Potential of Climate Change and Herbivory to Affect the Release and Atmospheric Reactions of BVOCs from Boreal and Subarctic Forests.

Compared to most other forest ecosystems, circumpolar boreal and subarctic forests have few tree species, and are prone to mass outbreaks of herbivorous insects. A short growing season with long days allows rapid plant growth, which will be stimulated by predicted warming of polar areas. Emissions of biogenic volatile organic compounds (BVOC) from soil and vegetation could be substantial on sunny and warm days and biotic stress may accelerate emission rates. In the atmosphere, BVOCs are involved in various gas-phase chemical reactions within and above forest canopies. Importantly, the oxidation of BVOCs leads to secondary organic aerosol (SOA) formation. SOA particles scatter and absorb solar radiation and grow to form cloud condensation nuclei (CCN) and participate in cloud formation. Through BVOC and moisture release and SOA formation and condensation processes, vegetation has the capacity to affect the abiotic environment at the ecosystem scale. Recent BVOC literature indicates that both temperature and herbivory have a major impact on BVOC emissions released by woody species. Boreal conifer forest is the largest terrestrial biome and could be one of the largest sources of biogenic mono- and sesquiterpene emissions due to the capacity of conifer trees to store terpene-rich resins in resin canals above and belowground. Elevated temperature promotes increased diffusion of BVOCs from resin stores. Moreover, insect damage can break resin canals in needles, bark, and xylem and cause distinctive bursts of BVOCs during outbreaks. In the subarctic, mountain birch forests have cyclic outbreaks of Geometrid moths. During outbreaks, trees are often completely defoliated leading to an absence of BVOC-emitting foliage. However, in the years following an outbreak there is extended shoot growth, a greater number of leaves, and greater density of glandular trichomes that store BVOCs. This can lead to a delayed chemical defense response resulting in the highest BVOC emission rates from subarctic forest in the 1-3 years after an insect outbreak. Climate change is expected to increase insect outbreaks at high latitudes due to warmer seasons and arrivals of invasive herbivore species. Increased BVOC emission will affect tropospheric ozone (O3) formation and O3 induced oxidation of BVOCs. Herbivore-induced BVOC emissions from deciduous and coniferous trees are also likely to increase the formation rate of SOA and further growth of the particles in the atmosphere. Field experiments measuring the BVOC emission rates, SOA formation rate and particle concentrations within and above the herbivore attacked forest stands are still urgently needed.

Terpene Composition Complexity Controls Secondary Organic Aerosol Yields from Scots Pine Volatile Emissions.

Secondary organic aerosol (SOA) impact climate by scattering and absorbing radiation and contributing to cloud formation. SOA models are based on studies of simplified chemical systems that do not account for the chemical complexity in the atmosphere. This study investigated SOA formation from a mixture of real Scots pine (Pinus sylvestris) emissions including a variety of monoterpenes and sesquiterpenes. SOA generation was characterized from different combinations of volatile compounds as the plant emissions were altered with an herbivore stress treatment. During active herbivore feeding, monoterpene and sesquiterpene emissions increased, but SOA mass yields decreased after accounting for absorption effects. SOA mass yields were controlled by sesquiterpene emissions in healthy plants. In contrast, SOA mass yields from stressed plant emissions were controlled by the specific blend of monoterpene emissions. Conservative estimates using a box model approach showed a 1.5- to 2.3-fold aerosol enhancement when the terpene complexity was taken into account. This enhancement was relative to the commonly used model monoterpene, "α-pinene". These results suggest that simplifying terpene complexity in SOA models could lead to underpredictions in aerosol mass loading.

Plant-derived Secondary Organic Material in the Air and Ecosystems.

Biogenic secondary organic aerosol (SOA) and deposited secondary organic material (SOM) are formed by oxidation of volatile organic compounds (VOCs) emitted by plants. Many SOA compounds have much longer chemical lifetimes than the original VOC, and may accumulate on plant surfaces and in soil as SOM because of their low volatility. This suggests that they may have important and presently unrecognized roles in plant adaptation. Using reactive plant terpenoids as a model we propose a three-tier (atmosphere-vegetation-soil) framework to better understand the ecological and evolutionary functions of SOM. In this framework, SOA in the atmosphere is known to affect solar radiation, SOM on the plant surfaces influences the interactive organisms, and wet and dry deposition of SOM on soil affects soil organisms.

Manipulation of VOC emissions with methyl jasmonate and carrageenan in the evergreen conifer Pinus sylvestris and evergreen broadleaf Quercus ilex.

Plant defence can be induced by exposing plants to the plant hormone jasmonic acid (JA) or its volatile ester, methyl jasmonate (MeJA). Carrageenans (Carr) - sulphated D-galactans extracted from red algae - can also induce plant defences. In this study, the effects of exogenous MeJA and Carr application (concentration 300 and 12.7 µmol, respectively) on volatile emissions from two widespread evergreen woody species, Pinus sylvestris (nine Turkish and one Finnish provenance) and Quercus ilex (Italian provenance) were investigated. We collected headspace samples from seedlings and analysed the quality and quantity of volatile compounds emitted by treated and control plants. In total, 19 monoterpenes, 10 sesquiterpenes, 10 green leaf volatiles (GLVs) and two aromatic compounds were emitted by P. sylvestris from all the provenances studied. Foliar MeJA application clearly affected the volatile profiles of trees from all the provenances. Effects of Carr were genotype specific. In Q. ilex, emissions of sesquiterpenes, GLVs and the homoterpene (E)-DMNT were all induced by MeJA application. However, emissions of most constitutively emitted monoterpenes were significantly reduced. Carr application also led to a significant reduction in monoterpene emissions, but without corresponding increases in other emissions. Our results indicate that exogenously applied MeJA and Carr can both significantly modify the volatile profiles of P. sylvestris and Q. ilex, but also that there are important provenance- and species-specific differences in the overall degree of elicitation and compositions of elicited compounds.

The influence of different nutrient levels on insect-induced plant volatiles in Bt and conventional oilseed rape plants.

Transgenic Bt (expressing the cry1Ac endotoxin gene) and conventional oilseed rape plants grown in different soils were used to study nutrient uptake and emission of volatiles after herbivore damage. All plants were greenhouse-grown in soils representing low-, medium- and high-nutrient levels. The concentrations of N, P, K, Mg and Zn were significantly affected by the transgene, while the main effect of soil type appeared in N, P, Ca, Mg, B, Mn and Zn concentrations in the plants. Plants with four to five leaves were infested with the third instar larvae of Bt-susceptible Plutella xylostella for 48 h, and samples of volatiles were collected and analysed. In the first experiment, the soil nutrient level had a significant effect on the emissions of (Z)-3-hexen-1-ol, (Z)-3-hexenyl acetate, hexyl acetate, (E)-4,8-dimethyl-1,3,7-non-atriene (DMNT), beta-elemene, gamma-bisabolene, alpha-bisabolene and (E)-nerolidol. The induction of these volatiles was significantly higher in infested conventional plants grown at a high-soil nutrient level compared to infested conventional plants at a low-soil nutrient level. In the second experiment, the soil nutrient level had a significant effect on the emissions of (Z)-3-hexen-1-ol, (Z)-3-hexenyl acetate and beta-elemene and, again, this was significantly higher in infested conventional plants grown at high-soil nutrient levels in comparison with infested plants at a low-soil nutrient level. In both experiments, the transgene effect was significant on the emissions of DMNT and (E,E)-alpha-farnesene. The differences in emissions between the two separate experiments suggest that growth conditions (particularly daylength) and sampling procedure may affect the ratio of compounds detected in the emission blend, even though the response to herbivory, nutrient availability and the transgene were similar.

Host location behavior of Cotesia plutellae Kurdjumov (Hymenoptera: Braconidae) in ambient and moderately elevated ozone in field conditions.

In field O(3)-enrichment experiments increased herbivore densities have been reported, which could be due to negatively affected host location behavior of natural enemies. We addressed the impact of doubling background O(3) on the host location of the parasitoid Cotesia plutellae by conducting 24-h trials in an open-air O(3)-fumigation system during two consecutive years. Two circles (radii 1.40 and 4.00 m) of Plutella xylostella-infested potted cabbage plants were placed in the O(3) and ambient plots. Female wasps were released into each plot from the center, and observed 5 times over a 24-h period to assess their host location capability. Thereafter, plants were kept in laboratory conditions until larvae pupation to determine parasitism rates. No significant differences were detected between ambient and O(3)-enriched environments either in the number of wasps found in the field, or in the percentages of parasitized larvae. This suggests that moderately elevated O(3) will not affect the behavior of this parasitoid.

Significance of wood terpenoids in the resistance of Scots pine provenances against the old house borer, Hylotrupes bajulus, and brown-rot fungus, Coniophora puteana.

We tested how terpenoid (i.e., monoterpenes and resin acids) composition and concentration in wood affects resistance against wood-borers and decaying fungi. Scots pine (Pinus sylvestris) wood from nine provenances having variable terpenoid profiles was studied against the old house borer, Hylotrupes bajulus, and the decay fungus, Coniophora puteana. Provenances represented a 1200-km N-S transect from Estonia to northern Finland, but they were all cultivated for 7 years in the same nursery field, in central Finland. Mean relative growth rate (MRGR) of small H. hajulus larvae positively correlated with the total monoterpene concentration of wood, and feeding was associated with high proportion of levopimaric+palustric acid in wood. Provenance did not affect the MRGR of small or big larvae, but big larvae consumed more wood and produced more frass on the northern Ylitornio trees than on the southern Rakvere and Ruokolahti trees. Low beta-pinene and total monoterpene concentration and low beta: alpha-pinene ratio in wood were all associated with a high number of eggs. The most northern Muonio provenance was the most favored as an oviposition site, differing significantly from Saaremaa, Tenhola, and Suomussalmi. Wood from Saaremaa, Tenhola, Ruokolahti, and Suomussalmi provenance was most resistant against decay fungus, differing significantly from that of Kinnula provenance. However, decay resistance was not clearly associated with the concentrations of wood terpenoids. These results suggest that monoterpene composition of wood affects resistance against wood-boring Cerambycid beetles, but resistance against wood-decaying fungi is not as clearly associated with wood terpenoids.

Secondary metabolite concentrations and terpene emissions of Scots pine xylem after long-term forest fertilization.

Secondary compounds are known to be associated with the resistance of conifer xylem against insects and fungi. The effects of long-term forest fertilization with nitrogen (N) or with N, calcium (Ca), and phosphorus (P) on secondary compounds in the xylem of 50-yr-old Scots pine (Pinus sylvestris L.) trees were examined. Xylem samples were collected from trees growing in three locations in southern Finland: Vilppula, Padasjoki, and Punkaharju. Forests were fertilized every fifth (Vilppula and Padasjoki) or tenth (Punkaharju) year since the 1950s. We compared concentrations of individual and total monoterpenes and resin acids in the heartwood and sapwood of Scots pine. Terpene emissions were analyzed from the sapwood and total phenolics from the heartwood. Fertilization did not have any significant effect on the concentrations and emissions of xylem monoterpenes. Concentrations of several individual terpenes in sapwood were positively correlated with the corresponding terpene emission. The concentrations of individual resin acids (i.e., abietic and dehydroabietic) decreased significantly in Punkaharju, but increased in the sapwood of N-fertilized trees compared with control ones at Padasjoki and Vilppula. The concentrations of resin acids in the heartwood were not significantly affected by fertilization. Both fertilization treatments decreased the total phenolic concentrations in the heartwood of trees growing in Padasjoki. There was a significant positive correlation between the total phenolics and total resin acid concentration. Overall, resin acids and phenolics seemed be more responsive than monoterpenes to N treatment. These results suggest that forest fertilization might cause slight changes in secondary compound concentrations of xylem, and thus might have significance in the decay resistance of wood.

Olfactory responses of Plutella xylostella natural enemies to host pheromone, larval frass, and green leaf cabbage volatiles.

The parasitoids Trichogramma chilonis (Hymenoptera: Trichogrammatidae) and Cotesia plutellae (Hymenoptera: Braconidae), and the predator Chrysoperla carnea (Neuroptera: Chrysopidae), are potential biological control agents for the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae). We present studies on the interactions between these bioagents and various host-associated volatiles using a Y olfactometer. T chilonis was attracted to a synthetic pheromone blend (Z11-16:Ald, Z11-16:Ac, and Z11-16:OH in a 1:1:0.01 ratio), to Z11-16:Ac alone, and to a 1:1 blend of Z11-16:Ac and Z11-16:Ald. C. plutellae responded to the blend and to Z11-16:Ac and Z11-16:Ald. Male and female C. carnea responded to the blend and to a 1:1 blend of the major components of the pheromone, although no response was elicited by single compounds. Among the four host larval frass volatiles tested (dipropyl disulfide, dimethyl disulfide, allyl isothiocyanate, and dimethyl trisulfide), only allyl isothiocyanate elicited significant responses in the parasitoids and predator, but C. plutellae and both sexes of C. carnea did respond to all four volatiles. Among the green leaf volatiles of cabbage (Brassica oleracea subsp. capitata), only Z3-6:Ac elicited significant responses from T. chilonis, C. plutellae, and C. carnea, but C. plutellae also responded to E2-6:Ald and Z3-6:OH. When these volatiles were blended with the pheromone, the responses were similar to those elicited by the pheromone alone, except for C. carnea males, which had an increased response. The effect of temperature on the response of the biological agents to a mixture of the pheromone blend and Z3-6:Ac was also studied. T. chilonis was attracted at temperatures of 25-35 degrees C, while C. plutellae and C. carnea responded optimally at 30-35 degrees C and 20-25 degrees C, respectively. These results indicate that the sex pheromone and larval frass volatiles from the diamondback moth, as well as volatile compounds from cabbage, may be used by these natural enemies to locate their diamondback moth host.

UV-B radiation and insect herbivory: preliminary observations on strawberry plants and the leaf beetle Galerucella sagittariae in Finland.

Enhanced UV-B (280-315 nm) radiation, as a result of stratospheric ozone depletion is known to increase UV-protective phenolics, especially flavonoids in plant tissues. We tested the hypothesis that the suitability of plant foliage to insect herbivores might be reduced due to higher amount of defence compounds produced by the host plant when exposed to enhanced UV-B radiation. Strawberry plants were exposed to supplementary UV-B and UV-A radiation in an open-field exposure system. The test animal was the leaf beetle (Galerucella sagittariae, Coleoptera, Chrysomelidae), which is known to damage strawberry leaves both in larval and adult stage. The tentative first-year results reported in this paper showed slight, but not statistically significant support for this hypothesis.

Norway spruce and spruce shoot aphid as indicators of traffic pollution.

Two-year-old Norway spruce (Picea abies (L.) Karst) seedlings were exposed to traffic emissions along roadsides with three different traffic densities and speed limits; highway, street and a quiet local road. The responses of the exposed seedlings as a host plant and those of spruce shoot aphid (Cinara pilicornis Hartig) were studied. The concentrations of soluble N and free amino acids, defence chemicals (total phenolics, monoterpenes) were analysed, and aphid growth and reproduction were studied. Along the highway, street and at the local road control site, the atmospheric concentrations of black carbon (BC) and oxides of N (NO(x)) were measured for 1 week during the experiment. The BC data indicate deposition of organic particulate compounds along the highway and street. The NO(x) concentrations along the highway and street showed great diurnal variation, but the average NO(x) concentrations were relatively low. Thus, no changes in N metabolism or growth of the exposed Norway spruce seedlings were found. Along the street, the concentrations of many individual free amino acids, such as proline, as well as total amino acid concentrations, were lower than at the associated control site. Correspondingly, there was also no increase in spruce shoot aphid mean relative growth rate. The aphid reproduction, however, increased along the highway and is suggested to be due to more conducive microclimatic conditions at the exposure site or lack of natural enemies. No changes in defence chemicals (total phenolics, monoterpenes) in relation to the traffic exposure were found. Instead, the microclimatic conditions (temperature, solar irradiation) seemed to affect the concentration of total phenolics.

Responses of spruce seedlings (Picea abies) to exhaust gas under laboratory conditions--I. Plant-insect interactions.

The effects of motor vehicle exhaust gas on Norway spruce seedlings (Picea abies (L.) Karst) and plant-insect interaction of spruce shoot aphid (Cinara pilicornis Hartig) was studied. The exhaust gas concentrations in the fumigation chambers were monitored and controlled by measuring the concentration of nitrogen oxides (NO(x)) with a computer aided feedback system. The concentrations of major exhaust gas components (black carbon [BC], fine particles, VOCs and carbonyl compounds) in the chamber air were also measured. Responses of Norway spruce seedlings to a 2 and 3-week exhaust gas exposure and subsequent performance of spruce shoot aphid were studied using realistic exposure regimes; 50, 100 and 200 ppb NO(x). The feedback control system based on NO(x) concentrations proved an adequate and practical means for controlling the concentration of exhaust gases and studying plant responses in controlled environment chambers. The exhaust exposure resulted in increased concentrations of proline, glutamine, threonine, aspartic acid, glycine and phenylalanine and decreased concentration of arginine, serine, alanine and glycine in young needles. No changes in soluble N concentrations were observed. The results are interpreted as a stress response rather than use of NO(x) as a nitrogen source. No changes in total phenolics and only transient changes in some individual terpene concentrations were detected. The exhaust gas exposure stressed the exposed seedlings, but had no significant effect on N metabolism or the production of defence chemicals. Aphid performance was not significantly affected. Soluble N, secondary metabolism and aphid performance were not sensitive to exhaust gas exposure during shoot elongation in Norway spruce.

The influence of elevated CO2 and O3 concentrations on Scots pine needles: changes in starch and secondary metabolites over three exposure years.

Scots pine (Pinus sylvestris L.) trees, aged about 20 years old, growing on a natural pine heath were exposed to two concentrations of CO2 (ambient CO2 and double-ambient CO2) and two O3 regimes (ambient O3 and double-ambient O3) and their combination in open-top chambers during growing seasons 1994, 1995 and 1996. Concentrations of foliar starch and secondary compounds are reported in this paper. Starch concentrations remained unaffected by elevated CO2 and/or O3 concentrations during the first 2 study years. But in the autumn of the last study year, a significantly higher concentration of starch was found in current-year needles of trees exposed to elevated CO2 compared with ambient air. There were large differences in concentrations of starch and secondary compounds between individual trees. Elevated concentrations of CO2 and/or O3 did not have any significant effects on the concentrations of foliar total monoterpenes, total resin acids or total phenolics. Significantly higher concentrations of monoterpenes and resin acids and mostly lower concentrations of starch were found in trees growing without chambers than in those growing in open-top chambers, while there were no differences in concentrations of total phenolics between trees growing without or in chambers. The results suggest that elevated concentrations of CO2 might increase foliar starch concentrations in Scots pine, while secondary metabolites remain unaffected. Realistically elevated O3 concentrations do not have clear effects on carbon allocation to starch and secondary compounds even after 3 exposure years.

Gramine and free amino acids as indicators of fluoride-induced stress in barley and its consequences to insect herbivory.

Barley leaves were sprayed with aqueous NaF, which caused accumulation of fluoride in the foliage, but no visible symptoms were detectable. No significant correlation was observed between foliar fluoride concentration and content of the indole alkaloid gramine after exposure to fluoride levels of 20 to 60 mg F liter-1. Fluoride exposure did not explicitly affect the performance of Carausius morosus or Rhopalosiphum paid on barley. After exposure to fluoride levels of 100 and 200 mg F liter-1, as NaF, fluoride treatment had a significant effect on gramine concentration of the first leaf of barley, being highest at a fluoride treatment of 200 mg liter-1, and there was a slight, but significant positive correlation between the log-transformed foliar fluoride concentration and log-transformed gramine concentration of the first leaf. Fluoride treatment increased levels of some individual free amino acids in barley foliage. Exposure of young barley to NaF in aqueous form caused accumulation of fluoride in barley foliage and resulted in increased levels of gramine in the first leaf and levels of some free amino acids in foliage. It is possible that the fluoride-induced concurrent increase in gramine concentration in barley leaves could override the eventual increase in nutritive value to herbivorous insects after fluoride exposure. More detailed biochemical studies of the induction of gramine production are needed to understand the fluoride effects in secondary metabolism of barley.

The response of spruce shoot aphid Cinara pilicornis hartig to ambient and filtered air at two elevations and pollution climates.

The effects of ambient air compared to filtered air on the reproduction of females and mean relative growth rate (MRGR) of nymphs of C. pilicornis on Norway spruce was determined in open-top chambers at Wengernalp in the Swiss Alps (1900 m a.s.l.) and at Schönenbuch near the city of Basle (400 m a.s.l.). The ambient concentration of O(3), the main pollutant at both sites, varied between 45 and 120 microg m(-3) (24-h mean) at both sites. A 5-8 day exposure of spruce saplings to ambient compared to filtered air enhanced the MRGR of nymphs of C. pilicornis of local and northern origins at Schönenbuch. The cumulative numbers of offspring of C. pilicornis were higher in ambient air chambers than in filtered air chambers at Schönenbuch, but not at Wengernalp. Filtration of ambient air did not affect significantly the levels of total free amino acids or reducing sugars in phloem sap or concentration of total phenolics in needles at the end of the growing season. The results suggest that ambient air with elevated O(3) and with high daily fluctuations, as it can be observed in Schönenbuch, affects aphid performance on conifers more than ambient air with also elevated, but rather constant levels of O(3), as it can be observed in mountain forest areas.

Conifer aphids in an air-polluted environment. I. Aphid density, growth and accumulation of sulphur and nitrogen by scots pine and Norway spruce seedlings.

The population dynamics of conifer aphids on pine and spruce seedlings growing in plastic pots were studied along a gradient from a pulp mill emitting mainly SO(2). At five locations, two apterous females of the grey pine aphid (Schizolachnus pineti L.) feeding on needles, and two apterous females of the spruce shoot aphid (Cinara pilicornis Hartig) feeding on the stem, were transferred to the leader shoot of 10 pine and spruce seedlings, respectively. The sulphur concentrations of the transplanted lichen Hypogymnia physodes (L.) Nyl. and needles of conifer seedlings were significantly increased in the close vicinity of the pulp mill. On pine seedlings, the numbers of S. pineti developed similarly at all study sites. However, in mid-July, the numbers of aphids started to decline near the emission source. In June, C. pilicornis reproduction on spruce was significantly faster at 0.2 and 0.5 km from the pollution source than at the more distant sites, but there was a rapid decline in aphid numbers in July. A negative correlation was observed between the numbers of C. pilicornis during the population peak in late June and the final length of the current year's leader shoot of spruce seedlings. Leader shoot growth of spruce seedlings infested by aphids was positively correlated with the distance from the pulp mill. There were no clear responses to pollution level in parasitization or predation rates of the aphids. The results suggest that all conifer aphids are not good indicators of pollution impact on conifers around a pollution source. There is a general agreement with the earlier reports that Cinara spp. numbers have a positive response to air pollution, especially to SO(2). This is probably due to promoted growth of nymphal aphids on the host plants suffering from pollution. However, high levels of air pollution may also cause rapid collapse of aphid populations. S. pineti, which lives on foliage, did not have a positive response to increased SO(2) levels.

Conifer aphids in an air-polluted environment. II. Host plant quality.

Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) seedlings were transported to five forest sites at increasing distances from a pulp mill emitting mainly SO(2). Levels of compounds which may have nutritional or defensive value for aphids on pine and spruce seedlings were studied. Glucose and fructose concentrations were significantly increased in pine and spruce needles near the pulp mill. There were no changes in sucrose and starch concentrations. In pine shoots, total free amino acid concentration and the concentrations of ornithine, lysine, histidine and arginine were significantly negatively correlated with the distance from the pulp mill, while in spruce only the individual amino acids glycine, ornithine, lysine and histidine showed a significant negative correlation with distance. There were no changes in total phenolic, catechin, total monoterpene and total resin acid concentrations. However, in pine seedlings monoterpenes beta-pinene and sabinene and in spruce seedlings resin acid palustric acid were significantly correlated with the distance from the pulp mill. The results indicate that SO(2) disturbs carbohydrate metabolism in spruce and pine seedlings. The elevated concentrations of arginine may be the result of the combinations of SO(2), NO(3) and NH(3) emissions of the pulp mill. The emissions did not have any impact on total amounts of defensive substances in trees. Thus, the possible susceptibility of conifers to herbivores appears to be due to changes in nutritive value rather than to reduced chemical resistance.

Effect of exposure to fluoride, nitrogen compounds and SO2 on the numbers of spruce shoot aphids on Norway spruce seedlings.

Development of spruce shoot aphid (Cinara pilicornis Hartig) populations was monitored in natural and artificial infestations of Norway spruce (Picea abies Karst.) seedlings, exposed to air pollutants in an experimental field. The pollutants, applied both singly and in mixtures, were gaseous sulphur dioxide, NaF (30 mg l-1 F) and Ca(NO3)2 or (NH4)2SO4 in aqueous solutions (200 mg l-1 N). Aphid numbers on 10 seedlings in each treatment and two control plots were counted at 2-week intervals. At the beginning of the experiment aphid numbers did not differ between treatments. Aphid populations peaked in late June and early July. All the pollutants and their combinations significantly increased the numbers of aphids per seedling. Four apterous females were transferred to spruce seedlings which were growing in containers in the same plots. After 4-5 weeks aphid numbers were significantly higher in the fluoride treatment and in the combined treatment of fluoride, nitrogen and SO2. The pollution treatments did not have a significant effect on shoot growth. Concentrations of F and S in needles were higher in treatments involving these pollutants. There were no significant differences in the concentrations of free amino acids in shoot stems between control and fluoride treatment. However, the relatively low concentration of arginine in the F treatment at the end of the growing season might indicate disturbances in the nitrogen metabolism of spruce seedlings.