Abstract There has been a significant increase in studies of how global change parameters affect interacting species or entire communities, yet the combined or interactive effects of increased atmospheric CO 2 and associated increases in global mean temperatures on chemically mediated trophic interactions are mostly unknown. Thus, predictions of climate-induced changes on plant-insect interactions are still based primarily on studies of individual species, individual global change parameters, pairwise interactions, or parameters that summarize communities. A clear understanding of community response to global change will only emerge from studies that examine effects of multiple variables on biotic interactions. We examined the effects of increased CO 2 and temperature on simple laboratory communities of interacting alfalfa, chemical defense, armyworm caterpillars, and parasitoid wasps. Higher temperatures and CO 2 caused decreased plant quality, decreased caterpillar development times, developmental asynchrony between caterpillars and wasps, and complete wasp mortality. The effects measured here, along with other effects of global change on natural enemies suggest that biological control and other top-down effects of insect predators will decline over the coming decades.

Citation: Dyer LA, Richards LA, Short SA, Dodson CD (2013) Effects of CO 2 and Temperature on Tritrophic Interactions. PLoS ONE 8(4): e62528. https://doi.org/10.1371/journal.pone.0062528 Editor: Nicholas J. Mills, University of California, Berkeley, United States of America Received: October 12, 2012; Accepted: March 21, 2013; Published: April 25, 2013 Copyright: © 2013 Dyer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by: Earthwatch Institute (www.Earthwatch.org), University of Nevada Reno (www.unr.edu), Department of Energy (National Institute for Global Environmental Change and National Institute for Climatic Change Research, http://niccr.nau.edu/), and National Science Foundation (nsf.gov) grants - DEB1020509 and DEB0849361. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Methods Experimental Overview The research described here was conducted from January 2006 to December 2009 at Tulane University (New Orleans, LA, USA) and the University of Nevada (Reno, NV, USA). We examined the effects of increased CO 2 and temperature on alfalfa chemical defenses and biomass, growth and survivorship of the generalist herbivore Spodoptera exigua, and growth and survivorship of the parasitoid Cotesia marginiventris in chamber experiments. We utilized a full factorial design manipulating temperature (ambient and elevated), CO 2 (ambient and elevated) and food chain length (plants only, plants+herbivores, plants+herbivores+parasitoids). Thus, alfalfa plants, grown from seeds, were subjected to 12 different treatment combinations. Temperature and CO 2 were manipulated both independently and simultaneously in sequential experiments with these combinations of treatments: ambient temperature and CO 2 , elevated temperature and ambient CO 2 , elevated CO 2 and ambient temperature, and elevated CO 2 and temperature. Ambient CO 2 was 380 ppm and elevated CO 2 was 650 ppm, which is within the range of the projected increase in ambient levels by 2080 [63], [64]. Ambient temperatures were the mean monthly high (30°C day) and low (18°C night) temperatures during July (mid growing season) in northern Colorado. The high temperature was 5°C above ambient temperature (35°C day–23°C night), the maximum projected increase due to global warming [63], [64]. The experiments took place inside 3 VWR CO 2 Incubators, which control CO 2 and temperature, with the light regime of 14 hour days and 10 hour nights. Levels of CO 2 and temperature were randomly assigned to each incubator for each trial. Within the incubators, food chain treatments were randomly assigned to separate cages (plastic microcosms, 24×24×32 cm), resulting in interspersion across space and time. There were 30 replicate cages utilized for each treatment combination, for a total of 360 cages. Cages that did not meet conditions of the experiment (i.e. larvae or plants were not ready for introduction of parasitoids or caterpillars) were discarded, and when possible these replicates were repeated at later dates. Plants Each cage contained nine pots with four alfalfa plants each (36 plants total) and when plants were harvested, all plants and associated insects within a cage were combined for biomass and other measures. Pots contained watered soils at the start of experiments, and incubator relative humidity was greater than 80%, so plants were not watered during the course of the experiment. At the end of each experiment, plants were harvested, weighed, and air-dried for analysis of total carbon, total nitrogen, and quantification of secondary metabolites; for these chemical analyses, plants were combined from 10 replicate cages (with the same treatment combinations) to allow for sufficient plant material. Insect Colonies Spodoptera exigua were purchased from a supplier (Agripest) and immatures of the parasitoid Cotesia marginiventris were collected from northern Colorado fields; both were maintained as colonies in the laboratory. Two newly eclosed (within 12 hours) first instar caterpillars were placed on each alfalfa plant after the appearance of the second trifoliate leaf (approximately 4 weeks after planting). This resulted in 72 caterpillars per cage. In parasitoid addition treatments, the caterpillars were allowed to feed undisturbed for 7 days, at which point a mated female parasitoid was added to each cage and remained in the cages for the duration of the experiment. We ensured that all caterpillars were in the third instar at this point, regardless of experimental treatment. Each trial ended when the last caterpillar pupated or died. Herbivore days to eclose, pupal mass, survival, and parasitism rate were recorded; cage means were calculated for days to eclose and pupal mass, while total survivorship and total percent parasitism for the 72 caterpillars was calculated as a single value for the cage. Chemical Analysis To analyze saponin content, we utilized a modified isolation and quantification procedure [65]. In preparation for chemical extraction, leaf samples were dried overnight in an oven at 40°C and ground to a coarse powder, and subsamples of the dry leaf powder were analyzed by the Nevada Stable Isotopes Lab for total carbon and nitrogen content. One hundred milligrams of dry leaf powder were placed into a centrifuge tube and compounds were extracted from the leaf material in 30 ml of 80% ethanol with stirring. The samples were then centrifuged and the sample plus solvent was separated from the leaf material and dried under a vacuum. The process was repeated to completely extract compounds from the leaves. The dried samples were then dissolved in 15 ml methanol and defatted by shaking the solution with 15 ml of hexanes (98.5% hexane plus a mixture of isomers) in a centrifuge tube. The hexane layer was pipetted off and the process was repeated. The hexanes plus lipids were dried under N 2 with heat. The defatted methanol layer was dried under a vacuum, and the samples were dissolved in 20 ml water. This solution was centrifuged to separate any remaining leaf material from the dissolved sample. C-18 SepPak cartridges (Waters Corp.) were then preconditioned with 15 ml acetone followed by 15 ml water. The water with dissolved sample was passed through the cartridge, and the elution was dried under a vacuum. The cartridge was then sequentially eluted with 20 ml each of 35%, 60%, 80% and 100% methanol. The elutions were transferred directly to a pre-weighed scintillation vials and dried under N 2 with heat. Samples were stored in the freezer. According to previous work with this method, the 35% fraction contains flavans, the 60% fraction is comprised of flavones, the 80% fraction contains saponins and the 100% fraction contains sapogenins. The water fraction has sugars and organic acids, and the hexanes have lipids. Samples were completely dried overnight in an oven at low temperature. Vials with samples were then weighed to determine the mass of each class of compounds contained in the leaf material. The weights of samples from the elutions were used in analyses. Content of elutions was confirmed by HPLC with a matrix-assisted laser desorption ionization source. The main components for the 80% elution fraction were the alfalfa saponins soyasapogenol B-3-O-Rhamnose-Galactose-Glucose carboxylic acid (mass is 965.5 for the sodiated ion +H) and Hederagenin-3-O- [Beta D glucose acid methyl ester] -28-O- [Beta D glucose] (mass is 847.5 for the sodiated ion +H). The main components for the sapogenin (100%) fractions were hederagenin (mass is 685.5 for the ion and 494.6 for the sodiated ion +H), and zahnic acid (mass is 508.6 for the sodiated ion +H), medicagenic acid (mass is 522.6 for the sodiated ion −2 H+). Statistical Analysis The focal statistical analyses were path analyses based on our causal hypotheses presented in Figure 1. In order to identify which variables were best to test in our focal path analyses, the main and interaction effects of temperature, CO 2 and parasitoid treatment on all response variables were estimated using analysis of variance (ANOVA). For ANOVAs, replication for plant chemistry was lower than for other response variables because plants from different cages (replicates with the same levels of all treatments) were combined to provide enough material for chemical analysis. Cage means of caterpillar and plant response variables, including survival and percent parasitism (for 72 caterpillars), were used as response variables, and residuals from these variables met assumptions of normality. In addition, we used a Mann-Whitney z statistic to test the specific hypothesis that percentage parasitism was associated with the 4 combinations of temperature and CO 2 . We examined direct and indirect effects of CO 2 and temperature on alfalfa biomass with path analysis (Proc CALIS, SAS Institute Inc., NC). We proposed 2 general models based on previous literature (Fig. 1) as well as an alternative, simpler model, all of which elucidated direct versus indirect effects of CO 2 and temperature on alfalfa biomass, quality, larval development, and parasitoid performance. While other important interaction pathways, including numerous indirect effects, can be proposed from the literature, several reviews suggest that the direction and magnitude of those effects are still too variable to predict [20], [29], [30]. Path models yielding a goodness of fit chi-square with a P-value greater than 0.5 were considered a good fit to the data.

Discussion The most notable result of our experiments was the indirect effect of increased temperature, which caused decreased development times for caterpillars, resulting in a dramatic negative effect on parasitism. Caterpillars developed rapidly at the higher temperatures and pupated before parasitoids were able to eclose from the late larval stages, resulting in death of the developing parasitoid. This developmental asynchrony could have been exacerbated by the timing of parasitoid introduction to the chambers – had they been introduced earlier, perhaps parasitoid success would have been higher in all treatments. However, the time of introduction was chosen to maximize parasitism based on the phenology of the lab colony – introduction of adults at third instar was optimal for successful parasitism for this particular colony. The 29.6% decline in parasitism recorded at the higher temperature is biologically significant [58] and such developmental mismatches can contribute to overall phenological asynchrony, since the parasitoid must pupate before its host, and if it does manage to eclose, this species has a very short adult stage for mating and finding an early instar host. In contrast to temperature, increases in CO 2 indirectly increased larval development times by decreasing plant quality, both of which were associated with lower levels of parasitism. For both temperature and CO 2 effects on trophic interactions, the host-parasitoid developmental mismatch could contribute to the phenological asynchrony predicted by other climate change scenarios [40] and is likely to synergize with similar global change parameters that delink parasitoids from their hosts. For example, extreme weather events, such as floods and droughts, are increasing with global warming, and these climatic events are likely to cause decreases in caterpillar parasitism rates due to delinking the phenologies of host-parasitoid populations [58]. The fact that increased temperature and CO 2 each cause temporal developmental shifts between parasitoids and hosts provides a clear mechanism by which alfalfa biomass is not enhanced by parasitoids under a changing climate: parasitoids simply cannot track the variable development and quality of their hosts. Interestingly, rather than increasing alfalfa biomass indirectly via killing their caterpillar hosts, parasitoids at high temperature caused a decrease in biomass via increased consumption by their caterpillar hosts with no associated mortality. In any biotic community, this effect on biomass could be maintained by immigration of mated adults from adjacent patches or by host shifts by other parasitoids, but in the absence of genetic variation in development rates, the parasitoids would go locally extinct and the direct effects of herbivory on plant biomass would be more important. This parasitoid-biomass result is more relevant to classical biological control, since the continual release of parasitoids could sustain this indirect negative effect on biomass in warmer and CO 2 enriched conditions. Literature syntheses on climate change and biological control [5], [27] indicate that parasitoid-host developmental mismatches could be common. Herbivore populations are affected by a combination of abiotic factors, natural enemies, and plant quality and availability. Changes in climate have direct effects on the autecology of herbivores via changes in growth rate, metabolic activity, survivorship, and related factors, but as shown in our chamber experiments, indirect effects can modify significantly the outcomes of consumer-resource interactions [66]. At first glance, the predictions from previous studies on associations between climate variables and consumer-resource relationships are adequate for predicting more complex interactions (i.e. comparing Figs. 1 and 4). However, quantifications of only the direct effects do not uncover important indirect mechanisms, such as developmental asynchrony and trade-offs between growth and plant quality. Experiments on simple tritrophic systems that include controlled manipulations of multiple variables that are changing globally provide data that allow for considerable insight into basic questions about the regulation of herbivore populations. More experiments, coupled with observational data and models, will help clarify the conditions under which factors like connectance, parasitism, herbivore, outbreaks, and ecosystem services will increase or decrease in response to interacting climate change variables [8]–[28]. Furthermore, understanding relationships between global climatic changes and tritrophic interactions is particularly important in agricultural systems, where herbivore outbreaks are predicted to increase. There are multiple consequences to the fact that the responses of biotic interactions to climate change are complex. This includes the possibility that effects acting via different direct or indirect pathways could cancel each other out or lead to changes not predicted by single factor models or experiments. For example, increases in plant biomass due to temperature can be counteracted by changes in parasitism, larval development, and increases in production of secondary metabolites, such that the overall effects of temperature (direct plus indirect) are negative (e.g., Fig. 4). It is clear that such interactions must be examined in order to produce realistic predictions for future impacts of climate change on biotic communities. Our results are limited to an unnatural experimental setting; most notably, the community is an unrealistically simple chain, the three trophic levels did not evolve together, and levels of temperature and CO 2 will gradually increase to our experimental levels over a number of decades. However, models and experiments are necessarily artificial, and in this case our experiments provided relevant insight into mechanisms by which trophic asynchrony can occur. Based on our experimental results here and accompanying models [4] and observational field studies [5], [30], [58], we conclude that any efforts to conserve natural enemies or to enhance natural biological control will be negatively affected by complex interactions between multiple climate change metrics and biotic communities. These effects are likely to be exacerbated by increases in extreme weather events [7], [10], [11], [58], contributing to increased insect outbreaks through a number of direct and indirect pathways.

Acknowledgments Thanks to M. Forister, T. Massad, C. Jeffrey, the chemical ecology group at UNR, N. Mills, and two anonymous reviewers for comments and edits. J. Ruberson graciously supplied insects for our lab colonies. T. Massad developed the methods for quantifying alfalfa saponins. The authors thank the staff at Earthwatch, M. Tobler, G. Rodriguez-Castaneda, M. Olson, and many Earthwatch volunteers for their hard work and enthusiasm for the project.

Author Contributions Conceived and designed the experiments: LAD. Performed the experiments: SAS LAD. Analyzed the data: LAR LAD. Contributed reagents/materials/analysis tools: CDD LAD LAR. Wrote the paper: LAD LAR SAS CDD.