An integrated spatio-temporal view of riverine biodiversity using environmental DNA metabarcoding.

Anthropogenically forced changes in global freshwater biodiversity demand more efficient monitoring approaches. Consequently, environmental DNA (eDNA) analysis is enabling ecosystem-scale biodiversity assessment, yet the appropriate spatio-temporal resolution of robust biodiversity assessment remains ambiguous. Here, using intensive, spatio-temporal eDNA sampling across space (five rivers in Europe and North America, with an upper range of 20-35 km between samples), time (19 timepoints between 2017 and 2018) and environmental conditions (river flow, pH, conductivity, temperature and rainfall), we characterise the resolution at which information on diversity across the animal kingdom can be gathered from rivers using eDNA. In space, beta diversity was mainly dictated by turnover, on a scale of tens of kilometres, highlighting that diversity measures are not confounded by eDNA from upstream. Fish communities showed nested assemblages along some rivers, coinciding with habitat use. Across time, seasonal life history events, including salmon and eel migration, were detected. Finally, effects of environmental conditions were taxon-specific, reflecting habitat filtering of communities rather than effects on DNA molecules. We conclude that riverine eDNA metabarcoding can measure biodiversity at spatio-temporal scales relevant to species and community ecology, demonstrating its utility in delivering insights into river community ecology during a time of environmental change.

100 years of anthropogenic impact causes changes in freshwater functional biodiversity.

Despite efforts from scientists and regulators, biodiversity is declining at an alarming rate. Unless we find transformative solutions to preserve biodiversity, future generations may not be able to enjoy nature's services. We have developed a conceptual framework that establishes the links between biodiversity dynamics and abiotic change through time and space using artificial intelligence. Here, we apply this framework to a freshwater ecosystem with a known history of human impact and study 100 years of community-level biodiversity, climate change and chemical pollution trends. We apply explainable network models with multimodal learning to community-level functional biodiversity measured with multilocus metabarcoding, to establish correlations with biocides and climate change records. We observed that the freshwater community assemblage and functionality changed over time without returning to its original state, even if the lake partially recovered in recent times. Insecticides and fungicides, combined with extreme temperature events and precipitation, explained up to 90% of the functional biodiversity changes. The community-level biodiversity approach used here reliably explained freshwater ecosystem shifts. These shifts were not observed when using traditional quality indices (e.g. Trophic Diatom Index). Our study advocates the use of high-throughput systemic approaches on long-term trends over species-focused ecological surveys to identify the environmental factors that cause loss of biodiversity and disrupt ecosystem functions.

Over long periods of time, environmental changes ­ such as chemical pollution and climate change ­ affect the diversity of organisms that live in an ecosystem, known as 'biodiversity'. Understanding the impact of these changes is challenging because they can happen slowly, their effect is only measurable after years, and historical records are limited. This can make it difficult to determine when specific changes happened, what might have driven them and what impact they might be having. One way to measure changes in biodiversity over time is by analysing traces of DNA shed by organisms. Plants, animals, and bacteria living in lakes leave behind genetic material that gets trapped and buried in the sediment at the bottom of lakes. Similarly, biocides ­ substances used to kill or control populations of living organisms ­ that run-off into lakes leach into the sediment and can be measured years later. Therefore, this sediment holds a record of life and environmental impacts in the lake over past centuries. Eastwood, Zhou et al. wanted to understand the relationship between environmental changes (such as the use of biocides and climate change) and shifts in lake biodiversity. To do so, the researchers studied a lake community that had experienced major environmental impacts over the last century (including nutrient pollution, chemical pollution and climate change), but which appeared to improve over the last few years of the 20^th century. Using machine learning to find connections over time between biodiversity and non-living environmental changes, Eastwood, Zhou et al. showed that, despite apparent recovery in water quality, the biodiversity of the lake was not restored to its original state. A combination of climate factors (such as rainfall levels and extreme temperatures) and biocide application (particularly insecticides and fungicides) explained up to 90% of the biodiversity changes that occurred in the lake. These changes had not been identified before using traditional techniques. The functional roles microorganisms played in the ecosystem (such as degradation and nitrogen metabolism) were also altered, suggesting that loss of biodiversity may lead to loss of ecosystem functions. The findings described by Eastwood, Zhou et al. can be used by environmental regulators to identify species or ecosystems at risk from environmental change and prioritise them for intervention. The approach can also be used to identify which chemicals pose the greatest threat to biodiversity. Additionally, the use of environmental DNA from sediment can provide rich historical biodiversity data, which can be used to train artificial intelligence-based models to improve predictions of how ecosystems will respond to complex environmental changes.

Harnessing water fleas for water reclamation: A nature-based tertiary wastewater treatment technology.

Urbanisation, population growth, and climate change have put unprecedented pressure on water resources, leading to a global water crisis and the need for water reuse. However, water reuse is unsafe unless persistent chemical pollutants are removed from reclaimed water. State-of-the-art technologies for the reduction of persistent chemical pollutants in wastewater typically impose high operational and energy costs and potentially generate toxic by-products (e.g., bromate from ozonation). Nature-base solutions are preferred to these technologies for their lower environmental impact. However, so far, bio-based tertiary wastewater treatments have been inefficient for industrial-scale applications. Moreover, they often demand significant financial investment and large infrastructure, undermining sustainability objectives. Here, we present a scalable, low-cost, low-carbon, and retrofittable nature-inspired solution to remove persistent chemical pollutants (pharmaceutical, pesticides and industrial chemicals). We showed Daphnia's removal efficiency of individual chemicals and chemicals from wastewater at laboratory scale ranging between 50 % for PFOS and 90 % for diclofenac. We validated the removal efficiency of diclofenac at prototype scale, showing sustained performance over four weeks in outdoor seminatural conditions. A techno-commercial analysis on the Daphnia-based technology suggested several technical, commercial and sustainability advantages over established and emerging treatments at comparable removal efficiency, benchmarked on available data on individual chemicals. Further testing of the technology is underway in open flow environments holding real wastewater. The technology has the potential to improve the quality of wastewater effluent, meeting requirements to produce water appropriate for reuse in irrigation, industrial application, and household use. By preventing persistent chemicals from entering waterways, this technology has the potential to maximise the shift to clean growth, enabling water reuse, reducing resource depletion and preventing environmental pollution.

The hologenome of Daphnia magna reveals possible DNA methylation and microbiome-mediated evolution of the host genome.

Properties that make organisms ideal laboratory models in developmental and medical research are often the ones that also make them less representative of wild relatives. The waterflea Daphnia magna is an exception, by both sharing many properties with established laboratory models and being a keystone species, a sentinel species for assessing water quality, an indicator of environmental change and an established ecotoxicology model. Yet, Daphnia's full potential has not been fully exploited because of the challenges associated with assembling and annotating its gene-rich genome. Here, we present the first hologenome of Daphnia magna, consisting of a chromosomal-level assembly of the D. magna genome and the draft assembly of its metagenome. By sequencing and mapping transcriptomes from exposures to environmental conditions and from developmental morphological landmarks, we expand the previously annotates gene set for this species. We also provide evidence for the potential role of gene-body DNA-methylation as a mutagen mediating genome evolution. For the first time, our study shows that the gut microbes provide resistance to commonly used antibiotics and virulence factors, potentially mediating Daphnia's environmental-driven rapid evolution. Key findings in this study improve our understanding of the contribution of DNA methylation and gut microbiota to genome evolution in response to rapidly changing environments.

Daphnia as a Sentinel Species for Environmental Health Protection: A Perspective on Biomonitoring and Bioremediation of Chemical Pollution.

Despite available technology and the knowledge that chemical pollution damages human and ecosystem health, chemical pollution remains rampant, ineffectively monitored, rarely prevented, and only occasionally mitigated. We present a framework that helps address current major challenges in the monitoring and assessment of chemical pollution by broadening the use of the sentinel species Daphnia as a diagnostic agent of water pollution. And where prevention has failed, we propose the application of Daphnia as a bioremediation agent to help reduce hazards from chemical mixtures in the environment. By applying "omics" technologies to Daphnia exposed to real-world ambient chemical mixtures, we show improvements at detecting bioactive components of chemical mixtures, determining the potential effects of untested chemicals within mixtures, and identifying targets of toxicity. We also show that using Daphnia strains that naturally adapted to chemical pollution as removal agents of ambient chemical mixtures can sustainably improve environmental health protection. Expanding the use of Daphnia beyond its current applications in regulatory toxicology has the potential to improve both the assessment and the remediation of environmental pollution.

Historical exposure to chemicals reduces tolerance to novel chemical stress in Daphnia (waterflea).

Until the last few decades, anthropogenic chemicals used in most production processes have not been comprehensively assessed for their risk and impact on wildlife and humans. They are transported globally and usually end up in the environment as unintentional pollutants, causing long-term adverse effects. Modern toxicology practices typically use acute toxicity tests of unrealistic concentrations of chemicals to determine their safe use, missing pathological effects arising from long-term exposures to environmentally relevant concentrations. Here, we study the transgenerational effect of environmentally relevant concentrations of five chemicals on the priority list of international regulatory frameworks on the keystone species Daphnia magna. We expose Daphnia genotypes resurrected from the sedimentary archive of a lake with a known history of chemical pollution to the five chemicals to understand how historical exposure to chemicals influences adaptive responses to novel chemical stress. We measure within- and transgenerational plasticity in fitness-linked life history traits following exposure of "experienced" and "naive" genotypes to novel chemical stress. As the revived Daphnia originate from the same genetic pool sampled at different times in the past, we are able to quantify the long-term evolutionary impact of chemical pollution by studying genome-wide diversity and identifying functional pathways affected by historical chemical stress. Our results suggest that historical exposure to chemical stress causes reduced genome-wide diversity, leading to lower cross-generational tolerance to novel chemical stress. Lower tolerance is underpinned by reduced gene diversity at detoxification, catabolism and endocrine genes in experienced genotypes. We show that these genes sit within pathways that are conserved and potential chemical targets in other species, including humans.

The Time Machine framework: monitoring and prediction of biodiversity loss.

Transdisciplinary solutions are needed to achieve the sustainability of ecosystem services for future generations. We propose a framework to identify the causes of ecosystem function loss and to forecast the future of ecosystem services under different climate and pollution scenarios. The framework (i) applies an artificial intelligence (AI) time-series analysis to identify relationships among environmental change, biodiversity dynamics and ecosystem functions; (ii) validates relationships between loss of biodiversity and environmental change in fabricated ecosystems; and (iii) forecasts the likely future of ecosystem services and their socioeconomic impact under different pollution and climate scenarios. We illustrate the framework by applying it to watersheds, and provide system-level approaches that enable natural capital restoration by associating multidecadal biodiversity changes to chemical pollution.

Evolutionary mechanisms underpinning fitness response to multiple stressors in Daphnia.

Multiple stressors linked to anthropogenic activities can influence how organisms adapt and evolve. So far, a consensus on how multiple stressors drive adaptive trajectories in natural populations has not been reached. Some meta-analysis reports show predominance of additive effects of stressors on ecological endpoints (e.g., fecundity, mortality), whereas others show synergistic effects more frequently. Moreover, it is unclear what mechanisms of adaptation underpin responses to complex environments. Here, we use populations of Daphnia magna resurrected from different times in the past to investigate mechanisms of adaptation to multiple stressors and to understand how historical exposure to environmental stress shapes adaptive responses of modern populations. Using common garden experiments on resurrected modern and historical populations, we investigate (1) whether exposure to one stress results in higher tolerance to a second stressor; (2) the mechanisms of adaptation underpinning long-term evolution to multistress (genetic evolution, plasticity, evolution of plasticity); and (3) the interaction effects of multiple stressors on fitness (synergism, antagonism, additivity). We measure the combined impact of different levels of resource availability (algae) and biocides on fitness-linked life-history traits and interpret these results in light of historical environmental exposures. We show that exposure to one stressor can alter tolerance to second stressors and that the interaction effect depends on the severity of either stressor. We also show that mechanisms of adaptation underpinning phenotypic evolution significantly differ in single-stress and multistress scenarios. These adaptive responses are driven largely by synergistic effects on fecundity and size at maturity, and additive effects on age at maturity. Exposure to multiple stressors shifts the trade-offs among fitness-linked life-history traits, with a stronger effect on Daphnia populations when low-resource availability and high biocide levels are experienced. Our study indicates that mitigation interventions based on single-stress analysis may not capture realistic threats.

Extensive standing genetic variation from a small number of founders enables rapid adaptation in Daphnia.

We lack a thorough understanding of the origin and maintenance of standing genetic variation that enables rapid evolutionary responses of natural populations. Whole genome sequencing of a resurrected Daphnia population shows that standing genetic variation in over 500 genes follows an evolutionary trajectory that parallels the pronounced and rapid adaptive evolution of multiple traits in response to predator-driven natural selection and its subsequent relaxation. Genetic variation carried by only five founding individuals from the regional genotype pool is shown to suffice at enabling the observed evolution. Our results provide insight on how natural populations can acquire the genomic variation, through colonization by a few regional genotypes, that fuels rapid evolution in response to strong selection pressures. While these evolutionary responses in our study population involved hundreds of genes, we observed no evidence of genetic erosion.

Protocol for assay of transposase accessible chromatin sequencing in non-model species.

The assay for transposase accessible chromatin (ATAC-seq) is a method for mapping genome-wide chromatin accessibility. Coupled with high-throughput sequencing, it enables integrative epigenomics analyses. ATAC-seq requires direct access to cell nuclei, a major challenge in non-model species such as small invertebrates, whose soft tissue is surrounded by a protective exoskeleton. Here, we present modifications of the ATAC-seq protocol for applications in small crustaceans, extending applications to non-model species. For complete information on the use and execution of this protocol, please refer to Buenrostro et al. (2013).

Roundup causes embryonic development failure and alters metabolic pathways and gut microbiota functionality in non-target species.

BACKGROUND: Research around the weedkiller Roundup is among the most contentious of the twenty-first century. Scientists have provided inconclusive evidence that the weedkiller causes cancer and other life-threatening diseases, while industry-paid research reports that the weedkiller has no adverse effect on humans or animals. Much of the controversial evidence on Roundup is rooted in the approach used to determine safe use of chemicals, defined by outdated toxicity tests. We apply a system biology approach to the biomedical and ecological model species Daphnia to quantify the impact of glyphosate and of its commercial formula, Roundup, on fitness, genome-wide transcription and gut microbiota, taking full advantage of clonal reproduction in Daphnia. We then apply machine learning-based statistical analysis to identify and prioritize correlations between genome-wide transcriptional and microbiota changes. RESULTS: We demonstrate that chronic exposure to ecologically relevant concentrations of glyphosate and Roundup at the approved regulatory threshold for drinking water in the US induce embryonic developmental failure, induce significant DNA damage (genotoxicity), and interfere with signaling. Furthermore, chronic exposure to the weedkiller alters the gut microbiota functionality and composition interfering with carbon and fat metabolism, as well as homeostasis. Using the "Reactome," we identify conserved pathways across the Tree of Life, which are potential targets for Roundup in other species, including liver metabolism, inflammation pathways, and collagen degradation, responsible for the repair of wounds and tissue remodeling. CONCLUSIONS: Our results show that chronic exposure to concentrations of Roundup and glyphosate at the approved regulatory threshold for drinking water causes embryonic development failure and alteration of key metabolic functions via direct effect on the host molecular processes and indirect effect on the gut microbiota. The ecological model species Daphnia occupies a central position in the food web of aquatic ecosystems, being the preferred food of small vertebrates and invertebrates as well as a grazer of algae and bacteria. The impact of the weedkiller on this keystone species has cascading effects on aquatic food webs, affecting their ability to deliver critical ecosystem services. Video Abstract.

Remasking of Candida albicans ß-Glucan in Response to Environmental pH Is Regulated by Quorum Sensing.

Candida albicans is a commensal yeast of the human gut which is tolerated by the immune system but has the potential to become an opportunistic pathogen. One way in which C. albicans achieves this duality is through concealing or exposing cell wall pathogen-associated molecular patterns (PAMPs) in response to host-derived environment cues (pH, hypoxia, and lactate). This cell wall remodeling allows C. albicans to evade or hyperactivate the host's innate immune responses, leading to disease. Previously, we showed that adaptation of C. albicans to acidic environments, conditions encountered during colonization of the female reproductive tract, induces significant cell wall remodeling resulting in the exposure of two key fungal PAMPs (ß-glucan and chitin). Here, we report that this pH-dependent cell wall remodeling is time dependent, with the initial change in pH driving cell wall unmasking, which is then remasked at later time points. Remasking of ß-glucan was mediated via the cell density-dependent fungal quorum sensing molecule farnesol, while chitin remasking was mediated via a small, heat-stable, nonproteinaceous secreted molecule(s). Transcript profiling identified a core set of 42 genes significantly regulated by pH over time and identified the transcription factor Efg1 as a regulator of chitin exposure through regulation of CHT2 This dynamic cell wall remodeling influenced innate immune recognition of C. albicans, suggesting that during infection, C. albicans can manipulate the host innate immune responses.IMPORTANCECandida albicans is part of the microbiota of the skin and gastrointestinal and reproductive tracts of humans and has coevolved with us for millennia. During that period, C. albicans has developed strategies to modulate the host's innate immune responses, by regulating the exposure of key epitopes on the fungal cell surface. Here, we report that exposing C. albicans to an acidic environment, similar to the one of the stomach or vagina, increases the detection of the yeast by macrophages. However, this effect is transitory, as C. albicans is able to remask these epitopes (glucan and chitin). We found that glucan remasking is controlled by the production of farnesol, a molecule secreted by C. albicans in response to high cell densities. However, chitin-remasking mechanisms remain to be identified. By understanding the relationship between environmental sensing and modulation of the host-pathogen interaction, new opportunities for the development of innovative antifungal strategies are possible.

The RNA helicase UPF1 associates with mRNAs co-transcriptionally and is required for the release of mRNAs from gene loci.

UPF1 is an RNA helicase that is required for nonsense-mediated mRNA decay (NMD) in eukaryotes, and the predominant view is that UPF1 mainly operates on the 3'UTRs of mRNAs that are directed for NMD in the cytoplasm. Here we offer evidence, obtained from Drosophila, that UPF1 constantly moves between the nucleus and cytoplasm by a mechanism that requires its RNA helicase activity. UPF1 is associated, genome-wide, with nascent RNAs at most of the active Pol II transcription sites and at some Pol III-transcribed genes, as demonstrated microscopically on the polytene chromosomes of salivary glands and by ChIP-seq analysis in S2 cells. Intron recognition seems to interfere with association and translocation of UPF1 on nascent pre-mRNAs, and cells depleted of UPF1 show defects in the release of mRNAs from transcription sites and their export from the nucleus.

Transgenerational response to early spring warming in Daphnia.

Temperature and photoperiod regulate key fitness traits in plants and animals. However, with temperature increase due to global warming, temperature cue thresholds are experienced at shorter photoperiods, disrupting the optimal seasonal timing of physiological, developmental and reproductive events in many species. Understanding the mechanisms of adaptation to the asynchrony between temperature and photoperiod is key to inform our understanding of how species will respond to global warming. Here, we studied the transgenerational mechanisms of responses of the cyclical parthenogen Daphnia magna to different photoperiod lengths co-occurring with warm temperature thereby assessing the impact of earlier spring warming on its fitness. Daphnia uses temperature and photoperiod cues to time dormancy, and to switch between sexual and asexual reproduction. Daphnia life cycle offers the opportunity to measure the relative contribution of plastic and genetic responses to environmental change across generations and over evolutionary time. We use transgenerational common garden experiments on three populations 'resurrected' from a biological archive experiencing temperature increase over five decades. Our results suggest that response to early spring warming evolved underpinned by a complex interaction between plastic and genetic mechanisms while a positive maternal contribution at matching environments between parental and offspring generation was also observed.

Adaptive responses along a depth and a latitudinal gradient in the endemic seagrass Posidonia oceanica.

Seagrass meadows provide important ecosystem services and are critical for the survival of the associated invertebrate community. However, they are threatened worldwide by human-driven environmental change. Understanding the seagrasses' potential for adaptation is critical to assess not only their ability to persist under future global change scenarios, but also to assess the persistence of the associated communities. Here we screened a wild population of Posidonia oceanica, an endemic long-lived seagrass in the Mediterranean Sea, for genes that may be target of environmental selection, using an outlier and a genome-wide transcriptome analysis. We identified loci where polymorphism or differential expression was associated with either a latitudinal or a bathymetric gradient, as well as with both gradients in an effort to identify loci associated with temperature and light. We found the candidate genes underlying growth and immunity to be divergent between populations adapted to different latitudes and/or depths, providing evidence for local adaptation. Furthermore, we found evidence of reduced gene flow among populations including adjacent populations. Reduced gene flow, combined with low sexual recombination, small effective population size, and long generation time of P. oceanica raises concerns for the long-term persistence of this species, especially in the face of rapid environmental change driven by human activities.

Predictability of the impact of multiple stressors on the keystone species Daphnia.

Eutrophication and climate change are two of the most pressing environmental issues affecting up to 50% of aquatic ecosystems worldwide. Mitigation strategies to reduce the impact of environmental change are complicated by inherent difficulties of predicting the long-term impact of multiple stressors on natural populations. Here, we investigated the impact of temperature, food levels and carbamate insecticides, in isolation and in combination, on current and historical populations of the freshwater grazer Daphnia. We used common garden and competition experiments on historical and modern populations of D. magna 'resurrected' from a lake with known history of anthropogenic eutrophication and documented increase in ambient temperature over time. We found that these populations response dramatically differed between single and multiple stressors. Whereas warming alone induced similar responses among populations, warming combined with insecticides or food limitation resulted in significantly lower fitness in the population historically exposed to pesticides. These results suggest that the negative effect of historical pesticide exposure is magnified in the presence of warming, supporting the hypothesis of synergism between chemical pollution and other stressors.

Evolution of thermal tolerance in multifarious environments.

Species extinction rates are many times greater than the direst predictions made two decades ago by environmentalists, largely because of human impact. Major concerns are associated with the predicted higher recurrence and severity of extreme events, such as heat waves. Although tolerance to these extreme events is instrumental to species survival, little is known whether and how it evolves in natural populations, and to what extent it is affected by other environmental stressors. Here, we study physiological and molecular mechanisms of thermal tolerance over evolutionary times in multifarious environments. Using the practice of "resurrection ecology" on the keystone grazer Daphnia magna, we quantified genetic and plastic differences in physiological and molecular traits linked to thermal tolerance in historical and modern genotypes of the same population. This population experienced an increase in average temperature and occurrence of heat waves, in addition to dramatic changes in water chemistry, over five decades. On genotypes resurrected across the five decades, we measured plastic and genetic differences in CTmax , body size, Hb content and differential expression of four heat shock proteins after exposure to temperature as single stress and in combination with food levels and insecticide loads. We observed evolution of the critical thermal maximum and plastic response in body size, HSP expression and Hb content over time in a warming only scenario. Molecular and physiological responses to extreme temperature in multifarious environments were not predictable from the response to warming alone. Underestimating the effect of multiple stressors on thermal tolerance can lead to wrong estimates of species evolvability and persistence.

Cracking the Code of Biodiversity Responses to Past Climate Change.

How individual species and entire ecosystems will respond to future climate change are among the most pressing questions facing ecologists. Past biodiversity dynamics recorded in the paleoecological archives show a broad array of responses, yet significant knowledge gaps remain. In particular, the relative roles of evolutionary adaptation, phenotypic plasticity, and dispersal in promoting survival during times of climate change have yet to be clarified. Investigating the paleo-archives offers great opportunities to understand biodiversity responses to future climate change. In this review we discuss the mechanisms by which biodiversity responds to environmental change, and identify gaps of knowledge on the role of range shifts and tolerance. We also outline approaches at the intersection of paleoecology, genomics, experiments, and predictive models that will elucidate the processes by which species have survived past climatic changes and enhance predictions of future changes in biological diversity.

Resurrection of Dormant Daphnia magna: Protocol and Applications.

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical data that may be used in predictive modelling to forecast evolutionary responses of natural ecosystems to future environmental changes. However, excluding a few exceptional cases, long-term studies are scarce because of logistic difficulties associated with accessing temporal samples. Temporal dynamics are frequently studied in the laboratory or in controlled mesocosm experiments with exceptional studies that reconstruct the evolution of natural populations in the wild. Here, a standard operating procedure (SOP) is provided to revive or resurrect dormant Daphnia magna, a widespread zooplankton keystone species in aquatic ecosystems, to dramatically advance the state-of-the-art longitudinal data collection in natural systems. The field of Resurrection Ecology was defined in 1999 by Kerfoot and co-workers, even though the first attempts at hatching diapausing zooplankton eggs date back to the late 1980s. Since Kerfoot's seminal paper, the methodology of resurrecting zooplankton species has been increasingly frequently applied, though propagated among laboratories only via direct knowledge transfer. Here, an SOP is described that provides a step-by-step protocol on the practice of resurrecting dormant Daphnia magna eggs. Two key studies are provided in which the fitness response of resurrected Daphnia magna populations to warming is measured, capitalizing on the ability to study historical and modern populations in the same settings. Finally, the application of next generation sequencing technologies to revived or still dormant stages is discussed. These technologies provide unprecedented power in dissecting the processes and mechanisms of evolution if applied to populations that have experienced changes in selection pressure over time.

Haemoglobin-mediated response to hyper-thermal stress in the keystone species Daphnia magna.

Anthropogenic global warming has become a major geological and environmental force driving drastic changes in natural ecosystems. Due to the high thermal conductivity of water and the effects of temperature on metabolic processes, freshwater ecosystems are among the most impacted by these changes. The ability to tolerate changes in temperature may determine species long-term survival and fitness. Therefore, it is critical to identify coping mechanisms to thermal and hyper-thermal stress in aquatic organisms. A central regulatory element compensating for changes in oxygen supply and ambient temperature is the respiratory protein haemoglobin (Hb). Here, we quantify Hb plastic and evolutionary response in Daphnia magna subpopulations resurrected from the sedimentary archive of a lake with known history of increase in average temperature and recurrence of heat waves. By measuring constitutive changes in crude Hb protein content among subpopulations, we assessed evolution of the Hb gene family in response to temperature increase. To quantify the contribution of plasticity in the response of this gene family to hyper-thermal stress, we quantified changes in Hb content in all subpopulations under hyper-thermal stress as compared to nonstressful temperature. Further, we tested competitive abilities of genotypes as a function of their Hb content, constitutive and induced. We found that Hb-rich genotypes have superior competitive abilities as compared to Hb-poor genotypes under hyper-thermal stress after a period of acclimation. These findings suggest that whereas long-term adjustment to higher occurrence of heat waves may require a combination of plasticity and genetic adaptation, plasticity is most likely the coping mechanism to hyper-thermal stress in the short term. Our study suggests that with higher occurrence of heat waves, Hb-rich genotypes may be favoured with potential long-term impact on population genetic diversity.