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.

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.

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.

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.

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.

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.

Multigenerational effects modify the tolerance of mosquito larvae to chlorpyrifos but not to a heat spike and do not change their synergism.

While interactions with global warming and multigenerational effects are considered crucial to improve risk assessment of pesticides, these have rarely been studied in an integrated way. While heat extremes can magnify pesticide toxicity, no studies tested how their combined effects may transmit to the next generation. We exposed mosquito larvae in a full factorial, two-generation experiment to a heat spike followed by chlorpyrifos exposure. As expected, the heat spike magnified the chlorpyrifos-induced lethal and sublethal effects within both generations. Only when preceded by the heat spike, chlorpyrifos increased mortality and reduced the population growth rate. Moreover, chlorpyrifos-induced reductions in heat tolerance (CTmax), acetylcholinesterase (AChE) activity and development time were further magnified by the heat spike. Notably, when parents were exposed to chlorpyrifos, the chlorpyrifos-induced lethal and sublethal effects in the offspring were smaller, indicating increased tolerance to chlorpyrifos. In contrast, there was no such multigenerational effect for the heat spike. Despite the adaptive multigenerational effect to the pesticide, the synergism with the heat spike was still present in the offspring generation. Generally, our results provide important evidence that short exposure to pulse-like global change stressors can strongly affect organisms within and across generations, and highlight the importance of considering multigenerational effects in risk assessment.

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.

Acute warming increases pesticide toxicity more than transgenerational warming by reducing the energy budget.

There is increasing awareness that the toxicity of pesticides can to a large extent be modulated by warming, and that temporal exposure scenarios may strongly affect the impact of two stressors. Nevertheless, we lack information on how the exposure duration to warming may shape pesticide toxicity under warming. Furthermore, despite that bioenergetic responses have the potential to generate mechanistic insights in how toxicants interact with warming, this has been understudied in ecotoxicology. To investigate whether warming duration modifies pesticide toxicity, mosquito larvae were exposed to a control temperature at 20 °C or three warming treatments at 24 °C (acute, developmental and transgenerational warming), and to four pesticide treatments (solvent control, and three chlorpyrifos concentrations) in a full factorial design. Chlorpyrifos increased mortality, growth rate and the energy consumed, and reduced the AChE (acetylcholinesterase) activity, the energy available, and the net energy budget (estimated as cellular energy allocation). The warming treatments did not affect mortality, AChE activity, and the energy consumed. However, acute warming increased the growth rate and decreased the energy available, while both acute and developmental warming decreased the cellular energy allocation. A first key finding was that the lethal and sublethal effects of chlorpyrifos were less strong under warming because of a higher degradation in the medium under warming. A second key finding was that, among the warming treatments, the pesticide toxicity was more increased under acute warming than under transgenerational warming. This could be explained by the negative impact of acute warming but not transgenerational warming on the net energy budget. The results in this study provide mechanistic insights that the exposure duration to warming can play an important role in modulating the impact of pesticides under warming. Therefore, including ecologically relevant temporal scenarios of exposure to warming is important in ecotoxicological studies.

Daily temperature variation lowers the lethal and sublethal impact of a pesticide pulse due to a higher degradation rate.

Daily temperature variation (DTV) is an important warming-related stressor that may magnify pesticide toxicity. Yet, it is unknown whether the pesticide impact under DTV is partly ameliorated by a faster pesticide degradation caused by cyclically higher temperatures under DTV. As synergisms may be more likely under energy-limiting conditions, the impact of the pesticide chlorpyrifos was tested under DTV on the mosquito Culex pipiens in the absence and presence of interspecific competition with the water flea Daphnia magna. Chlorpyrifos exposure at a constant temperature without interspecific competition caused considerable mortality, decreased development time, and increased pupal mass of C. pipiens. Competition with D. magna had negative sublethal effects, but it did not affect the toxicity of chlorpyrifos. In contrast, the presence of C. pipiens decreased the impact of chlorpyrifos on D. magna probably due to corporal absorption of chlorpyrifos by C. pipiens. A key finding was that chlorpyrifos no longer caused lethal effects on C. pipiens under DTV, despite DTV on its own being mildly lethal. Additionally, chlorpyrifos exposure under DTV decreased development time less and had no effect anymore on pupal mass compared to chlorpyrifos exposure at a constant temperature. Similarly, the negative chlorpyrifos impact on adult survival of D. magna was less under DTV than at the constant temperature. This could be explained by a faster chlorpyrifos degradation under DTV. This antagonism between pesticide exposure and DTV is likely widespread because organisms experience DTV, many pesticides are applied in pulses, and pesticide degradation is faster at higher temperatures.

Transgenerational exposure to warming reduces the sensitivity to a pesticide under warming.

Despite the increased attention for temporal aspects of stressor interactions and for effects of warming in ecotoxicological studies, we lack knowledge on how different exposure durations to warming may affect pesticide sensitivity. We tested how three types of exposure duration to 4 °C warming (acute, developmental and transgenerational exposure to 24 °C vs 20 °C) shape the effect of the pesticide chlorpyrifos on two ecologically relevant fitness-related traits of mosquito larvae: heat tolerance and antipredator behaviour. Transgenerational (from the parental generation) and developmental (from the egg stage) warming appeared energetically more stressful than acute warming (from the final instar), because (i) only the latter resulted in an adaptive increase of heat tolerance, and (ii) especially developmental and transgenerational warming reduced the diving responsiveness and diving time. Exposure to chlorpyrifos decreased the heat tolerance, diving responsiveness and diving time. The impact of chlorpyrifos was lower at 24 °C than at 20 °C indicating that the expected increase in toxicity at 24 °C was overruled by the observed increase in pesticide degradation. Notably, although our results suggest that transgenerational warming was energetically more stressful, it did reduce the chlorpyrifos-induced negative effects at 24 °C on heat tolerance and the alarm escape response compared to acute warming. Our results provide important evidence that the exposure duration to warming may determine the impact of a pesticide under warming, thereby identifying a novel temporal aspect of stressor interactions in risk assessment.

Mosquito larvae that survive a heat spike are less sensitive to subsequent exposure to the pesticide chlorpyrifos.

While extreme high temperatures are an important aspect of global warming, their effects on organisms are relatively understudied, especially in ecotoxicology. Sequential exposure to heat spikes and pesticides is a realistic scenario as both are typically transient stressors and are expected to further increase in frequency under global warming. We tested the effects of exposure to a lethal heat spike and subsequently to an ecologically relevant lethal pulse exposure of the pesticide chlorpyrifos in the larvae of mosquito Culex pipiens. The heat spike caused direct and delayed mortality, and resulted in a higher heat tolerance and activity of acetylcholinesterase, and a lower fat content in the survivors. The chlorpyrifos exposure caused mortality, accelerated growth rate, and decreased the heat tolerance and the activity of acetylcholinesterase. The preceding heat spike did not change how chlorpyrifos reduced the heat tolerance. Notably, the preceding heat spike did lower the lethal effect of the pesticide, which makes an important novel finding at the interface of ecotoxicology and global change biology, and adds a new dimension to the "climate-induced toxicant sensitivity" (CITS) concept. This may be due to both survival selection and cross-tolerance, and therefore likely a widespread phenomenon. Our results emphasize the importance of including extreme high temperatures as an important transient global change stressor in ecotoxicology.

Effects of predator cues and pesticide resistance on the toxicity of a (bio)pesticide mixture.

BACKGROUND: Populations of target species are typically exposed to pesticide mixtures and natural stressors such as predator cues, and are increasingly developing resistance to single pesticides. Nevertheless, we have poor knowledge whether natural stressors and the presence of pesticide resistance shape mixture toxicity. We tested the single and combined effects of the pesticide chlorpyrifos and the biopesticide Bacillus thuringiensis israelensis (Bti) on the survival of the Southern house mosquito (Culex quinquefasciatus, Say) and whether these effects were magnified by synthetic predator cues of Notonecta water bugs and differed between a chlorpyrifos-resistant (Ace-1R) and non-resistant (S-Lab) strain. RESULTS: Single exposure to Bti caused mortality in both strains (S-Lab ∼27%, Ace-1R ∼41%) and single exposure to chlorpyrifos caused only mortality in the S-Lab strain (∼33%), while predator cues did not induce mortality. The chlorpyrifos-resistant strain was 1.5-fold more sensitive to Bti, indicating a cost of resistance. The interaction types between chlorpyrifos and Bti (additive), between chlorpyrifos and predator cues (additive), and between Bti and predator cues (synergistic) were consistent in both strains. Despite predator cues making Bti approximately 8% more lethal, they did not change the additive interaction between Bti and chlorpyrifos in their mixture in either strain. CONCLUSION: These results indicate that the resistance against chlorpyrifos was not partly lifted when chlorpyrifos exposure was combined with Bti and predator cues. Identifying the interaction type within pesticide mixtures and how this depends on natural stressors is important to select control strategies that give a disadvantage to resistant individuals compared to non-resistant individuals. © 2019 Society of Chemical Industry.

Whether warming magnifies the toxicity of a pesticide is strongly dependent on the concentration and the null model.

How global warming changes the toxicity of contaminants is a research priority at the intersection of global change biology and ecotoxicology. While many pesticides are more toxic at higher temperatures this is not always detected. We studied whether deviations from this general pattern can be explained by concentration-dependent interaction effects and by testing the interaction against the inappropriate null model. We exposed larvae of the mosquito Culex pipiens to three concentrations of the pesticide chlorpyrifos (absence, low and high) in the absence and presence of 4 °C warming. Both the low and high chlorpyrifos concentration were lethal and generated negative sublethal effects: activity of acetylcholinesterase (AChE) and total fat content decreased, and oxidative damage to lipids increased, yet growth rate increased. Warming was slightly lethal, yet had positive sublethal effects: growth rate, total fat content and metabolic rate increased, and oxidative damage decreased. For four out of seven response variables the independent action model identified the expected synergistic interaction between chlorpyrifos and warming. Notably, for three variables (survival, AChE and fat content) this was strongly dependent on the chlorpyrifos concentration, and for two of these (AChE and fat content) not associated with a significant interaction in the general(ized) linear models. For survival and fat content, warming only potentiated chlorpyrifos (CPF) toxicity at the low CPF concentration, while the opposite was true for AChE. Our results highlight that taking into account concentration-dependence and appropriate null model testing is crucial to improve our understanding of the toxicity of contaminants in a warming world.

Resistance to a chemical pesticide increases vulnerability to a biopesticide: Effects on direct mortality and mortality by predation.

Pesticide mixtures are increasingly used to fight pest species that developed resistance to pesticides. To assess the pesticide control efficiency and to reduce ecological damage to non-target species, it is important to quantify the effect of these mixtures and compare them with the effect of their single pesticides on pest species, non-target species and their predator-prey interactions. We studied the effects of the chemical pesticide chlorpyrifos (CPF), the biopesticide Bacillus thuringiensis israelensis (Bti) and their mixture both on the direct mortality and on the mortality by predation. We focused on larvae of a CPF-resistant and a non-resistant strain of the vector mosquito Culex quinquefasciatus and its predator, the pygmy backswimmer Plea minutissima. In the CPF-Bti mixture, both pesticides interacted antagonistically for direct mortality. Exposure to the mixture caused equal direct mortality and equal mortality by predation in both strains. As expected, exposure to CPF resulted in less direct mortality and less mortality by predation in the CPF-resistant mosquito strain compared to the non-resistant strain. Notably, Bti caused a higher mortality in the mosquito larvae of the CPF-resistant strain compared to the non-resistant strain. Furthermore, the predator killed more mosquito larvae of the resistant strain compared to the non-resistant strain when exposed before to Bti alone. These observations identify a novel cost of resistance to a chemical pesticide in terms of increased vulnerability to a biopesticide.

Daily temperature variation magnifies the toxicity of a mixture consisting of a chemical pesticide and a biopesticide in a vector mosquito.

While many studies on the toxicity of pesticides looked at the effects of a higher mean temperature, effects of the realistic scenario of daily temperature variation are understudied. Moreover, despite the increasing interest for the toxicity of pesticide mixtures how this is influenced by temperature has been largely ignored. We tested whether daily temperature variation (DTV) magnifies the toxicity of two pesticides with a different mode of action, the organophosphate pesticide chlorpyrifos (CPF) and the biopesticide Bacillus thuringiensis var. israelensis (Bti), and of their mixture in the vector mosquito Culex pipiens. Single exposure to CPF and Bti increased mortality and reduced female development time, and exposure to CPF also increased female wing length. DTV was not lethal and did not change the toxicity of the individual pesticides. Yet, a key novel finding was that high DTV increased the mortality of the mixture by changing the interaction between both pesticides from additive to synergistic. Given that in nature daily temperature variation is omnipresent, this is important both for vector control and for ecological risk assessment. The higher toxicity of the mixture at high DTV compared to the typically used constant test temperatures in the laboratory urges caution when evaluating the environmental impact of pesticide mixtures.

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.