Environmental Pollution 157 (2009) 174–180

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Pine weevil feeding on Norway spruce bark has a stronger impact on

needle VOC emissions than enhanced ultraviolet-B radiation
James D. Blande*, Katariina Turunen, Jarmo K. Holopainen
Department of Environmental Science, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland

Chronic exposure to enhanced UV-B radiation has little effect on volatile emissions of Norway spruce.

a r t i c l e i n f o a b s t r a c t

Article history: Plants can respond physiologically to damaging ultraviolet-B radiation by altering leaf chemistry,
Received 1 April 2008 especially UV absorbing phenolic compounds. However, the effects on terpene emissions have received
Received in revised form 11 July 2008 little attention. We conducted two field trials in plots with supplemented UV-B radiation and assessed
Accepted 17 July 2008
the influence of feeding by pine weevils, Hylobius abietis L., on volatile emissions from 3-year old Norway
spruce trees (Picea abies L. Karst.). We collected emissions from branch tips distal to the feeding weevils,
Keywords:
and from whole branches including the damage sites. Weevil feeding clearly induced the emission of
Hylobius abietis
monoterpenes and sesquiterpenes, particularly linalool and (E)-b-farnesene, from branch tips, and the
Picea abies
Terpenoids sums of monoterpenes and sesquiterpenes emitted by whole branches were substantially increased. We
Volatile organic compounds discovered little effect of UV-B radiation up to 30% above the ambient level on volatile emissions from
Systemic responses branch tips distal to damage sites, but there was a possible effect on bark emissions from damage sites.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction showed no differences in insect feeding induced volatile organic

Anthropogenic release of halocarbons, e.g. chlorofluorocarbons,
has resulted in a thinning of the stratospheric ozone layer facili-
tating the passage of increased levels of short wavelength
(280–320 nm) ultraviolet-B (UV-B) radiation to the biosphere (Kerr,
1988, 1991; Madronich et al., 1995; McKenzie et al., 1999). Whilst
rarely damaging in the field, UV-B radiation induces many
morphological, physiological and biochemical changes in plants,
including the elevation of phenolic compound concentrations in
leaf tissue (e.g. Lavola, 1998; Rousseaux et al., 2004; Caputo et al.,
2006). These changes influence interactions between plants and
herbivores (Ballaré et al., 1996, 2001; Paul and Gwynn-Jones, 2003;
Caputo et al., 2006).
In tobacco, solar UV-B radiation and simulated insect herbivory
elicit overlapping responses both in terms of gene expression
(Izaguirre et al., 2003) and accumulation of phenolic compounds
(Caputo et al., 2006; Izaguirre et al., 2007). In particular, both
stimuli elicit a down-regulation of genes encoding proteins of the
photosynthetic apparatus, and up-regulation of genes that encode
enzymes of the octadecanoid pathway, leading to the formation of
jasmonic acid (JA) and other oxylipins (Izaguirre et al., 2003).
Soybeans grown under ambient and attenuated UV radiation levels
* Corresponding author. Tel.: þ358 17 163210; fax: þ358 17 163191.
E-mail addresses: James.Blande@uku.fi (J.D. Blande), ksturune@hytti.uku.fi
(K. Turunen), Jarmo.Holopainen@uku.fi (J.K. Holopainen).
compound (VOC) emissions and there were no behavioural
responses displayed by herbivore parasitoids, despite plants
displaying morphological and physiological changes in response to
UV radiation (Winter and Rostás, 2008).
To our knowledge the influence of above ambient UV-B radia-
tion on volatile products of the octadecanoid signalling pathway
and monoterpene and sesquiterpene emissions, all important
indirect defence responses in plants (e.g. Holopainen, 2004; Dicke
and Hilker, 2003), has not previously been addressed. However,
Harley et al. (1996) observed that isoprene-emitting Quercus gam-
belii Nutt., grown in the field with supplementary UV-B simulating
a 30% depletion of ozone, emitted isoprene in greater amounts than
under ambient conditions. Furthermore, Tiiva et al. (2007) reported
that supplementary UV-B radiation, simulating a 20% depletion of
stratospheric ozone, increased isoprene emissions from a subarctic
fen. Isoprene is thought to play a role in increasing the thermal
tolerance of photosynthesis (Sharkey et al., 2001), or in protecting
plants from oxidative damage (Loreto and Velikova, 2001).
For this study we selected Norway spruce (Picea abies), one of
the most economically and ecologically important tree species in
Finland, as a model on which to test the effects of above ambient
UV-B radiation on insect-induced volatile emissions. As Norway
spruce also grows in parts of southern Europe, including at high
altitudes, we may expect a greater resistance to UV-B than in
species restricted to Scandinavia. Indeed, current evidence suggests
that conifers have a fairly high degree of tolerance to UV-B radiation
(Day et al., 1992; Laakso and Huttunen, 1998; Turtola et al., 2006),

0269-7491/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2008.07.007
 J.D. Blande et al. / Environmental Pollution 157 (2009) 174–180
with thickness of epidermal tissue, and location, concentration and
quality of UV absorbing compounds determining to what extent
UV-B penetrates needles (Day et al., 1993). Young needles are more
vulnerable to UV-B (DeLucia et al., 1992), which can damage the
photosynthetic apparatus, and reduce photosynthetic capacity
(Šprtová et al., 1999).
VOC emissions are substantially modified quantitatively and
qualitatively by insect feeding, which can be mimicked using
defence elicitors, for example methyl jasmonate (MeJA). Applica-
tion of MeJA to Norway spruce saplings increases emissions of
sesquiterpenes by over 30-fold and monoterpenes by approxi-
mately 2-fold with greater than 100-fold increases in linalool
(Martin et al., 2003). The number of traumatic resin ducts and the
concentration of terpenes in stem tissue of conifers are also
increased after MeJA application, leading to a reduction in bark
beetle (Ips typographus L.) colonisation (Erbilgin et al., 2006) and
decreased bark area removed by the large pine weevil (Hylobius
abietis L.) (Heijari et al., 2005). Feeding by white pine weevils on
bark induces VOC emissions in Sitka spruce (Picea sitchensis (Bong.)
Carr), with a similar profile to that of methyl jasmonate-induced
plants (Miller et al., 2005). However, jasmonic acid and wounding
pre-treatments of Norway spruce did not significantly affect the
feeding activity, oviposition or adult emergence of the white pine
weevil (Pissodes strobi Peck) (Nicole et al., 2006). Conifer bark
defence against bark beetles has been thoroughly reviewed by
Franceschi et al. (2005).
We hypothesised that long-term exposure to enhanced UV-B
radiation would affect the VOC emissions from Norway spruce and
in particular monoterpene and sesquiterpene emissions syn-
thesised de novo following herbivore damage. We conducted two
field trials in an ultraviolet exposure facility to test this hypothesis.
We used weevils (H. abietis L., Coleoptera: Curculionidae) which
feed on tree bark and can reach outbreak levels on Scots pine and
Norway spruce seedlings in Finland to exert biotic stresses, and
used an air entrainment system to collect VOCs. We evaluated the
effects of weevil feeding and UV-B radiation on localised and
systemic induction of VOCs by collecting emissions from whole
branches and the tip of the branch located distal to the damage site.
2. Materials and methods
2.1. Trees and insects
Norway spruce (P. abies (L.) Karst.) seedlings originating from a registered seed
orchard (seed stock 113: Kangasniemi, Finland, 61 540 N, 26 400 E, 100 m a.s.l) were
grown for 3 years at a forest nursery (METLA Suonenjoen tutkimustaimitarha,
Suonenjoki, Finland). Seedlings were re-potted in 5 L black polyethylene pots
containing a 1:1 mixture of M-6 Sphagnum peat and quartz sand (grain size
0.5–1.2 mm, Maxit Oy, Helsinki, Finland) on May 18, 2004 and transferred to
wooden platforms at the Ruohoniemi experimental field site of the University of
Kuopio Research Garden (Turtola et al., 2006). During May, plants were watered and
nutritionally supplemented twice per week with 0.1% Superex 5 fertilizer (Kekkilä
Oyj, Tuusula, Finland). During the summer months (June–August) plants were
watered twice per week and nutritionally supplemented once per week. In 2006,
Table 1
The monthly means of biologically effective UV-B radiation (kJ m2 d1) during the two growing seasons
2005
Ambienta UV-Bb
Mean  SD H L Mean  SD H L
June 1.954  0.610 3.132 0.673 2.614  1.075 3.932
July 2.524  0.814 3.512 0.553 3.000  1.019 4.321
August 1.788  1.415 5.940 0.134 1.788  1.398 5.940
September 0.490  0.349 1.378 0.037 0.490  0.344 1.378
SD indicates the standard deviation of each monthly mean. H indicates the monthly high and L the monthly low.
a
Ambient values represent the reference to which the enhanced values were set.
b
UV-B values are an average of the four enhanced ozone plots.
175
when the feeding experiments were conducted, the fertilizer concentration was
increased to 0.2%.
In 2007 further measurements were taken from three year old saplings, grown
at ambient UV-B, of the same origin as the previous experimental trees. Saplings
were planted in 5 L polyethylene pots and grown in the University of Kuopio
Research Garden.
Pine weevils H. abietis L. were collected from spruce and pine logs at a sawmill
area in Suonenjoki (Iisveden Metsä Oy). Adult weevils were stored at 8  C in plastic
containers and provided with cut pine branches as a food source. Weevils were
starved for 24 h prior to the start of experiments to encourage feeding.
2.2. UV-B enhancement plots
The UV-irradiation facility is described in detail by Turtola et al. (2006). It
consisted of 12 plots, each including a rectangular wooden platform (3.0  1.2 m)
2.5 m above which an aluminium frame of the same dimensions was supported by
metal poles. Each frame held six equally spaced fluorescent tubes (Philips TL40/12,
Philips Lighting BV, Eindhoven, The Netherlands). The plots were arranged in
a randomised block design with four replicates of three treatments: ambient control,
enhanced UV-A control and enhanced UV-B. The ambient control UV-B levels were
achieved by naturally occurring solar radiation, lamps were not functional. The
enhanced UV-B treatment was obtained by covering the lamps with cellulose
diacetate filters (100 mm; Expopak Oy, Jäminkipohja, Finland), which eliminate UV-C
radiation with a wavelength below 290 nm. UV-B and a small amount of UV-A pass
through the filter, and consequently a UV-A control was required. The UV-A control
was achieved by covering lamps with a polyester film (125 mm; Mylar D, Trafomo AB,
Sweden), which eliminates both UV-B and UV-C radiations. The ambient UV-B levels
were measured from the centre of the first ambient control plot by a weatherproof
erythema detector (type PMA 1102 Analog, Solar Light Co., Glenside, PA, USA). Each
enhanced UV-B plot was irradiated relative to this control value, with enhanced UV-
B levels measured centrally in each plot (see Table 1 for irradiance levels). The lamp
intensity of each plot was adjusted once per minute to maintain the supplementary
UV radiation at 30% above the ambient level. When solar irradiation fell below
10 mW m2 the lamps were automatically switched off, and were removed during
the winter period. On each platform there were 10 saplings arranged in two parallel
lines of 5, the pots within each line were spaced 50 cm apart and the two lines were
spaced 70 cm apart.
2.3. Branch tip emission experiment
Nine plots were selected, three for each treatment. Within each plot two trees
were selected from one end of the platform to be infested with three weevils and
two control trees were selected from the opposite end of the platform (1.5 m from
the infested trees) to avoid any priming effect of the infested trees on control trees.
One infested tree and one control tree were established per plot on each of two
consecutive days at the end of June 2006. Both infested and control trees had a fabric
mesh sleeve fastened over the inner half of one branch. In addition, infested trees
had three weevils added to the sleeve. Volatiles were collected from the branch tips
distal to the sleeves five days (120 h) after the establishment of treatment on the
first batch of trees. Rain imposed a delay of one day on the second set of collections,
thus these trees had been infested for 6 days. Collections were made from control
and infested trees on each platform concurrently. Branch tips were enclosed in
pre-cleaned (oven baking: 120  C, 1 h) multi-purpose cooking bags (polyethylene
terephthalate (PET), vol. 3 L, Look, Terinex Ltd., UK) (Stewart-Jones and Poppy, 2006).
The bags were fastened around the branch enclosing all the needle growth on the
outermost half of the branch (Fig. 1a). Although the bags were 3 L in capacity, this
was reduced to approximately 2 L after fastening to trees. One bottom corner of the
bag was cut and an air inlet tube was inserted. Air was pumped through a filter and
an MnO4 scrubber to remove ozone and into the bags at a rate of 205 mL min1.
When the bags had expanded and the air had circulated the other bag corner was cut
and a stainless steel tube (ATD sample tubes, Perkin Elmer Corp, Norwalk, CT, USA)
filled with Tenax TA adsorbent mesh 60/80 (Supelco Inc., Bellefonte, PA, USA) was
inserted. Air was pulled through the tube by a vacuum pump (Model (N022AN.18)
2006
Ambienta UV-Bb
Mean  SD H L Mean  SD H L
0.872 3.491  2.232 7.766 0.431 3.988  2.043 9.059 0.533
0.553 2.247  0.790 3.546 0.615 2.857  0.929 4.566 0.866
0.134 1.523  0.542 2.638 0.503 1.883  0.641 3.300 0.580
0.037 0.320  0.243 0.828 0.037 0.413  0.312 1.085 0.044
176 J.D. Blande et al. / Environmental Pollution 157 (2009) 174–180

2.5. Laboratory standardised volatile collections, 2007

a
G
D In mid-June 2007 we collected volatiles from trees under laboratory conditions
to gain insight on the variation that could be attributed to variable field conditions.
H Volatile samples were taken from five infested and five control Norway spruce
saplings. Infested and control trees were set up in the field in the same way as in the
branch tip emission experiment. Five days after infestation the saplings were moved
to the laboratory and sampled. Samples were taken at bench level under the
B constant conditions of 300 mmol m2 s1 PAR and 23  C using the same collection
system as in the branch tip emission experiment. During sampling it was evident
that weather conditions had disrupted weevil feeding and only three infested trees
were found to have signs of feeding damage, trees without visible damage were
A removed from the sample for analysis. The dry weights and damaged areas were
measured as above.

2.6. GC–MS analysis

Samples were analysed by GC–MS (GC type 6890, MSD 5973, Hewlett Packard,
b Wilmington, DE, USA). Trapped compounds were thermally desorbed (Perkin Elmer
C ATD400 Automatic Thermal Desorption System) at 250  C for 10 min, cryofocused at
A 30  C in a liquid nitrogen cooled cryotrap and injected onto an HP-5 capillary
F
column (50 m  250 mm  0.25 mm, Hewlett Packard). The carrier gas used was
helium. The temperature program sequence was 50  C for 1 min, followed by
D increases of 5  C min1 to 210  C and 20  C min1 to 250  C with a final hold time of
E 1.5 min. Compounds were identified by comparison of the mass spectra with those
B in the Wiley library and with pure standards. For identification and quantification of
emissions, 19 commercially available reference substances were used. Known
quantities of each reference substance were diluted in methanol and injected into
a cleaned Tenax tube. Quantification of collected samples was done by comparison
of peak areas. The reference substances for (E)-b-ocimene, and (E,E)-a-farnesene
were not available, therefore the concentrations of these compounds were calcu-
Fig. 1. Schematic representations of the volatile collection apparatus. (a) Branch tip lated by assuming that the responses would be the same as the responses of
experiment, collection bags. (b) Whole branch experiment, cuvette. A ¼ Teflon sheath, (Z)-ocimene for (E)-b-ocimene, and (E)-caryophyllene for (E,E)-a-farnesene. Green
B ¼ Hylobius abietis weevils, C ¼ glass cuvette, D ¼ Tenax TA tube (air outlet), E ¼ Teflon leaf volatiles (GLVs) were identified and quantified in the same way with 10
tube (filtered air inlet), F ¼ motor with aluminium propeller, G ¼ polyethylene bag, and commercially available reference substances used. Emission rates were related to
H ¼ cloth mesh bag. dry weight of leaf biomass.

2.7. Statistical analysis

KNF Neuberger, Freiburg, Germany) at a rate of 200 mL min1. The flow through the
Monoterpene and sesquiterpene emissions were standardised relative to
Tenax tube was calibrated before each collection with a mini-Buck calibrator (Model
a temperature of 30  C using an algorithm developed by Guenther et al. (1993). For
M-5, A.P. Buck, Inc., Orlando, FL, USA). Sampling was conducted for 30 min.
monoterpene emissions we used a temperature coefficient (b [K1]) of 0.09
Temperature at the leaf surface inside the sampling bags and photosynthetically
recommended by Guenther et al. (1993) and shown to compare well with empirical
active radiation (PAR) levels were measured using a thermometer and a PAR sensor
monoterpene coefficients in recent experiments on loblolly pine (Helmig et al.,
(Li-Cor Quantum, Li-Cor, Inc., Lincoln, NE) respectively. The PAR was measured from
2006). As the sesquiterpene emissions were dominated by (E)-b-farnesene we used
a central place between the two collection bags. During sunny periods collection
the coefficient of 0.18, as used by Helmig et al. (2006) for modelling (E)-b-farnesene
bags and the PAR sensor were protected from direct sunlight and consequent
emissions from loblolly pine. The temperatures recorded during the whole branch
heating by a few layers of a thin white polypropylene fleece (Agryl 10, Dubois
experiment ranged from 25.89  C to 37.65  C with a mean of 30.9  C, and those for
Agrinovation Inc., Saint-Remi, Canada), simulating cloud cover. The fleece reduced
the branch tip experiment ranged from 21.7  C to 36.4  C with a mean of 26.2  C. All
PAR by approximately 50%, a reduction factor that was tentatively calculated by
analyses were conducted using SPSS for Windows statistical package (SPSS 14.0,
taking field-based PAR readings with and without the fleece at ambient PAR levels of
SPSS Inc, Chicago, IL, USA). The branch tip experiment data were analysed by
1000 mmol m2 s1. This fleece was used minimally, during strong sunlight (above
multivariate ANOVA with Bonferroni correction to test the influence of UV treat-
1000 mmol m2 s1) and only throughout the sample collection process.
ment, herbivore feeding, and interactive effects on volatile emissions, the data from
After volatile collection the branch was cut and the dry mass of the plant
the two different sample dates were pooled, meaning that the feeding period was
material within the sample bag was measured. Samples were dried at 60  C for 24 h
5–6 days. The areas of bark removed from each tree, and the area of bark removed
before weighing. The part of the branch with feeding damage was also removed and
per weevil were analysed by multivariate ANOVA, correlations between bark area
the area of bark removed by the weevils was calculated. This was done by drawing
removed and emissions of induced VOCs were tested by Pearson’s correlation
around the sites of damage onto a plain sheet of paper with a square of known area
coefficient. Data from the whole branch experiment are presented as mean-
on the page. We then scanned the page and used Adobe Photoshop Software to
s  standard error, the sample size is n ¼ 2 for each treatment combination. The
count the pixels in both the drawn and known areas and calculated the area of bark
laboratory volatile collections taken in 2007 were analysed by Mann–Whitney
removed by extrapolation.
U-tests to test for the influence of insect feeding, for the control replicates n ¼ 5 and
for the infested replicates n ¼ 3.

2.4. Whole branch emission experiment 3. Results

One tree from each plot was selected, i.e. four trees per treatment. In mid-June
2006 two trees per treatment were infested with three adult H. abietis weevils
enclosed in clip cages. The two remaining trees in each treatment were controls with
a clip cage but without weevils, giving a sample size of two for each combination
(n ¼ 2). After 48 h the weevils were removed and the VOC emissions from each
treated branch were collected using an air entrainment system. Collections were
made from two trees on adjacent plots concurrently. Branches were enclosed in
cuvettes (Fig. 1b) comprising a 1.5 L glass jar fitted with a Teflon sheath (B 11 cm, FEP
Teflon, DuPont Ltd., Stevenage, UK), total volume approx. 2 l. Cuvettes were held in
place using adjustable tripods, and fastened securely to the branches using plastic
clips, clothes pegs and PTFE tape. Each cuvette had an air inlet and outlet, through
which Teflon tubing was inserted. The same air-flow system as above was used, and
sampling was conducted for 30 min. Air in cuvettes was stirred using a custom-built
aluminium propeller attached to a small motor bracketed to the outside of the
cuvette. Leaf temperature and PAR levels were measured as above.
3.1. Branch tip experiment (distal emissions)
Under all UV conditions there was a trend for an increase in
emissions of several monoterpenes in response to insect feeding
including a-pinene, camphene, sabinene, b-pinene, myrcene,
limonene, and linalool. Emissions of the monoterpenes a-pinene,
b-pinene, myrcene and linalool, and the sesquiterpene (E)-b-far-
nesene (Fig. 2) and total monoterpenes and total sesquiterpenes
(Table 2) were shown by multivariate ANOVA to be significantly
increased by insect feeding (Fig. 2).
There was a significant effect of UV treatment on emission of
methyl salicylate, which was emitted in fairly low amounts in
 J.D. Blande et al. / Environmental Pollution 157 (2009) 174–180 177

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 variation between treatments (Fig. 3a). Correlation of emissions of

each compound induced by insect feeding with bark area removed
T †
was tested for groups of herbivore damaged trees in each treatment
I * * ‡ † * using Pearson’s correlation coefficient. Under ambient UV-B radi-
T×I † * † ation emissions of linalool correlated with the area of bark removed
(r ¼ 0.968; p ¼ 0.007; n ¼ 5), however no other significant correla-
500 tions were observed. The area of bark removed per weevil per day
Amb_cont
Emission rates (ng/gDW/h)

450 was not significantly different in any of the three treatments

Amb_herb
400 (Fig. 3b).
350
300 3.3. Whole branch experiment
250
200 Emission rates of total monoterpenes for all treatment combi-
150 nations were several fold (4–35) higher from whole branches than
100 branch tips (Table 2). In ambient and UV-A treatments the total
50 GLVs including (E)-2-hexanal, (Z)-3-hexen-1-ol, (Z)-3-hexenyl
0 acetate and (Z)-3-hexenyl butyrate were also emitted in greater
quantities from whole branches. Weevil feeding increased emis-
500 sions of monoterpenes, sesquiterpenes and GLVs in control and
Emission rates (ng/gDW/h)

450 UVA_cont UV-A treatments, but not in the UV-B treatment.

UVA_herb
400
350 3.4. Laboratory standardised volatile collections, 2007
300
250 Analysis by Mann–Whitney U-test showed significant insect-
200 induced emissions of monoterpenes including a-pinene, b-pinene,
150 and linalool, and the sesquiterpene (E)-b-farnesene (Table 3). This
100 matches quite closely the emission data collected in the branch tip
50 experiment in the field.
0
4. Discussion
500
Emission rates (ng/gDW/h)

450 UVB_cont In all our experiments several monoterpenes including

UVB_herb
400
350
300
250
200
150
100
50
0 pine weevil feeding on Sitka spruce, and Arimura et al. (2004)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Fig. 2. Distal emissions from Norway spruce (Picea abies) branches (not including the
feeding site), measured in the field. Figures (n ¼ 5, except UV-B control n ¼ 6) are
means  (SE). All values are emission rates (ng (gDW)1 h1) adjusted using the rele-
vant coefficients (see methods). Multivariate ANOVA was used to test the influence of
UV treatment (T), insect infestation (I) and the interactive effect (T*I). Significance is
denoted by * p < 0.05, y p < 0.01, and z p < 0.001. Treatments are denoted by
cont ¼ control, herb ¼ herbivore damaged, Amb ¼ ambient, UVA ¼ enhanced UV-A,
and UVB ¼ enhanced UV-B. The different compounds are numbered: (1) tricyclene, (2)
a-pinene, (3) camphene, (4) sabinene, (5) b-pinene, (6) myrcene, (7) 3-carene, (8)
limonene, (9) 1,8 cineole, (10) linalool, (11) camphor, (12) borneol, (13) a-terpineol,
(14) bornyl acetate, (15) (E)-b-farnesene, (16) (Z)-a-bisabolene, (17) nonanal, (18)
methyl salicylate.
response to insect feeding and enhanced UV-B radiation. There
were four significant interactive effects shown by the analysis,
indicating an interactive effect of UV treatment and weevil feeding
on the emission of b-pinene, nonanal, methyl salicylate (Fig. 2) and
total GLVs (Table 2).
3.2. Insect damage intensity
The area of branch bark removed by insects was calculated, and
is expressed in cm2 tree1, where all trees used in volatile sampling
analyses are included. One-way ANOVA showed no significant
a-pinene, b-pinene and myrcene were emitted constitutively by
control plants, with damaged plants emitting enhanced quantities
of these compounds. Both linalool and (E)-b-farnesene were
emitted in negligible quantities by control plants, but induced
substantially by weevil feeding (Fig. 2; Table 3). Previous studies
have shown that these two compounds are particularly important
inducible plant volatiles. Miller et al. (2005) provided data in
support of a de novo production of linalool in response to white
showed that insect-damaged hybrid poplar emitted linalool (and
(E,E)-a-farnesene) systemically in a diurnal pattern, indicating the
need for active photosynthesis for emission of these compounds.
Interestingly, Mumm et al. (2003), using Scots pine, demon-
strated systemic emission of (E)-b-farnesene in response to
oviposition by the sawfly Diprion piri L., and showed that an
increase in emissions of this compound attracted the egg parasitoid
Chrysonotomyia ruforum Krausse. This suggests that this inducible
compound plays an important role in attracting herbivore natural
enemies, which may function in our study system. In both Norway
spruce (Martin et al., 2003) and Sitka spruce (Miller et al., 2005)
linalool, (E)-b-farnesene and (E)-a-bisabolene were reported
among the most significantly induced emissions in response to
MeJA treatment. However, in this study (E)-a-bisabolene was
emitted in very small quantities in response to weevil feeding. In
Sitka spruce, substantial gene expression changes were induced by
mechanical wounding, feeding by spruce budworms, and by white
pine weevils, with considerable overlap (Ralph et al., 2006). In
Norway spruce, accumulation of mono- and sesquiterpene
synthases (TPS) in response to white pine weevil feeding has been
analysed (Nicole et al., 2006). A more pronounced accumulation
was observed in the bark than needles of somatic seedlings, while
in field grown Norway spruce there was induction of a sesqui-TPS-
like gene in the bark tissues of the terminal leader, reflecting the
possible induction of a specific bark TPS (Nicole et al., 2006).
178 J.D. Blande et al. / Environmental Pollution 157 (2009) 174–180

Table 2
Volatile emissions from Norway spruce (Picea abies) collected from whole branches, including feeding site, and from distal branch tip, not including feeding site

Total monoterpenes Total sesquiterpenes Total green leaf volatiles

Ambient Control 2699.2  272.6 10.9  2.0 110.60  23.86
Infested 22,793.3  2063.6 485.6  363.2 487.64  289.07

Whole brancha UV-A Control 4612.4  1873.0 17.1  6.0 112.11  50.62
Infested 9507.7  2044.8 108.5  74.3 168.02  29.23

UV-B Control 2636.9  1205.4 21.4  14.6 74.63  19.55

Infested 2473.0  480.4 25.0  7.7 60.85  10.17

Ambient Control 332.95  108.97 4.73  4.73 14.36  0.66

Infested 655.67  127.15 263.51  110.79 46.55  12.81

Branch tipb UV-A Control 413.51  272.11 5.63  3.36 19.79  3.69
Infested 703.27  322.81 132.37  53.22 31.3  5.46

UV-B Control 234.38  45.7 2.18  2.18 48.24  11.14

Infested 621.08  191.52 303.88  154.65 36.45  6.13

Multivariate ANOVA of BT experimentc T ns ns ns

I * ** ns
IT ns ns *

All values are emission rates (ng/gDW/h) adjusted using the relevant coefficients (see methods).
a
Whole branch (WB) data are means of 2 values (n ¼ 2)  (SE).
b
Branch tip (BT) data are means of 5 values (n ¼ 5) except UV-B control (n ¼ 6)  (SE).
c
Multivariate ANOVA was used to test the influence of UV Treatment (T), Insect infestation (I) and the interactive effect (T*I) for the BT experiment. Significance is denoted by
* p < 0.05, and ** p < 0.01, ns indicates p > 0.05.

The distal branch parts were rich in new needle growth with
a few older needles, while the whole branches included more
exposed bark and a greater number of mature needles. Emission
rates of terpenes from whole branches were much greater than
those from the distal branch parts with monoterpenes dominating
the emission profile. Monoterpenes are an important component of
conifer oleoresins (Bohlmann and Croteau, 1999; Trapp and
Croteau, 2001) which are mobilised to wound and infection sites to
protect against herbivore and pathogen invasion (Phillips and
Croteau, 1999; Franceschi et al., 2005; Keeling and Bohlmann,
2006). Sesquiterpenes are a relatively minor constituent of needle
and stem oleoresins in intact Norway spruce (Martin et al., 2002,
2003; Sallas et al., 2003; Nicole et al., 2006). It is probable that the
large monoterpene emissions that we observed (Table 2) are due to
passive volatilisation of monoterpenes from oleoresin at the
damage site, as reported for Sitka spruce by Miller et al. (2005). Our
recent unpublished observations of VOC emissions from Hylobius-
damaged bark of Norway spruce and Scots pine support this view.
Emission rates of monoterpenes from whole branch controls
were also higher than those of distal controls. Two hypotheses to
explain this include (1) that small areas of the branch bark had resin
accumulations, unnoticed during sampling, that passively emitted
monoterpenes, and (2) that the older needles were constitutively
emitting greater quantities of monoterpenes than the new needle
growth. In Sitka spruce the constitutive amounts of mono- and
sesquiterpenoids in young needles were lower than in mature
needles, though these amounts became much more even following
damage by white pine weevils (Miller et al., 2005).
In all our experiments the emission rates from undamaged
branches were within the range of monoterpene emissions
(0.2–7.8 mg g(LDW)1 h1) reviewed by Kesselmeier and Staudt
(1999). However, the monoterpene emissions from whole branches
after insect feeding in ambient and enhanced UV-A treatments
markedly exceeded this upper limit. So, although our sample size
for the whole branch emissions is lower than optimal, the data that
we have presented suggest that assessments of tree emission
potential and atmospheric impact should include data from insect-
damaged trees, which could be particularly relevant during insect
outbreaks.
There was little effect of UV-B radiation on emissions from the
branch tips, with methyl salicylate (MeSA), produced via the
shikimic acid pathway, the only compound significantly affected.
However, the analyses did indicate interactive effects of enhanced
UV-B and herbivore feeding on emissions of b-pinene, nonanal and
MeSA. Although there is a wealth of information showing that UV-B
exposure affects the nutritive quality of plant tissue and promotes
the production of UV-B protective phenolic compounds, also
produced via the shikimic acid pathway (e.g. Lavola, 1998; Rous-
seaux et al., 2004), the effects on terpene content have received less
attention. Our results suggest that supplemented UV-B radiation
does not set constraints on carbon allocation to the mevalonic acid
(MVA) or methylerythritol (MEP) pathways, which are responsible
for terpene production (e.g. Holopainen, 2004). MeSA emissions in
the elevated UV-B treatment indicate that possible increased
carbon distribution to protective phenolic pigments (e.g. Kotilainen
et al., 2008) under UV-B did not cause any constraints in volatile
compound production from the same pathway. A recent investi-
gation by Turtola et al. (2006) on Norway spruce showed no
significant effects of chronic UV-B exposure on wood and needle
terpene concentrations. However, the authors did not utilise
herbivores in their investigation, so the combined effects of UV-B
irradiance and herbivory, and thus de novo produced compounds,
were not considered. Our results are consistent with recent work by
Winter and Rostás (2008) showing no effects of ambient UV levels
on VOC emissions from soybean.
In our whole branch experiment there appeared to be herbi-
vore-induced emissions of monoterpenes, sesquiterpenes and GLVs
in the UV-A and ambient control treatments, but not from the UV-B
treated trees. However, the sample size is inadequate to conclude
a definite effect of UV-B, and could be related to the extent of insect
feeding, which was observed but not measured. Herbivore feeding
at the branch base can significantly induce elevated emissions of
reactive sesquiterpenes from the distal part of the branch. This is of
important significance for estimating the impact of herbivores on
volatile emissions released at the whole tree level from conifers to
the troposphere. Bonn and Moortgat (2003) proposed that
sesquiterpenes have a crucial role in secondary aerosol (SOA)
formation in the atmosphere. Thus, it is worth investigating if
 J.D. Blande et al. / Environmental Pollution 157 (2009) 174–180 179

Table 3
a 0,18 Distal emissions from Norway spruce (Picea abies) branches (not including the
feeding site) measured in the laboratory
0,16 Control Infested P
Mean bark area removed per

Mean  SE Median Mean  SE Median

0,14
Tricyclene 4.6  2.9 0 4.1  4.1 0 ns
weevil per day (cm2)

0,12 a-Pinene 35.4  17.9 20.1 304.3  96.8 279.2 0.03

Camphene 45.4  25.8 14.2 75.2  26.6 63.8 ns
0,1 Sabinene 1.6  1.6 0 28.5  14.5 38.4 ns
b-Pinene 22.3  12.8 4 267.4  87.5 321.9 0.03
Myrcene 16.8  6.0 18 45.9  18.6 30.4 ns
0,08
3-Carene 8.1  7.3 0 233.7  145.8 199.6 ns
Limonene 86.9  47.5 69.6 200.9  47.9 224.3 ns
0,06 1,8 Cineole 12.4  7.7 5.2 32.3  10.9 27.5 ns
Linalool 0 0 57.4  32.8 25.9 0.01
0,04 Camphor 0 0 0 0 ns
Borneol 0 0 0 0 ns
0,02 a-Terpineol 0 0 0 0 ns
Bornyl acetate 44.9  25.2 28.4 27.9  18.7 9.2 ns
0
Control UV-A UV-B Total monoterpenes 290.5  141.0 214.6 1423.9  398.4 1267.3 0.03

(E)-b-farnesene 0 0 323.0  263.6 81.3 0.01

b 4
(Z)-a-bisabolene 0 0 6.0  6.0 0 ns
Mean bark area removed per tree (cm2)

Total sesquiterpenes 4.4  4.4 0 328.9  261.0 99.2 0.02

3,5
Nonanal 12.6  1.9 12.7 14.5  3.7 11.3 ns
3 Methyl salicylate 0 00 0.9  0.5 1.3 ns

All values are emission rates (ng (gDW)1 h1) adjusted using the relevant coeffi-
2,5 cients (see methods) (ng (gDW)1 h1)  SE. Mann–Whitney U-tests were used to
test between infested (n ¼ 3) and control (n ¼ 5) categories. P-values >0.05 are non-
significant (ns).
2

1,5 Whilst undertaking this kind of field investigation factors other

than those tested can affect emissions from plants (Blande et al.,
1 2007). These may include the weather which we combat using
techniques such as protection from direct sunlight during collec-
0,5 tion, and naturally occurring insects which may be attracted to
UV-B treatments and have some feeding damage effect (Newsham
0 et al., 1996). However, we did not find a significant presence of
Control UV-A UV-B
spruce feeding insects, with the exception of some aphid infesta-
Fig. 3. a. The mean area of bark removed per tree for each treatment (cm2  SE). b. The tions later in the summer.
average area of branch bark removed per weevil per day (cm2  SE). Values were taken
from replicates in which all insects were living at the end of the experiment. Bars are
not significantly different. 5. Conclusions

weevil or bark beetle feeding on bark of taller Norway spruce trees
induces substantial emissions of sesquiterpenes at the canopy and
stand levels, and if it has the potential to result in promoted
formation of aerosols locally. Secondary aerosols are known to have
negative effects on solar radiation and it has been suggested that
aerosols could globally compensate CO2-induced global warming
with a net cooling effect (Andreae et al., 2005).
There were no significant differences in the bark area removed
by weevils in each treatment, suggesting no effect of the treatments
on insect feeding. However, although not statistically significant
there was slightly more bark removed from UV-B supplemented
trees, which matches the work by Warren et al. (2002) in which leaf
discs of Populus trichocarpa Torr. exposed to double ambient UV-B
were consumed by Chrysomela scripta F. larvae at a greater rate than
leaf discs from plants grown at ambient or zero UV-B irradiance
levels. Veteli et al. (2003) demonstrated that growth of second-
instar Phratora vitellinae L. (brassy willow beetle) larvae was not
affected by a 50% supplementation of ambient UV-B radiation on
Salix myrsinifolia Salisb., but larval growth was retarded on UV-B
treated leaves of Salix phylicifolia L. These observations are evidence
that at least in some field grown tree species UV-B elicits responses
that can influence herbivores, and that stronger UV-B enhancement
may elicit stronger effects.
In conclusion, moderately enhanced UV-B radiation does not
appear to affect the constitutive or insect-induced volatile emis-
sions from distal branch tips of Norway spruce saplings, though
more work on the effects on whole branch emissions should be
conducted. There were emissions of (E)-b-farnesene and linalool
from the distal branch tips in response to feeding by H. abietis,
supporting previous results with Sitka spruce and P. strobi (Miller
et al., 2005) and results with Norway spruce treated with methyl
jasmonate (Martin et al., 2003). These emissions were much lower
per plant biomass than those from whole branches, which may
have been due to passive emission of VOCs from resin localised to
the wound site. However, because these emissions include highly
reactive sesquiterpenes responsible for secondary aerosol forma-
tion in the atmosphere, and because similar emissions could be
induced in the foliage of larger conifer trees after bark damage,
more extensive studies should be established to quantify the role of
localised biotic stresses on whole tree emissions.
Acknowledgements
We thank Timo Oksanen for providing invaluable technical
assistance. We also acknowledge the discussions and constructive
interactions at the European Science Foundation VOCBAS
180 J.D. Blande et al. / Environmental Pollution 157 (2009) 174–180
workshop on Plant Volatile Analysis, Wageningen in March 2006.
JDB was funded by a Marie-Curie Research Training Network
fellowship (ISONET, MRTN-CT-2003-504720) and KT, JKH by
Academy of Finland decisions no. 202300 and no. 105209 (JKH).
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