Rapid evolution of consumptive and non-consumptive predator effects on prey population densities, bioenergetics and stoichiometry.

Predators can strongly influence prey populations not only through consumptive effects (CE) but also through non-consumptive effects (NCE) imposed by predation risk. Yet, the impact of NCE on bioenergetic and stoichiometric body contents of prey, traits that are shaping life histories, population and food web dynamics, is largely unknown. Moreover, the degree to which NCE can evolve and can drive evolution in prey populations is rarely studied. A 6-week outdoor mesocosm experiment with Caged-Fish (NCE) and Free-Ranging-Fish (CE and NCE) treatments was conducted to quantify and compare the effects of CE and NCE on population densities, bioenergetic and stoichiometric body contents of Daphnia magna, a keystone species in freshwater ecosystems. We tested for evolution of CE and NCE by using experimental populations consisting of D. magna clones from two periods of a resurrected natural pond population: a pre-fish period without fish and a high-fish period with high predation pressure. Both Caged-Fish and Free-Ranging-Fish treatments decreased the body size and population densities, especially in Daphnia from the high-fish period. Only the Free-Ranging-Fish treatment affected bioenergetic variables, while both the Caged-Fish and Free-Ranging-Fish treatments shaped body stoichiometry. The effects of CE and NCE were different between both periods indicating their rapid evolution in the natural resurrected population. Both the Caged-Fish and Free-Ranging-Fish treatments changed the clonal frequencies of the experimental Daphnia populations of the pre-fish as well as the high-fish period, indicating that not only CE but also NCE induced clonal sorting, hence rapid evolution during the mesocosm experiment in both periods. Our results demonstrate that CE as well as NCE have the potential to change not only the body size and population density but also the bioenergetic and stoichiometric characteristics of prey populations. Moreover, we show that these responses not only evolved in the studied resurrected population, but that CE and NCE also caused differential rapid evolution in a time frame of 6 weeks (ca. four to six generations). As NCE can evolve as well as can drive evolution, they may play an important role in shaping eco-evolutionary dynamics in predator-prey interactions.

Rapid evolution of unimodal but not of linear thermal performance curves in Daphnia magna.

Species may cope with warming through both rapid evolutionary and plastic responses. While thermal performance curves (TPCs), reflecting thermal plasticity, are considered powerful tools to understand the impact of warming on ectotherms, their rapid evolution has been rarely studied for multiple traits. We capitalized on a 2-year experimental evolution trial in outdoor mesocosms that were kept at ambient temperatures or heated 4°C above ambient, by testing in a follow-up common-garden experiment, for rapid evolution of the TPCs for multiple key traits of the water flea Daphnia magna. The heat-selected Daphnia showed evolutionary shifts of the unimodal TPCs for survival, fecundity at first clutch and intrinsic population growth rate toward higher optimum temperatures, and a less pronounced downward curvature indicating a better ability to keep fitness high across a range of high temperatures. We detected no evolution of the linear TPCs for somatic growth, mass and development rate, and for the traits related to energy gain (ingestion rate) and costs (metabolic rate). As a result, also the relative thermal slope of energy gain versus energy costs did not vary. These results suggest the overall (rather than per capita) top-down impact of D. magna may increase under rapid thermal evolution.

Thermal Performance Curves in a Polluted World: Too Cold and Too Hot Temperatures Synergistically Increase Pesticide Toxicity.

Ecotoxicological studies typically cover only a limited part of the natural thermal range of populations and ignore daily temperature fluctuations (DTFs). Therefore, we may miss important stressor interaction patterns and have poor knowledge on how pollutants affect thermal performance curves (TPCs), which is needed to improve insights into the fate of populations to warming in a polluted world. We tested the single and combined effects of pesticide exposure and DTFs on the TPCs of low- and high-latitude populations of Ischnura elegans damselfly larvae. While chlorpyrifos did not have any effect at the intermediate mean temperatures (20-24 °C), it became toxic (reflecting synergisms) at lower (≤16 °C, reduced growth) and especially at higher (≥28 °C, reduced survival and growth) mean temperatures, resulting in more concave-shaped TPCs. Remarkably, these toxicity patterns were largely consistent at both latitudes and hence across a natural thermal gradient. Moreover, DTFs magnified the pesticide-induced survival reductions at 34 °C. The TPC perspective allowed us to identify different toxicity patterns and interaction types (mainly additive vs synergistic) across the thermal gradient. This highlights the importance of using thermal gradients to make more realistic predictions about the impact of pesticides in a warming world and of warming in a polluted world.

Microplastics increases the heat tolerance of Daphnia magna under global warming via hormetic effects.

The ecological risk assessment of microplastics under global warming receives increasing attention. Yet, such studies mostly focused on increased mean temperatures (MT), ignoring another key component of global warming, namely daily temperature fluctuations (DTF). Moreover, we know next to nothing about the combined effects of multigenerational exposure to microplastics and warming. In this study, Daphnia magna was exposed to an environmentally relevant concentration of polystyrene microplastics (5 µg L-1) under six thermal conditions (MT: 20 â„ƒ, 24 â„ƒ; DTF: 0 â„ƒ, 5 â„ƒ, 10 â„ƒ) over two generations to investigate the interactive effects of microplastics and global warming. Results showed that microplastics had no effects on Daphnia at standard thermal conditions (constant 20 °C). Yet, microplastics increased the fecundity, heat tolerance, amount of energy storage, net energy budget and cytochrome P450 activity, and decreased the energy consumption when tested under an increased MT or DTF, indicating a hormesis effect induced by microplastics under warming. The unexpected increase in heat tolerance upon exposure to microplastics could be partly explained by the reduced energy consumption and/or increased energy availability. Overall, the present study highlighted the importance of including DTF and multigenerational exposure to improve the ecological risk assessment of microplastics under global warming.

A fast pace-of-life is traded off against a high thermal performance.

The integration of life-history, behavioural and physiological traits into a 'pace-of-life syndrome' is a powerful concept in understanding trait variation in nature. Yet, mechanisms maintaining variation in 'pace-of-life' are not well understood. We tested whether decreased thermal performance is an energetic cost of a faster pace-of-life. We characterized the pace-of-life of larvae of the damselfly Ischnura elegans from high-latitude and low-latitude regions when reared at 20°C or 24°C in a common-garden experiment, and estimated thermal performance curves for a set of behavioural, physiological and performance traits. Our results confirm a faster pace-of-life (i.e. faster growth and metabolic rate, more active and bold behaviour) in the low-latitude and in warm-reared larvae, and reveal increased maximum performance, Rmax, but not thermal optimum Topt, in low-latitude larvae. Besides a clear pace-of-life syndrome integration at the individual level, larvae also aligned along a 'cold-hot' axis. Importantly, a faster pace-of-life correlated negatively with a high thermal performance (i.e. higher Topt for swimming speed, metabolic rate, activity and boldness), which was consistent across latitudes and rearing temperatures. This trade-off, potentially driven by the energetically costly maintenance of a fast pace-of-life, may be an alternative mechanism contributing to the maintenance of variation in pace-of-life within populations.

Scared to evolve? Non-consumptive effects drive rapid adaptive evolution in a natural prey population.

Predators can strongly influence prey populations through both consumptive and non-consumptive effects. Nevertheless, most studies have focused on the consumptive effects in driving evolutionary changes. By integrating experimental evolution and resurrection ecology, we tested the roles of non-consumptive and consumptive effects in driving evolution in a Daphnia magna population that experienced strong changes in fish predation pressure. All resurrected genotypes were pooled, inoculated in outdoor mesocosms, and exposed to free-fish or caged-fish treatments. Non-consumptive effects induced rapid, repeatable changes in the clonal composition and associated genotypic trait changes that were similar in magnitude and direction to those imposed by killing. Both non-consumptive and consumptive effects caused a shift towards a dominance of the high-fish period clones that can perform better under fish predation, and this may be explained by the higher intrinsic growth rate of the high-fish period clones under predation risk. The genotypic trait changes (e.g. reduced body sizes, earlier maturation, more and smaller offspring) of the Daphnia in the mesocosm experiments were in the same direction as the adaptive trait shifts observed in situ through resurrection ecology. Our results demonstrate that non-consumptive effects can induce rapid adaptive evolution and may represent an overlooked driver of eco-evolutionary dynamics.

Thermal plasticity and evolution shape predator-prey interactions differently in clear and turbid water bodies.

Warming and eutrophication negatively affect freshwater ecosystems by modifying trophic interactions and increasing water turbidity. We need to consider their joint effects on predator-prey interactions and how these depend on the thermal evolution of both predator and prey. We quantified how 4°C warming and algae-induced turbidity (that integrates turbidity per se and increased food for zooplankton prey) affect functional response parameters and prey population parameters in a common-garden experiment. We did so for all combinations of high- and low-latitude predator (damselfly larvae) and prey (water fleas) populations to assess the potential impact of thermal evolution of predators and/or prey at a high latitude under warming using a space-for-time substitution. We then modelled effects on the system stability (i.e. tendency to oscillate) under different warming, turbidity and evolutionary scenarios. Warming and turbidity had little effect on the functional response parameters of high-latitude predators. In contrast, warming and turbidity reduced the handling times of low-latitude predators. Moreover, warming increased the search rates of low-latitude predators in clear water but instead decreased these in turbid water. Warming increased stability (i.e. prevented oscillations) in turbid water (except for the 'high-latitude predator and high-latitude prey' system), mainly by decreasing the prey's carrying capacity and partly also by decreasing search rates, while it did not affect stability in clear water. Algae-induced turbidity generally decreased stability, mainly by increasing the prey's carrying capacity and partly also by increasing search rates. This resembles findings that nutrient enrichment can reduce the stability of trophic systems. The expected stability of the high-latitude trophic system under warming was dependent on the turbidity level: our results suggest that thermal plasticity tends to destabilize the high-latitude trophic system under warming in clear water but not in turbid water, and that thermal evolution of the predator will stabilize the high-latitude system under warming in turbid water but less so in clear water. The extent to which thermal plasticity and evolution shape trophic system stability under warming may strongly differ between clear and turbid water bodies, with their contributions having a more stabilizing role in turbid water.

Cryptic eco-evolutionary feedback in the city: Urban evolution of prey dampens the effect of urban evolution of the predator.

Most research on eco-evolutionary feedbacks focuses on ecological consequences of evolution in a single species. This ignores the fact that evolution in response to a shared environmental factor in multiple species involved in interactions could alter the net cumulative effect of evolution on ecology. We empirically tested whether urbanization-driven evolution in a predator (nymphs of the damselfly Ischnura elegans) and its prey (the water flea Daphnia magna) jointly shape the outcome of predation under simulated heatwaves. Both interactors show genetic trait adaptation to urbanization, particularly to higher temperatures. We cross-exposed common-garden reared damselflies and Daphnia from replicated urban and rural populations, and quantified predation rates and functional response traits. Urban damselfly nymphs showed higher encounter and predation rates than rural damselflies when exposed to rural prey, but this difference disappeared when they preyed on urban Daphnia. This represents a case of a cryptic evo-to-eco feedback, where the evolution of one species dampens the effects of the evolution of another species on their interaction strength. The effects of evolution of each single species were strong: the scenario in which only the predator or prey was adapted to urbanization resulted in a c. 250% increase in encounter rate and a c. 25% increase in predation rate, compared to the rural predator-rural prey combination. Our results provide unique evidence for eco-evolutionary feedbacks in cities, and underscore the importance of a multi-species approach in eco-evolutionary dynamics research.

Evolution of pesticide tolerance and associated changes in the microbiome in the water flea Daphnia magna.

Exposure to pesticides can have detrimental effects on aquatic communities of non-target species. Populations can evolve tolerance to pesticides which may rescue them from extinction. However, the evolution of tolerance does not always occur and insights in the underlying mechanisms are scarce. One understudied mechanism to obtain pesticide tolerance in hosts are shifts toward pesticide-degrading bacteria in their microbiome. We carried out experimental evolution trials where replicated experimental populations of the water flea Daphnia magna were exposed to the pesticide chlorpyrifos or a solvent control, after which we performed acute toxicity assays to evaluate the evolution of chlorpyrifos tolerance. Additionally, we quantified changes in the microbiota community composition of whole body and gut samples to assess which sample type best reflected the pesticide tolerance of the Daphnia host. As expected, chlorpyrifos-selected clones became more tolerant to chlorpyrifos as shown by the higher EC5048 h (36% higher) compared with the control clones. This was associated with shifts in the microbiome composition whereby the abundance of known organophosphate-degrading bacterial genera increased on average ~4 times in the chlorpyrifos-selected clones. Moreover, the abundances of several genera, including the organophosphate-degrading bacteria Pseudomonas, Flavobacterium and Bacillus, were positively correlated with the EC5048 h of the host populations. These shifts in bacterial genera were similar in magnitude in whole body and gut samples, yet the total abundance of organophosphate-degrading bacteria was ~6 times higher in the whole body samples, suggesting that the gut is not the only body part where pesticide degradation by the microbiome occurs. Our results indicate that the microbiome is an important mediator of the development of tolerance to pesticides in Daphnia.

Resurrecting the metabolome: Rapid evolution magnifies the metabolomic plasticity to predation in a natural Daphnia population.

Populations rely on already present plastic responses (ancestral plasticity) and evolution (including both evolution of mean trait values, constitutive evolution, and evolution of plasticity) to adapt to novel environmental conditions. Because of the lack of evidence from natural populations, controversy remains regarding the interplay between ancestral plasticity and rapid evolution in driving responses to new stressors. We addressed this topic at the level of the metabolome utilizing a resurrected natural population of the water flea Daphnia magna that underwent a human-caused increase followed by a reduction in predation pressure within ~16 years. Predation risk induced plastic changes in the metabolome which were mainly related to shifts in amino acid and sugar metabolism, suggesting predation risk affected protein and sugar utilization to increase energy supply. Both the constitutive and plastic components of the metabolic profiles showed rapid, probably adaptive evolution whereby ancestral plasticity and evolution contributed nearly equally to the total changes of the metabolomes. The subpopulation that experienced the strongest fish predation pressure and showed the strongest phenotypic response, also showed the strongest metabolomic response to fish kairomones, both in terms of the number of responsive metabolites and in the amplitude of the multivariate metabolomic reaction norm. More importantly, the metabolites with higher ancestral plasticity showed stronger evolution of plasticity when predation pressure increased, while this pattern reversed when predation pressure relaxed. Our results therefore highlight that the evolution in response to a novel pressure in a natural population magnified the metabolomic plasticity to this stressor.

Evolution of cold tolerance and thermal plasticity in life history, behaviour and physiology during a poleward range expansion.

Many species that are moving polewards encounter novel thermal regimes to which they have to adapt. Therefore, rapid evolution of thermal tolerance and of thermal plasticity in fitness-related traits in edge populations can be crucial for the success and speed of range expansions. We tested for adaptation in cold tolerance and in life history, behavioural and physiological traits and their thermal plasticity during a poleward range expansion. We reconstructed the thermal performance curves of life history (survival, growth and development rates), behaviour (food intake) and cold tolerance (chill coma recovery time) in the aquatic larval stage of the damselfly Ischnura elegans that is currently showing a poleward range expansion in northern Europe. We studied larvae from three edge and three core populations using a common-garden experiment. Consistent with the colder annual temperatures, larvae at the expansion front evolved an improved cold tolerance. The edge populations showed no overall (across temperatures) evolution of a faster life history that would improve their range-shifting ability. Moreover, consistent with damselfly edge populations from colder latitudes, edge populations evolved at the highest rearing temperature (28°C) a faster development rate, likely to better exploit the rare periods with higher temperatures. This was associated with a higher food intake and a lower metabolic rate. In conclusion, our results suggest that the edge populations rapidly evolved adaptive changes in trait means and thermal plasticity to the novel thermal conditions at the edge front. Our results highlight the importance of considering besides trait plasticity and the evolution of trait means, also the evolution of trait plasticity to improve forecasts of responses to climate change.

Durante su expansión hacia los polos, las especies encuentran nuevos regímenes de temperatura a los que tienen que adaptarse. Por esto, una rápida evolución de la tolerancia térmica y de la plasticidad térmica de rasgos fisiológicos clave en las poblaciones del borde del área de distribución es crucial para el éxito y la velocidad de las expansiones de rango. En este estudio testamos la adaptación de la tolerancia la frío y la plasticidad térmica de rasgos de historia de vida, comportamiento y fisiológicos durante una expansión de rango hacia el norte. Reconstruimos las curvas de rendimiento térmico de rasgos de historia de vida (supervivencia, tasa de crecimiento y tasa de desarrollo) y comportamiento (ingestión de alimento), así como la tolerancia al frío (tiempo de recuperación del coma por frío) en la larva acuática de la especie de caballito del diablo Ischnura elegans, especie que muestra un rango de expansión actual en el norte de Europa. Estudiamos larvas de tres poblaciones de la región de expansión norte y tres poblaciones de la región central usando experimentos en laboratorio. En concordancia con las temperaturas más bajas, las larvas del área de expansión mostraron mayor tolerancia al frío. Sin embargo, estas larvas no mostraron en general (a lo largo de las temperaturas estudiadas) evolución hacia una historia de vida más rápida, que aumentaría su habilidad para expandirse. Además, en consistencia con la menor temperatura de la región de expansión, las larvas presentaron mayor tasa de desarrollo a la temperatura experimental más alta (28°C), probablemente para explotar mejor los infrecuentes períodos con altas temperaturas en dicha región. Esto estuvo asociado con una mayor ingestión de alimento y una menor tasa metabólica. En conclusión, nuestros resultados sugieren que los valores medios y la plasticidad de los rasgos estudiados de las poblaciones del frente de expansión evolucionaron rápidamente para adaptarse a las nuevas condiciones térmicas en dicha región. Asimismo, nuestros resultados destacan la importancia de considerar, además de la media y plasticidad de los rasgos, la evolución de esta plasticidad, con el fin de mejorar las predicciones de las respuestas de las especies al cambio climático.

Towards a unified study of multiple stressors: divisions and common goals across research disciplines.

Anthropogenic environmental changes, or 'stressors', increasingly threaten biodiversity and ecosystem functioning worldwide. Multiple-stressor research is a rapidly expanding field of science that seeks to understand and ultimately predict the interactions between stressors. Reviews and meta-analyses of the primary scientific literature have largely been specific to either freshwater, marine or terrestrial ecology, or ecotoxicology. In this cross-disciplinary study, we review the state of knowledge within and among these disciplines to highlight commonality and division in multiple-stressor research. Our review goes beyond a description of previous research by using quantitative bibliometric analysis to identify the division between disciplines and link previously disconnected research communities. Towards a unified research framework, we discuss the shared goal of increased realism through both ecological and temporal complexity, with the overarching aim of improving predictive power. In a rapidly changing world, advancing our understanding of the cumulative ecological impacts of multiple stressors is critical for biodiversity conservation and ecosystem management. Identifying and overcoming the barriers to interdisciplinary knowledge exchange is necessary in rising to this challenge. Division between ecosystem types and disciplines is largely a human creation. Species and stressors cross these borders and so should the scientists who study them.

Genetic compensation rather than genetic assimilation drives the evolution of plasticity in response to mild warming across latitudes in a damselfly.

Global warming is causing plastic and evolutionary changes in the phenotypes of ectotherms. Yet, we have limited knowledge on how the interplay between plasticity and evolution shapes thermal responses and underlying gene expression patterns. We assessed thermal reaction norm patterns across the transcriptome and identified associated molecular pathways in northern and southern populations of the damselfly Ischnura elegans. Larvae were reared in a common garden experiment at the mean summer water temperatures experienced at the northern (20°C) and southern (24°C) latitudes. This allowed a space-for-time substitution where the current gene expression levels at 24°C in southern larvae are a proxy for the expected responses of northern larvae under gradual thermal evolution to the predicted 4°C warming. Most differentially expressed genes showed fixed differences across temperatures between latitudes, suggesting that thermal genetic adaptation will mainly evolve through changes in constitutive gene expression. Northern populations also frequently showed plastic responses in gene expression to mild warming, while southern populations were much less responsive to temperature. Thermal responsive genes in northern populations showed to a large extent a pattern of genetic compensation, namely gene expression that was induced at 24°C in northern populations remained at a lower constant level in southern populations, and were associated with metabolic and translation pathways. There was instead little evidence for genetic assimilation of an initial plastic response to mild warming. Our data therefore suggest that genetic compensation rather than genetic assimilation may drive the evolution of plasticity in response to mild warming in this damselfly species.

Reduced stress defence responses contribute to the higher toxicity of a pesticide under warming.

There is a pressing need to identify the molecular mechanisms underlying the, often magnifying, interactive effects between contaminants and natural stressors. Here we test our hypothesis that lower general stress defence responses contribute to synergistic interactions between stressors. We focus on the widespread pattern that many contaminants are more toxic at higher temperatures. Specifically, we tested the effects of an environmentally realistic low-effect and high-effect concentration of the pesticide chlorpyrifos under warming at the gene expression level in the northern house mosquito Culex pipiens molestus (Forskal, 1775). By applying the independent action model for combined stressors on RNA-sequencing data, we identified interactive gene expression patterns under combined exposure to chlorpyrifos and warming for general stress defence responses: protection of macromolecules, antioxidant processes, detoxification and energy metabolism/allocation. Most of these general stress defence response genes showed upregulated antagonistic interactions (i.e., were less upregulated than expected under the independent action model). This indicates that when pesticide exposure was combined with warming, the general stress defence responses were no longer buffering increased stress levels, which may contribute to a higher sensitivity to toxicants under warming. These upregulated antagonistic interactions were stronger for the high-effect chlorpyrifos concentration, indicating that exposure to this concentration under warming was most stressful. Our results highlight that quantitative analysis of the frequency and strength of the interaction types of general stress defence response genes, specifically focusing on antagonistic upregulations and synergistic downregulations, may advance our understanding of how natural stressors modify the toxicity of contaminants.

Urbanization drives cross-taxon declines in abundance and diversity at multiple spatial scales.

The increasing urbanization process is hypothesized to drastically alter (semi-)natural environments with a concomitant major decline in species abundance and diversity. Yet, studies on this effect of urbanization, and the spatial scale at which it acts, are at present inconclusive due to the large heterogeneity in taxonomic groups and spatial scales at which this relationship has been investigated among studies. Comprehensive studies analysing this relationship across multiple animal groups and at multiple spatial scales are rare, hampering the assessment of how biodiversity generally responds to urbanization. We studied aquatic (cladocerans), limno-terrestrial (bdelloid rotifers) and terrestrial (butterflies, ground beetles, ground- and web spiders, macro-moths, orthopterans and snails) invertebrate groups using a hierarchical spatial design, wherein three local-scale (200 m × 200 m) urbanization levels were repeatedly sampled across three landscape-scale (3 km × 3 km) urbanization levels. We tested for local and landscape urbanization effects on abundance and species richness of each group, whereby total richness was partitioned into the average richness of local communities and the richness due to variation among local communities. Abundances of the terrestrial active dispersers declined in response to local urbanization, with reductions up to 85% for butterflies, while passive dispersers did not show any clear trend. Species richness also declined with increasing levels of urbanization, but responses were highly heterogeneous among the different groups with respect to the richness component and the spatial scale at which urbanization impacts richness. Depending on the group, species richness declined due to biotic homogenization and/or local species loss. This resulted in an overall decrease in total richness across groups in urban areas. These results provide strong support to the general negative impact of urbanization on abundance and species richness within habitat patches and highlight the importance of considering multiple spatial scales and taxa to assess the impacts of urbanization on biodiversity.

Strong species differences in life history do not predict oxidative stress physiology or sensitivity to an environmental oxidant.

Species typically align along a fast-slow life-history continuum, yet it is not clear to what extent oxidative stress physiology can be integrated with this continuum to form a 'pace-of-life syndrome', especially so in invertebrates. This is important, given the assumed role of oxidative stress in mediating life-history trade-offs, and the prediction that species with a faster pace should be more vulnerable to oxidative stress. We tested whether a species' life-history pace, here represented by its growth rate, can predict species-level differentiation in physiology and sensitivity to oxidative stress. Therefore, we exposed four species of Ischnura damselflies that strongly align along a fast-slow life-history continuum to different levels of ultraviolet (UV) radiation. We measured an extended set of physiological traits linked to the pace-of-life: standard metabolic rate, oxidative stress physiology (antioxidant enzymes and oxidative damage) and defence/condition traits (investment in immune function, energy storage and structural defence). Despite strong species differences in growth rate and physiology, growth rate did not predict species-level differentiation in physiology. Hence there was no support for the integration of metabolic rate, oxidative stress physiology or defence/condition traits into a species-level syndrome. Ultraviolet exposure affected nearly all traits: it reduced growth rate and increased metabolic rate, affected all oxidative stress physiology traits and increased the two defence traits (immune function, and melanin content). Nevertheless, the pace-of-life based on growth rate did not predict sensitivity to UV. Instead, the observed pattern of investment in structural UV defence (melanin) might have reduced the need for enzymatic antioxidant defence, this way potentially decoupling the covariation between the life-history pace and oxidative stress physiology. The absence of an integrated axis of life-history and physiological variation indicates no major constraints for the evolution of these traits among the studied damselfly species. Our study highlights that ecological differences between species may decouple covariation between species' life-history pace and their physiology, as well as their sensitivity to environmental stressors.

The Exposure Order Strongly Modifies How a Heat Spike Increases Pesticide Toxicity.

The exposure order may strongly affect the impact of stressors, yet is largely ignored for the frequently occurring combinations of toxicants with natural stressors. We tested how exposure order shaped the interactive effects of serial exposure to the pesticide chlorpyrifos and to a heat spike in the larvae of the mosquito Culex pipiens. Notably, the chlorpyrifos-induced mortality was much more magnified by the heat spike and a synergism was already detected at the low concentration when exposure to chlorpyrifos followed the heat spike. This suggests that the preceding heat spike weakened the larvae as reflected in their lower net energy budget, moreover the chlorpyrifos-induced inhibition of its target enzyme (acetylcholinesterase) was only magnified by the heat spike when it was the first stressor. Also the chlorpyrifos-induced reduction in heat tolerance was stronger when the pesticide pulse followed the heat spike, and was buffered by the heat spike when this was the second stressor. Our results provide the first evidence that the exposure order can strongly change the magnifying effect of an important climate change factor on the toxicity of a pesticide. This highlights the importance of exposure order in ecological risk assessment of toxicants under realistic combinations with natural stressors.

Effects of thermal evolution on the stoichiometric responses to nano-ZnO under warming are not general: insights from experimental evolution.

A key challenge for ecological risk assessment of contaminants under global warming is to predict effects at higher levels of biological organisation. One approach to reach this goal is to study how contaminants and warming cause changes in body stoichiometry as these may potentially cascade through food webs. Furthermore, though contaminants typically interact with warming, how rapid adaptation to higher temperatures affects these interactions is poorly studied. Here, we examined the effects of an important contaminant (ZnO nanoparticles, nZnO) and mild warming (4 °C) on body stoichiometry (C, N, P and their ratios) of an aquatic keystone species, the water flea Daphnia magna. To evaluate whether thermal evolution impacts the effects of nZnO at higher temperatures, we compared two sets of clones from a thermal selection experiment where Daphnia were kept in outdoor mesocosms at ambient or ambient +4 °C temperatures for 2 years. Exposure to nZnO decreased key body stoichiometric ratios (C:N, C:P and a trend for N:P) while warming increased the body C:N ratio. The stoichiometric changes to nZnO and warming were mostly independent and could be partly explained by changes in the macromolecules sugars and fat. Exposure to nZnO decreased C-rich sugars contributing to a reduced %C. Warming reduced body %C due to decreased C-rich sugars and fat levels, yet warming decreased body N% even more resulting in a higher C:N ratio. The stoichiometric responses to nZnO at the higher temperature did not differ between the two sets of clones, indicating experimental thermal evolution did not change the effects of nZnO under warming. Studying the stoichiometric responses to nZnO and warming of this keystone species may provide novel insights on the toxic effects of contaminants under warming. Moreover, understanding the influence of thermal evolution on the toxicity of contaminants is important for ecological risk assessment especially in a warming world.

Temperature variation makes an ectotherm more sensitive to global warming unless thermal evolution occurs.

To assess long-term impacts of global warming on species, there is growing interest in latitudinal intraspecific patterns in thermal adaptation. Yet, while both mean temperatures and daily temperature fluctuations (DTFs) are expected to increase under global warming, latitudinal differences in the effects of DTFs have not been documented. We tested whether low-latitude populations of an ectotherm deal better with greater DTF than high-latitude populations, especially at a high mean temperature close to the optimal temperature for growth where DTF causes exposure to extreme high temperatures. We evaluated the impact of DTFs when assessing the effect of gradual thermal evolution at the high latitude with a space-for-time substitution. We compared effects of both mean temperatures (20 and 24°C) and DTFs (constant = 0°C, low = 5°C and high = 10°C) on growth rates between low-latitude and high-latitude populations of the damselfly Ischnura elegans in a common-garden experiment. DTFs, if anything, reduced growth and were generally stressful as indicated by reductions in body condition, antioxidant defence and metabolic rate, and increases in oxidative damage. Most negative effects of DTFs were only present at a mean of 24°C when too high temperatures were reached during a daily cycle. Notably, while 4°C warming was beneficial in terms of growth rate at both latitudes at a constant temperature regime, this changed in a negative effect at high DTF. Moreover, this modulating effect of the mean temperature by DTF differed between latitudes indicating local thermal adaptation. While 4°C warming at low DTF still caused faster growth in low-latitude larvae, it already slowed growth in high-latitude larvae. This supports the emerging insight that warming would increase growth in high-latitude larvae in the absence of DTF, yet would decrease growth in the more realistic scenarios with DTF. In contrast, a space-for-time substitution approach suggested that under gradual thermal evolution, the evolved high-latitude larvae would no longer suffer a growth reduction in the presence of DTF. Our study provided important proof-of-principle that jointly integrating gradual thermal evolution and the expected increase in DTF generates opposing predictions of effects of global warming on this ectotherm.

Latitude-associated evolution and drivers of thermal response curves in body stoichiometry.

Trait-based studies are needed to understand the plastic and genetic responses of organisms to warming. A neglected organismal trait is elemental composition, despite its potential to cascade into effects on the ecosystem level. Warming is predicted to shape elemental composition through shifts in storage molecules associated with responses in growth, body size and metabolic rate. Our goals were to quantify thermal response patterns in body composition and to obtain insights into their underlying drivers and their evolution across latitudes. We reconstructed the thermal response curves (TRCs) for body elemental composition [C (carbon), N (nitrogen) and the C:N ratio] of damselfly larvae from high- and low-latitude populations. Additionally, we quantified the TRCs for survival, growth and development rates and body size to assess local thermal adaptation, as well as the TRCs for metabolic rate and key macromolecules (proteins, fat, sugars and cuticular melanin and chitin) as these may underlie the elemental TRCs. All larvae died at 36°C. Up to 32°C, low-latitude larvae increased growth and development rates and did not suffer increased mortality. Instead, growth and development rates of high-latitude larvae were lower and levelled off at 24°C, and mortality increased at 32°C. This latitude-associated thermal adaptation pattern matched the 'hotter-is-better' hypothesis. With increasing temperatures, low-latitude larvae decreased C:N, while high-latitude larvae increased C:N. These patterns were driven by associated changes in N contents, while C contents did not respond to temperature. Consistent with the temperature-size rule and the thermal melanism hypothesis, body size and melanin levels decreased with warming. While all traits and associated macromolecules (except for metabolic rate that showed thermal compensation) assumed to underlie thermal responses in elemental composition showed thermal plasticity, these were largely independent and none could explain the stoichiometric TRCs. Our results highlight that thermal responses in elemental composition cannot be explained by traditionally assumed drivers, asking for a broader perspective including the thermal dependence of elemental fluxes. Another key implication is that thermal evolution can reverse the plastic stoichiometric thermal responses and hence reverse how warming may shape food web dynamics through changes in body composition at different latitudes.