Isolation of ethanol-tolerant mutants of yeast by continuous selection

Summary Mutants of Saccharomyces uvarum, 5D-cyc with increased tolerance to ethanol have been isolated by a continuous selection technique which allows the culture itself to determine the intensity of selection via a feedback control circuit. The output of CO₂ from a continuous culture of the yeast was monitored using an infrared analyser and the signal from that analyser fed to a potentiometric controller which regulated the introduction of a concentrated ethanol solution into the culture vessel. The frequency of ethanol addition to the culture thus increased as the tolerance of the organisms improved.The use of this system permitted the selection of mutants of yeast which were viable in the presence of 12% w/v ethanol and which showed higher fermentation rates (as measured by CO₂ production) than the wild-type in the presence of 10% w/v ethanol and above. The technique of continuous selection with feedback should be generally applicable to the isolation of mutants of any microorganism to improved tolerance to any inhibitory condition of either its physical or chemical environment.     

The relative merits of open- and closed-path analysers for measurement of eddy fluxes

Theoretical and practical aspects of measuring eddy fluxes of trace gases using open-and closed-path analysers are presented. Trace gas fluxes measured with an open-path analyser require the concurrent measurement of sensible and latent heat fluxes to correct for density fluctuations in trace gas concentration caused by these fluxes. A closed-path analyser eliminates the corrections due to sensible heat flux, but not for water vapour, provided temperature fluctuations are completely removed without significantly reducing fluctuations in the trace gas mixing ratio. Theory for the design of heat exchangers and for the attenuation of concentration fluctuations during air flow through tubes is used to provide design criteria for closed-path systems. Spectral transfer functions are used to estimate flux losses caused by flow through the sampling tube and gas analyser. Other factors considered include cross-sensitivity of infrared CO₂ analysers to water vapour, and deterioration of system performance caused by contaminants on the walls of sampling tubes. Of two open-path, infrared CO₂ analysers tested, one showed a strong interaction between CO₂ and water vapour, while the other showed little sensitivity to the presence of water vapour, other than caused by dilution. A commercial closed-path CO₂ analyser also showed little cross-sensitivity to water vapour. Compared to results for a clean sampling tube, the spectral bandwidth for water vapour fluctuations decreased significantly after several weeks of sampling. No such deterioration in bandwidth was observed for CO₂. These findings are attributed to differential adsorption/desorption of water vapour by dust or salt on the tubing walls. Rain and dust must be removed from open-path analysers to obtain satisfactory measurements. Careful system design and maintenance is required for both open- and closed-path systems to ensure satisfactory long-term measurement of trace gas fluxes. With these precautions, both approaches will provide satisfactory flux measurements.     

A sensitive method for continuous measurement of the carbon dioxide evolution rate of soil samples

Summary A differential infrared CO₂ analyser combined with a 12 channel gas handling system have been used for the measurement of CO₂ evolution rates of soil samples. A constant flow of air over the soil was maintained during the incubation period. Automatic sequential measurement and recording of the increase of the CO₂ content of the flushed air of the 12 channels lasted 24 min with a dwell time of 2 min per channel. This technique has proven to be very useful for accurate and rapid measurement of the biological activities in untreated and treated soil.     

Attractiveness of CO2 released by root respiration fades on the background of root exudates

Plants are endangered at their roots by soil-dwelling rhizophagous insects. These below-ground living herbivores may orient to the source of carbon dioxide (CO₂), an ubiquitous volatile released by respiring plant roots. Here, we studied the interaction of CO₂ and other plant root-derived chemical stimuli with regard to the chemical orientation of the polyphagous larvae of Melolontha melolontha L. (Scarabaeidae). A soil arena was developed that enabled both determination of the actual soil CO₂ concentration and the behavioural response of an insect to (a) CO₂ gradients per se, (b) chemical stimuli released from respiring, undamaged roots of plants potted into vermiculite in this arena and (c) combinations of CO₂ gradients and root-derived stimuli. In a root-free arena, larvae of M. melolontha oriented to the source of synthetic CO₂. However, similar CO₂ gradients generated by host plant roots did not attract the larvae. Neither did a synthetic CO₂ gradient combined with aqueous extracts from rhizospheres with undamaged plant roots elicit an attractive effect. Our data suggest that orientation of cockchafer larvae within CO₂ gradients generated by respiring roots is ‘masked’ by an aqueous extract from a rhizosphere with undamaged roots. The results emphasise that effects of behaviour modifying plant-derived compounds need to be investigated against the background of naturally co-occurring chemicals. The significance of our results for orientation of soil living insects is discussed with respect to abiotic conditions in natural soil and the role of soil microorganisms for the attractiveness of plant roots.     

CO2 exchange in an organic field growing barley or grass in eastern Finland

The CO₂ dynamics were measured in an organic soil in eastern Finland during the growing season and wintertime, and the annual CO₂ balance was calculated for plots where barley or grass was grown. During the summer, the CO₂ dynamics were measured by transparent and opaque chambers using a portable infrared gas analyser for the CO₂ analyses. During the winter, the CO₂ release was measured by opaque chambers analysing the samples in the laboratory with a gas chromatograph. Statistical response functions for CO₂ dynamics were constructed to evaluate the annual CO₂ exchange from the climatic data. The net CO₂ exchange was calculated for every hour in the snow‐free season. The carbon balance varied extensively depending on the weather conditions, and type and phenology of vegetation. During the growing season, the grassland was a net source while the barley field was a net sink for CO₂. However, both soils were net sources for CO₂ when autumn, winter and spring were included also. The annual CO₂ emissions from the grassland and barley soil were 750 g CO₂‐C m^?2 and 400 g CO₂‐C m^?2, respectively. The carbon accumulated in root and shoot biomass during the growing season was 330 g m^?2 for grass and 520 g m^?2 for barley. The C in the aboveground plant biomass ranged from 43 to 47% of the carbon fixed in photosynthesis (P_G) and the proportion of C in the root biomass was 10% of the carbon fixed in photosynthesis. The bare soils had 10–60% higher net CO₂ emission than the vegetated soils. These results indicate that the carbon balance of organic soils is affected by the characteristics of the prevailing plant cover. The dry summer of 1997 may have limited the growth of grass in the late summer thus reducing photosynthesis, which could be one reason for the high CO₂ release from this grass field.     

Orientation response of haematophagous bugs to CO2: the effect of the temporal structure of the stimulus

Carbon dioxide is generally recognized as an important cue used by haematophagous insects to locate a food source. When the mammalian hosts of these insects breathe, they normally emanate considerable amounts of CO₂ at discrete intervals, i.e. with each exhalation. In this work, we analysed the effect of temporally pulsing CO₂ on the host-seeking behaviour of Triatoma infestans. We investigated the ability of T. infestans to follow continuous and intermittent air pulses of 0.25, 0.5 and 1 Hz that included different concentrations of CO₂. We found that insects were attracted to pulsed airstreams of 0.25 and 0.5 Hz transporting 400 ppm of CO₂ above the ambient levels and to continuous streams added with the same amount of CO₂. On the other hand, insects walked away from streams pulsed at rates of 1 Hz regardless of the amount of CO₂ they bear. The walking trajectories displayed by bugs to attractive CO₂-pulsed streams were as rectilinear and accurate as those to CO₂-continuous streams. Our results are discussed in the frame of the interaction between olfactory and mechanoreceptive inputs as affecting the behavioural response of bugs.     

Insects and fungi on a C3 sedge and a C4 grass exposed to elevated atmospheric CO2 concentrations in open-top chambers in the field

The effects of elevated atmospheric CO₂ concentration on plant-fungi and plant-insect interactions were studied in an emergent marsh in the Chesapeake Bay. Stands of the C₃ sedge Scirpus olneyi Grey, and the C₄ grass Spartina patens (Ait.) Muhl. have been exposed to elevated atmospheric CO₂ concentrations during each growing season since 1987. In August 1991 the severities of fungal infections and insect infestations were quantified. Shoot nitrogen concentration ([N]) and water content (WC) were determined. In elevated concentrations of atmospheric CO₂, 32% fewer S. olneyi plants were infested by insects, and there was a 37% reduction in the severity of a pathogenic fungal infection, compared with plants grown in ambient CO₂ concentrations. S. olneyi also had reduced [N], which correlated positively with the severities of fungal infections and insect infestations. Conversely, S. patens had increased WC but unchanged [N] in elevated concentrations of atmospheric CO₂ and the severity of fungal infection increased. Elevated atmospheric CO₂ concentration increased or decreased the severity of fungal infection depending on at least two interacting factors, [N] and WC; but it did not change the number of plants that were infected with fungi. In contrast, the major results for insects were that the number of plants infected with insects decreased, and that the amount of tissue that each insect ate also decreased.     

Potential effects of elevated carbon dioxide on leaf-feeding forest insects

The elevated concentration of atmospheric CO₂ may result in a decline of leaf nutritional quality (especially N) and an increase in some kinds of defensive secondary components (such as phenolics). The changes in the phytochemistry of trees, combined with the effect of elevated CO₂ per se, have a potential negative influence on insect herbivores. Here, we review the effect of elevated CO₂ on the performance of leaffeeding forest insects at individual-level and community-level. The elevated CO₂ per se have little influence on the metabolism of insects. Over half of the tree-insect experimental systems show that the performance of individual insect become poorer under high-CO₂ grown trees; but the others show that the insects have just little or no response to the treatments. The direction and magnitude of the changes in the performance of insects could be mediated by various factors. The effects of treatment are strongly species-dependent. The magnitude of changes in the phytochemistry, the sensitivity and adaptive capacity of insects to the poorer leaf quality, the differences in plant growth conditions and experimental methods, and the mediated effects of other environmental factors (such as soil nutrient availability, light, temperature, O₃) were all closely related to the final performance of insects. However, the larvae’s consumption usually increased under enriched CO₂ treatment, which was widely thought to be a compensatory response to poorer plant quality. The experiments on forest community-level found identically a reduction in herbivory, which was contrary to the results from small-scale experiments. The changes in insect population and the actual response of consumption by leaf-feeding forest insects under CO₂ enrichment remain unclear, and more field-based experiments need to be conducted. __________ Translated from Chinese Journal of Applied Ecology, 2006, 17(4): 720–726 [译自: 应用生态学报]     

Measurement of respiratory CO2 production of roots in an aquatic medium

Changes in pH, total alkalinity and O₂ concentration were followed in an aquatic medium with excised wheat roots (Tritkum aestivum L.). Concentrations of total inorganic carbon and free CO₂ were calculated from total alkalinity and pH according to carbonate equilibria. The total inorganic carbon was estimated by flow-injection infra-red gas analysis. Total alkalinity increased in the root medium during incubation. Respiratory CO₂ production was estimated best from the increase in total inorganic carbon measured with an infra-red gas analyser.     

Carbon Dioxide Production in Stored Maize as Affected by Moisture Content,Level of Broken Corn and Foreign Materials and Infestation by Sitophilus zeamais Motschulsky

Carbon dioxide gas production in maize, mixed with 0, 5 or 10% broken corn and foreign material (BCFM), and 0 or 100 adult maize weevils at 13, 16 or 19% moisture content (mc) was studied in 1.8 liter thermos containers which were held at 26.6°C and 60±5% r.h. for 80 days. CO₂ was measured at 7 day intervals using an infrared gas analyzer. At 13 or 16% mc, higher CO₂ production was measured in infested maize than in uninfested maize, and BCFM did not significantly affect CO₂ production. At 19% mc, CO₂ production was greatly increased regardless of the presence of insects and BCFM. CO₂ produced over 12 weeks was 110–166g/kg. The number of live maige weevils after 80 days was 538 in 13% mc, 344 in 16% mc and 48 in the 19% mc Therefore, respiration of fungi such as Aspergillus glaucus, Aspergillus candidus and Aspergillus terreus other than that of insects appeared to more greatly influence CO₂ production than did the insects at 19% mc The moisture content and presence of maize weevils were major factors affecting respiration during storage, but level of BCFM did not significantly affect CO₂ production. The CO₂ produced over 12 weeks was 135–147 g/kg in infested maize at 13% and 136–144 g/kg at 16% mc.     

Aphid and host‐plant genotype × genotype interactions under elevated CO2

1. Elevated CO₂ can alter plant physiology and morphology, and these changes are expected to impact diet quality for insect herbivores. While the plastic responses of insect herbivores have been well studied, less is known about the propensity of insects to adapt to such changes. Genetic variation in insect responses to elevated CO₂ and genetic interactions between insects and their host plants may exist and provide the necessary raw material for adaptation. 2. We used clonal lines of Rhopalosiphum padi (L.) aphids to examine genotype‐specific responses to elevated CO₂. We used the host plant Schedonorus arundinaceus (tall fescue; Schreb), which is capable of asexual reproduction, to investigate host plant genotype‐specific effects and possible host plant‐by‐insect genotype interactions. The abundance and density of three R. padi genotypes on three tall fescue genotypes under three concentrations of CO₂ (ambient, 700, and 1000 ppm) in a controlled greenhouse environment were examined. 3. Aphid abundance decreased in the 700 ppm CO₂ concentration, but increased in the 1000 ppm concentration relative to ambient. The effect of CO₂ on aphid density was dependent on host plant genotype; the density of aphids in high CO₂ decreased for two plant genotypes but was unchanged in one. No interaction between aphid genotype and elevated CO₂ was found, nor did we find significant genotype‐by‐genotype interactions. 4. This study suggests that the density of R. padi aphids feeding on tall fescue may decrease under elevated CO₂ for some plant genotypes. The likely impact of genotype‐specific responses on future changes in the genetic structure of plant and insect populations is discussed.     

Gas exchange pattern in the two‐spot ladybird beetle,Adalia bipunctata,differs in dry vs. moist air

Gas exchange patterns in the ladybird beetle, Adalia bipunctata (L.) (Coleoptera: Coccinellidae), were investigated using an infrared gaseous analyser (IRGA) and a coulometric O₂ respirometer (manometric–volumetric system). Before testing, the beetles were kept either in dry (dehydrated) or moist (hydrated) conditions for 1 day. Their subsequent gas exchange patterns did not depend on their state of humidity but rather were controlled by the humidity of the insect chamber during gas exchange measurement. If this chamber contained dry air, the beetles exhibited CO₂ release by burst, which we interpreted as cyclic gas exchange (CGE) with inter‐burst periods, but if the chamber was switched to contain moist air, then cyclic CO₂ release was soon abandoned and a pattern of continuous gas exchange appeared. Measurements with the coulometric respirometer in moist air showed that continuous gas exchange was often associated with weak abdominal pulsations, which we interpreted as active ventilation. Their metabolic rate was lower during gas exchange cycles than during continuous gas exchange. We revealed that in the ladybird beetle metabolic rate increased in moist air when the gas exchange pattern transitioned from cyclic to continuous.     

Photosynthesis in the water-stressed C4 grass Setaria sphacelata is mainly limited by stomata with both rapidly and slowly imposed water deficits

A comparison of the effects of a rapid and a slowly imposed water deficit on photosynthesis was performed in Setaria sphacelata var. splendida (Stapf) Clayton, a C₄ NADP‐ME grass. Gas exchange was measured in rapidly and slowly dehydrated adult leaves either under atmospheric CO₂ partial pressure with an infrared gas analyser or under saturating CO₂ partial pressure with a leaf disc oxygen electrode. These measurements were used to calculate stomatal and non‐stomatal limitations to photosynthesis. These were further investigated using modulated chlorophyll a fluorescence measurements and photosynthetic pigment quantification. The decrease of net photosynthesis, leaf conductance and water use efficiency was more pronounced under rapid stress than in slow stress. However, photosynthesis is always mainly limited by stomata in both types of stress, albeit the contribution of non‐stomatal limitations increases at severe water deficits in slow stress experiments. The substomatal CO₂ partial pressure significantly increased in both types of stress, suggesting an increased resistance due to an internal barrier to CO₂ diffusion. Physical alterations in the structure of the intercellular spaces due to leaf shrinkage may account for these results. The maximal photochemical efficiency of photosystem II (PSII) was remarkably resistant to stress, as the F_v/F_m ratio decreased only at severe water deficit. On the contrary, the effective photochemical efficiency of PSII (ΔF/F′_m) measured under high actinic light decreased linearly in both types of stress, although in a more pronounced way under rapid stress. A similar variation in photochemical quenching suggests that the decrease of ΔF/F′_m is mainly due to the closure of PSII reaction centres. The non‐photochemical quenching did not change significantly except under severe dehydration indicating that the energization state of thylakoids remained stable under stress. The decrease observed in photosynthetic pigments may be an adaptation to stress rather than a limiting factor to photosynthesis. Results suggests that, although intrinsic mesophyll metabolic inhibitions occur, stomatal limitation to CO₂ diffusion is the main reason for the decrease in photosynthesis.     

Elevated CO2 alters the feeding behaviour of the pea aphid by modifying the physical and chemical resistance of Medicago truncatula

Elevated CO₂ compromises the resistance of leguminous plants against chewing insects, but little is known about whether elevated CO₂ modifies the resistance against phloem‐sucking insects or whether it has contrasting effects on the resistance of legumes that differ in biological nitrogen fixation. We tested the hypothesis that the physical and chemical resistance against aphids would be increased in Jemalong (a wild type of Medicago truncatula) but would be decreased in dnf1 (a mutant without biological nitrogen fixation) by elevated CO₂. The non‐glandular and glandular trichome density of Jemalong plants increased under elevated CO₂, resulting in prolonged aphid probing. In contrast, dnf1 plants tended to decrease foliar trichome density under elevated CO₂, resulting in less surface and epidermal resistance to aphids. Elevated CO₂ enhanced the ineffective salicylic acid‐dependent defence pathway but decreased the effective jasmonic acid/ethylene‐dependent defence pathway in aphid‐infested Jemalong plants. Therefore, aphid probing time decreased and the duration of phloem sap ingestion increased on Jemalong under elevated CO₂, which, in turn, increased aphid growth rate. Overall, our results suggest that elevated CO₂ decreases the chemical resistance of wild‐type M. truncatula against aphids, and that the host's biological nitrogen fixation ability is central to this effect.     

Effects of elevated CO2 and temperature on development and consumption rates of Octotoma championi and O. scabripennis feeding on Lantana camara

We carried out a factorial experiment to explore the effect of doubled CO₂ concentration and a 3 °C temperature increase on the development of a complete generation of the beetles Octotoma championi Baly and O. scabripennis Guérin‐Méneville (Coleoptera: Chrysomelidae). These species are biological control agents of Lantana camara L. (Verbenaceae), with a leaf‐mining larval phase and free‐living, leaf‐chewing adults. Plants grown at elevated CO₂ had enhanced above‐ground biomass, thicker leaves, reduced nitrogen concentration, and increased C:N ratios. Under the high temperature treatment, plants grown at ambient CO₂ suffered wilting and premature leaf loss, despite daily watering; this effect was ameliorated at elevated CO₂. The wilting of plants in the ambient CO₂/high temperature treatment reduced the emergence success of the beetles, particularly O. championi. Development time was accelerated by approximately 10–13 days at the higher temperature, but was not affected by CO₂. Neither CO₂ nor temperature affected adult beetle weight. Consumption rates of free‐living beetles were not affected by either CO₂ or temperature. By contrast, in the short‐term trials using excised foliage, beetles given no choice between ambient and elevated CO₂‐grown foliage, consumed more from ambient plants. When beetles were offered a choice between foliage grown at the two CO₂ levels, O. championi did not display a significant preference but O. scabripennis consumed more ambient CO₂‐grown foliage when feeding at the lower temperature. This study indicates that under future conditions of higher temperatures, amelioration of water stress in host plants growing in elevated CO₂ may benefit some endophagous insects by reducing premature leaf loss. Under some circumstances, this benefit may outweigh the deleterious effects of lower leaf nitrogen. Our results also indicate that foliage consumption under elevated CO₂ by mobile, adult insects on whole plants may not be significantly increased, as was previously indicated by short‐term experiments using excised foliage.     

Transgenic Bacillus thuringiensis (Bt) cotton (Gossypium hirsutum) allomone response to cotton aphid, Aphis gossypii, in a closed-dynamics CO(2) chamber (CDCC)

Allocation of allomones of transgenic Bacillus thuringiensis Gossypium hirsutum (Bt cotton) (cv. GK-12) and non-Bt-transgenic cotton (cv. Simian-3) grown in elevated CO₂ in response to infestation by cotton aphid, Aphis gossypii Glover, was studied in a closed-dynamics CO₂ chamber. Significant increases in foliar condensed tannin and carbon/nitrogen ratio for GK-12 and Simian-3 were observed in elevated CO₂ relative to ambient CO_2, as partially supported by the carbon nutrient balance hypothesis, owing to limiting nitrogen and excess carbon in cotton plants in response to elevated CO₂. The CO₂ level significantly influenced the foliar nutrients and allomones in the cotton plants. Aphid infestation significantly affected foliar nitrogen and allomone compounds in the cotton plants. Allomone allocation patterns in transgenic Bt cotton infested by A. gossypii may have broader implications across a range of plant and herbivorous insects as CO₂ continues to rise. Gang Wu and Fa Jun Chen contributed equally to this work.     

Effects of elevated CO2 and O3 on leaf damage and insect abundance in a soybean agroecosystem

By altering myriad aspects of leaf chemistry, increasing concentrations of CO₂ and O₃ in the atmosphere derived from human activities may fundamentally alter the relationships between insect herbivores and plants. Because exposure to elevated CO₂ can alter the nutritional value of leaves, some herbivores may increase consumption rates to compensate. The effects of O₃ on leaf nutritional quality are less clear; however, increased senescence may also reduce leaf quality for insect herbivores. Additionally, changes in secondary chemistry and the microclimate of leaves may render plants more susceptible to herbivory in elevated CO₂ and O₃. Damage to soybean (Glycine max L.) leaves and the size and composition of the insect community in the plant canopy were examined in large intact plots exposed to elevated CO₂ (~550 μmol mol⁻¹) and elevated O₃ (1.2*ambient) in a fully factorial design with a Soybean Free Air Concentration Enrichment system (SoyFACE). Leaf area removed by folivorous insects was estimated by digital photography and insect surveys were conducted during two consecutive growing seasons, 2003 and 2004. Elevated CO₂ alone and in combination with O₃ increased the number of insects and the amount of leaf area removed by insect herbivores across feeding guilds. Exposure to elevated CO₂ significantly increased the number of western corn rootworm (Diabrotica virgifera) adults (foliage chewer) and soybean aphids (Aphis glycines; phloem feeder). No consistent effect of elevated O₃ on herbivory or insect population size was detected. Increased loss of leaf area to herbivores was associated with increased carbon-to-nitrogen ratio and leaf surface temperature. Soybean aphids are invasive pests in North America and new to this ecosystem. Higher concentrations of CO₂ in the atmosphere may increase herbivory in the soybean agroecosystem, particularly by recently introduced insect herbivores. Handling editor: Gary Felton.     

Leaf temperature of soybean grown under elevated CO2 increases Aphis glycines (Hemiptera: Aphididae) population growth

Abstract Plants grown under elevated carbon dioxide (CO₂) experience physiological changes that influence their suitability as food for insects. To determine the effects of living on soybean (Glycine max Linnaeus) grown under elevated CO₂, population growth of the soybean aphid (Aphis glycines Matsumura) was determined at the SoyFACE research site at the University of Illinois, Urbana‐Champaign, Illinois, USA, grown under elevated (550 μL/L) and ambient (370 μL/L) levels of CO₂. Growth of aphid populations under elevated CO₂ was significantly greater after 1 week, with populations attaining twice the size of those on plants grown under ambient levels of CO₂. Soybean leaves grown under elevated levels of CO₂ were previously demonstrated at SoyFACE to have increased leaf temperature caused by reduced stomatal conductance. To separate the increased leaf temperature from other effects of elevated CO₂, air temperature was lowered while the CO₂ level was increased, which lowered overall leaf temperatures to those measured for leaves grown under ambient levels of CO₂. Aphid population growth on plants grown under elevated CO₂ and reduced air temperature was not significantly greater than on plants grown under ambient levels of CO₂. By increasing Glycine max leaf temperature, elevated CO₂ may increase populations of Aphis glycines and their impact on crop productivity.     

Multi‐factor climate change effects on insect herbivore performance

The impact of climate change on herbivorous insects can have far‐reaching consequences for ecosystem processes. However, experiments investigating the combined effects of multiple climate change drivers on herbivorous insects are scarce. We independently manipulated three climate change drivers (CO₂, warming, drought) in a Danish heathland ecosystem. The experiment was established in 2005 as a full factorial split‐plot with 6 blocks × 2 levels of CO₂ × 2 levels of warming × 2 levels of drought = 48 plots. In 2008, we exposed 432 larvae (n = 9 per plot) of the heather beetle (Lochmaea suturalis Thomson ), an important herbivore on heather, to ambient versus elevated drought, temperature, and CO₂ (plus all combinations) for 5 weeks. Larval weight and survival were highest under ambient conditions and decreased significantly with the number of climate change drivers. Weight was lowest under the drought treatment, and there was a three‐way interaction between time, CO₂, and drought. Survival was lowest when drought, warming, and elevated CO₂ were combined. Effects of climate change drivers depended on other co‐acting factors and were mediated by changes in plant secondary compounds, nitrogen, and water content. Overall, drought was the most important factor for this insect herbivore. Our study shows that weight and survival of insect herbivores may decline under future climate. The complexity of insect herbivore responses increases with the number of combined climate change drivers.