDBH: Herms and Mattson, 1992. CNBH: Bryant et al., 1983. PCM:
Jones and Hartley, 1998; 1999. Earlier Mekrijärvi studies: Kuokkanen et al., 2001; Kuokkanen et al., In press; Mattson et al.,
Unpublished). For abbreviations, see Table 1.
Lindroth et al. 1993), and this could result
in nitrogen dilution.
Secondary chemicals of plants have been
considered to be greater contributors
influencing feeding of herbivores than
actual nutritional factors. This is especially
assumed for the feeding of generalist
insects. It has been suggested that, in
enriched CO2 environments, plants should
have increased allocation to carbon-based
defences (Table 2; Bryant et al., 1983;
Bazzaz et al., 1987; Jonasson et al., 1986;
but see Jones and Hartley 1999). However,
only rarely has this been observed for a
variety of plant species that uses carbonbased chemical defences (Lincoln et al.,
1993). In phenolic compounds, however, an
increase of 31% (on average) has been
found in 87% of the studies (Bezemer and
Jones, 1998). Interestingly, in our studies a
negative effect of CO2 was found; and the
concentrations
of
several
phenolic
compounds in the leaves were reduced (IV,
V). At elevated CO2, phenolic compounds
and nutrients may be diluted, and water
content decreased in the leaves, partly due
to increased carbon allocation to different
structures and to storage (e.g. thickening of
cell wall and increase of trichomes etc.).
Thus the growth demands of plants,
especially
at
elevated
temperature,
apparently exceeded the ability of plants to
synthetise CBSC as predicted by the
protein-competition model of Jones and
Hartley (1998, 1999).
CO2 enhancement increased the SLW,
which is a crude measure of leaf toughness
(Lindroth et al., 1993). SLW has been
shown to increase in e. g. red and white
oak, quaking aspen, sagebrush and many
other C3 plants (Lindroth et al., 1993;
Johnson and Lincoln, 1991; Lincoln et. al.,
1993 and references therein). If SLW is a
good estimator of leaf toughness, this may
mean that leaves from plants grown in
enriched CO2 may be more difficult for
some herbivorous insects to consume,
especially for early instars with weak
mandibles (Reavey, 1993). This may have
been the case for S. myrsinifolia and the
second instar larvae of P. vitellinae, since
In enriched CO2 atmospheres, plants
commonly have reduced concentrations of
foliar nitrogen, a phenomenon usually
referred to as the “nitrogen dilution effect”
or as an increase in the carbon-to-nitrogen
ratio (C:N). This was also the case here
(IV, V). This phenomenon, according to
Lincoln et al. (1993), depends on (1) the
carbon fixation pathway (i. e. C3 plants
have greater dilution than C4 plants), (2) the
plant species and community and (3) the
availability of other resources. This
phenomenon has been observed for
agricultural and non-domesticated species
in many habitats (Lincoln et al., 1993;
Bezemer and Jones, 1998). In almost all
studies concerning the effect of elevated
CO2, nitrogen levels of plant-tissue have
decreased by an average of 15% (Table 2;
Bezemer and Jones, 1998). A negative, but
not significant, trend under enriched CO2
has been observed in the water content of
plants (Table 2; Bezemer and Jones, 1998).
Here too the effect on the water content of
all the species studied was negative (IV, V),
but the effect was not statistically
significant.
For almost all of the species studied, the
carbohydrate concentrations of leaves have
been reported to rise by an average of 47%
(Bezemer and Jones; 1998). In the present
study the carbohydrate concentrations were
not measured, but the SLW showed an
increase in enriched CO2, which may be a
result of carbon accumulation in the leaves
(IV). Of the carbohydrates, starches may
enhance the herbivore’s ability to digest
leaves, whereas structural carbohydrates,
such as cellulose, hemicellulose, as well as
phenolic lignin, may retard feeding and
digestion (Lincoln et al. 1993). Since insect
herbivores tend to be nitrogen-limited
rather than carbon-limited (Mattson 1980),
an increase in foliar non-structural and
structural carbon concentration may reduce
insect performance if nitrogen is diluted.
This was probably the case with the species
studied here (IV, V). The accumulation of
non-structural carbohydrates at elevated
CO2 in leaves has been proven in many
cases (e. g. Johnson and Lincoln, 1991;
24
food-processing efficiencies and nitrogenconsumption rates. Their experiments
showed that even a narrow range of
temperature variation with variation in the
nitrogen content of food can have important
direct and interactive effects on the
performance of insects. Now, keeping in
mind that a combination of elevated
temperature and CO2 reduces the nitrogen
content of the leaves in all of the cases
studied (IV, V, Kuokkanen et al. 2001), the
predicted rise in temperature may shorten
the development times of insects; and the
indirect detrimental effect of nitrogen
dilution, higher SLW and lower water
content in their diet will not be enough to
compensate for the positive effect of the
rise in temperature rise, at least in the
systems studied.
All these results suggest that under the
enriched-CO2 atmospheres of the future,
both the direct effects of warmer
temperatures and indirect effects via
changes in plant quality may be alter the
performance of insects in a complex way.
growth of the latter was retarded when it
was fed with leaves originating from the
elevated CO2 treatment (IV). The other
possibility is the lowered quality of the
leaves offered as a food owing to nitrogen
dilution or reduced water content. This is
supported by the fact that adult beetles did
not clearly discriminate between willow
leaves grown in different temperature and
CO2 environments, but tended to eat more
leaf material from chambers with doubled
CO2
concentration,
thus
possibly
attempting to compensate for lower quality
food. At elevated CO2, adult beetles may
need to eat more leaf material in order to
reproduce, which may in turn prolong the
life cycle, increasing the risk of being eaten
and possibly affecting the ability to
overwinter
successfully.
Overall,
atmospheric change may significantly
modify the dynamic interaction between
willow and beetle populations.
The literature includes only a few studies
of the direct effects of temperature and
virtually none concerning the direct effects
of CO2 on herbivorous leaf feeding insects.
It is generally assumed that increased
temperature will enhance the ability of
insects to disperse and utilise food.
Lindroth et al. (1997) studied the direct
effects of temperature elevation and
concentration of dietary nitrogen on insect
performance. They found that higher
temperatures did not influence larval
survival, marginally increased final pupal
weights and strongly increased the longterm development rates of the gypsy moth
(Lymantria dispar L.). Short-term growth
and consumption rates were also enhanced,
and at higher temperatures food-processing
efficiency tended to increase. High
concentration of dietary nitrogen increased
survival rates and final pupal weights, but
increased the long-term development rates
only marginally. It also accelerated shortterm development and growth rates,
reduced consumption rates and improved
food digestibility. The insects responded to
low nitrogen-content diets primarily by
eating faster. The thermal regime also
interacted to influence growth rates, overall
5. Conclusions
5.1. UV-B
The results of this study and earlier studies
(Table 1) indicate that (1) the constitutive
level and quality of secondary chemicals in
native willow species or clones do not
predict their sensitivity to elevated UVradiation, (2) secondary chemical responses
to UV-radiation in willows are more clonespecific than species-specific and (3) the
leaves of field-grown willows treated with
UV-B radiation accumulate only those
phenolics that screen UV-B efficiently. The
results also suggest that (4) environmental
variation readily modifies the effects of
UV-B radiation on plant-insect interactions
and (5) specialist herbivores might be more
sensitive to chemical changes in their
secondary host plants than to changes in
their primary hosts. High level of
ultraviolet-B radiation may (6) decrease
egg and larval survival of insects, (7)
reduce larval growth, (8) prolong the
development of eggs and (9) prevent the
25
come in order to fill the voids between my
ears.
A great deal of this thesis work I owe to
my collaborators Mika Sipura (in
particular), Riitta Tegelberg, Pedro Aphalo,
Kari Kuokkanen, Jyrki Pusenius, OlliPekka Tikkanen, Pekka Niemelä and Seppo
Kellomäki, who provided me important
background information, labour and
facilities. Without you this work would
have been much more difficult or even
impossible. I also thank the staffs of
Mekrijärvi Research Station and Botanical
Gardens of the University of Joensuu. For
supporting this work financially, I thank the
Centre of Excellence in Forest Ecology and
Management and the Finnish Academy
Miikka Eriksson, Jaakko Pohjoismäki,
Pekka Pohjola, Markku Raijas and Ville
Raassina gave important help at some
stages of the fieldwork. Matti Savinainen
and Outi Nousiainen were important
persons in technical support. I also thank
Joann von Weissenberg for her wonderful
work in correcting my deficient English
development of larvae compared to the
situation without ultraviolet radiation. (10)
The responses of insects to UV-B radiation
are genetically determined.
5.2. CO2 and temperature
The results reported here and in earlier
studies (Table 2) indicate that elevated CO2
and temperature (1) enhance the growth of
plants and (2) lower the levels of nitrogen
and water in leaf tissue. The effects of
elevated CO2 (3) on levels of CBSC are
variable whereas temperature elevation
tends to lower CBSC levels in these
species. (4) These reactions are probably
due to enhanced plant growth and
subsequent limitation of the supply of
carbon (GDBH) or phenylalanine (PCM).
Some insect species feeding on these plants
(5) have reduced growth, and therefore (5)
may be subjected to predators for a longer
time under elevated CO2 atmospheres.
Higher temperatures, on the other hand,
may compensate for the negative effects of
increased CO2.
Acknowledgements
A great deal of this work I owe to my
family: my wife Pirkko, who took care of
me during this process, my son Tuukka
who always wanted to use the portable for
other purposes than my writing (mainly for
using the “Nature” CD-Rom) and the joy of
my life - my daughter Hanna, whose
development gave me a wonderful escape
from the scientific world from time to time
(more than often). I also thank my mother
and father who raised a boy who was
utterly interested in reading (mainly
comics, a couple of decades ago) and in
nature at the expense of any decent work.
I thank my supervisors, Jorma, Riitta and
Heikki, all of whom have given me
inspiration and support (in addition to
paying the bills) for completing this thesis.
Jorma, I thank you especially for the
fabulous conversations not concerning the
scientific world, namely dogs and hunting.
I hope I can join you on hunts and in
delightful conversations for many years to
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