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.

Metabolomics Simultaneously Derives Benchmark Dose Estimates and Discovers Metabolic Biotransformations in a Rat Bioassay.

Benchmark dose (BMD) modeling estimates the dose of a chemical that causes a perturbation from baseline. Transcriptional BMDs have been shown to be relatively consistent with apical end point BMDs, opening the door to using molecular BMDs to derive human health-based guidance values for chemical exposure. Metabolomics measures the responses of small-molecule endogenous metabolites to chemical exposure, complementing transcriptomics by characterizing downstream molecular phenotypes that are more closely associated with apical end points. The aim of this study was to apply BMD modeling to in vivo metabolomics data, to compare metabolic BMDs to both transcriptional and apical end point BMDs. This builds upon our previous application of transcriptomics and BMD modeling to a 5-day rat study of triphenyl phosphate (TPhP), applying metabolomics to the same archived tissues. Specifically, liver from rats exposed to five doses of TPhP was investigated using liquid chromatography-mass spectrometry and 1H nuclear magnetic resonance spectroscopy-based metabolomics. Following the application of BMDExpress2 software, 2903 endogenous metabolic features yielded viable dose-response models, confirming a perturbation to the liver metabolome. Metabolic BMD estimates were similarly sensitive to transcriptional BMDs, and more sensitive than both clinical chemistry and apical end point BMDs. Pathway analysis of the multiomics data sets revealed a major effect of TPhP exposure on cholesterol (and downstream) pathways, consistent with clinical chemistry measurements. Additionally, the transcriptomics data indicated that TPhP activated xenobiotic metabolism pathways, which was confirmed by using the underexploited capability of metabolomics to detect xenobiotic-related compounds. Eleven biotransformation products of TPhP were discovered, and their levels were highly correlated with multiple xenobiotic metabolism genes. This work provides a case study showing how metabolomics and transcriptomics can estimate mechanistically anchored points-of-departure. Furthermore, the study demonstrates how metabolomics can also discover biotransformation products, which could be of value within a regulatory setting, for example, as an enhancement of OECD Test Guideline 417 (toxicokinetics).

Multi-omics bioactivity profile-based chemical grouping and read-across: a case study with Daphnia magna and azo dyes.

Grouping/read-across is widely used for predicting the toxicity of data-poor target substance(s) using data-rich source substance(s). While the chemical industry and the regulators recognise its benefits, registration dossiers are often rejected due to weak analogue/category justifications based largely on the structural similarity of source and target substances. Here we demonstrate how multi-omics measurements can improve confidence in grouping via a statistical assessment of the similarity of molecular effects. Six azo dyes provided a pool of potential source substances to predict long-term toxicity to aquatic invertebrates (Daphnia magna) for the dye Disperse Yellow 3 (DY3) as the target substance. First, we assessed the structural similarities of the dyes, generating a grouping hypothesis with DY3 and two Sudan dyes within one group. Daphnia magna were exposed acutely to equi-effective doses of all seven dyes (each at 3 doses and 3 time points), transcriptomics and metabolomics data were generated from 760 samples. Multi-omics bioactivity profile-based grouping uniquely revealed that Sudan 1 (S1) is the most suitable analogue for read-across to DY3. Mapping ToxPrint structural fingerprints of the dyes onto the bioactivity profile-based grouping indicated an aromatic alcohol moiety could be responsible for this bioactivity similarity. The long-term reproductive toxicity to aquatic invertebrates of DY3 was predicted from S1 (21-day NOEC, 40 µg/L). This prediction was confirmed experimentally by measuring the toxicity of DY3 in D. magna. While limitations of this 'omics approach are identified, the study illustrates an effective statistical approach for building chemical groups.

Modernizing persistence-bioaccumulation-toxicity (PBT) assessment with high throughput animal-free methods.

The assessment of persistence (P), bioaccumulation (B), and toxicity (T) of a chemical is a crucial first step at ensuring chemical safety and is a cornerstone of the European Union's chemicals regulation REACH (Registration, Evaluation, Authorization, and Restriction of Chemicals). Existing methods for PBT assessment are overly complex and cumbersome, have produced incorrect conclusions, and rely heavily on animal-intensive testing. We explore how new-approach methodologies (NAMs) can overcome the limitations of current PBT assessment. We propose two innovative hazard indicators, termed cumulative toxicity equivalents (CTE) and persistent toxicity equivalents (PTE). Together they are intended to replace existing PBT indicators and can also accommodate the emerging concept of PMT (where M stands for mobility). The proposed "toxicity equivalents" can be measured with high throughput in vitro bioassays. CTE refers to the toxic effects measured directly in any given sample, including single chemicals, substitution products, or mixtures. PTE is the equivalent measure of cumulative toxicity equivalents measured after simulated environmental degradation of the sample. With an appropriate panel of animal-free or alternative in vitro bioassays, CTE and PTE comprise key environmental and human health hazard indicators. CTE and PTE do not require analytical identification of transformation products and mixture components but instead prompt two key questions: is the chemical or mixture toxic, and is this toxicity persistent or can it be attenuated by environmental degradation? Taken together, the proposed hazard indicators CTE and PTE have the potential to integrate P, B/M and T assessment into one high-throughput experimental workflow that sidesteps the need for analytical measurements and will support the Chemicals Strategy for Sustainability of the European Union.

Sex biased expression and co-expression networks in development, using the hymenopteran Nasonia vitripennis.

Sexual dimorphism requires regulation of gene expression in developing organisms. These developmental differences are caused by differential expression of genes and isoforms. The effect of expressing a gene is also influenced by which other genes are simultaneously expressed (functional interactions). However, few studies have described how these processes change across development. We compare the dynamics of differential expression, isoform switching and functional interactions in the sexual development of the model parasitoid wasp Nasonia vitripennis, a system that permits genome wide analysis of sex bias from early embryos to adults. We find relatively little sex-bias in embryos and larvae at the gene level, but several sub-networks show sex-biased functional interactions in early developmental stages. These networks provide new candidates for hymenopteran sex determination, including histone modification. In contrast, sex-bias in pupae and adults is driven by the differential expression of genes. We observe sex-biased isoform switching consistently across development, but mostly in genes that are already differentially expressed. Finally, we discover that sex-biased networks are enriched by genes specific to the Nasonia clade, and that those genes possess the topological properties of key regulators. These findings suggest that regulators in sex-biased networks evolve more rapidly than regulators of other developmental networks.

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.

Broccoli: Combining Phylogenetic and Network Analyses for Orthology Assignment.

Orthology assignment is a key step of comparative genomic studies, for which many bioinformatic tools have been developed. However, all gene clustering pipelines are based on the analysis of protein distances, which are subject to many artifacts. In this article, we introduce Broccoli, a user-friendly pipeline designed to infer, with high precision, orthologous groups, and pairs of proteins using a phylogeny-based approach. Briefly, Broccoli performs ultrafast phylogenetic analyses on most proteins and builds a network of orthologous relationships. Orthologous groups are then identified from the network using a parameter-free machine learning algorithm. Broccoli is also able to detect chimeric proteins resulting from gene-fusion events and to assign these proteins to the corresponding orthologous groups. Tested on two benchmark data sets, Broccoli outperforms current orthology pipelines. In addition, Broccoli is scalable, with runtimes similar to those of recent distance-based pipelines. Given its high level of performance and efficiency, this new pipeline represents a suitable choice for comparative genomic studies. Broccoli is freely available at https://github.com/rderelle/Broccoli.

Progress towards an OECD reporting framework for transcriptomics and metabolomics in regulatory toxicology.

Omics methodologies are widely used in toxicological research to understand modes and mechanisms of toxicity. Increasingly, these methodologies are being applied to questions of regulatory interest such as molecular point-of-departure derivation and chemical grouping/read-across. Despite its value, widespread regulatory acceptance of omics data has not yet occurred. Barriers to the routine application of omics data in regulatory decision making have been: 1) lack of transparency for data processing methods used to convert raw data into an interpretable list of observations; and 2) lack of standardization in reporting to ensure that omics data, associated metadata and the methodologies used to generate results are available for review by stakeholders, including regulators. Thus, in 2017, the Organisation for Economic Co-operation and Development (OECD) Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) launched a project to develop guidance for the reporting of omics data aimed at fostering further regulatory use. Here, we report on the ongoing development of the first formal reporting framework describing the processing and analysis of both transcriptomic and metabolomic data for regulatory toxicology. We introduce the modular structure, content, harmonization and strategy for trialling this reporting framework prior to its publication by the OECD.

Evolutionary transition from blood feeding to obligate nonbiting in a mosquito.

The spread of blood-borne pathogens by mosquitoes relies on their taking a blood meal; if there is no bite, there is no disease transmission. Although many species of mosquitoes never take a blood meal, identifying genes that distinguish blood feeding from obligate nonbiting is hampered by the fact that these different lifestyles occur in separate, genetically incompatible species. There is, however, one unique extant species with populations that share a common genetic background but blood feed in one region and are obligate nonbiters in the rest of their range: Wyeomyia smithii Contemporary blood-feeding and obligate nonbiting populations represent end points of divergence between fully interfertile southern and northern populations. This divergence has undoubtedly resulted in genetic changes that are unrelated to blood feeding, and the challenge is to winnow out the unrelated genetic factors to identify those related specifically to the evolutionary transition from blood feeding to obligate nonbiting. Herein, we determine differential gene expression resulting from directional selection on blood feeding within a polymorphic population to isolate genetic differences between blood feeding and obligate nonbiting. We show that the evolution of nonbiting has resulted in a greatly reduced metabolic investment compared with biting populations, a greater reliance on opportunistic metabolic pathways, and greater reliance on visual rather than olfactory sensory input. W. smithii provides a unique starting point to determine if there are universal nonbiting genes in mosquitoes that could be manipulated as a means to control vector-borne disease.

A comprehensive epigenomic analysis of phenotypically distinguishable, genetically identical female and male Daphnia pulex.

BACKGROUND: Daphnia species reproduce by cyclic parthenogenesis involving both sexual and asexual reproduction. The sex of the offspring is environmentally determined and mediated via endocrine signalling by the mother. Interestingly, male and female Daphnia can be genetically identical, yet display large differences in behaviour, morphology, lifespan and metabolic activity. Our goal was to integrate multiple omics datasets, including gene expression, splicing, histone modification and DNA methylation data generated from genetically identical female and male Daphnia pulex under controlled laboratory settings with the aim of achieving a better understanding of the underlying epigenetic factors that may contribute to the phenotypic differences observed between the two genders. RESULTS: In this study we demonstrate that gene expression level is positively correlated with increased DNA methylation, and histone H3 trimethylation at lysine 4 (H3K4me3) at predicted promoter regions. Conversely, elevated histone H3 trimethylation at lysine 27 (H3K27me3), distributed across the entire transcript length, is negatively correlated with gene expression level. Interestingly, male Daphnia are dominated with epigenetic modifications that globally promote elevated gene expression, while female Daphnia are dominated with epigenetic modifications that reduce gene expression globally. For examples, CpG methylation (positively correlated with gene expression level) is significantly higher in almost all differentially methylated sites in male compared to female Daphnia. Furthermore, H3K4me3 modifications are higher in male compared to female Daphnia in more than 3/4 of the differentially regulated promoters. On the other hand, H3K27me3 is higher in female compared to male Daphnia in more than 5/6 of differentially modified sites. However, both sexes demonstrate roughly equal number of genes that are up-regulated in one gender compared to the other sex. Since, gene expression analyses typically assume that most genes are expressed at equal level among samples and different conditions, and thus cannot detect global changes affecting most genes. CONCLUSIONS: The epigenetic differences between male and female in Daphnia pulex are vast and dominated by changes that promote elevated gene expression in male Daphnia. Furthermore, the differences observed in both gene expression changes and epigenetic modifications between the genders relate to pathways that are physiologically relevant to the observed phenotypic differences.

Multigenerational Exposures of Daphnia Magna to Pristine and Aged Silver Nanoparticles: Epigenetic Changes and Phenotypical Ageing Related Effects.

Engineered nanoparticles (NPs) undergo physical, chemical, and biological transformation after environmental release, resulting in different properties of the "aged" versus "pristine" forms. While many studies have investigated the ecotoxicological effects of silver (Ag) NPs, the majority focus on "pristine" Ag NPs in simple exposure media, rather than investigating realistic environmental exposure scenarios with transformed NPs. Here, the effects of "pristine" and "aged" Ag NPs are systematically evaluated with different surface coatings on Daphnia magna over four generations, comparing continuous exposure versus parental only exposure to assess recovery potential for three generations. Biological endpoints including survival, growth and reproduction and genetic effects associated with Ag NP exposure are investigated. Parental exposure to "pristine" Ag NPs has an inhibitory effect on reproduction, inducing expression of antioxidant stress related genes and reducing survival. Pristine Ag NPs also induce morphological changes including tail losses and lipid accumulation associated with aging phenotypes in the heart, abdomen, and abdominal claw. These effects are epigenetic remaining two generations post-maternal exposure (F2 and F3). Exposure to identical Ag NPs (same concentrations) aged for 6 months in environmentally realistic water containing natural organic matter shows considerably reduced toxicological effects in continuously exposed generations and to the recovery generations.

Perfluoroalkyl Substances Stimulate Insulin Secretion by Islet ß Cells via G Protein-Coupled Receptor 40.

The potential causal relationship between exposure to environmental contaminants and diabetes is troubling. Exposure of perfluoroalkyl substances (PFASs) is found to be associated with hyperinsulinemia and the enhancement of insulin secretion by islet ß cells in humans, but the underlying mechanism is still unclear. Here, by combining in vivo studies with both wild type and gene knockout mice and in vitro studies with mouse islet ß cells (ß-TC-6), we demonstrated clearly that 1 h exposure of perfluorooctanesulfonate (PFOS) stimulated insulin secretion and intracellular calcium level by activating G protein-coupled receptor 40 (GPR40), a vital free fatty acid regulated membrane receptor on islet ß cells. We further showed that the observed effects of PFASs on the mouse model may also exist in humans by investigating the molecular binding interaction of PFASs with human GPR40. We thus provided evidence for a novel mechanism for how insulin-secretion is disrupted by PFASs in humans.

Dynamics of Cadmium Acclimation in Daphnia pulex: Linking Fitness Costs, Cross-Tolerance, and Hyper-Induction of Metallothionein.

Acclimation increases tolerance to stress in individuals but is assumed to contribute fitness costs when the stressor is absent, though data supporting this widely held claim are sparse. Therefore, using clonal (i.e., genetically identical) cultures of Daphnia pulex, we isolated the contributions of acclimation to the regulation of the metal response gene, metallothionein 1 (MT1), and defined the reproductive benefits and costs of cadmium (Cd)-acclimation. Daphnia pulex were exposed for 50 parthenogenetic generations to environmentally realistic levels (1 µg Cd/L), and tolerance to Cd and other metals assessed during this period via standard toxicity tests. These tests revealed (1) increased tolerance to Cd compared to genetically identical nonacclimated cultures, (2) fitness costs in Cd-acclimated Daphnia when Cd was removed, and (3) cross-tolerance of Cd-acclimated Daphnia to zinc and silver, but not arsenic, thereby defining a functional role for metallothionein. Indeed, Cd-acclimated clones had significantly higher expression of MT1 mRNA than nonacclimated clones, when Cd exposed. Both the enhanced induction of MT1 and tolerant phenotype were rapidly lost when Cd was removed (1-2 generations), which is further evidence of acclimation costs. These findings provide evidence for the widely held view that acclimation is costly and are important for investigating evolutionary principles of genetic assimilation and the survival mechanisms of natural populations that face changing environments.

Mixtures of Aluminum and Indium Induce More than Additive Phenotypic and Toxicogenomic Responses in Daphnia magna.

Aquatic systems are contaminated by many metals but their effects as mixtures on organisms are not well understood. Here, we assessed effects of aluminum with fairly well-known modes of actions and indium, an understudied emerging contaminant from electronics, followed by studying equi-effective mixtures thereof. We report acute and adverse phenotypic effects in Daphnia magna adults and global transcriptomic effects employing RNA sequencing in neonates. The mixture induced more than additive activity in mortality and in physiological effects, including growth and reproduction. Similarly, transcriptomic effects were more than additive, as indicated by a markedly higher number of 463 differentially expressed transcripts in the mixture and by distinct classes of genes assigned to several biological functions, including metabolic processes, suggesting depleted energy reserves, which may be responsible for the observed impaired reproduction and growth. A gene set enrichment analysis (GSEA) of a priori known response pathways for aluminum confirmed activation of distinct molecular pathways by indium. Our study is highlighting more than additive effects at the transcriptional and physiological level and is providing a state-of-the art approach to mixture analysis, which is important for risk assessment of these metals and metal mixtures.

The transcriptomic and proteomic responses of Daphnia pulex to changes in temperature and food supply comprise environment-specific and clone-specific elements.

BACKGROUND: Regulatory adjustments to acute and chronic temperature changes are highly important for aquatic ectotherms because temperature affects their metabolic rate as well as the already low oxygen concentration in water, which can upset their energy balance. This also applies to severe changes in food supply. Thus, we studied on a molecular level (transcriptomics and/or proteomics) the immediate responses to heat stress and starvation and the acclimation to different temperatures in two clonal isolates of the model microcrustacean Daphnia pulex from more or less stressful environments, which showed a higher (clone M) or lower (clone G) tolerance to heat and starvation. RESULTS: The transcriptomic responses of clone G to acute heat stress (from 20 °C to 30 °C) and temperature acclimation (10 °C, 20 °C, and 24 °C) and the proteomic responses of both clones to acute heat, starvation, and heat-and-starvation stress comprised environment-specific and clone-specific elements. Acute stress (in particular heat stress) led to an early upregulation of stress genes and proteins (e.g., molecular chaperones) and a downregulation of metabolic genes and proteins (e.g., hydrolases). The transcriptomic responses to temperature acclimation differed clearly. They also varied depending on the temperature level. Acclimation to higher temperatures comprised an upregulation of metabolic genes and, in case of 24 °C acclimation, a downregulation of genes for translational processes and collagens. The proteomic responses of the clones M and G differed at any type of stress. Clone M showed markedly stronger and less stress-specific proteomic responses than clone G, which included the consistent expression of a specific heat shock protein (HSP60) and vitellogenin (VTG-SOD). CONCLUSIONS: The expression changes under acute stress can be interpreted as a switch from standard products of gene expression to stress-specific products. The expression changes under temperature acclimation probably served for an increase in energy intake (via digestion) and, if necessary, a decrease in energy expenditures (e.g, for translational processes). The stronger and less stress-specific proteomic responses of clone M indicate a lower degree of cell damage and an active preservation of the energy balance, which allowed adequate proteomic responses under stress, including the initiation of resting egg production (VTG-SOD expression) as an emergency reaction.

Mitotic activity patterns and cytoskeletal changes throughout the progression of diapause developmental program in Daphnia.

BACKGROUND: Diapause is a form of dormancy that is genetically predetermined to allow animals to overcome harsh environmental conditions. It is induced by predictive environmental cues bringing cellular activity levels into a state of suspended animation. Entering diapause requires organismal, molecular and cellular adaptation to severely reduced energy flows. Cells must therefore have evolved strategies that prepare them for periods with limited metabolic resources. However, changes that occur on the (sub-)cellular level have not been thoroughly described. RESULTS: We investigated mitotic activity and we monitored cytoskeletal network changes in successive stages of diapausing and non-diapausing Daphnia magna embryos using (immuno-)fluorescent labeling. We find that embryos destined to diapause show a delayed and 2.5x slower mitotic activity in comparison to continuously developing embryos. Development is halted when D. magna embryos reach ~ 3500 cells, whereupon mitotic activity is absent and cytoskeletal components are severely reduced, rendering diapause cells compact and condensed. CONCLUSION: In the initiation phase of diapause, the slower cell division rate points to prolonged interphase duration, preparing the cells for diapause maintenance. During diapause, cytoskeletal depletion and cellular condensation may be a means to save energy resources. Our data provide insights into the sub-cellular change of diapause in Daphnia.

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.

Arsenic Reduces Gene Expression Response to Changing Salinity in Killifish.

Toxicogenomic approaches can detect and classify adverse interactions between environmental toxicants and other environmental stressors but require more complex experimental designs and analytical approaches. Here we use novel toxicogenomic techniques to analyze the effect of arsenic exposure in wild killifish populations acclimating to changing salinity. Fish from three populations were acclimated to full strength seawater and transferred to fresh water for 1 or 24 h. Linear models of gene expression in gill tissue identified 31 genes that responded to osmotic shock at 1 h and 178 genes that responded at 24 h. Arsenic exposure (100 µg/L) diminished the responses (reaction norms) of these genes by 22% at 1 h ( p = 1.0 × 10-6) and by 10% at 24 h ( p = 3.0 × 10-10). Arsenic also significantly reduced gene coregulation in gene regulatory networks ( p = 0.002, paired Levene's test), and interactions between arsenic and salinity acclimation were uniformly antagonistic at the biological pathway level ( p < 0.05, binomial test). Arsenic's systematic interference with gene expression reaction norms was validated in a mouse multistressor experiment, demonstrating the ability of these toxicogenomic approaches to identify biologically relevant adverse interactions between environmental toxicants and other environmental stressors.

The Toxicogenome of Hyalella azteca: A Model for Sediment Ecotoxicology and Evolutionary Toxicology.

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.

Male meiosis in Crustacea: synapsis, recombination, epigenetics and fertility in Daphnia magna.

We present the first detailed cytological study of male meiosis in Daphnia (Crustacea: Branchiopoda: Cladocera)-an aquatic microcrustacean with a cyclical parthenogenetic life cycle. Using immunostaining of the testes in Daphnia magna for baseline knowledge, we characterized the different stages of meiotic division and spermiogenesis in relation to the distribution of proteins involved in synapsis, early recombination events and sister chromatid cohesion. We also studied post-translational histone modifications in male spermatocytes, in relation to the dynamic chromatin progression of meiosis. Finally, we applied a DNA fragmentation test to measure sperm quality of D. magna, with respect to levels of inbreeding. As a proxy for fertility, this technique may be used to assess the reproductive health of a sentinel species of aquatic ecosystems. Daphnia proves to be a model species for comparative studies of meiosis that is poised to improve our understanding of the cytological basis of sexual and asexual reproduction.