Article Cite This: Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Transgenerational Inheritance of DNA Hypomethylation in Daphnia magna in Response to Salinity Stress Guilherme Jeremias,†,¶ João Barbosa,†,¶ Seŕ gio M. Marques,†,‡ Karel A.C. De Schamphelaere,§ Filip Van Nieuwerburgh,∥ Dieter Deforce,∥ Fernando J.M. Gonçalves,†,‡ Joana Luísa Pereira,†,‡,# and Jana Asselman*,§,# †
Department of Biology, University of Aveiro, 3810-193, Aveiro, Portugal CESAM (Centre for Environmental and Marine Studies), University of Aveiro, 3810-193, Aveiro, Portugal § Laboratory for Environmental Toxicology and Aquatic Ecology (GhEnToxLab), Ghent University, 9000, Ghent, Belgium ∥ Laboratory of Pharmaceutical Biotechnology, Ghent University, 9000, Ghent, Belgium
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S Supporting Information *
ABSTRACT: Epigenetic mechanisms have been found to play important roles in environmental stress response and regulation. These can, theoretically, be transmitted to future unexposed generations, yet few studies have shown persisting stress-induced transgenerational effects, particularly in invertebrates. Here, we focus on the aquatic microcrustacean Daphnia, a parthenogenetic model species, and its response to salinity stress. Salinity is a serious threat to freshwater ecosystems and a relevant form of environmental perturbation affecting freshwater ecosystems. We exposed one generation of D. magna to high levels of salinity (F0) and found that the exposure provoked specific methylation patterns that were transferred to the three consequent nonexposed generations (F1, F2, and F3). This was the case for the hypomethylation of six protein-coding genes with important roles in the organisms’ response to environmental change: DNA damage repair, cytoskeleton organization, and protein synthesis. This suggests that epigenetic changes in Daphnia are particularly targeted to genes involved in coping with general cellular stress responses. Our results highlight that epigenetic marks are affected by environmental stressors and can be transferred to subsequent unexposed generations. Epigenetic marks could therefore prove to be useful indicators of past or historic pollution in this parthenogenetic model system. Furthermore, no life history costs seem to be associated with the maintenance of hypomethylation across unexposed generations in Daphnia following a single stress exposure.
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ations.16,17 Despite the potential use of epigenetics in environmental science, studies reporting contaminant-induced and persistent transgenerational epigenetic inheritance remain primarily limited to the human health context, using mammalian models such as mice.16,17 Most nonmammalian studies report transgenerational effects that disappear in the second or third generation.16,17 The difficulty of demonstrating true transgenerational effects in nonmammalian model systems could be attributed to the lack of genomic information for many ecotoxicologically relevant species combined with the sparsely methylated genomes of many invertebrates. So far, only one study has highlighted transgenerational effects of DNA methylation persisting until F3 with an invertebrate, in this case Daphnia.18 This can, in part, be attributed to the limited number of studies that have used sequencing
INTRODUCTION In the environment, organisms are faced with a multitude of stressors. This often requires that organisms adapt to stressors, so that they may cope with environmental stress through different strategies. For example, they may more immediately adapt by physiologically acclimating to the new conditions in the short term as an individual strategy, depending on phenotypic plasticity ranges.1−3 Alternatively, genotypic plasticity and concurrent microevolution may also mediate the tolerance of aquatic populations in the long term.4−9 Epigenetics may be a crucial contributing mechanism in these different strategies.10,11 Epigenetics is defined as the study of both mitotically and meiotically heritable changes in gene activity and expression without a change in the DNA sequence.12,13 In contrast to other molecular effects, epigenetic modifications can be inherited through successive generations, even in the absence of the initial stressor.14,15 Therefore, it has been suggested that epigenetic biomarkers may serve as a future risk assessment tool or an indication of past exposures as they can be transmitted to subsequent unexposed gener© XXXX American Chemical Society
Received: Revised: Accepted: Published: A
June 13, 2018 July 26, 2018 August 16, 2018 August 16, 2018 DOI: 10.1021/acs.est.8b03225 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Figure 1. Overview of the experimental design setup for the multigeneration experiment. F0, F1, F2, F3 represent generations. Arrows represent third brood offspring. White rectangles represent clean medium while gray rectangles represent NaCl dissolved in the culture medium at 4.1 g/L. Plus and minus indicate the history of each culture regarding exposure to NaCl (+) and maintenance in clean medium (−). Red boxes indicate treatments from which DNA was extracted for bisulfite sequencing.
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MATERIAL AND METHODS Daphnia Culturing. Monoclonal cultures of Daphnia magna (clone Beak) have been reared in the laboratory for more than 50 generations. Daphnids were cultured in ASTM hard water medium30 (salinity: 636 μS/cm, comparable to freshwater environments26) enriched with vitamins31 and supplemented with an organic additive (Ascophyllum nodosum extract).32 Cultures were maintained under a temperature of 20 ± 2 °C and a 16 h/8 h light/dark photoperiod (provided by cool fluorescent white lights). Culture medium was renewed and organisms were fed three times a week, with concentrated suspensions of Raphidocelis subcapitata (3 × 105 cells mL−1), which is cyclically cultured in Woods Hole MBL.33 Exposure and Sampling. Experiments were conducted under the previously described temperature and photoperiod conditions. Six monoclonal cultures of 70 neonates (
