Refining the evolutionary time machine: An assessment of whole genome amplification using single historical Daphnia eggs.

Whole genome sequencing is instrumental for the study of genome variation in natural populations, delivering important knowledge on genomic modifications and potential targets of natural selection at the population level. Large dormant eggbanks of aquatic invertebrates such as the keystone herbivore Daphnia, a microcrustacean widespread in freshwater ecosystems, provide detailed sedimentary archives to study genomic processes over centuries. To overcome the problem of limited DNA amounts in single Daphnia dormant eggs, we developed an optimized workflow for whole genome amplification (WGA), yielding sufficient amounts of DNA for downstream whole genome sequencing of individual historical eggs, including polyploid lineages. We compare two WGA kits, applied to recently produced Daphnia magna dormant eggs from laboratory cultures, and to historical dormant eggs of Daphnia pulicaria collected from Arctic lake sediment between 10 and 300 years old. Resulting genome coverage breadth in most samples was ~70%, including those from >100-year-old isolates. Sequence read distribution was highly correlated among samples amplified with the same kit, but less correlated between kits. Despite this, a high percentage of genomic positions with single nucleotide polymorphisms in one or more samples (maximum of 74% between kits, and 97% within kits) were recovered at a depth required for genotyping. As a by-product of sequencing we obtained 100% coverage of the mitochondrial genomes even from the oldest isolates (~300 years). The mitochondrial DNA provides an additional source for evolutionary studies of these populations. We provide an optimized workflow for WGA followed by whole genome sequencing including steps to minimize exogenous DNA.

Polystyrene nanoplastic induces oxidative stress, immune defense, and glycometabolism change in Daphnia pulex: Application of transcriptome profiling in risk assessment of nanoplastics.

Aquatic environments are generally contaminated with nanoplastic material. As a result, molecular mechanisms for sensitive species like Daphnia are needed, given that mechanistic nanoplastic toxicity is largely unknown. Here, global transcriptome sequencing (RNA-Seq) was performed on D. pulex neonates to quantitatively measure the expression level of transcripts. A total of 208 differentially expressed genes (DEGs) were detected in response to nanoplastic exposure for 96 h, with 107 being up-regulated and 101 down-regulated. The gene functions and pathways for oxidative stress, immune defense, and glycometabolism were identified. In this study, D. pulex neonates provide some molecular insights into nanoplastic toxicity. However, more studies on DEGs are needed to better understand the underlying mechanisms that result as a response to nanoplastic toxicity in aquatic organisms.

Adverse effects in Daphnia magna exposed to e-waste leachate: Assessment based on life trait changes and responses of detoxification-related genes.

The sharp increase in e-waste derived from great consumption of electronic products has become a potential global environmental challenge. Limited information is available about the potential detrimental impact of e-waste on aquatic organisms. The present study investigated the expression of detoxification-related genes and life-history parameter changes in Daphnia magna exposed to e-waste leachate, simultaneously integrating with the chemical analysis of typical pollutants from e-waste leachate. The study aims at assessing impacts of e-waste to aquatic invertebrates and providing insights into its toxic mechanisms. The results showed high concentration of heavy metals like Cu (1657.14 ± 259.3 µg g-1, DW) and persistent organic compounds like polybrominated diphenyl ethers (7831.32 ± 1273.86 ng g-1, DW) in stream sediments near e-waste dismantling areas. Chronic exposure to these pollutants can affect the growth and reproduction of D. magna, resulting in significant development retardation, decreased total egg production per female, and even smaller body size. Expression of some detoxification and reproduction-related genes including DappuHR96, CYP360a, P-gp, EcR, CYP314 and Vtg exhibited different response patterns depending on the e-waste leachate concentration. E-waste leachate may affect the expression of detoxification-related and growth and reproduction-related genes and disrupt the growth and reproduction processes of D. magna.

Assessment of Transcriptomic and Apical Responses of Daphnia magna Exposed to a Polyethylene Microplastic in a 21-d Chronic Study.

There is global concern regarding the fate and effects of microplastics in the environment, particularly in aquatic systems. In the present study, ethylene acrylic acid copolymer particles were evaluated in a chronic toxicity study with the aquatic invertebrate Daphnia magna. The study design included a natural particle control treatment (silica) to differentiate any potential physical effects of a particle from the intrinsic toxicity of the test material. In addition to the standard endpoints of survival, growth, and reproduction, the transcriptomic profiles of control and ethylene acrylic acid copolymer-exposed D. magna were evaluated at the termination of the 21-d toxicity study. No significant effects on D. magna growth, survival, or reproduction were observed in comparison with both particle and untreated control groups. Significant transcriptomic alterations were induced at the highest treatment level of 2.3 × 1012 particles of the ethylene acrylic acid copolymer/L in key pathways linked to central metabolism and energy reserves, oxidative stress, and ovulation and molting, indicating a global transcriptomic response pattern. To put the results in perspective is challenging at this time, because, to date, microplastic environmental monitoring approaches have not been equipped to detect particles in the nanosize range. However, our results indicate that ethylene acrylic acid copolymer microplastics in the upper nanosize range are not expected to adversely affect D. magna growth, survival, or reproductive outcomes at concentrations of up to 1012 particles/L. Environ Toxicol Chem 2020;39:1578-1589. © 2020 SETAC.

Assessment of oxidative stress of paracetamol to Daphnia magna via determination of Nrf1 and genes related to antioxidant system.

Paracetamol (APAP) is one of the most widely used anti-inflammatory and analgesic drugs in human being health care and has been universally detected in various aquatic environments. However, its potential adverse effects and toxic mechanisms on freshwater invertebrates still remain unclear. In the present study, the effects of APAP on the expressions of Nrf1 and the antioxidant related genes including GCLC, GST, GPX, CAT, TRX, TrxR and Prx1 in Daphnia magna (D. magna) were evaluated after 24, 48 and 96 h, and the changes of GPX, GST and CAT enzyme activities, as well as the GSH and MDA content under APAP exposure for 48 h were also determined. Results showed that paracetamol affected the expressions of Nrf1 and antioxidant related genes in D. magna, which were related to the exposure time and concentration of APAP. Nrf1 was inhibited at 48 h, but induced at 96 h under the APAP exposure, being about two fold of the control in 5.0 µg/L. CAT were significantly induced in all treatments. But Prx decreased in an concentration-dependent manner in all treatments. In comparison with the mRNA expression, antioxidant enzymes activity displayed less changes in D. magna. Overall, APAP exposure altered the expression of Nrf1 and genes related to antioxidant system and disturbed the redox homeostasis of D. magna.

Hierarchical Assessment of Mutation Properties in Daphnia magna.

Understanding the context-dependence of spontaneous mutations is crucial to predicting evolutionary trajectories. In this experiment, the impact of genetic background and trait-type on mutational susceptibility was investigated. Mutant and non-mutant lines of six unique genotypes from two populations of Daphnia magna were phenotypically assayed using a common-garden experiment. Morphological, life-history, and behavioral traits were measured and estimates of the mutation parameters were generated. The mutation parameters varied between the populations and among genotypes, suggesting differential susceptibility to mutation depending upon genomic background. Traits also varied in their susceptibility to mutation with behavioral traits evolving more rapidly than life-history and morphological traits. These results may reflect the unique selection histories of these populations.

Assessment of ecotoxicological effects of ciprofloxacin in Daphnia magna: life-history traits, biochemical and genotoxic effects.

Antibiotics (e.g. ciprofloxacin) have been detected in surface water and groundwater for several decades. In order to understand the potential impact of the continuous exposure of aquatic organisms to ciprofloxacin, a chronic assay was carried out with Daphnia magna. This approach allowed evaluation of the effects of ciprofloxacin on life-history and sub-individual parameters (antioxidant status and metabolic response: activities of catalase and glutathione S-transferases - GSTs; peroxidative damage; thiobarbituric acid reactive substances and genotoxic effects (genetic damage index, measured by the comet assay). Life-history parameters of D. magna showed no significant effects after ciprofloxacin exposure. Concerning oxidative stress and metabolism parameters, no significant alterations were reported for catalase and GSTs activities. However, a dual response was observed, with a significant decrease in lipid peroxidation levels at low ciprofloxacin concentrations (<0.013 mg/L), while a significant increase was verified at high ciprofloxacin concentrations (0.078 mg/L). The genotoxicity assay detected a significant increase in genetic damage index up to 0.013 mg/L of ciprofloxacin. The here-tested ciprofloxacin concentrations, which are ecologically relevant, did not cause significant impacts concerning the life-history parameters of D. magna; however, at the same levels of ciprofloxacin an oxidative stress and genotoxic damage scenarios were recorded.

Daphnia invest in sexual reproduction when its relative costs are reduced.

The timing of sex in facultatively sexual organisms is critical to fitness, due to the differing demographic consequences of sexual versus asexual reproduction. In addition to the costs of sex itself, an association of sex with the production of dormant life stages also influences the optimal use of sex, especially in environments where resting eggs are essential to survive unfavourable conditions. Here we document population dynamics and the occurrence of sexual reproduction in natural populations of Daphnia magna across their growing season. The frequency of sexually reproducing females and males increased with population density and with decreasing asexual clutch sizes. The frequency of sexually reproducing females additionally increased as population growth rates decreased. Consistent with population dynamic models showing that the opportunity cost of sexual reproduction (foregoing contribution to current population growth) diminishes as populations approach carrying capacity, we found that investment in sexual reproduction was highest when asexual population growth was low or negative. Our results support the idea that the timing of sex is linked with periods when the relative cost of sex is reduced due to low potential asexual growth at high population densities. Thus, a combination of ecological and demographic factors affect the optimal timing of sexual reproduction, allowing D. magna to balance the necessity of sex against its costs.

Assessment of biochemical mechanisms of tolerance to chlorpyrifos in ancient and contemporary Daphnia pulicaria genotypes.

The evolution of tolerance to environmental contaminants in non-target taxa has been largely studied by comparing extant populations experiencing contrasting exposure. Previous research has demonstrated that "resurrected" genotypes from a population of Daphnia pulicaria express temporal variation in sensitivity to the insecticide chlorpyrifos. Ancient genotypes (1301-1646AD.) were on average more sensitive to this chemical compared to the contemporary genotypes (1967-1977AD.). To determine the physiological mechanisms of tolerance, a series of biochemical assays was performed on three ancient and three contemporary genotypes; these six genotypes exhibited the most sensitive and most tolerant phenotypes within the population, respectively. Metabolic tolerance mechanisms were evaluated using acute toxicity testing, while target-site tolerance was assessed via in vitro acetylcholinesterase (AChE) assays. Acute toxicity tests were conducted using i) the toxic metabolite chlorpyrifos-oxon (CPF-oxon) and ii) CPF-oxon co-applied with piperonyl butoxide (PBO), a known Phase-I metabolic inhibitor. Both series of toxicity tests reduced the mean variation in sensitivity between tolerant and sensitive genotypes. Exposure to CPF-O reduced the disparity from a 4.7-fold to 1.6-fold difference in sensitivity. The addition of PBO further reduced the variation to a 1.2-fold difference in sensitivity. In vitro acetylcholinesterase assays yielded no significant differences in constitutive activity or target-site sensitivity. These findings suggest that pathways involving Phase-I detoxification and/or bioactivation of chlorpyrifos play a significant role in dictating the microevolutionary trajectories of tolerance in this population.

Assessment of mitochondrial functions in Daphnia pulex clones using high-resolution respirometry.

The objectives of our study were to adapt a method to measure mitochondrial function in intact mitochondria from the small crustacean Daphnia pulex and to validate if this method was sensitive enough to characterize mitochondrial metabolism in clones of the pulex complex differing in ploidy levels, mitochondrial DNA haplotypes, and geographic origins. Daphnia clones belonging to the Daphnia pulex complex represent a powerful model to delineate the link between mitochondrial DNA evolution and mitochondrial phenotypes, as single genotypes with divergent mtDNA can be grown under various experimental conditions. Our study included two diploid clones from temperate environments and two triploid clones from subarctic environments. The whole animal permeabilization and measurement of respiration with high-resolution respirometry enabled the measurement of the functional capacity of specific mitochondrial complexes in four clones. When expressing the activity as ratios, our method detected significant interclonal variations. In the triploid subarctic clone from Kuujjurapik, a higher proportion of the maximal physiological oxidative phosphorylation (OXPHOS) capacity of mitochondria was supported by complex II, and a lower proportion by complex I. The triploid subarctic clone from Churchill (Manitoba) showed the lowest proportion of the maximal OXPHOS supported by complex II. Additional studies are required to determine if these differences in mitochondrial functions are related to differences in mitochondrial haplotypes or ploidy level and if they might be associated with fitness divergences and therefore selective value.

High-resolution melting analysis: a genotyping tool for population studies on Daphnia.

Determining genetic variation at the DNA level within and between natural populations is important for understanding the role of natural selection on phenotypic traits, but many techniques of screening for genetic variation are either cost intensive, not sensitive enough or too labour- and time-consuming. Here, we demonstrate high-resolution melting analysis (HRMA) as a cost-effective and powerful tool for screening variable target genes in natural populations. HRMA is based on monitoring the melting of PCR amplicons. Owing to saturating concentrations of a dye that binds at high concentrations to double-stranded DNA, it is possible to genotype high numbers of samples rapidly and accurately. We analysed digestive trypsins of two Daphnia magna populations as an application example for HRMA. One population originated from a pond containing toxic cyanobacteria that possibly produce protease inhibitors and the other from a pond without such cyanobacteria. The hypothesis was that D. magna clones from ponds with cyanobacteria have undergone selection by these inhibitors, which has led to different trypsin alleles. We first sequenced pooled genomic PCR products of trypsins from both populations to identify variable DNA sequences of active trypsins. Second, we screened variable DNA sequences of each D. magna clone from both populations for single nucleotide polymorphisms via HRMA. The HRMA results revealed that both populations exhibited phenotypic differences in the analysed trypsins. Our results indicate that HRMA is a powerful genotyping tool for studying the variation of target genes in response to selection within and between natural Daphnia populations.

Testing hypotheses on the resistance to metals by Daphnia longispina: differential acclimation, endpoints association, and fitness costs.

Pollution by metals may lead to an increased tolerance in the exposed population through adaptive microevolution, with resistant genotypes becoming more abundant than in reference sites. This work investigated the outcomes associated with selection for resistance by testing three hypotheses to assess the following: Do resistant versus sensitive clonal lineages of Daphnia longispina differentially acclimate to metals during a long-term sublethal exposure, is there a significant correlation between lethal and sublethal responses, and does resistance to metals entail costs to fitness under uncontaminated conditions? No evidence of acclimation was observed. The median effective dilutions of acid mine drainage for reproduction were similar for successive broods within clones during long-term exposures. Lethal and sublethal responses were not correlated, indicating that mechanisms regulating the two types of response were more than likely different. Finally, fitness costs associated with the resistance to lethal levels of metals were not detected, but resistance to sublethal levels of Cu was found to be correlated with a lower intrinsic growth rate under control conditions.

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.

Assessment of chemical mixtures and groundwater effects on Daphnia magna transcriptomics.

Small organisms can be used as biomonitoring tools to assess chemicals in the environment. Chemical stressors are especially hard to assess and monitor when present as complex mixtures. Here, fifteen polymerase chain reaction assays targeting Daphnia magna genes were calibrated to responses elicited in D. magna exposed for 24 h to five different doses each of the munitions constituents 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, trinitrobenzene, dinitrobenzene, or 1,3,5-trinitro-1,3,5-triazacyclohexane. A piecewise-linear model for log-fold expression changes in gene assays was used to predict response to munitions mixtures and contaminated groundwater under the assumption that chemical effects were additive. The correlations of model predictions with actual expression changes ranged from 0.12 to 0.78 with an average of 0.5. To better understand possible mixture effects, gene expression changes from all treatments were compared using high-density microarrays. Whereas mixtures and groundwater exposures had genes and gene functions in common with single chemical exposures, unique functions were also affected, which was consistent with the nonadditivity of chemical effects in these mixtures. These results suggest that, while gene behavior in response to chemical exposure can be partially predicted based on chemical exposure, estimation of the composition of mixtures from chemical responses is difficult without further understanding of gene behavior in mixtures. Future work will need to examine additive and nonadditive mixture effects using a much greater range of different chemical classes in order to clarify the behavior and predictability of complex mixtures.

Successfully resisting a pathogen is rarely costly in Daphnia magna.

BACKGROUND: A central hypothesis in the evolutionary ecology of parasitism is that trade-offs exist between resistance to parasites and other fitness components such as fecundity, growth, survival, and predator avoidance, or resistance to other parasites. These trade-offs are called costs of resistance. These costs fall into two broad categories: constitutive costs of resistance, which arise from a negative genetic covariance between immunity and other fitness-related traits, and inducible costs of resistance, which are the physiological costs incurred by hosts when mounting an immune response. We sought to study inducible costs in depth using the crustacean Daphnia magna and its bacterial parasite Pasteuria ramosa. RESULTS: We designed specific experiments to study the costs induced by exposure to this parasite, and we re-analysed previously published data in an effort to determine the generality of such costs. However, despite the variety of genetic backgrounds of both hosts and parasites, and the different exposure protocols and environmental conditions used in these experiment, this work showed that costs of exposure can only rarely be detected in the D. magna-P. ramosa system. CONCLUSIONS: We discuss possible reasons for this lack of detectable costs, including scenarios where costs of resistance to parasites might not play a major role in the co-evolution of hosts and parasites.

Genetic costs of tolerance to metals in Daphnia longispina populations historically exposed to a copper mine drainage.

The present study was conducted to assess three microevolutionary aspects of adaptation to pollution in Daphnia longispina populations historically exposed to an acid mine drainage from an abandoned pyrite mine: pollution mediated effects in acute tolerance to copper (Cu) and zinc (Zn); pollution-mediated effects on genetic variability of tolerant and physiological traits related to fitness (feeding rates); and fitness costs of tolerance measured as genetic trade-offs between tolerance and feeding rates under none and low levels of contamination. These objectives were addressed by comparing broad sense heritabilities and genetic correlations using up to 20 distinct clonal lineages randomly obtained from two populations: one located in a water reservoir contaminated by the acid mine drainage, and the other located in a nearby clean water reservoir. Results showed that only sensitive and resistant lineages to Cu were present in the reference and contaminated site, respectively. For Zn, however, both populations had a similar distribution pattern of sensitivities. Heritability values for tolerant and feeding traits across metal exposure levels was similar in both populations being in most cases greater than 50%. Fitness costs of tolerance were illustrated by lower feeding rates of the tolerant population compared to the reference one and negative genetic correlations between mean clonal feeding rates and median clonal survival time in control conditions (no added Cu or Zn). The results obtained thus support the view that tolerance to pollution is ecologically costly.

MGEScan-non-LTR: computational identification and classification of autonomous non-LTR retrotransposons in eukaryotic genomes.

Computational methods for genome-wide identification of mobile genetic elements (MGEs) have become increasingly necessary for both genome annotation and evolutionary studies. Non-long terminal repeat (non-LTR) retrotransposons are a class of MGEs that have been found in most eukaryotic genomes, sometimes in extremely high numbers. In this article, we present a computational tool, MGEScan-non-LTR, for the identification of non-LTR retrotransposons in genomic sequences, following a computational approach inspired by a generalized hidden Markov model (GHMM). Three different states represent two different protein domains and inter-domain linker regions encoded in the non-LTR retrotransposons, and their scores are evaluated by using profile hidden Markov models (for protein domains) and Gaussian Bayes classifiers (for linker regions), respectively. In order to classify the non-LTR retrotransposons into one of the 12 previously characterized clades using the same model, we defined separate states for different clades. MGEScan-non-LTR was tested on the genome sequences of four eukaryotic organisms, Drosophila melanogaster, Daphnia pulex, Ciona intestinalis and Strongylocentrotus purpuratus. For the D. melanogaster genome, MGEScan-non-LTR found all known 'full-length' elements and simultaneously classified them into the clades CR1, I, Jockey, LOA and R1. Notably, for the D. pulex genome, in which no non-LTR retrotransposon has been annotated, MGEScan-non-LTR found a significantly larger number of elements than did RepeatMasker, using the current version of the RepBase Update library. We also identified novel elements in the other two genomes, which have only been partially studied for non-LTR retrotransposons.

The cost of being common: evidence from natural Daphnia populations.

The Red Queen coevolutionary hypothesis predicts that parasites drive oscillations in host genotype frequencies due to frequency-dependent selection where common hosts are at disadvantage. However, examples of this phenomenon in natural populations are scarce. To examine if the Red Queen theory operates in the wild, we studied the genetic structure of populations of the crustacean waterflea (Daphnia), in relation to their infection levels, for which we collected multiple samples from a variety of lakes. The most common clone in a given population was often underinfected. This advantage, however, did not remain stable over time. Instead, the most common clone decreased in frequency over subsequent generations, indicating that parasites can track common clones. Such decreases were not observed in uninfected populations. Moreover, host clonal evenness was higher across the set of infected lakes compared to uninfected lakes; suggesting that any common clone is selected against when parasites are present. These results strongly suggest that Red Queen dynamics do operate in the wild.

Both costs and benefits of sex correlate with relative frequency of asexual reproduction in cyclically parthenogenic Daphnia pulicaria populations.

Sexual reproduction is generally believed to yield beneficial effects via the expansion of expressed genetic variation, which increases the efficiency of selection and the adaptive potential of a population. However, when nonadditive gene action is involved, sex can actually impede the adaptive progress of a population. If selection promotes coupling disequilibria between genes of similar effect, recombination and segregation can result in a decrease in expressed genetic variance in the offspring population. In addition, when nonadditive gene action underlies a quantitative trait, sex can produce a change in trait means in a direction opposite to that favored by selection. In this study we measured the change in genotypic trait means and genetic variances across a sexual generation in four populations of the cyclical parthenogen Daphnia pulicaria, which vary predictably in their incidence of sexual reproduction. We show that both the costs and benefits of sex, as measured by changes in means and variances in life-history traits, increase substantially with decreasing frequency of sex.

Costs, benefits and the evolution of inducible defences: a case study with Daphnia pulex.

Phenotypic plasticity is one major source of variation in natural populations. Inducible defences, which can be considered threshold traits, are a form of plasticity that generates ecological and evolutionary consequences. A simple cost-benefit model underpins the maintenance and evolution of these threshold, inducible traits. In this model, a rank-order switch in expected fitness, defined by costs and benefits of induction between defended and undefended morphs, predicts the risk level at which individuals should induce defences. Here, taking predator-induced morphological defences in Daphnia pulex as a threshold trait, we provide the first comprehensive investigation into the costs and benefits of a threshold trait, and how they combine to reflect fitness and predict the switchpoint at which induction should occur. We develop reaction norms that show genetic variation in switchpoints. Further experiments show that induction can confer a survival benefit and a cost in terms of lifetime reproductive success. Together, these two traits combine to estimate expected fitness and can predict the switchpoint between an undefended and a defended strategy. The predictions match the reaction norm data for clones that experience these costs and benefits, and correspond well to independent field data on induction. However, predictions do not, and cannot, match for clones that do not gain a benefit from induction. This study confirms that a simple theory, based on life history costs and benefits, is a sufficient framework for understanding the ecology and evolution of inducible, threshold traits.