Transgenerational Inheritance of DNA Hypomethylation in Daphnia

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Ecotoxicology and Human Environmental Health

Transgenerational inheritance of DNA hypomethylation in Daphnia magna in response to salinity stress Guilherme Jeremias, João Barbosa, Sergio Marques, Karel AC De Schamphelaere, Filip Van Nieuwerburgh, Dieter Deforce, Fernando Gonçalves, Joana Pereira, and Jana Asselman Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03225 • Publication Date (Web): 16 Aug 2018 Downloaded from http://pubs.acs.org on August 17, 2018

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Environmental Science & Technology

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Transgenerational inheritance of DNA hypomethylation in Daphnia magna in

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response to salinity stress

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Guilherme Jeremias1†, João Barbosa1†, Sérgio M. Marques1,2, Karel A.C. De Schamphelaere3, Filip

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Van Nieuwerburgh4, Dieter Deforce4, Fernando J.M. Gonçalves1,2, Joana Luísa Pereira1,2§, Jana

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Asselman3*§

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1

Department of Biology, University of Aveiro, 3810-193, Aveiro, Portugal.

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2

CESAM (Centre for Environmental and Marine Studies), University of Aveiro, 3810-193, Aveiro, Portugal.

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Laboratory for Environmental Toxicology and Aquatic Ecology (GhEnToxLab), Ghent University, 9000, Ghent,

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Belgium.

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†

Laboratory of Pharmaceutical Biotechnology, Ghent University, 9000, Ghent, Belgium. Authors contributed equally, thus both should be considered first authors; § Authors contributed equally.

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*Corresponding author

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Jana Asselman. Laboratory for Environmental Toxicology and Aquatic Ecology (GhEnToxLab),

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Ghent University, Ghent, B-9000, Belgium.

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Email: [email protected].; Phone: 0032 9 264 38 97;

1 ACS Paragon Plus Environment


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Abstract

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Epigenetic mechanisms have been found to play important roles in environmental stress response and

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regulation. These can, theoretically, be transmitted to future unexposed generations, yet few studies

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have shown persisting stress-induced transgenerational effects, particularly in invertebrates. Here, we

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focus on the aquatic microcrustacean Daphnia, a parthenogenetic model species, and its response to

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salinity stress. Salinity is a serious threat to freshwater ecosystems and a relevant form of

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environmental perturbation affecting freshwater ecosystems. We exposed one generation of D.

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magna to high levels of salinity (F0) and found that the exposure provoked specific methylation

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patterns that were transferred to the three consequent non-exposed generations (F1, F2 and F3). This

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was the case for the hypomethylation of six protein-coding genes with important roles in the

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organisms’ response to environmental change: DNA damage repair, cytoskeleton organization and

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protein synthesis. This suggests that epigenetic changes in Daphnia are particularly targeted to genes

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involved in coping with general cellular stress responses. Our results highlight that epigenetic marks

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are affected by environmental stressors and can be transferred to subsequent unexposed generations.

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Epigenetic marks could therefore prove to be useful indicators of past or historic pollution in this

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parthenogenetic model system. Furthermore, no life history costs seem to be associated with the

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maintenance of hypomethylation of across unexposed generations in Daphnia following a single

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stress exposure.

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Keywords: Freshwater ecosystems, Epigenetic transgenerational inheritance, DNA methylation,

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Salinity, Daphnia magna

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Introduction

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In the environment, organisms are faced with a multitude of stressors. This often requires that

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organisms adapt to stressors, so that they may cope with environmental stress through different

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strategies. For example, they may more immediately adapt by physiologically acclimating to the new

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conditions in the short term as an individual strategy, depending on phenotypic plasticity ranges 1-3.

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Alternatively, genotypic plasticity and concurrent microevolution may also mediate the tolerance of

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aquatic populations in the long-term 4-9. 10, 11

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Epigenetics may be a crucial contributing mechanism in these different strategies

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Epigenetics is defined as the study of both mitotically and meiotically heritable changes in gene

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activity and expression without a change in the DNA sequence 12, 13. In contrast to other molecular

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effects, epigenetic modifications can be inherited through successive generations, even in the absence

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of the initial stressor 14, 15. Therefore, it has been suggested that epigenetic biomarkers may serve as

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a future risk assessment tool or an indication of past exposures as they can be transmitted to

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subsequent unexposed generations

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science, studies reporting contaminant-induced and persistent transgenerational epigenetic

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inheritance remain primarily limited to the human health context, using mammalian models such as

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mice 16, 17. Most non-mammalian studies report transgenerational effects that disappear in the second

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or third generation

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mammalian model systems could be attributed to the lack of genomic information for many

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ecotoxicologically relevant species combined with the sparsely methylated genomes of many

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invertebrates. So far, only one study has highlighted transgenerational effects of DNA methylation

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persisting until F3 with an invertebrate, in this case Daphnia 18. This can, in part, be attributed to the

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limited number of studies that have used sequencing approaches to determine DNA methylation

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patterns at the gene level, particularly to study epigenetic mechanisms in an ecotoxicological context

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18-20

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and potential pathways and which mechanisms are susceptible to epigenetic modifications and may

16, 17

.

. Despite the potential use of epigenetics in environmental

16, 17

. The difficulty of demonstrating true transgenerational effects in non-

. Yet, technologies such as whole genome sequencing approaches allow to identify which genes



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prove to be extremely useful in determining the potential transgenerational effects of these

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modifications.

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Here, we focus on the potential of epigenetic mechanisms as response to salinity stress.

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Phenomena such as increased temperature, evaporation in waterbodies, and sea level rising as well

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anthropogenic sources through the run-off of road salt, lead to increased salinity levels in freshwater

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ecosystems 21, 22. Salinity stress is typically reflected in reduced growth rates, development rates and

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fecundity

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parthenogenetic organism, which in laboratory is clonally propagated. This has the specific advantage

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that, for epigenetic studies, epigenetic modifications can be studied independent of genetic variation

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and that potential meiotic transfer of epigenetic marks is not studied 25. Daphnia magna can be found

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in both fresh and brackish water habitats. As far as salinity tolerance is concerned, D. magna is

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deemed euryhaline. A field study across Spain observed Daphnia magna in water bodies with salt

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ranges between 3.8 and 38 g.L-1 26 while Daphnia pulex is reported to tolerate ranges between 0.11

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and 9.95 g/L 27. Laboratory studies have reported acute effects of salinity on Daphnia magna ranging

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between 0.49 to 11.3 g/L 28. Previous research has shown that a short exposure to salinity 29 affected

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global DNA methylation levels in Daphnia magna yet the potential for transgenerational inheritance

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and the underlying molecular mechanisms remain unknown. Here, we explore the potential for

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transgenerational inheritance of DNA methylation patterns until the F3 generation after initial salinity

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exposure as well as the underlying molecular mechanisms. In this way, we can quantify (1)

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methylation changes that have directly arisen in the F0 generation exposed from neonate to adult (F0)

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and (2) methylation changes that are maintained through fully unexposed generations (F0 F3).

23, 24

. We particularly focus on the aquatic crustacean Daphnia. Daphnia is a

. At the same time, this also implies that meiotic recombination is not occurring during reproduction

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Material and Methods

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Daphnia culturing


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Monoclonal cultures of Daphnia magna (clone Beak) have been reared in the laboratory for more

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than 50 generations. Daphnids were cultured in ASTM hard water medium

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comparable to freshwater environments

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organic additive (Ascophyllum nodosum extract) 32. Cultures were maintained under a temperature of

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20 ± 2ºC and a 16hL:8hD photoperiod (provided by cool fluorescent white lights). Culture medium

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was renewed and organisms were fed three times a week, with concentrated suspensions of

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Raphidocelis subcapitata (3 x 105 cells·mL-1), which is cyclically cultured in Woods Hole MBL 33.

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) enriched with vitamins

31

30

(salinity: 636 µS/cm,

and supplemented with an

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Exposure and sampling

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Experiments were conducted under the previously described temperature and photoperiod conditions.

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Six monoclonal cultures of 70 neonates (< 24 h old, collected from the 3rd-5th brood in bulk

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monoclonal cultures) were established in plastic buckets filled with 4 L test solution (57 mL per

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daphnid), three for each control (0 g/L NaCl, i.e. blank ASTM medium) or salinity treatment (4.1 g/L

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NaCl in ASTM medium). The NaCl concentration used in the experiment was determined based on

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the results of a standardized short-term toxicity experiment to study the organisms’ current sensitivity

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to the chemical (Supportive information methods and results, Figure S1); it was set to ensure no

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effects on survival and taking into account earlier salinity studies with the same isolate 28. This is on

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the lower end of the reported values in the field, but in the middle of the range of values reported in

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literature for acute effect concentrations for salinity with Daphnia magna 2. Daphnids exposure lasted

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until the neonates initiating the experiment matured and reached their third brood. The third brood

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was used as it has been shown to lead to more consistent results in multigenerational experiments in

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Daphnia 34. Subsequently, mothers were harvested, pooled per replicate bucket and stored at -80ºC,

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for DNA extraction, immediately after releasing the third brood, always ensuring that their brood

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pouch was empty to avoid extracting DNA originating from eggs. All subsequent generations were

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started with 3rd brood neonates (< 24h old) and maintained in control medium until releasing their

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own 3rd brood, after which mothers were harvested and new generations established with the released 5 ACS Paragon Plus Environment


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neonates (Figure 1). This procedure was repeated for all four generations present in the experimental

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design (Figure 1). Life-history data were documented throughout the experiment. Namely, mortality,

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broods release and offspring number were recorded on a daily basis per bucket, so that age of first

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reproduction, net reproductive rate and per capita rate of population increase, on the basis of the

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Euler-Lotka equation, could be calculated.

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DNA extraction was performed from daphnids’ frozen tissue using the MasterPureTM

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Complete DNA and RNA Purification Kit (Epicentre, Madison, WI, USA), according to

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manufacturer’s instructions. DNA was extracted for all replicates (plastic buckets) for all salinity

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treatments (F0, F1, F2, F3) as well as the original control treatment (F0). The specific hypotheses

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were that (1) methylation changes induced by the F0 salinity treatment can be identified when

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comparing with the F0 control, and (2) maintained for subsequent unexposed salinity generations (F1,

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F2 and F3). While control F1, F2 and F3 treatments would provide interesting additional information

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and would exclude potential confounding factors such as time and generation between the direct

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comparisons F0 control and F1 to F3 exposed generations, the information that they provide is not

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essential to test the hypotheses postulated here. However, these control F1, F2 and F3 treatments were

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maintained to quantify life history effects. A NanoDrop 1000 spectrophotometer (NanoDrop

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Technologies, Wilmington, DE, USA) was used to briefly verify initial DNA quality. Quality criteria

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consisted of 260/230 ratios above 1.7, and 260/280 ratios between 1.8 and 2.1. Concentration of the

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extracted DNA was measured using the 'Quant-it Picogreen dsDNA assay kit' (Thermo Fisher

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Scientific, MA, USA). Genomic DNA was fragmented using MspI, followed by library preparation,

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bisulfite conversion and sequencing using an Illumina NextSeq 500

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protocol is described in supplementary information (supportive information methods and results).

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. The detailed sequencing

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Figure 1. Overview of the experimental design setup for the multi-generation experiment. F0, F1,

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F2, F3 represent generations. Arrows represent 3rd brood offspring. White rectangles represent clean

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medium while grey rectangles represent NaCl dissolved in the culture medium at 4.1 g/L. Plus and

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minus quoting immediately indicate the history of each culture regarding exposure to NaCl (+) and

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maintenance in clean medium (-). Red boxes indicate treatments from which DNA was extracted for

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bisulfite sequencing.

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Bioinformatic analysis

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Quality control of the raw data was executed with FastQC (version 0.11.5, Babraham Bioinformatics).

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Based on these results, the data was trimmed with Trim Galore!, a wrapper tool around Cutadapt and

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FastQC (version 0.4.4, Babraham Bioinformatics). For our data, bases with a quality Phred scale

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score below 30 were trimmed, adapter sequences (Illumina) were removed and RRBS mode was

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activated. Subsequently, Bismark version 0.18.1

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Daphnia magna genome assembly and gene models

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alignment was 64%, which is in line previous studies

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efficiency of bisulfite sequencing data 39. Analysis of the data with Bismark resulted in methylation

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calls for every single cytosine analysed on each strand.

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To determine the false positive rate, the hypothetical methylation rate of the mitochondrial genome

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was calculated. In invertebrates, mitochondrial DNA has been shown to lack methylation 40 and can

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was used for the mapping of our samples. The 37

were used as a reference. The average

19, 38

and attributes to the lower mapping



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therefore be used to evaluate the bisulfite conversion error. Indeed, any hypothetically methylated

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cytosines detected bioinformatically in the unmethylated mitochondrial DNA are the consequence of

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unmethylated cytosines that were not converted to thymines by the bisulfite. This is referred to as the

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bisulfite conversion error. Results showed an average bisulfite conversion error rate of approximately

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0.4% for our samples. To differentiate true positives from false positives, a model based on the

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binomial distribution B(n,p) was used, in which n referred to the coverage depth of each potential mC

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and p to the false positive rate. P-value was adjusted to 0.05 by BH 38, 41, 42. Adjusted P-values < 0.05

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were considered true positives. Afterwards, bedtools intersect was used to annotate the cytosines

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within genic regions. Raw data and processed methylation calls are deposited in NCBI GEO under

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the accession number: GSE107340 (2017). The data are confidential until acceptance for publication

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but can be accessed by reviewers using the following token: upkxieqwvlsvncp.

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Statistical analysis

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Statistics was performed with R 43. These raw data were used to extract potential patterns in age at

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first reproduction, net reproductive rate and per capita rate of population increase (r), calculated based

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on the Euler-Lotka equation. A nested ANOVA was used to quantify main effects on life-history

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traits. T-test was used for extensive two-way comparisons. Normality and homogeneity of variance

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were validated using the Shapiro test and Levene Test respectively. Given the small sample size, t-

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tests comparisons were validated using the non-parametric Kruskal Wallis test. To quantify gene

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methylation levels, we calculated the total number of methylated cytosines in each gene and

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normalised it by the total number of cytosines present in that gene 38, 42. A gene was defined using

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the published Daphnia magna gene models 37 and included both exonic and intronic regions. We used

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the BSmooth algorithm to estimate methylation levels across the different biological replicates and

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to account for differences in coverage across replicates

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methylated genes between treatments was assessed by DMLtest function for cytosines within genic

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regions, from the DSS package using default parameters 45, 46. We used a statistical test to determine

44

. Next, the existence of differentially


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19, 42

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differential methylation of genes between the different treatments

. In brief, a Wald test was

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performed for each gene under the null hypothesis that the means of both treatments were equal.

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Subsequently, genes with statistically significant methylation were highlighted by callDML function

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of the DSS package at a significance level of 0.05. Differentially methylated genes were sorted by

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statistical significance.

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Alternative splicing analysis

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Within NCBI, RNA seq data of an acute salinity exposure experiment with Daphnia magna was

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publically available 19. While the exposure concentration (5 g/L NaCl), exposure duration (4 hours)

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and clone were different, it was the most similar available RNA seq data set. Specifically, we

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extracted the following samples from the SRA archive: SRX1057851, SRX1057821, SRX1057818,

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SRX1057817, SRX1057816, SRX1057815. The reads were mapped to the mRNA sequences of the

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genes with significant differential methylation in three or four generations (Table S5 and Table S7),

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known to have alternative splice variants. The gene models and their alternative splice variants were

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from

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http://arthropods.eugenes.org/EvidentialGene/daphnia/daphnia_magna/Genes/earlyaccess/. Samples

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were aligned using bowtie2 47. Then, for each gene, read counts per exon were analyzed in EdgeR

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according to Asselman et al. 19 to test for differential exon usage. This step was performed using the

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function diffSpliceDGE and it is based on the differences in log-fold changes between exons for the

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same gene. Significant differences were identified at a false discovery rate of 0.01 and indicate

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alternative splicing events.

the

appropriate

gff

annotation

file

(dmagset7finalt9b.puban.mrna)

at

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Results and Discussion

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Life history responses

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At life history level, we observed no main effect of NaCl across all generations for the per capita rate

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of population increase (r), the age at first reproduction (AFR) and the net reproductive rate (P9 ACS Paragon Plus Environment


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values>0.05, Table S1, Figure 2, Figure S2). We did observe a significant difference across

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generations for the net reproductive rate (Table S1), as well as a significant effect of NaCl within a

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single generation on the net reproductive rate (Figure 2). Indeed, a significant decrease in net

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reproductive rate in the F0 generation was recorded, for which animals exposed to salinity had a net

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reproductive rate of 63% relative to the net reproductive rate of the control (p-value t-test = 0.046,

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Table S2). This decline in net reproductive rate did not persist in subsequent unexposed generations.

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In contrast, for the generations originating from the F0 salinity exposed generations, we observed a

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significant increase in net reproductive rate in the F1 generation compared to the F0 generation. This

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increase in reproduction was maintained in the F2 and F3 compared to the F0 generation but did not

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increase further with increasing generations, (p-value t-test F0-F1 = 0.005, Table S2). The net

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reproductive rate was significantly different from the control treatment in the F2 generation, for which

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animals had a net reproductive rate of 133% relative to the net reproductive rate of the control (p-

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value t-test = 0.008, Table S2). In the control treatment, the net reproductive rate did not increase in

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the F1 and F2 generations, but only in the F3 generation (p-value t-test = 0.005, Table S2).

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Figure 2. Net reproductive rate for each generation and each treatment (white bars represent the

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generations propagated by the original F0 control treatment, grey bars represent the generations 10 ACS Paragon Plus Environment

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propagated by the original F0 salinity treatment. All F1, F2 and F3 generations were reared in control

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medium and not exposed to salinity). Error bars highlight standard error and stars denote significant

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effects of the salinity treatments compared to the control treatment within the same generation at a

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significance level of 0.05.

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General methylation responses

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Overall, we observed consistent coverage of reads across the different samples at the gene level

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(Figure S3). We used reduced representation bisulfite sequencing, which targets regions rich in CpGs

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but does not cover the entire genome

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Orsini et al.

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18486 showing zero methylation, i.e. no methylated reads in any replicate in any treatment of which

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less than 10% (1735 genes) was not sampled in all treatments. The remaining 6606 genes which

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showed at least one methylated read in at least 1 replicate in 1 treatment are likely candidates for

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differential methylation. These results are in line with previously reported DNA methylation levels

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in Daphnia and invertebrates in general

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many genes lacking any methylation while a small proportion of genes is methylated.

37

35

. As such, out of the 29,930 gene models as published by

, 4138 or 14% of the gene models were not sampled in any replicate. We observed

38, 48

. This consists of low global methylation levels, with

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For each of the different comparisons, we observed around 100 genes that were differentially

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methylated (Table S3). We observed 105 genes that were differentially methylated between the

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exposed F0 generation and the control of which 52 genes that were uniquely differentially methylated

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between F0 and control (Figure 3, Table S4), indicating a direct exposure effect. No

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overrepresentation of gene families was observed, as the highest number of genes of a single gene

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family within this list was 2 (Table S4). The majority of the uniquely differentially methylated genes

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(39) between the F0 control and F0 salinity showed a decrease in methylation upon exposure to

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salinity (Table S4). This decline or hypomethylation is in line with previous reported results, where

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a decline in global methylation levels was observed for a Daphnia genotype exposed to 5 g/L of NaCl

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for 48 hours 29. At the global level, we observed no significant difference between the methylation 11 ACS Paragon Plus Environment


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levels of F0 control (1.2 ± 0.1 %) and F0 salinity (1.27 ±0.06 %). This may be due to difference in

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salinity concentration, exposure duration or even genotype.

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Figure 3. Venn Diagram overlapping the genes differentially methylated between NaCl treatments

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and the control through the different generations (p

Transgenerational Inheritance of DNA Hypomethylation in Daphnia

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