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Host-plant preference of an insect herbivore mediated by UV-B and CO2 in relation to plant secondary metabolites
Biochemical Systematics and Ecology 26 (1998) 1 — 12
Host-plant preference of an insect herbivore mediated by UV-B and CO in relation 2 to plant secondary metabolites Anu Lavola!,*, Riitta Julkunen-Tiitto!, Heikki Roininen!, Pedro Aphalo" ! Department of Biology, University of Joensuu, P.O. Box 111, SF-80101 Joensuu, Finland " Faculty of Forestry, University of Joensuu, P.O. Box 111, SF-80101 Joensuu, Finland Received 1 April 1997; accepted 1 September 1997
Abstract Leaves of European silver birch (Betula pendula Roth) seedlings subjected both to ambient and increased levels of CO concentration (350 and 700 ppm) with no UV-B and supplementary 2 UV-B radiation were offered to winter moth (Operophtera brumata L.) larvae in laboratory choice experiments. According to chemical analysis of the leaves, the high CO concentration 2 decreased the levels of phenolic acids and two flavonoids. The UV-B treatment increased the content of flavonoids. Winter moth larvae consumed most of the leaves of birch seedlings which had been exposed to UV-B radiation in both CO environments. Therefore, the effect of the 2 main leaf flavonoids, myricitrin and quercitrin, on the feeding of winter moth larvae was tested by an artificial diet choice experiment. The addition of the two main flavonoid glycosides into artificial diet had no stimulatory effect on larval feeding. Consequently, increases in flavonoid content may not be directly responsible for the larval preference for UV-B exposed leaves. ( 1998 Elsevier Science Ltd. All rights reserved. Keywords: Operophtera brumata; winter moth; Betula pendula; silver birch; CO concentration; ultraviolet2 B radiation; secondary metabolites; flavonoids; host-plant preference
1. Introduction The quality of plant tissue for insect herbivores is determined by its composition of primary and secondary metabolites, which depends on both the genetic identity and
* Corresponding author. Fax: #358 13 2513590; e-mail: [email protected]. 0305-1978/98/$19.00 ( 1998 Elsevier Science Ltd. All rights reserved. PII: S0 30 5 - 19 7 8( 9 7 )00 1 04 -X
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the growth conditions of the plant (e.g. Larsson et al., 1986; Bryant et al., 1987; Traw et al., 1996). Carbon dioxide concentration and UV-B radiation (280—320 nm) are factors that are changing globally in the environment and have direct biological effects on plants (e.g. Krupa and Kickert, 1989). The chemical changes in plants due to these factors can alter the susceptibility and the attractiveness of plants to herbivores (Lindroth et al., 1993; McCloud and Berenbaum, 1994; Ballare´ et al., 1996). Enhancement of CO and UV-B have been hypothesized to cause opposite re2 sponses in plants (e.g. Caldwell et al., 1995; Rozema et al., 1997). Elevated atmospheric CO concentration produces in plants the so-called “nitrogen dilution effect”: en2 hanced carbon assimilation promotes a decrease in the proportion of nitrogen and an increase in that of carbohydrates. This change is thought to force the herbivore to consume more plant material to obtain necessary levels of nitrogen (e.g. Lincoln et al., 1986; Fajer et al., 1989; Lindroth et al., 1993, 1995). Additionally, the excess availability of carbon may lead to the accumulation of carbon-based allelochemicals in the plant, which may also affect herbivory by reducing survival (e.g. Bryant et al., 1983, 1987; Roth and Lindroth, 1994; Lindroth et al., 1995). Correspondingly, enhanced UV-B radiation may not only have a direct effect on herbivores but also an indirect one via alteration of the chemical composition of host-plant tissues (e.g. Berenbaum, 1988; McCloud and Berenbaum, 1994). UV-B radiation has been shown to cause an increase in UV-protective secondary metabolites, mainly flavonoids, in various terrestrial plants (e.g. Tevini et al., 1981; Teramura, 1983; Caldwell et al., 1995). However, in contrast to CO -exposed plants, the carbon:nitrogen ratio of UV-B exposed plants 2 may decrease, which together with the increase in secondary metabolites may reduce the feeding rate and consumption of herbivores (Tevini et al., 1981; Teramura, 1983; Hatcher and Paul, 1994; Grant-Petersson and Renwick, 1996). In general, plant phenols influence preference and performance in many different herbivores, including insects. Phenols may have negative, neutral, or positive effects on herbivores depending on the feeding guild (e.g. Harborne, 1988; Bernays et al., 1989). Some ubiquitous plant allelochemicals, such as tannins and some flavonoids, possess antiherbivore properties and can increase herbivore resistance (e.g. Hedin and Waage, 1986; Berenbaum, 1988; Bernays et al., 1989), while some other more specific allelochemicals, such as certain flavonoids and small-molecular-weight phenolic glucosides, may act as stimulants for herbivore feeding (e.g. Nielsen et al., 1979; Bernays et al., 1991; Kolehmainen et al., 1995). European silver birch (Betula pendula Roth) is a common deciduous tree in the northern hemisphere of Europe (e.g. Atkinson, 1992) and is listed as one of several host plants for the polyphagous winter moth (Operophtera brumata L.) in Fennoscandia (e.g. Tenow, 1972; Tikkanen et al., 1997). Betula species contain mainly phenolics (phenolic glycosides, flavonoids, condensed tannins) and terpenoids as their secondary chemicals (Julkunen-Tiitto et al., 1996). Recent research has shown that the secondary chemical composition of birch responds both to enhanced CO concentra2 tion and UV-B radiation, but the responses are mostly compound-specific (Lavola and Julkunen-Tiitto, 1994; Roth and Lindroth, 1994; Lavola, 1997a,b; Lavola et al., 1997).
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The objective of this research was to study how CO and UV-B-induced changes in 2 secondary metabolites may influence the quality of birch leaves as food of a generalist insect herbivore, the winter moth. Therefore, in laboratory experiments the larvae of O. brumata were given a choice among the leaves of the host plant, B. pendula, which had been exposed simultaneously to either ambient or enhanced CO conditions and 2 with no or supplementary UV-B radiation. In addition, the effect of the two main flavonoid glycosides in the birch leaves on larval food preference was tested using an artificial diet.
2. Materials and methods 2.1.1. Treatment of seedlings Seeds of Betula pendula from a controlled orchard originating from seed collected in central Finland were germinated in a greenhouse and transplanted into pots (RayLeach cells, 165 cm3) containing birch-forest soil, peat and vermiculite (1:1.5:3 v/v). The seedlings were watered each day. Fertilizer was applied upon watering every second day: at first for 2 weeks with 20 ml of a 10%, then for the following 2 weeks with 50% and then with 100% nutrient solution balanced for birch by Ingestad (1962) (1.42 mg N d~1 plant~1). The six-week-old seedlings were transferred for two months into two clear plastic growth chambers located in a greenhouse, one with an ambient CO level (350 ppm) 2 and another one with increased CO level (700 ppm) (for details of CO exposure 2 2 design see Silvola and Ahlholm (1993)). In the chambers, the temperature was on average 22°C and the photoperiod was 18 h long. The additional PAR supplied using four metal halide discharge lamps (Osram Power Star, HQI-T 400 W, Germany) per chamber was about 300 lmol m~2 s~1 (measured with a Li-Cor LI-90SB quantum sensor). After 2 weeks of acclimation to the CO environments, the UV-B irradiation was 2 started. The seedlings were irradiated with UV-B supplied by three fluorescent lamps (UVB-313, Q-panel, U.S.A.) for 3 h per day centred around solar noon. The lamps were filtered with cellulose acetate film (removes all the radiation below 290 nm) for increased UV-B radiation and with polyester (removes all the radiation below 313 nm) for the control seedlings. Thus, in both chambers ("both CO concentra2 tions), half of the seedlings received no UV-B radiation (control seedlings) and half of the seedlings received a moderately increased daily dose of biologically effective UV-B radiation (8.16 kJ m~2 UV-B for exposed seedlings, Caldwell’s generalized plant BE damage, action"1 at 300 nm, measured with Macam SR9910 spectroradiometer). Plants for larval choice experiments were taken after 2, 3, and 4 weeks from the beginning of the UV-B irradiation. 2.1.2. Analysis of chemical components in seedlings The chemical analyses of the leaves were made from eight individual seedlings used in each larval choice experiment (n"8 leaves per harvest). The second fully expanded leaf from the top of these seedlings was dried at room temperature, extracted with
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A. Lavola et al./Biochemical Systematics and Ecology 26 (1998) 1—12
methanol and analysed by high performance liquid chromatography (HPLC), according to Meier et al. (1988) and Julkunen-Tiitto et al. (1996). The HPLC instrument (Hewlett-Packard, Avondale, Pennsylvania, U.S.A.) used consisted of a quaternary pump (HP 1050), an autosampler (HP 1050), and a photodiode-array detector (HP 1040A) combined with HP ChemStation. In the separation of phenolics a 3 lm HP Hypersil ODS column (60]4.6 mm I.D.) was used. Eluation solvents A (aqueous 1.5% tetrahydrofuran#0.25% orthophosphoric acid) and B (methanol) were used in the following gradient: 0—4 min 2—12% of B in A, 4—30 min 12—36% of B in A, 30—45 min 36—56% of B in A. The flow rate was 2 ml min~1 and the injection volume 20 ll. The identification of the secondary components in leaves was based on their retention time and spectral data (monitored by UV-220 and UV-360), and the quantification was based on commercial standards (Julkunen-Tiitto et al., 1996). Five flavonoid glycosides (myricitrin, quercitrin, hyperin, myricetin-3-galactoside, kaempferol-3-rhamnoside) and three phenolic acids (chlorogenic acid and two unidentified cinnamic acid derivatives) were determined by HPLC. Condensed tannin (proanthocyanidin) concentrations were determined from methanol extracts by the vanillin:HCl test (Julkunen-Tiitto, 1985). 2.1.3. Larval choice experiments The overwintering eggs of winter moth were collected from Klimovo village in Karelian Isthmus (60°40@N, 29°25@E). They were hatched in a growth chamber and the emerged larvae were transferred to Prunus padus (L.) leaves to grow, because newly hatched larvae did not survive on young birch leaves. After 1 week the larvae were fed with a mixture of B. pendula and P. padus leaves. The larval choice experiments were carried out in Petri dishes 15 cm in diameter in a growth chamber (temperature 22°C, humidity 80%). A moistened filter paper disk was placed in the bottom of the dish, on which the first fully expanded leaf of birch seedlings from each treatment was randomly arranged in a circle. Thus, there were four leaves in a Petri dish: UV-B irradiated and unirradiated leaves grown under both 350 and 700 ppm concentrations of CO . In a Petri dish, either three 2 individuals of early-instar larvae (second and third instars) or one individual of late-instar larvae (fourth and fifth instars) were placed in the middle of the leaf circle and the arena was covered with the Petri dish lid. The larvae were allowed to feed in each experiment for about 24 h. The leaves were then pressed and dried, and the amount of feeding (mm~2 removed) in each leaf was determined (Quantimet 500#Image Analysis System). Three larval choice experiments were conducted with exposed leaves. In the first experiment, after 2 weeks of UV-B irradiation, the leaves were offered to earlyinstar larvae (16 replicate Petri dishes). In the second choice experiment, after 3 weeks of UV-B irradiation, the leaves were offered to late-instar larvae (16 replicate Petri dishes). In the third choice experiment, after 4 weeks of UV-B irradiation, the leaves were offered to both early- and late-instar larvae (16 and 13 replicates, respectively). The effect of flavonoids on the larval feeding was also tested in a similar choice experiment. In Petri dishes (n"30), three types of agar-cakes were offered to two
A. Lavola et al./Biochemical Systematics and Ecology 26 (1998) 1—12
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early-instar larvae. All agar-cakes consisted of 1.5% agar and 0.1% fructose and the flavonoid-containing cakes had additional constituents of the two main leaf flavonoids (commercial standards): either 0.1% quercitrin and 0.05% myricitrin or 0.5% quercitrin and 0.1% myricitrin, representing low and high flavonoid concentrations found in native birch leaves, respectively. The experiment was terminated after 24 h and the amount of feeding was determined under a microscope as the percentage of cake surface eaten. Cakes were set into four different %-classes (0%, 1—4%, 5—7%, 8—10%) on the basis of the area removed from the cake. 2.1.4. Statistical analysis The effects of UV-B and CO treatments on plant phytochemicals in three harvests 2 were tested by three-factor ANOVA. Results of food choice tests with leaves and with agar-cakes were statistically tested by the Friedman test (Conover, 1980). ANOVA was used also in statistical evaluation of differences in feeding between the different choice experiments and between larval age and feeding.
3. Results 3.1. Secondary chemistry of seedlings In our experimental birches, the tannins constituted the main proportion of the leaf phenolics, 3—8% of the leaf dry weight, while the proportions of the lavanoid glycosides was 1.5—3% and the phenolic acids was 0.5—1% of the leaf dry weight. The amounts of leaf flavonoids and phenolic acids changed significantly due to enhancement of UV-B radiation and CO concentration, while the amount of condensed 2 tannins remained unaffected (Table 1). On a dry weight basis, the second fully expanded leaf of UV-B-exposed seedlings had a 15—95% higher amount of quercitrin, 60—240% higher amount of hyperin, 20—150% higher amount of myricetin-3-galactoside and 10—70% higher amount of kaempferol-3-rhamnoside than the seedlings not exposed to UV-B. In the elevated CO concentration, seedlings had reduced content 2 of phenolic acids and some flavonoids (quercitrin, kaempferol-3-rhamnoside) as compared with ambient CO (Table 1). However, the accumulation pattern of phen2 olics under UV-B radiation was not significantly changed by the enhancement of CO 2 (no UV-Bx CO interactions). 2 3.2. Larval feeding The leaves from the plants grown under enhanced UV-B radiation under both CO 2 environments (350 and 700 ppm) were eaten to a greater extent than the leaves which received no UV-B radiation (Fig. 1a, b). For early instar larvae, the amount of tissue removed from the UV-B-treated leaves was significantly higher than that of untreated leaves in both CO levels (Friedman test; X2"11.671, p"0.0086, df"3,93) (Fig. 1a). 2 Also, the consumption of late-instar larvae differed nearly significantly among treatments (Friedman test; X2"7.419, p"0.0597, df"3,84) and the amount of feeding
7.41$0.80 3.36$0.37 2.60$0.33 1.92$0.21 0.58$0.13 0.51$0.05 56.7$7.37 63.1$7.12
Other phenolics: Chlorogenic ac 350 700 Cinnamic ac I 350 700 Cinnamic ac II 350 700 Tannins 350 700
Kae-3-rha
Myr-3-gal
Hyperin
Quercitrin
6.39$0.52 4.33$0.32 2.26$0.23 1.48$0.10 0.75$0.13 0.45$0.03 67.5$4.94 67.2$6.23
11.3$1.55 9.8$1.23 14.8$1.75 10.5$1.19 1.43$0.18 1.03$0.13 1.24$0.27 0.69$0.08 1.14$0.13 0.93$0.08
4.63$0.64 3.26$0.37 2.18$0.29 1.75$0.23 0.48$0.05 0.35$0.04 75.8$4.13 74.7$13.7
13.3$1.15 11.6$1.23 7.28$0.89 7.59$0.66 0.51$0.11 0.61$0.11 0.56$0.14 0.82$0.18 0.65$0.08 0.69$0.05
!UV-B
!UV-B
#UV-B
Harvest II
Harvest I
13.6$1.88 11.0$1.55 12.9$0.63 6.56$0.34 0.73$0.13 0.39$0.07 0.78$0.24 0.52$0.08 1.01$0.09 0.56$0.05
CO ppm2
350 700 350 700 350 700 350 700 350 700
Flavonoids: Myricitrin
Phenolics
5.01$0.60 4.18$0.47 2.76$0.39 1.88$0.25 0.72$0.09 0.49$0.06 73.4$6.48 82.9$5.92
12.2$1.26 12.5$1.03 14.2$1.45 9.92$1.54 1.47$0.33 1.26$0.24 1.37$0.32 1.32$0.28 0.98$0.08 0.77$0.04
#UV-B
3.47$1.26 2.97$0.38 1.57$0.23 1.79$0.24 0.54$0.06 0.40$0.05 38.4$5.17 49.1$4.37
9.20$1.83 12.6$1.50 6.88$1.27 7.53$1.00 0.40$0.08 0.73$0.08 0.53$0.11 0.88$0.16 0.55$0.08 0.59$0.05
!UV-B
Harvest III
4.13$0.52 3.22$0.30 1.96$0.21 1.77$0.22 0.57$0.08 0.54$0.04 51.5$4.11 60.2$4.71
13.1$1.51 11.3$1.32 13.6$2.09 11.2$1.19 1.37$0.17 1.18$0.12 1.32$0.35 1.05$0.18 0.94$0.09 0.71$0.06
#UV-B
ns
*** ns
ns
ns
***
*
ns
*
*
ns
ns
*
ns
ns
ns
***
**
***
***
ns
ns
ns
***
ns
***
ns
***
ns
***
***
***
ns
ns
ns
ns
ns
*
ns
ns
ns
ns
Harvest CO xH UV-BxCO xH 2 2
ns
ns
ns
UV-B CO 2
ANOVA results
Table 1 The mean concentration of leaf secondary metabolites (mg g~1 d.wt$sd) in the three harvests and the results of ANOVA (p(0.001"***, p(0.01"**, p(0.05"*, p'0.05"non-significant (ns)). UV-Bx CO and UV-Bx harvest interactions were non-significant in all cases (myr"myricitrin, kae"kaem2 pferol, gal"galactoside, rha"rhamnoside, ac"acid)
6 A. Lavola et al./Biochemical Systematics and Ecology 26 (1998) 1—12
A. Lavola et al./Biochemical Systematics and Ecology 26 (1998) 1—12
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Fig. 1. The percentages (%) of leaf area eaten on differently treated leaves by early-instar larvae (a) and by late-instar larvae (b) in larval choice experiments. The number of touched leaves in each treatment is shown in columns (for early-instar larvae n"32, for late-instar larvae n"29).
on the leaves exposed to both UV-B radiation and elevated CO concentration was 2 slightly higher than that on leaves grown in ambient conditions (Fig. 1b). The leaf preference was similar in each choice experiment and for different sized larvae: there was no statistical difference in the feeding pattern among differently treated leaves between different choice experiments (ANOVA; F "2.387, 3,240 p"0.0697) or between early and late instars of larvae (ANOVA; F "0.174, 1,242 p"0.677). Also, the relative frequencies of leaves having some feeding damage were quite similar among different choice experiments (see Fig. 1a, b). However, the early-instar larvae were observed to behave more actively in food selection: they moved around the dishes and tasted a bit of each leaf they encountered before starting to feed. In contrast, the late-instar larvae placed themselves below a leaf shortly after
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A. Lavola et al./Biochemical Systematics and Ecology 26 (1998) 1—12
Table 2 The preference of winter moth larvae for low (0.15%) and high (0.6%) flavonoid concentrations expressed as a number of cakes eaten in each %-class and the mean ranks for treatments (Friedman test; X2"6.9, p"0.032, df"2,58) % Eaten
0 1—4 5—7 8—10 Mean rank
Control
6 13 9 2 2.17
Flavonoid concentration Low
High
9 9 8 4 2.17
11 10 7 2 1.67
the experiment started, and fed on that leaf for a while before changing their position. However, there were no treatment-based differences in leaf size (ANOVA; F "0.74, p"0.390 for UV-B treatments and F "2.25, p"0.134 for CO 1,240 1,240 2 treatments, no interactions), which could have affected the food selection. In the choice experiment using artifical substrate, the combinations of the two different concentrations of flavonoids myricitrin and quercitrin had no stimulating influence on larval feeding. Larvae consumed equally the lower flavonoid concentration cakes (0.15%) and control ones but they consumed significantly less high flavonoid concentration cakes (0.6%) than control ones (Friedman test; X2"6.9, p"0.032, df"2,58) (Table 2).
4. Discussion The chemical analyses of leaves support earlier observations that the concentration of secondary metabolites in birch species is influenced by UV-B radiation and CO 2 concentration. Likewise earlier (Lavola, 1997a; Lavola et al., 1997), the amount of flavonoids and some phenolic acids increased in birch leaves due to UV-B irradiation. Accumulation of phenolic compounds is a common response in plants to UV-B radiation, since UV-B usually activates the biosynthetic routes of secondary metabolites (e.g. Li et al., 1993; Caldwell et al., 1995). In this experiment, the CO enhancement reduced the amount of flavonoids and 2 phenolic acids in leaves. In earlier studies, the high CO levels have caused increase in 2 the amount of main secondary metabolites, tannins and flavonoids, in birch and similarly decrease in the amount of some minor phenolic compounds (phenolic glucosides) (Lavola and Julkunen-Tiitto, 1994; Roth and Lindroth, 1994; Lindroth et al., 1995; Traw et al., 1996). Although the introduction of CO is suggested to 2 increase the production of carbon-based secondary metabolites, especially the endproducts of secondary metabolite routes, it is not the general plant response to high CO concentrations (e.g. Reichardt et al., 1991; Ayres, 1993; Lindroth et al., 1993). 2
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Overall, the secondary chemical responses of birch seedlings to changes in different climatic factors are very compound dependent (Lavola et al., 1994, 1997; Lavola, 1997a,b) probably based on ontogenic differences in the carbon allocation of individual seedlings (Bryant and Julkunen-Tiitto, 1995). In choice experiments, the behaviour of O. brumata larvae, especially of early instars, implied active food selection. This is seen in higher feeding preference of larvae for UV-B-exposed birch leaves, which supports the observations of the marked effects of UV-B radiation on herbivory. As in deciduous shrubs in the field (Gwynn-Jones et al., 1997), UV-B-exposed plants experienced greater loss than in control ones with no interactive effect of enhanced CO concentration on herbivory. However, there 2 seems to be differences in herbivory among host-plant species and in consumption between different aged larvae due to UV-B radiation (Grant-Petersson and Renwick, 1996; Gwynn-Jones et al., 1997). Contrary to our results, in some cases the consumption and performance of larvae has decreased on host plants grown under increased UV-B radiation, and suggested to correlate with increased nitrogen or flavonoid concentrations of exposed plants (Hatcher and Paul, 1994; McCloud and Berenbaum, 1994; Ballare´ et al., 1996; Grant-Petersson and Renwick, 1996). However, herbivory of plants grown under elevated CO levels often increases, as the feeding of late-instar 2 larvae in this experiment may also imply, because of the “dilution effect” in CO 2 exposed plants (e.g. Lincoln et al., 1986; Lindroth et al., 1993, 1995; Roth and Lindroth, 1994). Consequently, insect performance and feeding preference on a host plant may correspond with different properties of leaves, such as texture, nutritional quality and secondary metabolite content (Larsson et al., 1986; Bryant et al., 1987; Lindroth et al., 1988). In many studies, increases in the amount of secondary metabolites has caused decreases in the rates of food consumption and the growth of insect herbivores (Feeny, 1970; Larsson et al., 1986; Bryant et al., 1987; Lindroth and Bloomer, 1991; Traw et al., 1996). However, the function of UV-B inducible flavonoids in plant—insect interactions is related to their chemical structure. According to Hedin and Waage (1986), flavonol aglycones possess antifeedant activity, which increases as the number of hydroxyl groups increases. The antifeedant activity of flavonoids may also depend on their glycosylation, since rhamnosides can be more effective (or even toxic) than glucosides and galactosides, which may even act as phagostimulants (Nielsen et al., 1979; Hedin and Waage, 1986; Harborne, 1988; Bernays et al., 1991; Hedin et al., 1992; Larsson et al., 1992). The main flavonoids in birch, myricitrin and quercitrin, are rhamnosides and they contain three and two hydroxyl groups in their B-ring, respectively (e.g. Hedin and Waage, 1986). Both of them have efficient antigrowth and antifeeding activities against some insects (e.g. Elliger et al., 1980; Dreyer and Jones, 1981) but, on the other hand, quercitrin has also been shown to stimulate feeding (e.g. Harborne, 1988). However, in this study the addition of these flavonoid glycosides to artificial diet of the larvae did not increase its consumption. Thus, the increase in the amount of these main flavonoids in leaves may not cause the higher preference of larvae for the UV-B treated leaves. This does not exclude the phagostimulative effect of some minor flavonoid glycoside, such as hyperin (quercetin-3-galactoside), which also increased
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highly under UV-B radiation (Table 1), or the effect of these flavonoids in combination with other leaf constituents, such as phenolic glucosides (e.g. Nielsen et al., 1979; Harborne, 1988; Lindroth et al., 1988; Roininen and Tahvanainen, 1989). However, it has been suggested that for early feeding generalist herbivores (like O. brumata) the total composition of phytochemicals and other traits of leaf quality (water content, toughness) may be more significant in the food selection than a change in any specific leaf component (e.g. Feeny, 1970; Lindroth and Bloomer, 1991). In conclusion, our results indicate significant changes in the chemical composition of plants and in the pattern of insect herbivory under enhanced UV-B radiation and CO levels. However, the UV-B induced accumulation of main flavonoids may not be 2 directly responsible for the increased larval consumption on UV-B-exposed leaves. Therefore, the impacts of UV-B and CO enhancement on herbivory via phytochemi2 cals of host plant are not easily generalized, since their mediation to herbivory may depend on the species-specific adaptations and specialization of each herbivore to the chemical composition of its host plant (e.g. Tahvanainen et al., 1985; Grant-Petersson and Renwick, 1996; Van der Meijden, 1996).
Acknowledgements This work was partly supported by the Academy of Finland and the Maj and Tor Nessling Foundation. The authors thank Ms Tania de la Rosa and Ph.D. Tarja Lehto for collaboration, and Professors Jorma Tahvanainen and Carlos Ballare´ for their comments on the manuscript.
References Atkinson, M.D., 1992. Betula pendula Roth (B. verrucosa Ehrh.) and B. pubescens Ehrh. J. Ecol. 80, 837—870. Ayres, M.P., 1993. Plant defense, herbivory, and climate change. In Biotic Interactions and Global Change, eds., P.M. Kareiva, J.G. Kingsolver and R. B. Huey, pp. 75—94. Ballare´, C.L., Scopel, A.L., Stapleton, A.E., Yanovsky, M.J., 1996. Solar ultraviolet-B radiation affects seedling emergence, DNA integrity, plant morphology, growth rate, and attractiveness to herbivore insects in Datura ferox. Plant Physiol. 112, 161—170. Berenbaum, M., 1988. Effects of electromagnetic radiation on insect—plant interactions. In Plant Stress—Insect Interactions, ed. E.A. Heinrichs, pp. 167—185. Wiley, New York. Bernays, E.A., Cooper Driver, G., Bilgener, M., 1989. Herbivores and plant tannins. Adv. Ecol. Res. 19, 263—302. Bernays, E.A., Howard, J.J., Champagne, D., Estesen, B.J., 1991. Rutin: a phagostimulant for the polyphagous acridid Schistocerca americana. Entomol. Exp. Appl. 60, 19—28. Bryant, J.P., Julkunen-Tiitto, R., 1995. Ontogenic development of chemical defense by seedling resin birch: energy cost of defense production. J. Chem. Ecol. 21, 883—896. Bryant, J.P., Chapin, F.S. III, Klein, D.R., 1983. Carbon/nutrient balance of boreal plants in relation to vertebrate herbivory. Oikos 40, 357—368. Bryant, J.P., Clausen, T.P., Reichardt, P.B., McCarthy, M.C., Werner, R.A., 1987. Effect of nitrogen fertilization upon the secondary chemistry and nutritional value of quaking aspen (Populus tremuloides Michx.) leaves for the large aspen tortrix (Choristoneura conflictana (Walker)). Oecologia 73, 513—517.
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Caldwell, M.M., Teramura, A.H., Tevini, M., Bornman, J.F., Bjo¨rn, L.O., Kulandaivelu, G., 1995. Effects of increased solar ultraviolet radiation on terrestrial plants. Ambio 24, 166—173. Conover, W.J., 1980. Practical Nonparametric Statistics. Wiley, New York. Dreyer, D.L., Jones, K. C., 1981. Feeding deterrency of flavonoids and related phenolics towards Schizaphis graminum and Myzus persicae: aphid feeding deterrents in wheat. Phytochemistry 20, 2489—2493. Elliger, C.A., Chan, B.C., Waiss, A.C. Jr, 1980. Flavonoids as larval growth inhibitors. Naturwissenschaften 67, 358—360. Fajer, E.D., Bowers, M.D., Bazzaz, F.A., 1989. The effects of enriched carbon dioxide atmospheres on plant—insect herbivore interactions. Science 243, 1198—1200. Feeny, P., 1970. Seasonal changes in oak leaf tannins and nutrients as a cause of spring feeding by winter moth caterpillars. Ecology 51, 565—581. Grant-Petersson, J., Renwick, J.A.A., 1996. Effects of ultraviolet-B exposure of Arabidopsis thaliana on herbivory by two crucifer-feeding insects (Lepidoptera). Environ. Entomol. 25, 135—142. Gwynn-Jones, D., Lee, J.A., Callaghan, T.V., 1997. Effects of enhanced UV-B radiation and elevated carbon dioxide concentrations on a sub-arctic forest heath ecosystem. Plant Ecology 128, 242—249. Harborne, J., 1988. Flavonoids in the environment: structure—activity relationships. In Plant Flavonoids in Biology and Medicine II: Biochemical, Cellular, and Medicinal Properties, eds V. Cody, E. Middleton Jr, J. Harborne and A. Beretz, pp. 17—27. Alan R. Liss, New York. Hatcher, P.E., Paul, N.D., 1994. The effect of elevated UV-B radiation on herbivory of pea by Autographa gamma. Entomol. Exp. Appl. 71, 227—233. Hedin, P.A. and Waage, S.K., 1986. Roles of flavonoids in plant resistance to insects. In Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological, and Structure—Activity Relationship, eds V. Cody, E. Middleton Jr and J. Harborne, pp. 87—100. Alan R. Liss, New York. Hedin, P.A., Jenkins, J.N., Parrott, W.L., 1992. Evaluation of flavonoids in Gossypium arboreum (L.) cottons as potential source of resistance to tobacco budworm. J. Chem. Ecol. 18, 105—114. Ingestad, T., 1962. Macro element nutrition of pine, spruce and birch seedlings in nutrient solutions. Medd. fra> n Statens Skogsforskingsinstitut 51, 16—21. Julkunen-Tiitto, R., 1985. Phenolic constituents in the leaves of northern willows: methods for the analysis of certain phenolics. J. Agric. Food Chem. 33, 213—217. Julkunen-Tiitto, R., Rousi, M., Bryant, J., Sorsa, S., Keina¨nen, M., Sikanen, H., 1996. Chemical diversity of several Betulaceae species: comparison of phenolics and terpenoids in northern birch stems. Trees 11, 16—22. Kolehmainen, J., Julkunen-Tiitto, R., Roininen, H., Tahvanainen, J., 1995. Phenolic glucosides as feeding cues for willow-feeding leaf beetles. Entomol. Exp. Appl. 74, 235—243. Krupa, S.V., Kickert, R.N., 1989. The greenhouse effect: impacts of ultraviolet-B (UV-B) radiation, carbon dioxide (CO ), and ozone (O ) on vegetation. Environ. Pollut. 61, 263—293. 2 3 Larsson, S., Wire´n, A., Lundgren, L., Ericsson, T., 1986. Effects of light and nutrient stress on leaf phenolic chemistry in Salix dasyclados and susceptibility to Galerucella lineola (Coleoptera).Oikos 47, 205—210. Larsson, S., Lundgren, L., Ohmart, C.P., Gref, R., 1992. Weak responses of pine sawfly larvae to high needle flavonoid concentrations in scots pine. J. Chem. Ecol. 18, 271—282. Lavola, A., 1998a. Accumulation of flavonoids and related compounds in birch induced by UV-B irradiance. Tree Physiology (in press). Lavola, A., 1998b. Soluble carbohydrates and secondary phytochemicals in Betula as affected by SO 2 pollution. Water Air Soil Pollut. (in press). Lavola, A., Julkunen-Tiitto, R., 1994. The effect of elevated CO and fertilization on primary and secondary 2 metabolites in birch, Betula pendula (Roth). Oecologia 99, 315—321. Lavola, A., Julkunen-Tiitto, R., Pa¨a¨kko¨nen, E., 1994. Does ozone stress change the primary or secondary metabolites of birch (Betula pendula Roth)?. New Phytologist 126, 637—642. Lavola, A., Julkunen-Tiitto, R., Aphalo, P., de la Rosa, T. and Lehto, T., 1997. The effect of UV-B radiation on UV-absorbing secondary metabolites in birch seedlings grown under simulated forest soil conditions. New Phytologist 137, 617—621. Li, J., Ou-Lee, T.M., Raba, R., Amundson, R.G., Last, R.L., 1993. Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation. Plant Cell 5, 171—179.
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Lincoln, D.E., Couvet, D., Sionit, N., 1986. Response of an insect herbivore to host plants grown in carbon dioxide enriched atmospheres. Oecologia 69, 556—560. Lindroth, R.L., Bloomer, M.S., 1991. Biochemical ecology of the forest tent caterpillar: responses to dietary protein and phenolic glycosides. Oecologia 86, 408—413. Lindroth, R.L., Scriber, J.M., Hsia, M.T.S., 1988. Chemical ecology of the tiger swallowtail: mediation of host use by phenolic glycosides. Ecology 69, 814—822. Lindroth, R.L., Kinney, K.K., Platz, C.L., 1993. Responses of deciduous trees to elevated atmospheric CO : 2 productivity, phytochemistry, and insect performance. Ecology 74, 763—777. Lindroth, R.L., Arteel, G.E., Kinney, K.K., 1995. Responses of three saturniid species to paper birch grown under enriched CO atmospheres. Funct. Ecol. 9, 306—311. 2 McCloud, E.S., Berenbaum, M.R., 1994. Stratospheric ozone depletion and plant—insect interactions: effects of UVB radiation on foliage quality of Citrus jambhiri for ¹richoplusia ni. J. Chem. Ecol. 20, 525—539. Meier, B., Julkunen-Tiitto, R., Tahvanainen, J., Sticher, O., 1988. Comparative high-performance liquid and gas—liquid chromatographic determination of phenolic glucosides in Salicaceae species. J. Chromatogr. 442, 175—186. Nielsen, J.K., Larsen, L.M., Sørensen, H., 1979. Host plant selection of the horseradish flea beetle Phyllotreta armoraciae (Coleoptera: Chrysomelidae): identification of two flavonol glycosides stimulating feeding in combination with glucosinolates. Ent. Exp. Appl. 26, 40—48. Reichardt, P.B., Chapin, F.S. III, Bryant, J.B., Mattes, B.R., Clausen, T.P., 1991. Carbon/nutrient balance as a predictor of plant defense in Alaskan balsam poplar: potential importance of metabolite turnover. Oecologia 88, 401—406. Roininen, H., Tahvanainen, J., 1989. Host selection and larval performance of two willow-feeding sawflies. Ecology 70, 129—136. Roth, S.K., Lindroth, R.L., 1994. Effects of CO -mediated changes in paper birch and white pine chemistry 2 on gypsy moth performance. Oecologia 98, 133—138. Rozema, J., Lenssen, G.M., van de Staaji, J.W.M., Tosserams, M., Visser, A.J., Broekman, R.A., 1997. Effects of UV-B radiation on terrestrial plants and ecosystems: interaction with CO enrichment. Plant Ecology 2 128, 182—191. Silvola, J., Ahlholm, U., 1993. Effects of CO concentration and nutrient status on growth, growth rhythm 2 and biomass partitioning in a willow, Salix phylicifolia. Oikos 67, 227—234. Tahvanainen, J., Julkunen-Tiitto, R., Kettunen, J., 1985. Phenolic glycosides govern the food selection pattern in willow feeding beetles. Oecologia 67, 52—56. Tenow, O., 1972. The outbreaks of Oporinia autumnata Bhk. and Operophtera spp. (Lep., Geometridae) in Scandinavian mountain chain and northern Finland 1862—1968. Zool. Bid. 2, 1—107. Teramura, A.H., 1983. Effects of ultraviolet-B radiation on the growth and yield of crop plants. Physiol. Plant. 58, 415—427. Tevini, M., Iwanzik, W., Thoma, U., 1981. Some effects of enhanced UV-B irradiation on the growth and composition of plants. Planta 153, 388—394. Tikkanen, O.-P., Roininen, H., Niemela¨, P., Tahvanainen, J. and Zinojev, A., 1997. The host plant use of Operophtera brumata L. (winter moth) during an outbreak in southeastern Fennoscandia. J. Appl. Entomol. (in press). Traw, M.B., Lindroth, R.L., Bazzaz, F.A., 1996. Decline in gypsy moth (¸ymantria dispar) performance in an elevated CO atmosphere depends on host plant species. Oecologia 108, 113—120. 2 Van der Meijden, E., 1996. Plant defence, an evolutionary dilemma: contrasting effects of (specialist and generalist) herbivores and natural enemies. Ent. Exp. Appl. 80, 307—310.
Host-plant preference of an insect herbivore mediated by UV-B and CO in relation 2 to plant secondary metabolites Anu Lavola!,*, Riitta Julkunen-Tiitto!, Heikki Roininen!, Pedro Aphalo" ! Department of Biology, University of Joensuu, P.O. Box 111, SF-80101 Joensuu, Finland " Faculty of Forestry, University of Joensuu, P.O. Box 111, SF-80101 Joensuu, Finland Received 1 April 1997; accepted 1 September 1997
Abstract Leaves of European silver birch (Betula pendula Roth) seedlings subjected both to ambient and increased levels of CO concentration (350 and 700 ppm) with no UV-B and supplementary 2 UV-B radiation were offered to winter moth (Operophtera brumata L.) larvae in laboratory choice experiments. According to chemical analysis of the leaves, the high CO concentration 2 decreased the levels of phenolic acids and two flavonoids. The UV-B treatment increased the content of flavonoids. Winter moth larvae consumed most of the leaves of birch seedlings which had been exposed to UV-B radiation in both CO environments. Therefore, the effect of the 2 main leaf flavonoids, myricitrin and quercitrin, on the feeding of winter moth larvae was tested by an artificial diet choice experiment. The addition of the two main flavonoid glycosides into artificial diet had no stimulatory effect on larval feeding. Consequently, increases in flavonoid content may not be directly responsible for the larval preference for UV-B exposed leaves. ( 1998 Elsevier Science Ltd. All rights reserved. Keywords: Operophtera brumata; winter moth; Betula pendula; silver birch; CO concentration; ultraviolet2 B radiation; secondary metabolites; flavonoids; host-plant preference
1. Introduction The quality of plant tissue for insect herbivores is determined by its composition of primary and secondary metabolites, which depends on both the genetic identity and
* Corresponding author. Fax: #358 13 2513590; e-mail: [email protected]. 0305-1978/98/$19.00 ( 1998 Elsevier Science Ltd. All rights reserved. PII: S0 30 5 - 19 7 8( 9 7 )00 1 04 -X
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A. Lavola et al./Biochemical Systematics and Ecology 26 (1998) 1—12
the growth conditions of the plant (e.g. Larsson et al., 1986; Bryant et al., 1987; Traw et al., 1996). Carbon dioxide concentration and UV-B radiation (280—320 nm) are factors that are changing globally in the environment and have direct biological effects on plants (e.g. Krupa and Kickert, 1989). The chemical changes in plants due to these factors can alter the susceptibility and the attractiveness of plants to herbivores (Lindroth et al., 1993; McCloud and Berenbaum, 1994; Ballare´ et al., 1996). Enhancement of CO and UV-B have been hypothesized to cause opposite re2 sponses in plants (e.g. Caldwell et al., 1995; Rozema et al., 1997). Elevated atmospheric CO concentration produces in plants the so-called “nitrogen dilution effect”: en2 hanced carbon assimilation promotes a decrease in the proportion of nitrogen and an increase in that of carbohydrates. This change is thought to force the herbivore to consume more plant material to obtain necessary levels of nitrogen (e.g. Lincoln et al., 1986; Fajer et al., 1989; Lindroth et al., 1993, 1995). Additionally, the excess availability of carbon may lead to the accumulation of carbon-based allelochemicals in the plant, which may also affect herbivory by reducing survival (e.g. Bryant et al., 1983, 1987; Roth and Lindroth, 1994; Lindroth et al., 1995). Correspondingly, enhanced UV-B radiation may not only have a direct effect on herbivores but also an indirect one via alteration of the chemical composition of host-plant tissues (e.g. Berenbaum, 1988; McCloud and Berenbaum, 1994). UV-B radiation has been shown to cause an increase in UV-protective secondary metabolites, mainly flavonoids, in various terrestrial plants (e.g. Tevini et al., 1981; Teramura, 1983; Caldwell et al., 1995). However, in contrast to CO -exposed plants, the carbon:nitrogen ratio of UV-B exposed plants 2 may decrease, which together with the increase in secondary metabolites may reduce the feeding rate and consumption of herbivores (Tevini et al., 1981; Teramura, 1983; Hatcher and Paul, 1994; Grant-Petersson and Renwick, 1996). In general, plant phenols influence preference and performance in many different herbivores, including insects. Phenols may have negative, neutral, or positive effects on herbivores depending on the feeding guild (e.g. Harborne, 1988; Bernays et al., 1989). Some ubiquitous plant allelochemicals, such as tannins and some flavonoids, possess antiherbivore properties and can increase herbivore resistance (e.g. Hedin and Waage, 1986; Berenbaum, 1988; Bernays et al., 1989), while some other more specific allelochemicals, such as certain flavonoids and small-molecular-weight phenolic glucosides, may act as stimulants for herbivore feeding (e.g. Nielsen et al., 1979; Bernays et al., 1991; Kolehmainen et al., 1995). European silver birch (Betula pendula Roth) is a common deciduous tree in the northern hemisphere of Europe (e.g. Atkinson, 1992) and is listed as one of several host plants for the polyphagous winter moth (Operophtera brumata L.) in Fennoscandia (e.g. Tenow, 1972; Tikkanen et al., 1997). Betula species contain mainly phenolics (phenolic glycosides, flavonoids, condensed tannins) and terpenoids as their secondary chemicals (Julkunen-Tiitto et al., 1996). Recent research has shown that the secondary chemical composition of birch responds both to enhanced CO concentra2 tion and UV-B radiation, but the responses are mostly compound-specific (Lavola and Julkunen-Tiitto, 1994; Roth and Lindroth, 1994; Lavola, 1997a,b; Lavola et al., 1997).
A. Lavola et al./Biochemical Systematics and Ecology 26 (1998) 1—12
3
The objective of this research was to study how CO and UV-B-induced changes in 2 secondary metabolites may influence the quality of birch leaves as food of a generalist insect herbivore, the winter moth. Therefore, in laboratory experiments the larvae of O. brumata were given a choice among the leaves of the host plant, B. pendula, which had been exposed simultaneously to either ambient or enhanced CO conditions and 2 with no or supplementary UV-B radiation. In addition, the effect of the two main flavonoid glycosides in the birch leaves on larval food preference was tested using an artificial diet.
2. Materials and methods 2.1.1. Treatment of seedlings Seeds of Betula pendula from a controlled orchard originating from seed collected in central Finland were germinated in a greenhouse and transplanted into pots (RayLeach cells, 165 cm3) containing birch-forest soil, peat and vermiculite (1:1.5:3 v/v). The seedlings were watered each day. Fertilizer was applied upon watering every second day: at first for 2 weeks with 20 ml of a 10%, then for the following 2 weeks with 50% and then with 100% nutrient solution balanced for birch by Ingestad (1962) (1.42 mg N d~1 plant~1). The six-week-old seedlings were transferred for two months into two clear plastic growth chambers located in a greenhouse, one with an ambient CO level (350 ppm) 2 and another one with increased CO level (700 ppm) (for details of CO exposure 2 2 design see Silvola and Ahlholm (1993)). In the chambers, the temperature was on average 22°C and the photoperiod was 18 h long. The additional PAR supplied using four metal halide discharge lamps (Osram Power Star, HQI-T 400 W, Germany) per chamber was about 300 lmol m~2 s~1 (measured with a Li-Cor LI-90SB quantum sensor). After 2 weeks of acclimation to the CO environments, the UV-B irradiation was 2 started. The seedlings were irradiated with UV-B supplied by three fluorescent lamps (UVB-313, Q-panel, U.S.A.) for 3 h per day centred around solar noon. The lamps were filtered with cellulose acetate film (removes all the radiation below 290 nm) for increased UV-B radiation and with polyester (removes all the radiation below 313 nm) for the control seedlings. Thus, in both chambers ("both CO concentra2 tions), half of the seedlings received no UV-B radiation (control seedlings) and half of the seedlings received a moderately increased daily dose of biologically effective UV-B radiation (8.16 kJ m~2 UV-B for exposed seedlings, Caldwell’s generalized plant BE damage, action"1 at 300 nm, measured with Macam SR9910 spectroradiometer). Plants for larval choice experiments were taken after 2, 3, and 4 weeks from the beginning of the UV-B irradiation. 2.1.2. Analysis of chemical components in seedlings The chemical analyses of the leaves were made from eight individual seedlings used in each larval choice experiment (n"8 leaves per harvest). The second fully expanded leaf from the top of these seedlings was dried at room temperature, extracted with
4
A. Lavola et al./Biochemical Systematics and Ecology 26 (1998) 1—12
methanol and analysed by high performance liquid chromatography (HPLC), according to Meier et al. (1988) and Julkunen-Tiitto et al. (1996). The HPLC instrument (Hewlett-Packard, Avondale, Pennsylvania, U.S.A.) used consisted of a quaternary pump (HP 1050), an autosampler (HP 1050), and a photodiode-array detector (HP 1040A) combined with HP ChemStation. In the separation of phenolics a 3 lm HP Hypersil ODS column (60]4.6 mm I.D.) was used. Eluation solvents A (aqueous 1.5% tetrahydrofuran#0.25% orthophosphoric acid) and B (methanol) were used in the following gradient: 0—4 min 2—12% of B in A, 4—30 min 12—36% of B in A, 30—45 min 36—56% of B in A. The flow rate was 2 ml min~1 and the injection volume 20 ll. The identification of the secondary components in leaves was based on their retention time and spectral data (monitored by UV-220 and UV-360), and the quantification was based on commercial standards (Julkunen-Tiitto et al., 1996). Five flavonoid glycosides (myricitrin, quercitrin, hyperin, myricetin-3-galactoside, kaempferol-3-rhamnoside) and three phenolic acids (chlorogenic acid and two unidentified cinnamic acid derivatives) were determined by HPLC. Condensed tannin (proanthocyanidin) concentrations were determined from methanol extracts by the vanillin:HCl test (Julkunen-Tiitto, 1985). 2.1.3. Larval choice experiments The overwintering eggs of winter moth were collected from Klimovo village in Karelian Isthmus (60°40@N, 29°25@E). They were hatched in a growth chamber and the emerged larvae were transferred to Prunus padus (L.) leaves to grow, because newly hatched larvae did not survive on young birch leaves. After 1 week the larvae were fed with a mixture of B. pendula and P. padus leaves. The larval choice experiments were carried out in Petri dishes 15 cm in diameter in a growth chamber (temperature 22°C, humidity 80%). A moistened filter paper disk was placed in the bottom of the dish, on which the first fully expanded leaf of birch seedlings from each treatment was randomly arranged in a circle. Thus, there were four leaves in a Petri dish: UV-B irradiated and unirradiated leaves grown under both 350 and 700 ppm concentrations of CO . In a Petri dish, either three 2 individuals of early-instar larvae (second and third instars) or one individual of late-instar larvae (fourth and fifth instars) were placed in the middle of the leaf circle and the arena was covered with the Petri dish lid. The larvae were allowed to feed in each experiment for about 24 h. The leaves were then pressed and dried, and the amount of feeding (mm~2 removed) in each leaf was determined (Quantimet 500#Image Analysis System). Three larval choice experiments were conducted with exposed leaves. In the first experiment, after 2 weeks of UV-B irradiation, the leaves were offered to earlyinstar larvae (16 replicate Petri dishes). In the second choice experiment, after 3 weeks of UV-B irradiation, the leaves were offered to late-instar larvae (16 replicate Petri dishes). In the third choice experiment, after 4 weeks of UV-B irradiation, the leaves were offered to both early- and late-instar larvae (16 and 13 replicates, respectively). The effect of flavonoids on the larval feeding was also tested in a similar choice experiment. In Petri dishes (n"30), three types of agar-cakes were offered to two
A. Lavola et al./Biochemical Systematics and Ecology 26 (1998) 1—12
5
early-instar larvae. All agar-cakes consisted of 1.5% agar and 0.1% fructose and the flavonoid-containing cakes had additional constituents of the two main leaf flavonoids (commercial standards): either 0.1% quercitrin and 0.05% myricitrin or 0.5% quercitrin and 0.1% myricitrin, representing low and high flavonoid concentrations found in native birch leaves, respectively. The experiment was terminated after 24 h and the amount of feeding was determined under a microscope as the percentage of cake surface eaten. Cakes were set into four different %-classes (0%, 1—4%, 5—7%, 8—10%) on the basis of the area removed from the cake. 2.1.4. Statistical analysis The effects of UV-B and CO treatments on plant phytochemicals in three harvests 2 were tested by three-factor ANOVA. Results of food choice tests with leaves and with agar-cakes were statistically tested by the Friedman test (Conover, 1980). ANOVA was used also in statistical evaluation of differences in feeding between the different choice experiments and between larval age and feeding.
3. Results 3.1. Secondary chemistry of seedlings In our experimental birches, the tannins constituted the main proportion of the leaf phenolics, 3—8% of the leaf dry weight, while the proportions of the lavanoid glycosides was 1.5—3% and the phenolic acids was 0.5—1% of the leaf dry weight. The amounts of leaf flavonoids and phenolic acids changed significantly due to enhancement of UV-B radiation and CO concentration, while the amount of condensed 2 tannins remained unaffected (Table 1). On a dry weight basis, the second fully expanded leaf of UV-B-exposed seedlings had a 15—95% higher amount of quercitrin, 60—240% higher amount of hyperin, 20—150% higher amount of myricetin-3-galactoside and 10—70% higher amount of kaempferol-3-rhamnoside than the seedlings not exposed to UV-B. In the elevated CO concentration, seedlings had reduced content 2 of phenolic acids and some flavonoids (quercitrin, kaempferol-3-rhamnoside) as compared with ambient CO (Table 1). However, the accumulation pattern of phen2 olics under UV-B radiation was not significantly changed by the enhancement of CO 2 (no UV-Bx CO interactions). 2 3.2. Larval feeding The leaves from the plants grown under enhanced UV-B radiation under both CO 2 environments (350 and 700 ppm) were eaten to a greater extent than the leaves which received no UV-B radiation (Fig. 1a, b). For early instar larvae, the amount of tissue removed from the UV-B-treated leaves was significantly higher than that of untreated leaves in both CO levels (Friedman test; X2"11.671, p"0.0086, df"3,93) (Fig. 1a). 2 Also, the consumption of late-instar larvae differed nearly significantly among treatments (Friedman test; X2"7.419, p"0.0597, df"3,84) and the amount of feeding
7.41$0.80 3.36$0.37 2.60$0.33 1.92$0.21 0.58$0.13 0.51$0.05 56.7$7.37 63.1$7.12
Other phenolics: Chlorogenic ac 350 700 Cinnamic ac I 350 700 Cinnamic ac II 350 700 Tannins 350 700
Kae-3-rha
Myr-3-gal
Hyperin
Quercitrin
6.39$0.52 4.33$0.32 2.26$0.23 1.48$0.10 0.75$0.13 0.45$0.03 67.5$4.94 67.2$6.23
11.3$1.55 9.8$1.23 14.8$1.75 10.5$1.19 1.43$0.18 1.03$0.13 1.24$0.27 0.69$0.08 1.14$0.13 0.93$0.08
4.63$0.64 3.26$0.37 2.18$0.29 1.75$0.23 0.48$0.05 0.35$0.04 75.8$4.13 74.7$13.7
13.3$1.15 11.6$1.23 7.28$0.89 7.59$0.66 0.51$0.11 0.61$0.11 0.56$0.14 0.82$0.18 0.65$0.08 0.69$0.05
!UV-B
!UV-B
#UV-B
Harvest II
Harvest I
13.6$1.88 11.0$1.55 12.9$0.63 6.56$0.34 0.73$0.13 0.39$0.07 0.78$0.24 0.52$0.08 1.01$0.09 0.56$0.05
CO ppm2
350 700 350 700 350 700 350 700 350 700
Flavonoids: Myricitrin
Phenolics
5.01$0.60 4.18$0.47 2.76$0.39 1.88$0.25 0.72$0.09 0.49$0.06 73.4$6.48 82.9$5.92
12.2$1.26 12.5$1.03 14.2$1.45 9.92$1.54 1.47$0.33 1.26$0.24 1.37$0.32 1.32$0.28 0.98$0.08 0.77$0.04
#UV-B
3.47$1.26 2.97$0.38 1.57$0.23 1.79$0.24 0.54$0.06 0.40$0.05 38.4$5.17 49.1$4.37
9.20$1.83 12.6$1.50 6.88$1.27 7.53$1.00 0.40$0.08 0.73$0.08 0.53$0.11 0.88$0.16 0.55$0.08 0.59$0.05
!UV-B
Harvest III
4.13$0.52 3.22$0.30 1.96$0.21 1.77$0.22 0.57$0.08 0.54$0.04 51.5$4.11 60.2$4.71
13.1$1.51 11.3$1.32 13.6$2.09 11.2$1.19 1.37$0.17 1.18$0.12 1.32$0.35 1.05$0.18 0.94$0.09 0.71$0.06
#UV-B
ns
*** ns
ns
ns
***
*
ns
*
*
ns
ns
*
ns
ns
ns
***
**
***
***
ns
ns
ns
***
ns
***
ns
***
ns
***
***
***
ns
ns
ns
ns
ns
*
ns
ns
ns
ns
Harvest CO xH UV-BxCO xH 2 2
ns
ns
ns
UV-B CO 2
ANOVA results
Table 1 The mean concentration of leaf secondary metabolites (mg g~1 d.wt$sd) in the three harvests and the results of ANOVA (p(0.001"***, p(0.01"**, p(0.05"*, p'0.05"non-significant (ns)). UV-Bx CO and UV-Bx harvest interactions were non-significant in all cases (myr"myricitrin, kae"kaem2 pferol, gal"galactoside, rha"rhamnoside, ac"acid)
6 A. Lavola et al./Biochemical Systematics and Ecology 26 (1998) 1—12
A. Lavola et al./Biochemical Systematics and Ecology 26 (1998) 1—12
7
Fig. 1. The percentages (%) of leaf area eaten on differently treated leaves by early-instar larvae (a) and by late-instar larvae (b) in larval choice experiments. The number of touched leaves in each treatment is shown in columns (for early-instar larvae n"32, for late-instar larvae n"29).
on the leaves exposed to both UV-B radiation and elevated CO concentration was 2 slightly higher than that on leaves grown in ambient conditions (Fig. 1b). The leaf preference was similar in each choice experiment and for different sized larvae: there was no statistical difference in the feeding pattern among differently treated leaves between different choice experiments (ANOVA; F "2.387, 3,240 p"0.0697) or between early and late instars of larvae (ANOVA; F "0.174, 1,242 p"0.677). Also, the relative frequencies of leaves having some feeding damage were quite similar among different choice experiments (see Fig. 1a, b). However, the early-instar larvae were observed to behave more actively in food selection: they moved around the dishes and tasted a bit of each leaf they encountered before starting to feed. In contrast, the late-instar larvae placed themselves below a leaf shortly after
8
A. Lavola et al./Biochemical Systematics and Ecology 26 (1998) 1—12
Table 2 The preference of winter moth larvae for low (0.15%) and high (0.6%) flavonoid concentrations expressed as a number of cakes eaten in each %-class and the mean ranks for treatments (Friedman test; X2"6.9, p"0.032, df"2,58) % Eaten
0 1—4 5—7 8—10 Mean rank
Control
6 13 9 2 2.17
Flavonoid concentration Low
High
9 9 8 4 2.17
11 10 7 2 1.67
the experiment started, and fed on that leaf for a while before changing their position. However, there were no treatment-based differences in leaf size (ANOVA; F "0.74, p"0.390 for UV-B treatments and F "2.25, p"0.134 for CO 1,240 1,240 2 treatments, no interactions), which could have affected the food selection. In the choice experiment using artifical substrate, the combinations of the two different concentrations of flavonoids myricitrin and quercitrin had no stimulating influence on larval feeding. Larvae consumed equally the lower flavonoid concentration cakes (0.15%) and control ones but they consumed significantly less high flavonoid concentration cakes (0.6%) than control ones (Friedman test; X2"6.9, p"0.032, df"2,58) (Table 2).
4. Discussion The chemical analyses of leaves support earlier observations that the concentration of secondary metabolites in birch species is influenced by UV-B radiation and CO 2 concentration. Likewise earlier (Lavola, 1997a; Lavola et al., 1997), the amount of flavonoids and some phenolic acids increased in birch leaves due to UV-B irradiation. Accumulation of phenolic compounds is a common response in plants to UV-B radiation, since UV-B usually activates the biosynthetic routes of secondary metabolites (e.g. Li et al., 1993; Caldwell et al., 1995). In this experiment, the CO enhancement reduced the amount of flavonoids and 2 phenolic acids in leaves. In earlier studies, the high CO levels have caused increase in 2 the amount of main secondary metabolites, tannins and flavonoids, in birch and similarly decrease in the amount of some minor phenolic compounds (phenolic glucosides) (Lavola and Julkunen-Tiitto, 1994; Roth and Lindroth, 1994; Lindroth et al., 1995; Traw et al., 1996). Although the introduction of CO is suggested to 2 increase the production of carbon-based secondary metabolites, especially the endproducts of secondary metabolite routes, it is not the general plant response to high CO concentrations (e.g. Reichardt et al., 1991; Ayres, 1993; Lindroth et al., 1993). 2
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Overall, the secondary chemical responses of birch seedlings to changes in different climatic factors are very compound dependent (Lavola et al., 1994, 1997; Lavola, 1997a,b) probably based on ontogenic differences in the carbon allocation of individual seedlings (Bryant and Julkunen-Tiitto, 1995). In choice experiments, the behaviour of O. brumata larvae, especially of early instars, implied active food selection. This is seen in higher feeding preference of larvae for UV-B-exposed birch leaves, which supports the observations of the marked effects of UV-B radiation on herbivory. As in deciduous shrubs in the field (Gwynn-Jones et al., 1997), UV-B-exposed plants experienced greater loss than in control ones with no interactive effect of enhanced CO concentration on herbivory. However, there 2 seems to be differences in herbivory among host-plant species and in consumption between different aged larvae due to UV-B radiation (Grant-Petersson and Renwick, 1996; Gwynn-Jones et al., 1997). Contrary to our results, in some cases the consumption and performance of larvae has decreased on host plants grown under increased UV-B radiation, and suggested to correlate with increased nitrogen or flavonoid concentrations of exposed plants (Hatcher and Paul, 1994; McCloud and Berenbaum, 1994; Ballare´ et al., 1996; Grant-Petersson and Renwick, 1996). However, herbivory of plants grown under elevated CO levels often increases, as the feeding of late-instar 2 larvae in this experiment may also imply, because of the “dilution effect” in CO 2 exposed plants (e.g. Lincoln et al., 1986; Lindroth et al., 1993, 1995; Roth and Lindroth, 1994). Consequently, insect performance and feeding preference on a host plant may correspond with different properties of leaves, such as texture, nutritional quality and secondary metabolite content (Larsson et al., 1986; Bryant et al., 1987; Lindroth et al., 1988). In many studies, increases in the amount of secondary metabolites has caused decreases in the rates of food consumption and the growth of insect herbivores (Feeny, 1970; Larsson et al., 1986; Bryant et al., 1987; Lindroth and Bloomer, 1991; Traw et al., 1996). However, the function of UV-B inducible flavonoids in plant—insect interactions is related to their chemical structure. According to Hedin and Waage (1986), flavonol aglycones possess antifeedant activity, which increases as the number of hydroxyl groups increases. The antifeedant activity of flavonoids may also depend on their glycosylation, since rhamnosides can be more effective (or even toxic) than glucosides and galactosides, which may even act as phagostimulants (Nielsen et al., 1979; Hedin and Waage, 1986; Harborne, 1988; Bernays et al., 1991; Hedin et al., 1992; Larsson et al., 1992). The main flavonoids in birch, myricitrin and quercitrin, are rhamnosides and they contain three and two hydroxyl groups in their B-ring, respectively (e.g. Hedin and Waage, 1986). Both of them have efficient antigrowth and antifeeding activities against some insects (e.g. Elliger et al., 1980; Dreyer and Jones, 1981) but, on the other hand, quercitrin has also been shown to stimulate feeding (e.g. Harborne, 1988). However, in this study the addition of these flavonoid glycosides to artificial diet of the larvae did not increase its consumption. Thus, the increase in the amount of these main flavonoids in leaves may not cause the higher preference of larvae for the UV-B treated leaves. This does not exclude the phagostimulative effect of some minor flavonoid glycoside, such as hyperin (quercetin-3-galactoside), which also increased
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highly under UV-B radiation (Table 1), or the effect of these flavonoids in combination with other leaf constituents, such as phenolic glucosides (e.g. Nielsen et al., 1979; Harborne, 1988; Lindroth et al., 1988; Roininen and Tahvanainen, 1989). However, it has been suggested that for early feeding generalist herbivores (like O. brumata) the total composition of phytochemicals and other traits of leaf quality (water content, toughness) may be more significant in the food selection than a change in any specific leaf component (e.g. Feeny, 1970; Lindroth and Bloomer, 1991). In conclusion, our results indicate significant changes in the chemical composition of plants and in the pattern of insect herbivory under enhanced UV-B radiation and CO levels. However, the UV-B induced accumulation of main flavonoids may not be 2 directly responsible for the increased larval consumption on UV-B-exposed leaves. Therefore, the impacts of UV-B and CO enhancement on herbivory via phytochemi2 cals of host plant are not easily generalized, since their mediation to herbivory may depend on the species-specific adaptations and specialization of each herbivore to the chemical composition of its host plant (e.g. Tahvanainen et al., 1985; Grant-Petersson and Renwick, 1996; Van der Meijden, 1996).
Acknowledgements This work was partly supported by the Academy of Finland and the Maj and Tor Nessling Foundation. The authors thank Ms Tania de la Rosa and Ph.D. Tarja Lehto for collaboration, and Professors Jorma Tahvanainen and Carlos Ballare´ for their comments on the manuscript.
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