Early transcriptional response pathways in Daphnia magna are coordinated in networks of crustacean-specific genes.

Natural habitats are exposed to an increasing number of environmental stressors that cause important ecological consequences. However, the multifarious nature of environmental change, the strength and the relative timing of each stressor largely limit our understanding of biological responses to environmental change. In particular, early response to unpredictable environmental change, critical to survival and fitness in later life stages, is largely uncharacterized. Here, we characterize the early transcriptional response of the keystone species Daphnia magna to twelve environmental perturbations, including biotic and abiotic stressors. We first perform a differential expression analysis aimed at identifying differential regulation of individual genes in response to stress. This preliminary analysis revealed that a few individual genes were responsive to environmental perturbations and they were modulated in a stressor and genotype-specific manner. Given the limited number of differentially regulated genes, we were unable to identify pathways involved in stress response. Hence, to gain a better understanding of the genetic and functional foundation of tolerance to multiple environmental stressors, we leveraged the correlative nature of networks and performed a weighted gene co-expression network analysis. We discovered that approximately one-third of the Daphnia genes, enriched for metabolism, cell signalling and general stress response, drives transcriptional early response to environmental stress and it is shared among genetic backgrounds. This initial response is followed by a genotype- and/or condition-specific transcriptional response with a strong genotype-by-environment interaction. Intriguingly, genotype- and condition-specific transcriptional response is found in genes not conserved beyond crustaceans, suggesting niche-specific adaptation.

The heat is on: Genetic adaptation to urbanization mediated by thermal tolerance and body size.

Worldwide, urbanization leads to tremendous anthropogenic environmental alterations, causing strong selection pressures on populations of animals and plants. Although a key feature of urban areas is their higher temperature ("urban heat islands"), adaptive thermal evolution in organisms inhabiting urban areas has rarely been studied. We tested for evolution of a higher heat tolerance (CTMAX ) in urban populations of the water flea Daphnia magna, a keystone grazer in freshwater ecosystems, by carrying out a common garden experiment at two temperatures (20°C and 24°C) with genotypes of 13 natural populations ordered along a well-defined urbanization gradient. We also assessed body size and haemoglobin concentration to identify underlying physiological drivers of responses in CTMAX . We found a higher CTMAX in animals isolated from urban compared to rural habitats and in animals reared at higher temperatures. We also observed substantial genetic variation in thermal tolerance within populations. Overall, smaller animals were more heat tolerant. While urban animals mature at smaller size, the effect of urbanization on thermal tolerance is only in part caused by reductions in body size. Although urban Daphnia contained higher concentrations of haemoglobin, this did not contribute to their higher CTMAX . Our results provide evidence of adaptive thermal evolution to urbanization in the water flea Daphnia. In addition, our results show both evolutionary potential and adaptive plasticity in rural as well as urban Daphnia populations, facilitating responses to warming. Given the important ecological role of Daphnia in ponds and lakes, these adaptive responses likely impact food web dynamics, top-down control of algae, water quality, and the socio-economic value of urban ponds.

Energy storage and fecundity explain deviations from ecological stoichiometry predictions under global warming and size-selective predation.

A key challenge for ecologists is to predict how single and joint effects of global warming and predation risk translate from the individual level up to ecosystem functions. Recently, stoichiometric theory linked these levels through changes in body stoichiometry, predicting that both higher temperatures and predation risk induce shifts in energy storage (increases in C-rich carbohydrates and reductions in N-rich proteins) and body stoichiometry (increases in C : N and C : P). This promising theory, however, is rarely tested and assumes that prey will divert energy away from reproduction under predation risk, while under size-selective predation, prey instead increase fecundity. We exposed the water flea Daphnia magna to 4 °C warming and fish predation risk to test whether C-rich carbohydrates increase and N-rich proteins decrease, and as a result, C : N and C : P increase under warming and predation risk. Unexpectedly, warming decreased body C : N, which was driven by reductions in C-rich fat and sugar contents while the protein content did not change. This reflected a trade-off where the accelerated intrinsic growth rate under warming occurred at the cost of a reduced energy storage. Warming reduced C : N less and only increased C : P and N : P in the fish-period Daphnia. These evolved stoichiometric responses to warming were largely driven by stronger warming-induced reductions in P than in C and N and could be explained by the better ability to deal with warming in the fish-period Daphnia. In contrast to theory predictions, body C : N decreased under predation risk due to a strong increase in the N-rich protein content that offsets the increase in C-rich fat content. The higher investment in fecundity (more N-rich eggs) under predation risk contributed to this stronger increase in protein content. Similarly, the lower body C : N of pre-fish Daphnia also matched their higher fecundity. Warming and predation risk independently shaped body stoichiometry, largely by changing levels of energy storage molecules. Our results highlight that two widespread patterns, the trade-off between rapid development and energy storage and the increased investment in reproduction under size-selective predation, cause predictable deviations from current ecological stoichiometry theory.

Real-life effectiveness of sacubitril/valsartan in older Belgians with heart failure, reduced ejection fraction and most severe symptoms.

We assessed the real-world effectiveness of sacubitril/valsartan in patients with chronic heart failure (HF) and reduced ejection fraction (HFrEF) with an emphasis on those with older age (≥ 75 years) or with New York Heart Association (NYHA) class IV, for whom greater uncertainty existed regarding clinical outcomes. We conducted a retrospective cohort study based on patient-level linkage of electronic healthcare datasets. Data from all adults with HFrEF in Belgium receiving a prescription for sacubitril/valsartan between 01-November-2016 and 31-December-2018 were collected, with a follow-up of > 6 years. The total study population comprised 5446 patients, older than the PARADIGM-HF trial participants, and with higher NYHA class (all P < 0.0001). NYHA class improved following sacubitril/valsartan initiation (P < 0.0001 baseline vs. reassessment). Most concomitant medications were reduced. Remarkably, the risk of hospitalization for a cardiovascular reason and for HF was reduced by > 26% in the overall cohort, and in subgroups of patients ≥ 75 years, with NYHA class III/IV (all P < 0.0001) or with NYHA class IV (P < 0.05), vs. baseline. All-cause mortality did not increase in real-world patients with NYHA class III/IV. The results support the long-term beneficial effects of sacubitril/valsartan in older patients and in those experiencing the most severe symptoms.

Microbial community analysis of a methane-producing biocathode in a bioelectrochemical system.

A methane-producing biocathode that converts CO(2) into methane was studied electrochemically and microbiologically. The biocathode produced methane at a maximum rate of 5.1 L CH(4)/m(2) projected cathode per day (1.6 A/m(2)) at -0.7 V versus NHE cathode potential and 3.0 L CH(4)/m(2) projected cathode per day (0.9 A/m(2)) at -0.6 V versus NHE cathode potential. The microbial community at the biocathode was dominated by three phylotypes of Archaea and six phylotypes of bacteria. The Archaeal phylotypes were most closely related to Methanobacterium palustre and Methanobacterium aarhusense. Besides methanogenic Archaea, bacteria seemed to be associated with methane production, producing hydrogen as an intermediate. Biomass density varied greatly with part of the carbon electrode covered with a dense biofilm, while only clusters of cells were found on other parts. Based on our results, we discuss how inoculum enrichment and changing operational conditions may help to increase biomass density and to select for microorganisms that produce methane.

Gene expression profiling of three different stressors in the water flea Daphnia magna.

Microarrays are an ideal tool to screen for differences in gene expression of thousands of genes simultaneously. However, often commercial arrays are not available. In this study, we performed microarray analyses to evaluate patterns of gene transcription following exposure to two natural and one anthropogenic stressor. cDNA microarrays compiled of three life stage specific and three stressor-specific EST libraries, yielding 1734 different EST sequences, were used. We exposed juveniles of the water flea Daphnia magna for 48, 96 and 144 h to three stressors known to exert strong selection in natural populations of this species i.e. a sublethal concentration of the pesticide carbaryl, infective spores of the endoparasite Pasteuria ramosa, and fish predation risk mimicked by exposure to fish kairomones. A total of 148 gene fragments were differentially expressed compared to the control. Based on a PCA, the exposure treatments were separated into two main groups based on the extent of the transcriptional response: a low and a high (144 h of fish or carbaryl exposure and 96 h of parasite exposure) stress group. Firstly, we observed a general stress-related transcriptional expression profile independent of the treatment characterized by repression of transcripts involved in transcription, translation, signal transduction and energy metabolism. Secondly, we observed treatment-specific responses including signs of migration to deeper water layers in response to fish predation, structural challenge of the cuticle in response to carbaryl exposure, and disturbance of the ATP production in parasite exposure. A third important conclusion is that transcription expression patterns exhibit stress-specific changes over time. Parasite exposure shows the most differentially expressed gene fragments after 96 h. The peak of differentially expressed transcripts came only after 144 h of fish exposure, while carbaryl exposure induced a more stable number of differently expressed gene fragments over time.

Physiological and molecular effect assessment versus physico-chemistry based mode of action schemes: Daphnia magna exposed to narcotics and polar narcotics.

Structural analogues are assumed to elicit toxicity via similar predominant modes of action (MOAs). Currently, MOA categorization of chemicals in environmental risk assessment is mainly based on the physicochemical properties of potential toxicants. It is often not known whether such classification schemes are also supported by mechanistic biological data. In this study, the toxic effects of two groups of structural analogues (alcohols and anilines) with predefined MOA (narcotics and polar narcotics) were investigated at different levels of biological organization (gene transcription, energy reserves, and growth). Chemical similarity was not indicative of a comparable degree of toxicity and a similar biological response. Categorization of the test chemicals based on the different biological responses (growth, energy use, and gene transcription) did not result in a classification of the predefined narcotics versus the predefined polar narcotics. Moreover, gene transcription based clustering profiles were indicative of the observed effects at higher level of biological organization. Furthermore, a small set of classifier genes could be identified that was discriminative for the clustering pattern. These classifier genes covaried with the organismal and physiological responses. Compared to the physico-chemistry based MOA classification, integrated biological multilevel effect assessment can provide the necessary MOA information that is crucial in high-quality environmental risk assessment. Our findings support the view that transcriptomics tools hold considerable promise to be used in biological response based mechanistic profiling of potential (eco)toxicants.

Single nucleotide polymorphism discovery from expressed sequence tags in the waterflea Daphnia magna.

BACKGROUND: Daphnia (Crustacea: Cladocera) plays a central role in standing aquatic ecosystems, has a well known ecology and is widely used in population studies and environmental risk assessments. Daphnia magna is, especially in Europe, intensively used to study stress responses of natural populations to pollutants, climate change, and antagonistic interactions with predators and parasites, which have all been demonstrated to induce micro-evolutionary and adaptive responses. Although its ecology and evolutionary biology is intensively studied, little is known on the functional genomics underpinning of phenotypic responses to environmental stressors. The aim of the present study was to find genes expressed in presence of environmental stressors, and target such genes for single nucleotide polymorphic (SNP) marker development. RESULTS: We developed three expressed sequence tag (EST) libraries using clonal lineages of D. magna exposed to ecological stressors, namely fish predation, parasite infection and pesticide exposure. We used these newly developed ESTs and other Daphnia ESTs retrieved from NCBI GeneBank to mine for SNP markers targeting synonymous as well as non synonymous genetic variation. We validate the developed SNPs in six natural populations of D. magna distributed at regional scale. CONCLUSIONS: A large proportion (47%) of the produced ESTs are Daphnia lineage specific genes, which are potentially involved in responses to environmental stress rather than to general cellular functions and metabolic activities, or reflect the arthropod's aquatic lifestyle. The characterization of genes expressed under stress and the validation of their SNPs for population genetic study is important for identifying ecologically responsive genes in D. magna.

Evolutionary ecotoxicology of pesticide resistance: a case study in Daphnia.

Natural populations that are exposed to pesticides in their environment may at the same time be exposed to natural stressors like parasites and predators, which may interact with pesticide exposure. This may not only impact target pest species but also a wide variety of non-target species. This review reports on a joint research program in the water flea Daphnia magna, a non-target species often used as model organism in ecology and ecotoxicology. The focus is on different aspects that are of key importance to understand the evolutionary ecology of pesticide exposure: (1) the capacity of natural populations to genetically adapt to pesticide exposure (2) the added complexity of synergistic effects caused by simultaneous exposure to natural stressors, and (3) the potential interference of evolutionary costs of adaptation to pesticide exposure. Our results showed that natural populations were able to rapidly evolve resistance to the pesticide carbaryl but at the expense of fitness costs. Individuals selected for carbaryl resistance had higher survival rates when exposed to the pesticide but also a greater susceptibility to the challenge imposed by the bacterial endoparasite Pasteuria ramosa. The evolved resistance to carbaryl was in some cases only expressed in the absence of fish kairomones. Further, it became clear that the responses to both exposure to single and combined stressors was for several life history variables strongly dependent upon past exposure to carbaryl. This indicates that past exposures to pesticides are important and can not be neglected when evaluating responses to current stressors.

Local exposure shapes spatial patterns in infectivity and community structure of Daphnia parasites.

1. Spatial patterns in parasite community structure are probably driven by the availability of infectious stages. This is because hosts become infected through picking up infectious stages from their environment. Several studies have, however, reported strong genotype by genotype interactions and parasite-mediated selection in hosts. This leads to the prediction of a parasite by host population interaction with respect to infection rates and intensities, which may also shape spatial patterns in parasite community structure. 2. Using the water flea Daphnia magna and its microparasites as a model, we carried out a laboratory experiment to test explicitly to what extent parasite community structure in host populations is determined by the availability of infectious stages in the sediment they are exposed to, and to what extent host population identity and location play a role. 3. We exposed 10 D. magna host populations each to sediment of their own habitat and sediment of the other nine habitats, and monitored the parasite community of the resulting experimental populations. 4. Sediment seems to be a strong determinant of parasite infection rates, while there was no overall effect of host population. Sympatric parasite and host population combinations did in most cases not result in significantly different infection rates than allopatric parasite and host combinations. Our results indicate that spore availability could be the key variable determining parasite community structure in natural Daphnia populations.

Rapid evolution in response to warming does not affect the toxicity of a pollutant: Insights from experimental evolution in heated mesocosms.

While human-induced stressors such as warming and pollutants may co-occur and interact, evolutionary studies typically focus on single stressors. Rapid thermal evolution may help organisms better deal with warming, yet it remains an open question whether thermal evolution changes the toxicity of pollutants under warming. We investigated the effects of exposure to a novel pollutant (zinc oxide nanoparticles, nZnO) and 4°C warming (20°C vs. 24°C) on key life history and physiological traits of the water flea Daphnia magna, a keystone species in aquatic ecosystems. To address the role of thermal evolution, we compared these effects between clones from an experimental evolution trial where animals were kept for two years in outdoor mesocosms at ambient temperatures or ambient +4°C. The nZnO was more toxic at 20°C than at 24°C: only at 20°C, it caused reductions in early fecundity, intrinsic growth rate and metabolic activity. This was due to a higher accumulated zinc burden at 20°C than at 24°C, which was associated with an upregulation of a metallothionein gene at 20°C but not at 24°C. Clones from the heated mesocosms better dealt with warming than clones from the ambient mesocosms, indicating rapid thermal evolution. Notably, rapid thermal evolution did not change the toxicity of nZnO, neither at 20°C nor at 24°C, suggesting no pleiotropy or metabolic trade-offs were at work under the current experimental design. Evaluating whether thermal evolution influences the toxicity of pollutants is important for ecological risk assessment. It provides key information to extrapolate laboratory-derived toxicity estimates of pollutants both in space to warmer regions and in time under future global warming scenarios. In general, studying how the evolution of tolerance to one anthropogenic stressor influence tolerance to other anthropogenic stressors should get more attention in a rapidly changing world where animals increasingly face combinations of stressors.

Thermal evolution offsets the elevated toxicity of a contaminant under warming: A resurrection study in Daphnia magna.

Synergistic interactions between temperature and contaminants are a major challenge for ecological risk assessment, especially under global warming. While thermal evolution may increase the ability to deal with warming, it is unknown whether it may also affect the ability to deal with the many contaminants that are more toxic at higher temperatures. We investigated how evolution of genetic adaptation to warming affected the interactions between warming and a novel stressor: zinc oxide nanoparticles (nZnO) in a natural population of Daphnia magna using resurrection ecology. We hatched resting eggs from two D. magna subpopulations (old: 1955-1965, recent: 1995-2005) from the sediment of a lake that experienced an increase in average temperature and in recurrence of heat waves but was never exposed to industrial waste. In the old "ancestral" subpopulation, exposure to a sublethal concentration of nZnO decreased the intrinsic growth rate, metabolic activity, and energy reserves at 24°C but not at 20°C, indicating a synergism between warming and nZnO. In contrast, these synergistic effects disappeared in the recent "derived" subpopulation that evolved a lower sensitivity to nZnO at 24°C, which indicates that thermal evolution could offset the elevated toxicity of nZnO under warming. This evolution of reduced sensitivity to nZnO under warming could not be explained by changes in the total internal zinc accumulation but was partially associated with the evolution of the expression of a key metal detoxification gene under warming. Our results suggest that the increased sensitivity to the sublethal concentration of nZnO under the predicted 4°C warming by the end of this century may be counteracted by thermal evolution in this D. magna population. Our results illustrate the importance of evolution to warming in shaping the responses to another anthropogenic stressor, here a contaminant. More general, genetic adaptation to an environmental stressor may ensure that synergistic effects between contaminants and this environmental stressor will not be present anymore.

Stoichiometric responses to nano ZnO under warming are modified by thermal evolution in Daphnia magna.

Effects of stressors on body stoichiometry are important as these may cascade through food webs. Contamination and global warming are two key anthropogenic stressors, yet their effects on body stoichiometry have been rarely tested. Further, while thermal evolution may increase the ability to deal with warming, it is unknown how thermal evolution modifies the effect of contaminants under warming. Using resurrection ecology, we studied two Daphnia magna subpopulations (old/recent) of which the recent subpopulation evolved a higher heat tolerance. We exposed both subpopulations to a sublethal concentration of nano zinc oxide (nZnO) and 4 °C warming and quantified their effects on body stoichiometry: carbon (C), nitrogen (N), phosphorus (P) contents and their ratios (C:N, C:P, N:P). In the old subpopulation, nZnO only marginally decreased the C content and had no effect on N and P contents and their ratios. In contrast, in the recent subpopulation nZnO strongly increased the body P content (+51%) and reduced the C:P (-34%) and N:P (-34%) ratios at 24 °C but not at 20 °C. Moreover, these stoichiometric changes were not explained by changes of corresponding macromolecules as assumed by theory. Our results indicate that the stoichiometric responses to nZnO in Daphnia are temperature-dependent and modified by rapid evolution. The observed changes in body stoichiometry may affect the food quality of this important prey and have the potential to cascade through food webs and shape nutrients cycling.

Rapid evolution leads to differential population dynamics and top-down control in resurrected Daphnia populations.

There is growing evidence of rapid genetic adaptation of natural populations to environmental change, opening the perspective that evolutionary trait change may subsequently impact ecological processes such as population dynamics, community composition, and ecosystem functioning. To study such eco-evolutionary feedbacks in natural populations, however, requires samples across time. Here, we capitalize on a resurrection ecology study that documented rapid and adaptive evolution in a natural population of the water flea Daphnia magna in response to strong changes in predation pressure by fish, and carry out a follow-up mesocosm experiment to test whether the observed genetic changes influence population dynamics and top-down control of phytoplankton. We inoculated populations of the water flea D. magna derived from three time periods of the same natural population known to have genetically adapted to changes in predation pressure in replicate mesocosms and monitored both Daphnia population densities and phytoplankton biomass in the presence and absence of fish. Our results revealed differences in population dynamics and top-down control of algae between mesocosms harboring populations from the time period before, during, and after a peak in fish predation pressure caused by human fish stocking. The differences, however, deviated from our a priori expectations. An S-map approach on time series revealed that the interactions between adults and juveniles strongly impacted the dynamics of populations and their top-down control on algae in the mesocosms, and that the strength of these interactions was modulated by rapid evolution as it occurred in nature. Our study provides an example of an evolutionary response that fundamentally alters the processes structuring population dynamics and impacts ecosystem features.

Prognostic relevance of tumor-infiltrating lymphocytes and immune checkpoints in pediatric medulloblastoma.

Pediatric medulloblastomas are the most frequently diagnosed embryonal tumors of the central nervous system. Current therapies cause severe neurological and cognitive side effects including secondary malignancies. Cellular immunotherapy might be key to improve survival and to avoid morbidity. Efficient killing of tumor cells using immunotherapy requires to overcome cancer-associated strategies to evade cytotoxic immune responses. Here, we examined the immune response and immune evasion strategies in pediatric medulloblastomas. Cytotoxic T-cells, infiltrating medulloblastomas with variable activation status, showed no correlation with overall survival of the patients. We found limited numbers of PD1+ T-cells and complete absence of PD-L1 on medulloblastomas. Medulloblastomas downregulated immune recognition molecules MHC-I and CD1 d. Intriguingly, expression of granzyme inhibitors SERPINB1 and SERPINB4 was acquired in 23% and 50% of the tumors, respectively. Concluding, pediatric medulloblastomas exploit multiple immune evasion strategies to overcome immune surveillance. Absence of PD-L1 expression in medulloblastoma suggest limited or no added value for immunotherapy with PD1/PD-L1 blockers.

Conserved Transcription Factors Steer Growth-Related Genomic Programs in Daphnia.

Ecological genomics aims to understand the functional association between environmental gradients and the genes underlying adaptive traits. Many genes that are identified by genome-wide screening in ecologically relevant species lack functional annotations. Although gene functions can be inferred from sequence homology, such approaches have limited power. Here, we introduce ecological regulatory genomics by presenting an ontology-free gene prioritization method. Specifically, our method combines transcriptome profiling with high-throughput cis-regulatory sequence analysis in the water fleas Daphnia pulex and Daphnia magna. It screens coexpressed genes for overrepresented DNA motifs that serve as transcription factor binding sites, thereby providing insight into conserved transcription factors and gene regulatory networks shaping the expression profile. We first validated our method, called Daphnia-cisTarget, on a D. pulex heat shock data set, which revealed a network driven by the heat shock factor. Next, we performed RNA-Seq in D. magna exposed to the cyanobacterium Microcystis aeruginosa. Daphnia-cisTarget identified coregulated gene networks that associate with the moulting cycle and potentially regulate life history changes in growth rate and age at maturity. These networks are predicted to be regulated by evolutionary conserved transcription factors such as the homologues of Drosophila Shavenbaby and Grainyhead, nuclear receptors, and a GATA family member. In conclusion, our approach allows prioritising candidate genes in Daphnia without bias towards prior knowledge about functional gene annotation and represents an important step towards exploring the molecular mechanisms of ecological responses in organisms with poorly annotated genomes.

Bioelectrochemical Power-to-Gas: State of the Art and Future Perspectives.

Bioelectrochemical power-to-gas (BEP2G) is considered a potentially convenient way of storing renewable surplus electricity in the form of methane. In methane-producing bioelectrochemical systems (BESs), carbon dioxide and electrical energy are converted into methane, using electrodes that supply either electrons or hydrogen to methanogenic archaea. This review summarizes the performance of methane-producing BESs in relation to cathode potential, electrode materials, operational strategies, and inoculum. Analysis and estimation of energy input and production rates show that BEP2G may become an attractive alternative for thermochemical methanation, and biochemical methanogenesis. To determine if BEP2G can become a future power-to-gas technology, challenges relating to cathodic energy losses, choice of a suitable electron donor, efficient reactor design/operation, and experience with large reactors need to be overcome.

Daphnia magna transcriptome by RNA-Seq across 12 environmental stressors.

The full exploration of gene-environment interactions requires model organisms with well-characterized ecological interactions in their natural environment, manipulability in the laboratory and genomic tools. The waterflea Daphnia magna is an established ecological and toxicological model species, central to the food webs of freshwater lentic habitats and sentinel for water quality. Its tractability and cyclic parthenogenetic life-cycle are ideal to investigate links between genes and the environment. Capitalizing on this unique model system, the STRESSFLEA consortium generated a comprehensive RNA-Seq data set by exposing two inbred genotypes of D. magna and a recombinant cross of these genotypes to a range of environmental perturbations. Gene models were constructed from the transcriptome data and mapped onto the draft genome of D. magna using EvidentialGene. The transcriptome data generated here, together with the available draft genome sequence of D. magna and a high-density genetic map will be a key asset for future investigations in environmental genomics.

Experimental evolution reveals high insecticide tolerance in Daphnia inhabiting farmland ponds.

Exposure of nontarget populations to agricultural chemicals is an important aspect of global change. We quantified the capacity of natural Daphnia magna populations to locally adapt to insecticide exposure through a selection experiment involving carbaryl exposure and a control. Carbaryl tolerance after selection under carbaryl exposure did not increase significantly compared to the tolerance of the original field populations. However, there was evolution of a decreased tolerance in the control experimental populations compared to the original field populations. The magnitude of this decrease was positively correlated with land use intensity in the neighbourhood of the ponds from which the original populations were sampled. The genetic change in carbaryl tolerance in the control rather than in the carbaryl treatment suggests widespread selection for insecticide tolerance in the field associated with land use intensity and suggests that this evolution comes at a cost. Our data suggest a strong impact of current agricultural land use on nontarget natural Daphnia populations.