Pollution offsets the rapid evolution of increased heat tolerance in a natural population.

Despite the increasing evidence for rapid thermal evolution in natural populations, evolutionary rescue under global warming may be constrained by the presence of other stressors. Highly relevant in our polluted planet, is the largely ignored evolutionary trade-off between heat tolerance and tolerance to pollutants. By using two subpopulations (separated 40 years in time) from a resurrected natural population of the water flea Daphnia magna that experienced a threefold increase in heat wave frequency during this period, we tested whether rapid evolution of heat tolerance resulted in reduced tolerance to the widespread metal zinc and whether this would affect heat tolerance upon exposure to the pollutant. Our results revealed rapid evolution of increased heat tolerance in the recent subpopulation. Notably, the sensitivity to the metal tended to be stronger (reduction in net energy budget) or was only present (reductions in heat tolerance and in sugar content) in the recent subpopulation. As a result, the rapidly evolved higher heat tolerance of the recent subpopulation was fully offset when exposed to zinc. Our results highlight that the many reports of evolutionary rescue to global change stressors may give a too optimistic view as our warming planet is polluted by metals and other pollutants.

Integrating climate model projections into environmental risk assessment: A probabilistic modeling approach.

The Society of Environmental Toxicology and Chemistry (SETAC) convened a Pellston workshop in 2022 to examine how information on climate change could be better incorporated into the ecological risk assessment (ERA) process for chemicals as well as other environmental stressors. A major impetus for this workshop is that climate change can affect components of ecological risks in multiple direct and indirect ways, including the use patterns and environmental exposure pathways of chemical stressors such as pesticides, the toxicity of chemicals in receiving environments, and the vulnerability of species of concern related to habitat quality and use. This article explores a modeling approach for integrating climate model projections into the assessment of near- and long-term ecological risks, developed in collaboration with climate scientists. State-of-the-art global climate modeling and downscaling techniques may enable climate projections at scales appropriate for the study area. It is, however, also important to realize the limitations of individual global climate models and make use of climate model ensembles represented by statistical properties. Here, we present a probabilistic modeling approach aiming to combine projected climatic variables as well as the associated uncertainties from climate model ensembles in conjunction with ERA pathways. We draw upon three examples of ERA that utilized Bayesian networks for this purpose and that also represent methodological advancements for better prediction of future risks to ecosystems. We envision that the modeling approach developed from this international collaboration will contribute to better assessment and management of risks from chemical stressors in a changing climate. Integr Environ Assess Manag 2024;20:367-383. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Evaluating the effects of climate change and chemical, physical, and biological stressors on nearshore coral reefs: A case study in the Great Barrier Reef, Australia.

An understanding of the combined effects of climate change (CC) and other anthropogenic stressors, such as chemical exposures, is essential for improving ecological risk assessments of vulnerable ecosystems. In the Great Barrier Reef, coral reefs are under increasingly severe duress from increasing ocean temperatures, acidification, and cyclone intensities associated with CC. In addition to these stressors, inshore reef systems, such as the Mackay-Whitsunday coastal zone, are being impacted by other anthropogenic stressors, including chemical, nutrient, and sediment exposures related to more intense rainfall events that increase the catchment runoff of contaminated waters. To illustrate an approach for incorporating CC into ecological risk assessment frameworks, we developed an adverse outcome pathway network to conceptually delineate the effects of climate variables and photosystem II herbicide (diuron) exposures on scleractinian corals. This informed the development of a Bayesian network (BN) to quantitatively compare the effects of historical (1975-2005) and future projected climate on inshore hard coral bleaching, mortality, and cover. This BN demonstrated how risk may be predicted for multiple physical and biological stressors, including temperature, ocean acidification, cyclones, sediments, macroalgae competition, and crown of thorns starfish predation, as well as chemical stressors such as nitrogen and herbicides. Climate scenarios included an ensemble of 16 downscaled models encompassing current and future conditions based on multiple emission scenarios for two 30-year periods. It was found that both climate-related and catchment-related stressors pose a risk to these inshore reef systems, with projected increases in coral bleaching and coral mortality under all future climate scenarios. This modeling exercise can support the identification of risk drivers for the prioritization of management interventions to build future resilient reefs. Integr Environ Assess Manag 2024;20:401-418. © 2023 Norwegian Institute for Water Research and The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Effects of heat and pesticide stress on life history, physiology and the gut microbiome of two congeneric damselflies that differ in stressor tolerance.

The combined impact of toxicants and warming on organisms is getting increased attention in ecotoxicology, but is still hard to predict, especially with regard to heat waves. Recent studies suggested that the gut microbiome may provide mechanistic insights into the single and combined stressor effects on their host. We therefore investigated effects of sequential exposure to a heat spike and a pesticide on both the phenotype (life history and physiology) and the gut microbiome composition of damselfly larvae. We compared the fast-paced Ischnura pumilio, which is more tolerant to both stressors, with the slow-paced I. elegans, to obtain mechanistic insights into species-specific stressor effects. The two species differed in gut microbiome composition, potentially contributing to their pace-of-life differences. Intriguingly, there was a general resemblance between the stressor response patterns in the phenotype and in the gut microbiome, whereby both species responded broadly similar to the single and combined stressors. The heat spike negatively affected the life history of both species (increased mortality, reduced growth rate), which could be explained not only by shared negative effects on physiology (inhibition of acetylcholinesterase, increase of malondialdehyde), but also by shared effects on gut bacterial species' abundances. The pesticide only had negative effects (reduced growth rate, reduced net energy budget) in I. elegans. The pesticide generated shifts in the bacterial community composition (e.g. increased abundance of Sphaerotilus and Enterobacteriaceae in the gut microbiome of I. pumilio), which potentially contributed to the relatively higher pesticide tolerance of I. pumilio. Moreover, in line with the response patterns in the host phenotype, the effects of the heat spike and the pesticide on the gut microbiome were mainly additive. By contrasting two species differing in stress tolerance, our results suggest that response patterns in the gut microbiome may improve our mechanistic understanding of single and combined stressor effects.

Adverse effects of the pesticide chlorpyrifos on the physiology of a damselfly only occur at the cold and hot extremes of a temperature gradient.

Ecotoxicological studies considerably improved realism by assessing the toxicity of pollutants at different temperatures. Nevertheless, they may miss key interaction patterns between pollutants and temperature by typically considering only part of the natural thermal gradient experienced by species and ignoring daily temperature fluctuations (DTF). We therefore tested in a common garden laboratory experiment the effects of the pesticide chlorpyrifos across a range of mean temperatures and DTF on physiological traits (related to oxidative stress and bioenergetics) in low- and high-latitude populations of Ischnura elegans damselfly larvae. As expected, the impact of chlorpyrifos varied along the wide range of mean temperatures (12-34 °C). None of the physiological traits (except the superoxide anion levels) were affected by chlorpyrifos at the intermediate mean temperatures (20-24 °C). Instead, most of them were negatively affected by chlorpyrifos (reduced activity levels of the antioxidant defense enzymes superoxide dismutase [SOD], catalase [CAT] and peroxidase [PER], and a reduced energy budget) at the very high (≥28 °C) or extreme high temperatures (≥32 °C), and to lesser extent at the lower mean temperatures (≤16 °C). Notably, at the lower mean temperatures the negative impact of chlorpyrifos was often only present or stronger under DTF. Although the chlorpyrifos effects on the physiological traits greatly depended on the experimentally imposed thermal gradient, patterns were mainly consistent across the natural latitude-associated thermal gradient, indicating the generality of our results. The thermal patterns in chlorpyrifos-induced physiological responses contributed to the observed toxicity patterns in life history (reduced survival and growth at low and high mean temperatures). Taken together, our results underscore the importance of evaluating pesticide toxicity along a temperature gradient and of taking a mechanistic approach with a focus on physiology, to improve our understanding of the combined effects of pollutants and temperature in natural populations.

Synergistic survival-related effects of larval exposure to an aquatic pollutant and food stress get stronger during and especially after metamorphosis and shape fitness of terrestrial adults.

To improve the ecological risk assessment of aquatic pollutants it is needed to study their effects not only in the aquatic larval stage, but also in the terrestrial adult stage of the many animals with a complex life cycle. This remains understudied, especially with regard to interactive effects between aquatic pollutants and natural abiotic stressors. We studied effects of exposure to the pesticide DNP (2,4-Dinitrophenol) and how these were modulated by limited food availability in the aquatic larvae, and the possible delayed effects in the terrestrial adults of the damselfly Lestes viridis. Our results revealed that DNP and low food each had large negative effects on the life history, behaviour and to a lesser extent on the physiology of not only the larvae, but also the adults. Food limitation magnified the negative effects of DNP as seen by a strong decline in larval survival, metamorphosis success and adult lifespan. Notably, the synergism between the aquatic pollutant and food limitation for survival-related traits was stronger in the non-exposed adults than in the exposed larvae, likely because metamorphosis is stressful itself. Our results highlight that identifying effects of aquatic pollutants and synergisms with natural abiotic stressors, not only in the aquatic larval but also in the terrestrial adult stage, is crucial to fully assess the ecological impact of aquatic pollutants and to reveal the impact on the receiving terrestrial ecosystem through a changed aquatic-terrestrial subsidy.

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.

Thermal and latitudinal patterns in pace-of-life traits are partly mediated by the gut microbiome.

The integration of life-history, physiological and behavioural traits into the pace-of-life generates a powerful framework to understand trait variation in nature both along environmental gradients and in response to environmental stressors. While the gut microbiome has been hypothesized as a candidate mechanism to underlie differentiation in the pace-of-life, this has been rarely studied. We investigated the role of the gut microbiome in contributing to the differentiation in pace-of-life and in thermal adaptation between populations of Ischnura elegans damselfly larvae inhabiting warmer low latitudes and colder high latitudes. We carried out a common-garden experiment, whereby we manipulated the exposure of the damselfly larvae to two key global warming factors: 4 °C warming and a 30 °C heat wave. Comparing the bacterial composition of the food source and the bacterioplankton indicated that damselfly larvae differentially take up bacteria from the surrounding environment and have a resident and functionally relevant microbiome. The gut microbiome differed between larvae of both latitudes, and this was associated with the host's latitudinal differentiation in activity, a key pace-of-life trait. Under heat wave exposure, the gut microbial community composition of high-latitude larvae converged towards that of the low-latitude larvae, with an increase in bacteria that likely are important in providing energy to cope with the heat wave. This suggests an adaptive latitude-specific shift in the gut microbiota matching the better ability of low-latitude hosts to deal with heat extremes. In general, our study provides evidence for the gut microbiome contributing to latitudinal differentiation in both the pace-of-life and in heat adaptation in natural populations.

Genetic variation of the interaction type between two stressors in a single population: From antagonism to synergism when combining a heat spike and a pesticide.

Despite the surging interest in the interactions between toxicants and non-chemical stressors, and in evolutionary ecotoxicology, we have poor knowledge whether these patterns differ among genotypes within a population. Warming and toxicants are two widespread stressors in aquatic systems that are known to modify each other's effects. We studied to what extent effects of sequential exposure to a heat spike and the pesticide esfenvalerate differed among genotypes in the water flea Daphnia magna. Esfenvalerate had similar negative effects on survival and body size across genotypes, and for most genotypes it increased time to maturation, yet the effects on the reproductive performance were only detected in some genotypes and were inconsistent in direction. Across genotypes, the heat spike increased the heat tolerance, yet the negative effects of the heat spike on survival, reproductive performance and body size, and the positive effects on grazing rate and the shortened time to maturation were only seen in some genotypes. Notably, the interaction type between both stressors differed among genotypes. In contrast to our expectation, the impact of esfenvalerate was only magnified by the heat spike in some genotypes and only for a subset of the traits. For survival and time to maturation, the interaction type for the same stressor combination covered all three categories: additions, synergisms and antagonisms. This illustrates that categorizing the interaction type between stressors at the level of populations may hide considerable intrapopulation variation among genotypes. Opposite to our expectation, the more pesticide-tolerant genotypes showed a stronger synergism between both stressors. Genotype-dependent interaction patterns between toxicants and non-chemical stressors may explain inconsistencies among studies and challenges ecological risk assessment based on single genotypes. The observed genetic differences in the responses to the (combined) stressors may fuel the evolution of the stressor interaction pattern, a largely ignored topic in evolutionary ecotoxicology.

Daily temperature fluctuations can magnify the toxicity of pesticides.

We review the effect of daily temperature fluctuations (DTF), a key thermal factor predicted to increase under climate change, on pesticide toxicity. The effect of DTF on pesticide toxicity may be explained by: (i) a DTF-specific mechanism (caused by Jensen's inequality) and (ii) general mechanisms underlying an increased pesticide toxicity at both higher (increased energetic costs, pesticide uptake and metabolic conversion) and lower constant temperatures (lower organismal metabolic and associated elimination rates, increased sodium channel modulated nervous system vulnerability and energetic costs). Furthermore, DTF may enhance pesticide-induced reductions in heat tolerance due to stronger effects on oxygen demand (increase) and oxygen supply (decrease). Our literature review showed considerable support that DTF increase the negative impact of pesticides on insects, especially in terms of decreased survival. Therefore, we suggest that considering DTF in ecotoxicological studies may be of great importance to better protect biodiversity in our warming world.

Warming, temperature fluctuations and thermal evolution change the effects of microplastics at an environmentally relevant concentration.

Microplastics are sometimes considered not harmful at environmentally relevant concentrations. Yet, such studies were conducted under standard thermal conditions and thereby ignored the impacts of higher mean temperatures (MT), and especially daily temperature fluctuations (DTF) under global warming. Moreover, an evolutionary perspective may further benefit the future risk assessment of microplastics under global warming. Here, we investigated the effects of two generations of exposure to an environmentally relevant concentration of polystyrene microplastics (5 µg L-1) under six thermal conditions (2 MT × 3 DTF) on the life history, physiology, and behaviour of Daphnia magna. To assess the impact of thermal evolution we thereby compared Daphnia populations from high and low latitudes. At the standard ecotoxic thermal conditions (constant 20 °C) microplastics almost had no effect except for a slight reduction of the heartbeat rate. Yet, at the challenging thermal conditions (higher MT and/or DTF), microplastics affected each tested variable and caused an earlier maturation, a higher fecundity and intrinsic growth rate, a decreased heartbeat rate, and an increased swimming speed. These effects may be partly explained by hormesis and/or an adaptive response to stress in Daphnia. Moreover, exposure to microplastics at the higher mean temperature increased the fecundity and intrinsic growth rate of cold-adapted high-latitude Daphnia, but not of the warm-adapted low-latitude Daphnia, suggesting that thermal evolution in high-latitude Daphnia may buffer the effects of microplastics under future warming. Our results highlight the critical importance of DTF and thermal evolution for a more realistic risk assessment of microplastics under global warming.

Higher mean and fluctuating temperatures jointly determine the impact of the pesticide chlorpyrifos on the growth rate and leaf consumption of a freshwater isopod.

There is growing evidence that both increases in mean temperature and the widespread daily temperature fluctuations (DTF) may increase pesticide toxicity. Nevertheless, the likely more stressful, realistic combination of the two warming-related stressors has rarely been considered in ecotoxicology. Moreover, we have little knowledge on whether these stressor combinations could impair ecosystem functioning. We examined the effect of the pesticide chlorpyrifos under an increased mean temperature (+4 °C, from 18 °C to 22 °C) and in the presence of DTF (constant and 8 °C) on two life-history traits (mortality and growth rate) and one ecologically important behavioural trait (feeding rate) in the freshwater isopod Asellus aquaticus. The chlorpyrifos concentration used, 0.2 µg/L, did not cause mortality in any thermal condition, nor did it cause sublethal effects at the mean temperature of 18 °C. A key finding was that growth rate was strongly reduced by the pesticide only under the combination of both a higher mean temperature and DTF, highlighting the importance of testing toxicity under this realistic thermal scenario. The leaf consumption of chlorpyrifos-exposed isopods increased at the higher mean temperature when this was kept constant, however, it lowered again towards control levels when DTF was induced, thereby contributing to the growth reduction at this most stressful condition. These alterations of growth and leaf degradation rates may impact nutrient recycling, a key ecosystem function. Our results highlight the importance of integrating both increases in mean temperature and in DTF to improve current and future ecological risk assessment of pesticides.

Negative bioenergetic responses to pesticides in damselfly larvae are more likely when it is hotter and when temperatures fluctuate.

To make more realistic predictions about the current and future effects of pesticides, we need to better understand physiological mechanisms associated with the widespread higher toxicity of many pesticides under increasing mean temperatures and daily temperature fluctuations (DTFs). One overlooked, yet insightful, mechanism are bioenergetic responses as these provide information about the balance between energy gains and costs. Therefore, we studied how the bioenergetic responses to the insecticide chlorpyrifos were affected by a higher mean temperature and a higher DTF in Ischnura elegans damselfly larvae. To quantify bioenergetic responses we measured energy availability (Ea), energy consumption (Ec) and total net energy budget (cellular energy allocation, CEA). Exposure to chlorpyrifos considerably reduced CEA values when a high mean temperature was combined with a high DTF (up to -18%). Notably, chlorpyrifos had little effect on CEA at a constant 20 °C, meaning that the bioenergetic impact of chlorpyrifos would have been underestimated if we had only tested under standard testing conditions. The chlorpyrifos-induced reductions in CEA under warming were driven by reductions in Ea (up to -16%, mainly through large reductions in sugar and fat contents) while Ec was unaffected by chlorpyrifos. Treatment groups with a lower CEA value showed a higher mortality and a lower growth rate, indicating bioenergetic responses are contributing to the higher toxicity of chlorpyrifos under warming. Our study highlights the importance of evaluating the effects of pesticides under an increase in both mean temperature and DTF to improve the ecological risk assessment of pesticides under global warming.

Temperature variation magnifies chlorpyrifos toxicity differently between larval and adult mosquitoes.

To improve risk assessment there is increasing attention for the effect of climate change on the sensitivity to contaminants and vice versa. Two important and connected topics have been largely ignored in this context: (i) the increase of daily temperature variation (DTV) as a key component of climate change, and (ii) differences in sensitivity to climate change and contaminants between developmental stages. We therefore investigated whether DTV magnified the negative effects of the organophosphate insecticide chlorpyrifos on mortality and heat tolerance and whether this effect was stronger in aquatic larvae than in terrestrial adults of the mosquito Culex pipiens. Exposure to chlorpyrifos at a constant temperature imposed mortality and reduced the heat tolerance in both larvae and adult males, but not in adult females. This provides the first evidence that the TICS ("toxicant-induced climate change sensitivity") concept can be sex-specific. DTV had no direct negative effects. Yet, consistent with the CITS ("climate-induced toxicant sensitivity") concept, DTV magnified the toxicity of the pesticide in terms of larval mortality. This was not the case in the adult stage indicating the CITS concept to be dependent on the developmental stage. Notably, chlorpyrifos reduced the heat tolerance of adult females only in the presence of DTV, thereby providing support for the reciprocal effects between DTV and contaminants, hence the coupling of the TICS and CITS concepts. Taken together, our results highlight the importance of integrating DTV and the developmental stage to improve risk assessment of contaminants under climate change.

Shrinking Body Size and Physiology Contribute to Geographic Variation and the Higher Toxicity of Pesticides in a Warming World.

To improve current and future risk assessment of pesticides under global warming, mechanistic insights and consideration of daily temperature fluctuations (DTFs) are needed. One overlooked mechanism how both higher mean temperatures and DTFs may increase toxicity is by reducing body size (temperature-size-rule). We studied whether a higher mean temperature and DTF magnified chlorpyrifos toxicity in Ischnura elegans damselfly larvae, and whether this was mediated by temperature-induced reductions in body size and/or physiological changes. The lethal effects of chlorpyrifos were magnified at the high mean temperature (up to ∼15%) and under DTF (up to ∼33%), and especially at their combination (up to ∼46%) indicating synergisms. This highlights that not only considering DTFs, but also their interaction with higher mean temperatures is pivotal for realistic predictions of pesticide toxicity. Both higher temperatures and DTFs resulted in smaller larvae, which were more sensitive to chlorpyrifos. Notably, the DTF-induced smaller body sizes, as well as the higher oxidative damage to lipids, contributed to the higher chlorpyrifos toxicity under DTF. By integrating the temperature-size rule and size-pesticide sensitivity pattern we provide proof-of-principle for a novel, likely general mechanism contributing to geographic variation and the higher toxicity of pesticides in a warming world.

Using natural laboratories to study evolution to global warming: contrasting altitudinal, latitudinal, and urbanization gradients.

Demonstrating the likelihood of evolution in response to global warming is important, yet challenging. We discuss how three spatial thermal gradients (latitudinal, altitudinal, and urbanization) can be used as natural laboratories to inform about the gradual thermal evolution of populations by applying a space-for-time substitution (SFTS) approach. We compare thermal variables and confounding non-thermal abiotic variables, methodological approaches and evolutionary aspects associated with each type of gradient. On the basis of an overview of recent insect studies, we show that a key assumption of SFTS, local thermal adaptation along these gradients, is often but not always met, requiring explicit validation. To increase realism when applying SFTS, we highlight the importance of integrating daily temperature fluctuations, multiple stressors and multiple interacting species. Finally, comparative studies, especially across gradient types, are important to provide more robust inferences of evolution under gradual global warming. Integrating these research directions will further strengthen the still underused, yet powerful SFTS approach to infer gradual evolution under global warming.

Increased Daily Temperature Fluctuations Overrule the Ability of Gradual Thermal Evolution to Offset the Increased Pesticide Toxicity under Global Warming.

The widespread evidence that global warming can increase species sensitivities to chemical toxicants, and vice versa, and the recent insight that thermal evolution may mitigate these effects is crucial to predict the future impact of toxicants in a warming world. Nevertheless, a major component of global warming, the predicted increase in daily temperature fluctuations (DTFs), has been ignored at the interface of evolutionary ecotoxicology and global change biology. We studied whether 4 °C warming and a 5 °C DTF increase (to 10 °C DTF) magnified the negative impact of the insecticide chlorpyrifos (CPF) in larvae of low- and high-latitude populations of the damselfly Ischnura elegans. While 4 °C warming only increased CPF-induced mortality in high-latitude larvae, the high (10 °C) DTF increased CPF-induced larval mortality at both latitudes. CPF reduced the heat tolerance; however, this was buffered by latitude-specific thermal adaptation to both mean temperature and DTF. Integrating our results in a space-for-time substitution indicated that gradual thermal evolution in high-latitude larvae may offset the negative effects of CPF on heat tolerance under warming, unless the expected DTF increase is taken into account. Our results highlight the crucial importance of jointly integrating DTFs and thermal evolution to improve risk assessment of toxicants under global warming.

Current and future daily temperature fluctuations make a pesticide more toxic: Contrasting effects on life history and physiology.

There is increasing concern that climate change may make organisms more sensitive to chemical pollution. Many pesticides are indeed more toxic at higher mean temperatures. Yet, we know next to nothing about the effect of another key component of climate change, the increase of daily temperature fluctuations (DTFs), on pesticide toxicity. Therefore, we tested the effect of the pesticide chlorpyrifos under different levels of DTF (constant = 0 °C, low = 5 °C (current maximum level) and high = 10 °C (predicted maximum level under global warming)) around the same mean temperature on key life history and physiological traits of Ischnura elegans damselfly larvae in a common-garden experiment. At all levels of DTF, chlorpyrifos exposure was stressful: it reduced energy storage (fat content) and the activity of its target enzyme acetylcholinesterase, while it increased the activity of the detoxification enzyme cytochrome P450 monooxygenase. Notably, chlorpyrifos did not cause mortality or reduced growth rate at the constant temperature (0 °C DTF), yet increased mortality 6x and reduced growth rate with ca. 115% in the presence of DTF. This indicates that daily short-term exposures to higher temperatures can increase pesticide toxicity. Our data suggest that when 5 °C DTF will become more common in the studied high-latitude populations, this will increase the toxicity of CPF, and that a further increase from 5° DTF to 10 °C DTF may not result in a further increase of pesticide toxicity. Our results highlight the biological importance of including daily temperature fluctuations in ecological risk assessment of pesticides and as an extra dimension in the climate-induced toxicant sensitivity concept.

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.

Voltinism-associated differences in winter survival across latitudes: integrating growth, physiology, and food intake.

Species that span large latitudinal gradients face strong differences in voltinism and in winter conditions within their range. Latitudinal gradients in winter survival and especially their underlying mechanisms and association with voltinism patterns are poorly studied. We tested in the damselfly Enallagma cyathigerum whether high-latitude populations were better in dealing with the longer winters compared to central- and low-latitude populations and whether this was associated with changes in voltinism. We thereby evaluated whether higher initial levels and/or lower reductions during winter of energy storage (measured as fat content) and investment in immune function [measured as the activity of phenoloxidase (PO)], and/or stronger compensatory responses in food intake contributed to the higher winter survival in high-latitude populations. To this end, we simulated a long high-latitude winter at 4 °C under manipulated food conditions. Across food levels, winter survival was highest in Swedish larvae, intermediate in Belgian larvae, and lowest in Spanish larvae, indicating latitude-specific thermal adaptation that could be partly linked to differences in voltinism. The semi-voltine Swedish larvae were growing slower before winter and as a result accumulated the highest fat content and PO activity when the winter started compared to the univoltine, faster growing Belgian, and Spanish larvae. Fat content and PO activity declined during the winter, yet equally across latitudes, and were not buffered by compensatory food intake. Our data identified possible underlying physiological mechanisms of winter survival and support the hypothesis that widespread latitude-associated voltinism shifts may be a selective factor contributing to latitudinal shifts in winter survival.