DNA methylation is an evolutionary ancient epigenetic modification that is phylogenetically widespread. Comparative studies of the methylome across a diverse range of non-conventional and conventional model organisms is expected to help reveal how the landscape of DNA methylation and its functions have evolved. Here, we explore the DNA methylation profile of two species of the crustacean Daphnia using whole genome bisulfite sequencing. We then compare our data with the methylomes of two insects and two mammals to achieve a better understanding of the function of DNA methylation in Daphnia. Using RNA-sequencing data for all six species, we investigate the correlation between DNA methylation and gene expression. DNA methylation in Daphnia is mainly enriched within the coding regions of genes, with the highest methylation levels observed at exons 2–4. In contrast, vertebrate genomes are globally methylated, and increase towards the highest methylation levels observed at exon 2, and maintained across the rest of the gene body. Although DNA methylation patterns differ among all species, their methylation profiles share a bimodal distribution across the genomes. Genes with low levels of CpG methylation and gene expression are mainly enriched for species specific genes. In contrast, genes associated with high methylated CpG sites are highly transcribed and evolutionary conserved across all species. Finally, the positive correlation between internal exons and gene expression potentially points to an evolutionary conserved mechanism, whereas the negative regulation of gene expression via methylation of promoters and exon 1 is potentially a secondary mechanism that has been evolved in vertebrates.

Daphnia are key model organisms for mechanistic studies of phenotypic plasticity, adaptation and microevolution, which have led to an increasing demand for genomics resources. A key step in any genomics analysis, such as high-throughput sequencing, is the availability of sufficient and high quality DNA. Although commercial kits exist to extract genomic DNA from several species, preparation of high quality DNA from Daphnia spp. and other chitinous species can be challenging. Here, we optimise methods for tissue homogenisation, DNA extraction and quantification customised for different downstream analyses (e.g., LC-MS/MS, Hiseq, mate pair sequencing or Nanopore). We demonstrate that if Daphnia magna are homogenised as whole animals (including the carapace), absorbance-based DNA quantification methods significantly over-estimate the amount of DNA, resulting in using insufficient starting material for experiments, such as preparation of sequencing libraries. This is attributed to the high refractive index of chitin in Daphnia’s carapace at 260 nm. Therefore, unless the carapace is removed by overnight proteinase digestion, the extracted DNA should be quantified with fluorescence-based methods. However, overnight proteinase digestion will result in partial fragmentation of DNA therefore the prepared DNA is not suitable for downstream methods that require high molecular weight DNA, such as PacBio, mate pair sequencing and Nanopore. In conclusion, we found that the MasterPure DNA purification kit, coupled with grinding of frozen tissue, is the best method for extraction of high molecular weight DNA as long as the extracted DNA is quantified with fluorescence-based methods. This method generated high yield and high molecular weight DNA (3.10 ± 0.63 ng/µg dry mass, fragments >60 kb), free of organic contaminants (phenol, chloroform) and is suitable for large number of downstream analyses.

Short-term exposures at critical stages of development can lead to delayed adverse effects long after the initial stressor has been removed, a concept referred to as developmental origin of adult disease. This indicates that organisms’ phenotypes may epigenetically reflect their past exposure history as well as reflecting chemicals currently present in their environment. This concept has significant implications for environmental monitoring. However, there is as yet little or no implementation of epigenetics in environmental risk assessment. In a proof-of-principle study we exposed Daphnia magna to 5-azacytidine, a known DNA de-methylating agent. Exposures covered combinations of prenatal and postnatal exposures as well as different exposure durations and recovery stages. Growth, the transcription of genes and levels of metabolites involved in regulating DNA methylation, and methylation levels of several genes were measured. Our data shows that prenatal exposures caused significant changes in the methylome of target genes, indicating that prenatal stages of Daphnia are also susceptible to same level of change as post-natal stages of Daphnia. While the combination of pre- and postnatal exposures caused the most extreme reduction in DNA methylation compared to the control group. Furthermore, some of the changes in the methylation patterns were persistent even after the initial stressor was removed. Our results suggest that epigenetic biomarkers have the potential to be used as indicators of past chemical exposure history of organisms and provide strong support for implementing changes to the current regimes for chemical risk assessment to mimic realistic environmental scenarios.

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.

The present study investigates the genotoxic, mutagenic, and cytotoxic potential of surface waters in urban streams using Allium cepa and analyzes the applicability of this assay for environmental monitoring. Water samples were collected from three streams located in the urban area of a municipality in the south of Brazil. For each stream, two samples were collected, one upstream and one downstream of the pollution discharge site. Physicochemical evaluation indicated that all samples had various degrees of environmental impact, but substantial impact was seen for the downstream samples of the Preto and Pedras streams. All samples increased the frequency of chromosome aberrations (P < 0.05). The sample from Pedras downstream site also caused a decrease in mitotic index (P < 0.08) and increase in micronuclei (P < 0.08) frequency, indicating potential cytotoxicity and mutagenicity. The Pedras stream receives mixed industrial and urban wastewater, while the Lajeado and Preto streams receive wastewater predominantly domestic in nature, which may partially explain the difference in toxicity among the samples. Moreover, the Allium cepa seeds/seedlings were shown to be extremely sensitive in detecting the genotoxicity of environmental water samples and can be applied as the first tool for environmental health hazard identification and prediction.

AIM: This study aimed to assess the quality of water and sediment of urban streams (Lajeado, Preto, Pedras and Lewis-Pedroso) located in Santa Cruz do Sul County, RS, Brazil, using the microcrustacean Ceriodaphnia dubia as test-organism. METHODS: Quarterly scientific excursions to the streams were held on August and November 2011, February and May of 2012 in order to collect water and sediment samples, in the upper reaches (P1, P3, P5, P7) and lower reaches (P2, P4, P6, P8), totalizing 8 points. To evaluate the toxicity (acute and chronic), the microcrustacean C. dubia was used. RESULTS AND CONCLUSION: The results indicated high toxicity levels detected in samples P2, P6 and P8 (lower reaches), as they caused the mortality of 100% of organisms in the water samples (P6 and P8) and sediment samples (P2 and P8), denoting acute effect. Yet, all upstream sites showed chronic effects in sediment samples, at least for one collection period, with the highest significant toxicity level among all samples (55.2%), which indicates the presence of contamination even in upper areas. These results indicated a strong degradation of the water and sediment quality of urban streams coming from the wastewater and industrial discharges of the urban area, which can cause damage to the biota as well as the public health, due to the multiples uses of water that the local population does, highlighting many of them as inappropriate to the water quality detected, such as the primary contact recreation (balneability).

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RESUMOConsiderando que os efluentes hospitalares possuem carga poluidora potencialmente tóxica e genotóxica, o objetivo do presente trabalho foi desenvolver a metodologia do Ensaio Cometa com um organismo-teste amplamente utilizados em teste de toxicidade, Daphnia magna, a fim de avaliar a genotoxicidade de efluente provindo do setor de lavanderia de um hospital do Vale do Rio Pardo, RS. Foram realizadas coletas mensais do efluente entre os meses de maio e julho de 2011. Após a padronização do Ensaio Cometa, os organismos foram expostos a concentrações subletais do efluente (0,195; 0,39; 0,78; 1,56%) por um período de 48 h. O ensaio foi realizado com modificações. Os resultados mostraram diferenças significativas (p<0,01) entre o controle negativo e todas as concentrações de efluente testadas. Isso sugere que mesmo diluído a 0,195%, o efluente de lavanderia hospitalar em questão apresenta compostos com potencial de causar lesão na molécula de DNA. Assim, D. magna mostrou-se adequada para essa avaliação, bem como o Ensaio Cometa, que é essencial para complementar outros ensaios, já que apresenta maior sensibilidade, fornecendo resultados importantes para a completa avaliação de efluentes.Palavras-chave: Ensaio Cometa. Daphnia magna. Genotoxicidade. Efluente hospitalar. ABSTRACTConsidering that hospital waste waters present potentially toxic and genotoxic pollutants, the aim of this study was developed and applied to Daphnia magna cells the Comet Assay, as it is an animal generally employed in toxicity tests. The experiment was designed to evaluate