Elevated CO2 independently reduced nitrogen levels and had no effect on carbon-based metabolites. The CO2-mediated decline in nitrogen levels was consistent with previous CO2–aspen studies (e.g. Roth et al., 1997, 1998; Lindroth & Kinney, 1998). Generally, CO2 enrichment has been shown to increase levels of starch, phenolic glycosides and condensed tannins in trembling aspen, although not all metabolites may be uniformly affected (e.g. Roth et al., 1997, 1998; Lindroth & Kinney, 1998; Lindroth et al., 2002b). Several explanations exist for the modest response in carbon-based metabolite levels observed in this study. First, levels of carbon-based metabolites have been shown to be less responsive to elevated CO2 under conditions of high nutrient availability, such as exist at the FACE site (Dickson et al., 2000), than under conditions of low nutrient availability (Kinney et al., 1997; Mansfield et al., 1999; Lindroth et al., 2001b). Second, most CO2–tree studies use a CO2 concentration higher than that used in this study (700–650 µL/L vs. 560 µL/L) (Roth & Lindroth, 1994; Lindroth et al., 1995; Agrell et al., 1999, 2000; McDonald et al., 1999). Finally, CO2-mediated accumulation of carbon-based metabolites varies among aspen genotypes (Mansfield et al., 1999; Lindroth et al., 2001a, 2002b) and the genotype used in this study may be particularly unresponsive. We must point out, however, that in a concurrent study using a different set of aspen (clone 216), trees responded to elevated levels of CO2 by increasing levels of salicortin and tremulacin and decreasing levels of condensed tannins (Lindroth et al., 2002b). Why the two sets of trees responded differently between the two studies is unclear, given that the only differences between the studies were the position of the trees within the ring and the tree species with which they were interplanted (aspen–birch vs. mixed aspen genotypes).
Elevated O3, like elevated CO2, altered concentrations of some foliar constituents, and these effects were further modified by interactions with CO2 and time. The exacerbated decrease in nitrogen levels under the CO2 + O3 treatment may be due to the effect of each pollutant on ribulose bisphosphate carboxylase (Rubisco) concentrations. Elevated CO2 can reduce Rubisco levels (reviewed by Saxe et al., 1998), a response that has been demonstrated for aspen at the FACE site (Takeuchi et al., 2001). O3 can inhibit the synthesis of Rubisco (Pell et al., 1994; Bortier et al., 2000). Overall, idenepsying general patterns of the effect of O3-exposure on foliar nitrogen levels is difficult because previous research has demonstrated that O3 can cause foliage to have higher, lower, or unaltered levels of nitrogen (Koricheva et al., 1998). With respect to starch, research typically reports an O3-mediated decrease in concentrations (Bücker & Ballach, 1992; Friend & Tomlinson, 1992; Lavola et al., 1994), which is attributed to the conversion of starch into soluble sugars used to repair O3 injury (Lavola et al., 1994). In this study, aspen trees exhibited symptoms (e.g. reduced growth, leaf necrosis) characteristic of O3 exposure but this damage did not reduce starch levels. With respect to secondary metabolites, O3 exposure tended to reduce tremulacin concentrations. The reason for this decrease is unknown but may include a reduction in biosynthesis due to decreased photosynthate availability or enzyme activity. Alternatively, O3 may accelerate turnover rates of tremulacin. Tremulacin levels under the CO2 + O3 treatment were similar to those in the control treatment, signifying that CO2 enrichment can ameliorate the O3-mediated reduction of some metabolites. O3 interacted with CO2 and time to affect tannin levels. Tannin levels under the CO2 + O3 treatment were significantly higher than those in the other fumigation treatments for the first collection, but did not differ for the remaining collection dates. We are uncertain of the cause of this response, although one reason may be developmental changes in susceptibility to CO2 and O3 exposure, with early season foliage more strongly affected by co-exposure than late season foliage. Our results indicate that when applied in combination, CO2 and O3 can exacerbate reductions in concentrations of some phytochemicals (e.g. nitrogen) while negating the effects of either pollutant acting alone for others (e.g. tremulacin).
Colonization rates were dramatically reduced by elevated CO2 and O3. This suppression, however, tended to be ameliorated when pollutants were administered in combination, resulting in colonization rates similar to those observed when administered alone. Other researchers have investigated the effects of CO2 and O3 on insect oviposition (Jones & Coleman, 1988; Stange et al., 1995; Stange, 1997; Jackson et al., 2000) but this study is the first to assess the combined effects of these pollutants on oviposition. Previous research typically reported that both elevated CO2 and O3 reduce oviposition (e.g. Jones & Coleman, 1988; Thompson & Drake, 1994; Stange, 1997; but see Jackson et al., 1999). For example, a pyralid moth (Cactoblastis cactorum) reduced oviposition rates when exposed to 720 µL/L of CO2 (Stange, 1997). Similarly, a chrysomelid beetle (Plagiodera versicolora) preferred to oviposit on charcoal-filtered, opposed to O3-exposed, cottonwood leaves (Jones & Coleman, 1988). In our study, ovipositing females were not directly exposed to either pollutant because oviposition occurs during the evening (M. Auerbach, pers. comm.), several hours after cessation of fumigation. Furthermore, leaf age, a factor known to influence P. tremuloidiella colonization (Auerbach, 1991; Auerbach & Alberts, 1992), was similar among all four treatments. A more likely explanation for the reduced colonization rates is changes to the leaf surface. Elevated levels of CO2 and O3 were shown by other researchers at the FACE site to alter the molecular composition and production of, and to degrade, aspen epicuticular waxes (Karnosky et al., 1999; K. Percy, pers. comm.). Epicuticular waxes are known to be important oviposition stimulants for some insect species (Eigenbrode & Espelie, 1995). Alternatively, reduced colonization could also be due to pollutant-mediated alterations in other oviposition stimulants or deterrents.
Elevated CO2 and O3 treatments had relatively minor effects on larval performance, and the magnitude of these depended on treatment, performance variable and sex. Survivorship (egg and larval) was not affected by either CO2 or O3. Elevated CO2 also did not independently influence insect development, feeding or pupal mass. These results differ from earlier research in that previous studies conducted with leafminers and free-feeding folivores typically report changes in development time, consumption or pupal mass (Lincoln et al., 1993; Salt et al., 1995; Watt et al., 1995; Docherty et al., 1996; Lindroth, 1996a,b; Bezemer & Jones, 1998; Smith & Jones, 1998; Coviella & Trumble, 1999; Stiling et al., 1999). Our results are similar to another study with P. tremuloidiella where no difference in consumption and only a marginal difference in pupal mass was found between insects in ambient and enriched CO2 (Mansfield et al., 1999). In our study, the lack of a CO2 effect on larval performance is probably a consequence of similar foliar chemistry between the CO2 and control treatments.
In contrast to CO2, O3 independently and interactively affected larval performance, and these results varied between males and females. Larvae tended to consume more leaf tissue under elevated O3 than did those reared under ambient O3, a response also demonstrated in other studies. For example, Coleman & Jones (1988) found that imported willow leaf beetle (Plagiodera versicolora) increased consumption when reared on willow foliage fumigated with O3. In our study the increase in consumption, at least in the CO2 + O3 treatment, could be due to a decrease in foliar nitrogen, which is the most limiting nutrient for herbivorous insects (e.g. Mattson, 1980). Regarding development time and pupal mass, we found a moderate increase in male development time in both the O3 and CO2 + O3 treatments, relative to those reared in control rings. Male pupal mass along with female development time and pupal mass, however, were unresponsive to elevated O3. Why male development time increased and female development time did not in response to O3 exposure remains unclear, although the effect on males was small and only marginally significant.
To conclude, elevated levels of CO2 and O3, alone and in combination, had modest effects on foliar chemistry and these changes produced at most only slight changes in larval performance. Our most striking result was that CO2 and O3 reduced colonization rates by nearly half, relative to the respective ambient treatments, demonstrating that these pollutants can markedly affect P. tremuloidiella oviposition. Because leaf age is an important determinant of P. tremuloidiella oviposition (Auerbach, 1991; Auerbach & Alberts, 1992) and because elevated levels of CO2 and O3 can alter the leaf phenology of some tree species (Gunthardt-Goerg et al., 1993; Ceulemans & Mousseau, 1994; Saxe et al., 1998; Norby et al., 1999), we suggest that CO2- and O3-mediated changes in leaf phenology may influence colonization rates beyond the changes due simply to altered oviposition stimulants or deterrents. If the leaf phenology of some aspen genotypes is affected more by these pollutants than is that of others, then CO2 and O3 sensitivity may ultimately influence host preference in this system. Additional research is required to investigate the cause of the CO2- and O3-mediated reduction in colonization and to determine if these results vary among genotypes and across environments. Finally, this research emphasizes the need for studies to investigate both population- and individual-level parameters to determine the full effects of CO2 and O3 on insect performance.