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Bisulfite sequencing with Daphnia highlights a role for epigenetics in regulating stress response to Microcystis through preferential differential methylation of serine and threonine amino acids Jana Asselman, Dieter I.M. De Coninck, Eline Beert, Colin R. Janssen, Luisa Orsini, Michael E. Pfrender, Ellen Decaestecker, and Karel A.C. De Schamphelaere Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03870 • Publication Date (Web): 16 Dec 2016 Downloaded from http://pubs.acs.org on December 18, 2016
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Environmental Science & Technology
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Bisulfite sequencing with Daphnia highlights a
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role for epigenetics in regulating stress response to
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Microcystis through preferential differential
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methylation of serine and threonine amino acids
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Jana Asselman †,‡,*, Dieter IM De Coninck†, Eline Beert§, Colin R Janssen†, Luisa Orsini#,
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Michael E Pfrender‡,∥, Ellen Decaestecker§, Karel AC De Schamphelaere†
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†
Laboratory for Environmental Toxicology and Aquatic Ecology (GhEnToxLab), Ghent
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University, Ghent, B-9000, Belgium ‡
Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556,
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USA §
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#
Laboratory of Aquatic Biology, KU Leuven–Kulak, Kortrijk, B-8500, Belgium
Environmental Genomics Group, School of Biosciences, University of Birmingham,
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Birmingham, B15 2TT, United Kingdom ∥Environmental
Change Initiative, University of Notre Dame, Notre Dame, IN 46556, USA
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* Corresponding author:
[email protected] contact information: Laboratory of
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Environmental Toxicology and Aquatic Ecology, Campus Coupure, Building F – 2nd Floor
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Coupure Links 653, B-9000 Ghent, Belgium. Tel: 0032 9 264 37 64.
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ABSTRACT
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Little is known about the influence that environmental stressors may have on genome-wide
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methylation patterns, and to what extent epigenetics may be involved in environmental stress
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response. Yet, studies of methylation patterns under stress could provide crucial insights on
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stress response and toxicity pathways. Here, we focus on genome-wide methylation patterns
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in the micro-crustacean Daphnia magna, a model organism in ecotoxicology and risk
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assessment, exposed to the toxic cyanobacterium Microcystis aeruginosa. Bisulfite
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sequencing of exposed and control animals highlighted differential methylation patterns in
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Daphnia upon exposure to Microcystis primarily in exonic regions. These patterns are
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enriched for serine/threonine amino acid codons and genes related to protein synthesis,
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transport and degradation. Furthermore, we observed that genes with differential methylation
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corresponded well with genes susceptible to alternative splicing in response to Microcystis
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stress. Overall, our results suggest a complex mechanistic response in Daphnia characterized
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by interactions between DNA methylation and gene regulation mechanisms. These results
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underscore that DNA methylation is modulated by environmental stress and can also be an
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integral part of the toxicity response in our study species.
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INTRODUCTION
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The field of ecotoxicology and environmental risk assessment focus on understanding how
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organisms respond to environmental stressors, including anthropogenic and biotic stressors.
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Studying organismal response to environmental stressors requires a deep understanding of
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the molecular mechanisms involved. Of particular interest are epigenetic mechanisms, given
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that these induce additional phenotypic variability upon environmental stress exposure [1, 2].
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In the past five years, significant progress has been made in unraveling the role of
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methylation in invertebrates. Indeed, the increasing number of available invertebrate
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methylomes and meta-analyses of genome sequences have improved our understanding of
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DNA methylation [3, 4, 5, 6, 7, 8]. Genome-wide differential methylation studies in
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invertebrates have primarily focused on differences among tissues, developmental stages,
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castes or species [3, 5, 9, 10]. DNA methylation in invertebrates seems to be primarily
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targeted to gene bodies in CpG context, while promoter or intergenic methylation and
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methylation in non CpG contexts are less frequently reported [3, 11, 12]. Although the role
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of DNA methylation in invertebrate responses to environmental stressors is difficult to
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assess, preliminary studies suggest that alternative splicing, gene family size and gene
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functions play an integral part [13, 12, 14]. At present, existing studies lack sufficient
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resolution and depth to identify specific genes and mechanisms susceptible to changes in
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methylation upon environmental exposure [15, 16, 17, 18, 19, 20].
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Here, we focus on genome-wide methylation patterns in the micro-crustacean Daphnia
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magna, exposed to the toxic cyanobacterium Microcystis aeruginosa with the aim of
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identifying genes and molecular mechanisms responsible for toxicity response in Daphnia.
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Daphnia is a model organism in ecology, ecotoxicology and environmental risk assessment
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due to its ecological position, life cycle and physical properties [21]. Cyanobacteria in
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general, and Microcystis in particular, are known to have a poor nutritional quality compared 3 ACS Paragon Plus Environment
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to green algae, the common food source for Daphnia [22, 23, 24]. Feeding and life-history
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responses of Daphnia to M. aeruginosa are well documented and range from feeding
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inhibition to decreased reproduction and increased mortality [25, 26, 27]. We have
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previously observed significant changes in global methylation levels when Daphnia were fed
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with different diets including Microcystis [19]. In addition, gene expression studies have
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identified potential mechanisms involved in cyanobacterial toxicity [28, 29]. Yet, it is
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unclear how Microcystis affects DNA methylation and to what extent changes in methylation
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may be involved in the mechanistic response to Microcystis stress. Here, we hypothesized
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that exposure to Microcystis would affect the methylation status of known genes involved in
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toxicity responses and that as such DNA methylation plays an active role in the stress
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response to Microcystis in Daphnia.
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METHODS
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Study species. The strain Xinb3, used to generate the first genome assembly of D. magna
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(NCBI accession number: LRGB00000000 [31]), was used to test methylation patterns
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associated with Microcystis exposure. The isolate was clonally propagated for at least 50
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generations under standardized culture conditions in the lab prior to exposure to reduce the
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influence of maternal effects. Second broods were always used to establish the next
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generation. Animals were cultured in ADaM medium [32] under controlled conditions (20 ±
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1°C, 16h:8h light-dark cycle at a light intensity of 14 µmoles·m-2·s-1). They were fed daily
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with an algal mixture consisting of Scenedesmus obliquus and Cryptomonas sp. (SAG 26.80
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http://sagdb.uni-goettingen.de) at a final concentration of 108 cells per liter in a 95:5 ratio on
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cell basis at a density of 20 animals per liter for the first 5 days and afterwards 1.5*108 cells
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per liter in a 95:5 ratio on cell basis at a density of 10 animals per liter.
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Experimental setup. Neonates of the second brood (
