MARGEN-00423; No of Pages 12

Marine Genomics xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Marine Genomics

Genome Perspectives

Epigenomics in marine fishes

David C.H. Metzger ⁎, Patricia M. Schulte
Department of Zoology, University of British Columbia, Vancouver, BC V6T 1Z4, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Epigenetic mechanisms are an underappreciated and often ignored component of an organism's response to en-
Received 5 January 2016 vironmental change and may underlie many types of phenotypic plasticity. Recent technological advances in
Received in revised form 20 January 2016 methods for detecting epigenetic marks at a whole-genome scale have launched new opportunities for studying
Accepted 21 January 2016
epigenomics in ecologically relevant non-model systems. The study of ecological epigenomics holds great prom-
Available online xxxx
ise to better understand the linkages between genotype, phenotype, and the environment and to explore mech-
Keywords:
anisms of phenotypic plasticity. The many attributes of marine fish species, including their high diversity, variable
DNA methylation life histories, high fecundity, impressive plasticity, and economic value provide unique opportunities for studying
Epigenetics epigenetic mechanisms in an environmental context. To provide a primer on epigenomic research for fish biolo-
Epigenomics gists, we start by describing fundamental aspects of epigenetics, focusing on the most widely studied and most
Environment well understood of the epigenetic marks: DNA methylation. We then describe the techniques that have been
Fishes used to investigate DNA methylation in marine fishes to date and highlight some new techniques that hold
great promise for future studies. Epigenomic research in marine fishes is in its early stages, so we first briefly dis-
cuss what has been learned about the establishment, maintenance, and function of DNA methylation in fishes
from studies in zebrafish and then summarize the studies demonstrating the pervasive effects of the environ-
ment on the epigenomes of marine fishes. We conclude by highlighting the potential for ongoing research on
the epigenomics of marine fishes to reveal critical aspects of the interaction between organisms and their
environments.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction These longer-lasting forms of plasticity can be grouped together

Environmentally-induced phenotypic plasticity is a critical compo-
nent of organismal responses to a changing environment. Understand-
ing the molecular mechanisms underlying this plasticity is an important
question in basic biology that also has important applications in areas as
diverse as aquaculture and environmental monitoring. Much of the
work on the mechanisms of environmentally-induced plasticity in fish-
es has focused on the reversible phenotypic plasticity that is character-
istic of acclimation responses, and this type of plasticity has been a
major focus of studies in marine genomics with many studies using
techniques such as cDNA microarray to examine changes in RNA levels
in response to environmental change (Cossins, 2006; Gracey et al.,
2004; Kalujnaia et al., 2007; Logan and Buckley, 2015). Less well under-
stood are the longer-lasting phenotypic changes that occur in response
to environmental changes that are experienced during development,
a phenomenon known as developmental plasticity (Alvarado et al.,
2015; Pfennig et al., 2010; Scott and Johnston, 2012). Some of these ac-
quired phenotypes can even last for multiple generations, in what has
been termed transgenerational plasticity.
⁎ Corresponding author at: Department of Zoology, University of British Columbia, 6270
University Blvd., Vancouver, British Columbia V6T 1Z4, Canada.
E-mail address: dmetzger@zoology.ubc.ca (D.C.H. Metzger).
under the umbrella term of “epigenetics”. Much of the research on epi-
genetic mechanisms has been performed in model systems (Bird, 2002;
Li and Zhang, 2014), but recent epigenetic studies in ecologically rele-
vant non-model organisms have begun to demonstrate the promise of
epigenetic and epigenomic approaches in fields such as ecology, evolu-
tionary biology and environmental biology (Bonasio, 2015; Bossdorf
et al., 2008; Ledón-Rettig, 2013). Here, we provide a primer for the ap-
plication of epigenetic and epigenomic research in marine fishes.
1.1. What is epigenetics?
Conrad Waddington was first credited with using the term “epige-
netics” in the 1940s to describe the developmental mechanisms that
can give rise to alternative phenotypes (Waddington, 1942), but since
this time there has been almost continuous refinement of this concept
(Deans and Maggert, 2015). More contemporary definitions are intrin-
sically tied to aspects of phenotypic plasticity, or how a single genotype
can produce multiple phenotypes in response to changes in the envi-
ronment. Thus, the broadest definitions of epigenetics include any pro-
cess that results in heritable phenotypic changes such as variation in
gene expression. Several attempts have been made, however, to refine
the definition of epigenetics in a way that distinguishes epigenetic
mechanisms from broader processes that regulate gene expression

http://dx.doi.org/10.1016/j.margen.2016.01.004
1874-7787/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: Metzger, D.C.H., Schulte, P.M., Epigenomics in marine fishes, Mar. Genomics (2016), http://dx.doi.org/10.1016/
j.margen.2016.01.004
2 D.C.H. Metzger, P.M. Schulte / Marine Genomics xxx (2016) xxx–xxx
and phenotypic plasticity. The most widely accepted contemporary def-
initions of epigenetics echo the one put forward by Deans and Maggert
(2015) who define epigenetics as “The study of phenomena and mech-
anisms that cause chromosome-bound, heritable changes to gene
expression that are not dependent on changes to DNA sequence.”
In this context, the word heritability is defined as involving both
meiotic and mitotic inheritance (Wu and Morris, 2001), and thus ac-
cording to these definitions, epigenetic mechanisms need not be
confined to processes that are inherited across generations, but
must at least be inherited across cell divisions. However, not all
epigeneticists subscribe to this definition. For example, the field of
epigenomics (or the study of epigenetics at a whole-genome level)
tends to follow a broader definition that includes additional forms
of phenotypic plasticity. For example, the NIH epigenomic roadmap
program uses the following definition: “epigenetics refers to both
heritable changes in gene activity and expression (in the progeny
of cells or of individuals) and also stable, long-term alterations in
the transcriptional potential of a cell that are not necessarily herita-
ble” (http://www.roadmapepigenomics.org/overview).
Because there is still disagreement about how to most appropriately
define the field of epigenetics, the mechanisms that constitute an epige-
netic signal or process are also controversial (Deans and Maggert, 2015;
Schaefer and Nadeau, 2015). Depending on the author, the list of poten-
tial epigenetic mechanisms may include covalent modification of DNA
through methylation of cytosines (Lister et al., 2009), covalent modifica-
tion of histones (Bannister and Kouzarides, 2011) via acetylation, phos-
phorylation, methylation and other mechanisms, as well as gene
regulation by non-coding RNAs (as microRNAs (miRNA), endogenous
small interfering RNAs (endo-siRNA), and PIWI-interacting RNAs
(piRNA) (Costa, 2008). In this review we focus on DNA methylation be-
cause it was the first epigenetic mark to be discovered, it has become
one of the most widely studied of the potential epigenetic mechanisms,
and it clearly fulfills the criterion of being a chromosome-bound, herita-
ble change that is not dependent on a change to DNA sequence (Deans
and Maggert, 2015).
1.2. DNA methylation as an epigenetic mark
DNA methylation is a covalent modification in which a methyl group
(CH3) is added to position 5 of the pyrimidine ring of a cytosine (5mC).
In eukaryotic cells, the majority of methylated cytosines occur on
cytosine-phosphate-guanine (CpG) dinucleotides resulting in comple-
mentary methylation of CpG motifs on both strands of DNA. Methylated
cytosines can also occur on non-CpG sites such as CpHpG and CpHpH
motifs (where H = A, C or T). However, these epigenetic marks are
fundamentally different from CpG methylation because they are
strand-specific. These strand-specific marks are rare in animals, al-
though they are prominent in the epigenetic landscape of plants (Law
and Jacobsen, 2010).
The addition or removal of methyl groups can regulate the transcrip-
tional activity of neighboring genes by altering the structure and function
of chromatin. High levels of methylation at the promoter of a gene are as-
sociated with repression of transcription (Li and Zhang, 2014). This re-
pression of transcription by promoter methylation is thought to be due
to the formation of inactive heterochromatin (Martinowich et al., 2003;
Razin, 1998). In addition, methylation of enhancer elements can also re-
press transcription by preventing the binding of the transcription factors
that would otherwise induce expression. Alternatively, methylated DNA
can recruit methyl-binding proteins that repress transcription (Klose and
Bird, 2006). DNA methylation of intragenic regions, however, is associ-
ated with active expression of nearby genes (Jjingo et al., 2012;
Zemach et al., 2010) and the regulation of alternative splice variants
(Laurent et al., 2010; Lyko et al., 2010; Park et al., 2011).
There is dramatic variation in the prevalence and patterns of DNA
methylation among taxa (Feng et al., 2010). For example, in Arabidopsis
only ~22% of CpG sites are methylated, while the CpG sites in vertebrate
Please cite this article as: Metzger, D.C.H., Schulte, P.M., Epigenomics in marine fishes, Mar. Genomics (2016), http://dx.doi.org/10.1016/
j.margen.2016.01.004
genomes are generally methylated to high levels (N 80%). Invertebrates
exhibit a wide range of CpG methylation levels, with the “model” inver-
tebrates Drosophila melanogaster and Caenorhabditis elegans essentially
lacking DNA methylation and the honeybee having very low CpG meth-
ylation levels (~.7%; Lyko et al., 2010). Many other invertebrates have
intermediate levels of CpG methylation (around 50%) with a “mosaic”
pattern of methylation such that the genome consists of interspersed re-
gions of methylated and unmethylated DNA of similar length (Feng
et al., 2010). In contrast, vertebrate genomes consist of long tracts in
which the CpG dinucleotides are methylated to high levels (N 80%)
punctuated by short unmethylated regions. These unmethylated re-
gions typically fall in areas called CpG islands (CGI), which have a GC-
rich base composition with a CpG dinucleotide approximately every
10 base pairs. CpG islands tend to be preferentially located in the prox-
imal promoter regions of genes (Fig. 1; Deaton and Bird, 2011), and this
accounts for an observed “dip” in the levels of DNA methylation around
the transcription start sites of actively transcribed genes (Jones, 2012;
Zemach et al., 2010).
There is also epigenetic variation among individuals within a species
in DNA methylation patterns (e.g. Massicotte et al., 2011; Liebl et al.,
2013). This individual-level variation in DNA methylation can be main-
tained throughout the lifespan of an organism and can even be trans-
mitted across generations, providing an added source of heritable
variation that is independent of genetic variation. Thus, the potential
impact of epigenetic variation has become an important topic in the
fields of ecology and evolution (Bossdorf et al., 2008; Ledón-Rettig,
2013), as it has been suggested to be important for evolutionary adapta-
tion to altered environments (Flores et al., 2013).
DNA methylation patterns can also vary within a single individ-
ual across life stages or among cell types (Flanagan et al., 2006;
Massicotte et al., 2011; Richardson, 2003). DNA methylation dur-
ing development is thought to play an important role in cellular
differentiation and is involved in maintaining cell-type specific
transcriptional activity in the genome (Li, 2002; Monk et al., 1987). Var-
iation in DNA methylation patterns that are induced by environmental
factors can alter the developmental trajectory of an individual resulting
in the expression of alternative phenotypes from a single genotype (i.e.
developmental plasticity) (e.g. Wolff et al., 1998; Kucharski et al., 2008;
Shao et al., 2014).
Most of what is known about the mechanisms that establish and
maintain DNA methylation patterns in animals has been derived from
studies in mammals. In mammals, DNA methylation patterns are
established by the de novo DNA methylation activity of the DNA
methyltransferases, DNMT3A and DNMT3B, which add methyl groups
to previously unmethylated cytosines (Law and Jacobsen, 2010). Main-
tenance of DNA methylation across cell divisions is achieved by the ad-
dition of methyl groups to the newly synthesized strand of DNA by the
DNA methyltransferase DNMT1 which is recruited to DNA by the SET-
and RING-associated domain contain protein UHRF1 (Bostick et al.,
Fig. 1. Patterns of CpG island methylation in proximal promoter regions. CpG islands are
regions with high concentrations of CpG motifs. Shore and shelf regions are located
immediately flanking CpG islands. A High level of methylation in a CpG island near the
transcription start site of a gene is associated with inactivation of transcription. Bent
arrow indicates transcription start site; empty circles indicate unmethylated CpG sites;
filled circles indicate methylated CpG sites.
 D.C.H. Metzger, P.M. Schulte / Marine Genomics xxx (2016) xxx–xxx 3

2007; Sharif et al., 2007). Two other genes in the mammalian genome are
related to the described DNA methyltransferase genes but do not possess
DNA methyltransferase activity. DNMT3L is a methylation cofactor that
has been associated with genomic imprinting (Suetake et al., 2004).
Another methyltransferase, DNMT2, predominantly methylates RNA
molecules, specifically tRNA, as opposed to DNA (Goll et al., 2006).
While the establishment of DNA methylation marks in the genome
occurs through active processes, de-methylation can occur via both ac-
tive and passive processes. Passive de-methylation occurs when DNA
methylation is not maintained through DNA replication and cell division
as a result of the lack of DNMT1 activity (Li and Zhang, 2014). Active
de-methylation requires the recruitment of protein complexes that con-
vert methylated cytosines to the methylation intermediates that are
then removed by base excision repair mechanisms. The intermediate
forms of methylated cytosines include 5-hydroxy-methyl cytosine
(5hmC), 5-formyl-cytosine (5fC), and 5-carboxyl-cytosine (5caC) (Shen
et al., 2014). A family of enzymes known as the ten-eleven translocation
(TET) enzymes catalyzes the stepwise oxidation of 5mC to 5fC and then
5caC, which can then be converted to unmethylated cytosines (Shen
et al., 2014). These oxidized derivatives were originally thought to be
transient demethylation intermediates, but recent studies suggest that
these marks are more persistent in the genome than originally thought,
and may have functional significance in their own right (Branco et al.,
2011; Kroeze et al., 2015; Véron and Peters, 2011).
2. Methods for assessing DNA methylation
There are a number of excellent reviews summarizing the major
techniques available for assessing levels of DNA methylation (Harrison
and Parle-McDermott, 2011; Laird, 2010; Plongthongkum et al., 2014;
Shen and Waterland, 2007; Umer and Herceg, 2013). Here we focus
on the techniques that have been used in marine fishes, or that show
considerable promise for application in future studies. We divide these
techniques based on their level of resolution from techniques that pro-
vide low-resolution information about whole-genome methylation
levels to techniques that provide information about methylation status
at the single base-pair level. Each of these approaches has advantages
and disadvantages (Table 1). Choosing the method that is best suited
for a particular study generally comes down to making a complex
trade-off that weighs the cost, the resolution of the assay, and the extent
of the genome that is monitored.
2.1. Low resolution assessment of global DNA methylation
The most quantitative method that can be used to evaluate whole-
genome DNA methylation levels at low resolution is reverse-phase
high performance liquid chromatography (RP-HPLC) (Kuo et al.,
1980). In RP-HPLC, DNA is digested down to single nucleotides, and
since cytosines and methylated cytosines have different molecular
masses, they can be distinguished and quantified by HPLC or liquid
chromatography coupled with tandem mass spectrometry (LC–MS/
MS). Global levels of DNA methylation can also be quantified using
ELISA-based methods. In these methods, purified genomic DNA is
hybridized to assay wells and an antibody specific to methylated cy-
tosines is used to detect methylated DNA. A secondary enzyme-
linked antibody is used for colorimetric quantification. These assays
are available as commercial kits, all of which are suitable for use in
non-model organisms such as marine fishes. These methods provide
a global overview of levels of DNA methylation, which can be useful
for detecting large-scale changes in methylation such as those that
occur during development and tumorigenesis. However, these low-
resolution techniques are unable to detect the many important epi-
genetic changes that occur at specific loci, which do not affect the
overall level of methylation of the genome. Responses to environ-
mental change are likely to involve the upregulation of some genes
and the down regulation of others, which would be predicted to involve
either demethylation or hypermethylation of specific loci. Low resolu-
tion methods, which detect the average change in methylation across
the genome, cannot capture the complexity of changes in DNA methyl-
ation at this scale.

Table 1
Advantages and disadvantages of selected methods for assessing DNA methylation.

Method Resolution Advantages Disadvantages

Reverse phase HPLC Low (whole genome) • No sequence information required • Only very large differences can be detected
(RP-HPLC) • Inexpensive • Very low resolution
ELISA Low (whole genome) • No sequence information required • Only very large differences can be detected
• High throughput • Very low resolution
• Inexpensive
MS-AFLP Low–moderate • No sequence information required • Low resolution
• Inexpensive • Provides +/− information (qualitative)
• Some information about location of meth- • Screens anonymous loci (candidates can be sequenced)
ylated sites can be obtained (if candidate • Reproducibility can be poor
loci sequenced)
Bisulfite sequencing of a candidate Single base pair • All CpG sites in targeted region can be an- • Low coverage of the genome
locus alyzed • Low throughput
• Generates information about location of • Identifying appropriate candidate regions is challenging
methylated sites • Sequence information is required for primer design
• Inexpensive
EpiRADseq Single base pair • High resolution • Expensive
• Quantitative • Requires genomic sequence information
• Genome-wide (~2 million CpG sites ex- • Only examines methylation at HpaII sites
amined per sample; ~6% of total sites)a • Fully methylated sites are not captured
Reduced representation bisulfite Single base pair • High resolution • Expensive
sequencing (RRBS) • Quantitative • Requires genomic sequence information
• Genome-wide (~3 million CpG examined • Only examines sites within ~100 bp of an enzyme rec-
per sample; ~10% of total sites)a ognition site
MeDIP-Seq/MDBSeq 100–300 bp • Fairly high resolution • Expensive
(Single base pair when • Quantitative • Requires genomic sequence information
combined with bisulfite • Genome-wide (~6 million CpG examined • Quantification complex
sequencing) per sample; ~20% of total sites)a
Whole-genome bisulfite Single base pair • High resolution • Extremely expensive
sequencing (WGBS) • Quantitative • Requires sophisticated bioinformatics
• Genome-wide (all CpG sites examined) • Requires assembled genome sequence
a
Based on a 3 Gb genome size.

Please cite this article as: Metzger, D.C.H., Schulte, P.M., Epigenomics in marine fishes, Mar. Genomics (2016), http://dx.doi.org/10.1016/
j.margen.2016.01.004
4 D.C.H. Metzger, P.M. Schulte / Marine Genomics xxx (2016) xxx–xxx
2.2. Methods with intermediate resolution
The most widely-used method for detecting DNA methylation pat-
terns at intermediate resolution is methylation sensitive amplified poly-
morphism analysis (Reyna-López et al., 1997). This method (which goes
by various abbreviations such as MSAP, MS-AFLP etc.) is a modification
of amplified fragment length polymorphism (AFLP) analysis. MS-AFLP
relies on parallel digestion of genomic DNA by methylation-sensitive
and non-methylation sensitive isoschizomeric restriction enzymes
such as HpaII and MspI. The resulting DNA fragments are then ligated
to adaptors and amplified using fluorescently labeled primers. The
resulting PCR products can then be run on a gel or analyzed on a
capillary sequencer. By comparing the banding pattern between DNA
digested with MspI and HpaII it is possible to determine the methylation
state of a particular locus. These differentially methylated bands can
then by isolated and sequenced to identify differentially methylated
genes. In addition, the overall banding patterns can provide information
about variation in methylation patterns among individuals or popula-
tions (e.g. Liebl et al., 2013).
The main advantage of MS-AFLP is that it can be applied in species
where genomic information is limited, and it requires no specialized
equipment other than that found in many labs equipped for molecular
biology. It is also cost-effective, so large numbers of samples can be an-
alyzed. Since only loci of interest need be sequenced, MS-AFLP has the
advantage that sequencing resources are not wasted on regions of the
genome that do not differ in methylation levels among samples. The
most important limitation of MS-AFLP is that the banding patterns pro-
duced can be quite difficult to interpret, because inconsistent restriction
digestion or PCR can easily be confused with changes in methylation
levels. As a result, this method has been challenging to replicate across
labs (Fulneček and Kovařík, 2014). An additional concern for studies
in ecological or evolutionary epigenomics is that genetic variation
among individuals can be confused with epigenetic variation because
differences in banding pattern can be produced by sequence variation
as well as by epigenomic variation. Finally, MS-AFLP treats methylation
as a +/− state and cannot distinguish between (for example) a site that
is 50% methylated and a site that is 100% methylated within a tissue.
These differences in relative levels of methylation could have important
phenotypic consequences, and this type of variation cannot be detected
by MS-AFLP. As a result, MS-AFLP is gradually being replaced with other
methods (Schrey et al., 2013).
2.3. Methods with single base-pair resolution
Most methods for measuring DNA methylation with single base-pair
resolution rely on bisulfite conversion of genomic DNA. When DNA is
treated with sodium bisulfite or metabisulfite, the unmethylated cyto-
sine bases (C) in the DNA are chemically converted into the base uracil
(U), whereas methylated cytosines (5mC) are protected from this bisul-
fite conversion. During subsequent PCR amplification, a thymine
(T) base is incorporated at any position that has been converted to a
U, which changes the original sequence from a C to a T in unmethylated
positions, whereas methylated positions remain as C's (Fig. 2). By com-
paring the bisulfite converted and untreated genomic DNA sequences it
is possible to detect the location of methylated cytosines. Individual
cells within a tissue can vary in whether a particular position is methyl-
ated or not, and the initial DNA isolation step that is the starting point of
bisulfite conversion is usually a sample that contains multiple cells.
Thus, results from these assays provide information on the percentage
of cells that have a methylated cytosine at a particular position.
Bisulfite treatment can be used to investigate methylation levels at a
particular candidate locus. Bisulfite-converted genomic DNA can be am-
plified using primers directed to a particular candidate region of inter-
est, followed by detection of the altered bases. The most commonly
used methods for detecting bisulfite-converted sites are real-time quan-
titative PCR (Eads et al., 2000), cloning followed by Sanger sequencing
Please cite this article as: Metzger, D.C.H., Schulte, P.M., Epigenomics in marine fishes, Mar. Genomics (2016), http://dx.doi.org/10.1016/
j.margen.2016.01.004
of multiple clones (Frommer et al., 1992), methylation sensitive dot
blot assays (MS-DBA)(Clément and Benhattar, 2005) and pyrosequenc-
ing (Dupont et al., 2004).
The primary advantage of taking a single-locus approach is the rela-
tively low cost and the fact that this method is fairly accessible for any
laboratory equipped for molecular biology. The primary disadvantage
of a single-locus approach is the difficulty of identifying appropriate
genomic regions of interest. In addition, sufficient sequence informa-
tion must be available so that appropriate primers can be designed.
Note that designing PCR primers for use on bisulfite-converted
DNA can be challenging, because bisulfite conversion alters the se-
quence of unmethylated cytosines, while leaving methylated cytosines
untouched. Therefore it is important to design the PCR primers away
from any regions that have the potential to be methylated. This is partic-
ularly challenging in species with unsequenced genomes.
The most comprehensive method for assessing patterns of DNA
methylation at single base-pair resolution is bisulfite conversion of ge-
nomic DNA followed by whole-genome shotgun sequencing (whole-
genome bisulfite sequencing WGBS; Laird, 2010). However, the cost of
comprehensive sequencing remains prohibitive for organisms with
large genomes, such as most marine fishes, and thus WGBS is usually
only applied to questions that can be answered using small numbers
of biological replicates. For example, current recommendations are
that 10× genome coverage and at least two biological replicates should
be used at a minimum for bisulfite sequencing (Ziller et al., 2014).
Studies looking to address epigenomic questions in an ecological or evo-
lutionary context (including many questions in the epigenomics of ma-
rine fishes) typically require larger sample sizes, which makes WGBS
prohibitively expensive. As the costs of genome sequencing decrease,
however, WGBS will become more accessible, and this technique is like-
ly to have much wider application. An additional barrier to applying this
method stems from the challenges of the associated bioinformatics,
which can be difficult even in model organisms (Adusumalli et al.,
2015; Bock, 2012) and are a particular barrier in species that lack a se-
quenced genome, such as many marine fishes. However, there has
been some recent development of bioinformatics methods that should
allow the application of these techniques even in the absence of a se-
quenced genome (Bewick et al., 2015).
A variety of methods have been devised to mitigate the high cost of
WGBS. In general, these methods reduce costs by focusing sequencing
effort on the methylated portions of the genome. This enrichment is
performed either using affinity-based or restriction-enzyme based
approaches. In the methods that rely on affinity-based enrichment,
DNA is first immunoprecipitated with an antibody that recognizes
methylated regions of the genome and the immunoprecipitated DNA
is then sequenced. For example, in MeDIPseq (methylated DNA immu-
noprecipitation and sequencing; Borgel et al., 2010) genomic DNA is
first sheared and then immunoprecipitated with an antibody that rec-
ognizes methylated cytosines. This technique is particularly suitable
for use in non-model organisms as the antibody should recognize
methylated cytosines in all genomes, and has been used successfully
in zebrafish (Lee et al., 2015). An alternative technique called MDBseq
(methyl CpG binding domain immunoprecipitation and sequencing;
Aberg et al., 2012) uses an antibody that recognizes the Methyl-CpG-
binding domain protein 2, which is a nuclear protein that binds spe-
cifically to methylated DNA. Because the MDBseq method relies on
antibody recognition of a specific protein, inter-specific variation of
the protein motif could make it difficult to adapt the technique to
non-model species. The primary disadvantage of these techniques
is that in the absence of a bisulfite conversion step, their resolution
is limited to the size of the immunoprecipitated DNA fragments (usual-
ly 100-300 bp).
There are several restriction-enzyme based approaches that enrich
for areas likely to be targets of DNA methylation, including reduced
representation bisulfite sequencing (RRBS; Gu et al., 2010; Jeddeloh
et al., 2008) and EpiRADseq (Schield et al., 2015). These methods
 D.C.H. Metzger, P.M. Schulte / Marine Genomics xxx (2016) xxx–xxx
Fig. 2. Bisulfite sequencing. Treatment of genomic DNA with sodium bisulfite converts unmethylated cytosines (C) into uracil (U), while methylated cytosines are protected. During PCR
and sequencing uracil is converted to thymine (T). Sequence comparison of untreated and bisulfite converted DNA sequences allows the detection of methylated sites.
allow sequencing efforts to be focused on methylated regions, which re-
duces costs relative to WGBS, but still provides single-base pair resolu-
tion detection of methylation status at a subset of locations within the
genome.
In RRBS, genomic DNA is first digested using a methylation-
insensitive restriction enzyme such as MspI that specifically target CpG
motifs. These fragments are then bisulfite treated and sequenced. This
allows sequencing efforts to be concentrated primarily on regions of
the genome that are likely to be methylated (Gu et al., 2010; Jeddeloh
et al., 2008). Although the RRBS technique helps to mitigate the cost
of sequencing per individual, the downstream analysis of RRBS data in
the absence of a reference genome can be complicated. However,
there are currently a variety of tools are available to help perform
these analyses in species without a reference genome (e.g. Chen et al.,
2010; Stockwell et al., 2014).
EpiRADseq (Schield et al., 2015) has recently been developed as an
alternative to RRBS that can be applied in the absence of genome
sequence information. EpiRADseq is a modification of a technique
known as ddRADseq (Peterson et al., 2012), and does not rely on bisul-
fite conversion of genomic DNA, but still provides single base pair reso-
lution of methylated sites. In EpiRADseq genomic DNA is first doubled-
digested with two restriction enzymes that recognize different se-
quences, one of which is methylation insensitive (such as PstI) and
one of which is methylation-sensitive (such as HpaII). Because the
methylation-sensitive restriction enzyme will only cut un-methylated
DNA, sites that are methylated will be under-represented in the
resulting sequences. Therefore, methylation state can be inferred by
comparing the frequencies at which loci are sampled. As a result, this
technique only provides information about methylation state at the en-
zyme cut-sites, and thus provides information at a reduced genome-
coverage relative to RRBS. EpiRADseq can be applied in the absence of
a genome sequence, although the downstream bioinformatics is some-
what more challenging in this case. Proof of principle of the utility of this
method, even in the absence of genomic information, has been demon-
strated using a water flea, Daphnia ambigua (Schield et al., 2015), but to
date the method has not yet been applied in other organisms.
The techniques outlined above are all designed to detect methylated
cytosines (5mC). A variety of other approaches are available for the
detection of demethylation intermediates such as 5hmC and 5fC.
Please cite this article as: Metzger, D.C.H., Schulte, P.M., Epigenomics in marine fishes, Mar. Genomics (2016), http://dx.doi.org/10.1016/
j.margen.2016.01.004
5
5hMeDIP-seq utilizes antibodies to target 5hmC. Other techniques
such as oxidative bisulfite sequencing (oxBS-Seq) (Booth et al., 2012),
which targets 5hmC, and reduced bisulfite sequencing (Booth et al.,
2014), which targets 5fC, rely on modifications of bisulfite sequencing.
To date these techniques have not been applied in any fish species
other than zebrafish.
3. DNA methylation in zebrafish
Much of what is currently understood about mechanisms and pat-
terns of DNA methylation in fish comes from studies conducted in
zebrafish, and there are several excellent reviews that focus on this spe-
cies (Goll and Halpern, 2011; Kamstra et al., 2015). Here, we briefly re-
view what is known about DNA methylation in zebrafish. In the next
section we summarize the relatively few studies that have been per-
formed in marine and diadromous fishes.
3.1. Mechanisms of DNA methylation in zebrafish
As in mammals, the major proteins involved in DNA methylation in
fishes are the DNA methyltransferases (DNMTs). The zebrafish genome
contains 8 DNA methyltransferase orthologs: one dnmt1 ortholog, one
dnmt2 ortholog, two dnmt3a orthologs, and four dnmt3b orthologs, al-
though they lack an ortholog of the mammalian dnmt3L isoform. The
teleosts are known to have undergone a whole-genome duplication rel-
ative to mammals (Jaillon et al., 2004), thus zebrafish might be expected
to have two copies of each mammalian Dnmt family member. This sug-
gests that the duplicates of dnmt1 and dnmt2 were lost during the
rediploidization following the whole genome-duplication event, and
that dnmt3b has undergone an additional round or rounds of duplica-
tion in zebrafish following the teleost whole-genome duplication.
The complex evolutionary history of the dnmt3 gene family has re-
sulted in challenges in the naming conventions for these genes, and
there are several proposed names for the zebrafish dnmt3 genes, as
they were originally named without reference to phylogenetic relation-
ships (Shimoda et al., 2005). Here, we follow the new phylogenetic
naming convention suggested by Campos et al. (2012). dnmt3a1 and
dnmt3a2 in zebrafish (also referred to as dnmt6 and dnmt8) are most
similar to the mammalian dnmt3a gene. dnmt3a1 and dnmt3a2 in
6 D.C.H. Metzger, P.M. Schulte / Marine Genomics xxx (2016) xxx–xxx
zebrafish share a generally similar developmental expression pattern
with the mammalian dnmt3a, further supporting the phylogenetic rela-
tionships among these genes (Smith et al., 2011). The two zebrafish iso-
forms differ in length at the 5′ end by 150 amino acids, whereas in
mouse the single dnmt3a gene is expressed as two isoforms, one short
and one long (Goll and Halpern, 2011).
The zebrafish dnmt3b1, dnmt3b2, dnmt3b3, and dnmt3b4 (also re-
ferred to as dnmt4 dnmt7 dnmt3, and dnmt5 respectively) are most sim-
ilar to the mammalian dnmt3b gene, and share a similar developmental
expression pattern, suggesting that they are related isoforms (Smith
et al., 2011). The fact that zebrafish have four dnmt3b isoforms suggests
duplications in addition to the teleost whole-genome duplication in this
gene for zebrafish. The current model for the evolution of the zebrafish
dnmt3b genes (Campos et al., 2012) is that the teleost whole-genome
duplication produced dnmt3b2 and another dnmt3b paralogue that
then went through two consecutive duplications that resulted in
dnmt3b1, dnmt3b3 and dnmt3b4. Interestingly, the Threespine Stickle-
back genome contains five dnmt3 genes (two dnmt3a homologs and
three dnmt3b homologs). This suggests that at least one of the duplica-
tion events that resulted in the four zebrafish dmnt3b isoforms is
lineage-specific.
The functions of the various dnmt genes in zebrafish remain poorly
understood. Knockdown of dnmt1 has been shown to result in changes
in methylation patterns and developmental abnormalities (Anderson
et al., 2009; Rhee et al., 2012). Expression of dnmt3 genes has been
shown to vary during development and can be affected by developmental
temperature, with a unique pattern for each isoform (Campos et al.,
2012). The lack of a dnmt3L gene in zebrafish is consistent with the re-
duced importance of imprinting in fishes. For example, in contrast to
mammals, homozygous diploid clones are viable in fishes demonstrating
that imprinting must be fundamentally different in fishes compared to
mammals (Streisinger et al., 1981). DNMT3L is also absent from amphib-
ian and bird genomes, suggesting that this gene may have arisen in mam-
mals along with the evolution of X-inactivation (Yokomine et al., 2006).
In addition to the presence of DNA methyltransferases, the zebrafish
genome also contains many of the other genes known to be involved
DNA methylation in mammals such as the CXXC finger protein 1 (cfp1),
methyl binding proteins mecp2 and mbd2, the Piwi-related proteins
ziwi and zili, and the dnmt1 cofactor uhrf1 which is required for the main-
tenance of DNA methylation patterns (Goll and Halpern, 2011).
3.2. Patterns of DNA methylation in zebrafish
As is the case for our understanding of the mechanisms of DNA
methylation in fish, current studies of patterns of DNA methylation
are dominated by work in zebrafish. Overall levels of DNA methylation
appear to be similar among vertebrates, with 80% of CpG dinucleotides
in the larval zebrafish genome being methylated compared to about 74%
in the mouse (Feng et al., 2010). Similarly, as is the case for other verte-
brates, zebrafish genomes have prominent unmethylated CpG islands
located near the transcription start sites of actively transcribed genes.
Differential methylation of distal enhancer elements has also been
shown to play a biologically important role in zebrafish (Lee et al.,
2015). The de-methylation intermediate 5hmC has also been observed
in zebrafish, with the highest levels being observed in brain tissue
(Kamstra et al., 2015). Non-CpG methylation, such as CpHpG and
CpHpH, has also been detected in zebrafish but at low levels (2.13% in
embryos) and these sites are evenly distributed throughout the ge-
nome. By comparison, non-CpG methylation in mouse embryos is
0.59% (Feng et al., 2010).
In mammals, methylation patterns are thought to be globally erased
and reset soon after fertilization. Whether or not this occurs in zebrafish
is not clear. The earliest studies of this phenomenon in zebrafish yielded
contradictory results (Macleod et al., 1999; Mhanni and McGowan,
2004), whereas the most recent work suggests that intermediate levels
of erasure occur (Jiang et al., 2013; Potok et al., 2013). Zebrafish sperm
Please cite this article as: Metzger, D.C.H., Schulte, P.M., Epigenomics in marine fishes, Mar. Genomics (2016), http://dx.doi.org/10.1016/
j.margen.2016.01.004
DNA is hyper-methylated (95%) relative to oocyte DNA (75%). Follow-
ing fertilization, 16-cell embryo DNA methylation levels are intermedi-
ate to those of sperm and oocytes, but whether this is the result of
erasure is unclear (Jiang et al., 2013; Potok et al., 2013). The lack of clar-
ity regarding the level of erasure of methylation may be due, at least in
part, to the asynchronous development of zebrafish oocytes (Kimmel
et al., 1995) and the associated difficulty of obtaining enough develop-
mentally synchronized embryos to obtain the quantity of genomic
DNA necessary for this type of analysis. Perhaps using a fish species in
which more synchronous fertilization can be achieved would allow for
a more detailed analysis of whole genome DNA methylation levels ear-
lier in development.
After the 16 cell stage, whole-embryo DNA methylation levels
increase at each cell division, approaching the methylation levels
observed in sperm DNA by the time the embryo reaches gastrulation.
It has been proposed that patterns of DNA methylation in developing
embryos are reset to the patterns observed in sperm. However, parthe-
nogenic fertilization of zebrafish oocytes results in the same whole-
genome DNA methylation patterns as traditional fertilization (Potok
et al., 2013), suggesting that components within the oocyte provide all
of the information required to establish methylation patterns in the off-
spring. It is possible that the demethylated state of the oocyte is indica-
tive of a more totipotent state of the oocyte compared to the more
differentiated state of the embryo, with increased whole genome meth-
ylation levels. Alternatively, piRNAs could survive the UV irradiation
treatment of sperm DNA prior to gynogenic fertilization acting as the
primary regulators of DNA methylation reprogramming to a paternal
pattern (Potok et al., 2013). Whether the changes in methylation levels
and patterns during development are conserved across fishes or are
unique to zebrafish remains relatively unexplored.
4. Studies of DNA methylation in marine fishes
Although DNA methylation has been studied for several decades in
zebrafish (e.g. Macleod et al., 1999), much less is known about DNA
methylation in marine fishes. Database searches for “DNA methylation”
and “fish” yielded less than 30 studies in any marine or diadromous fish
species (Table S1). The earliest studies that include data on marine fish-
es focussed on the identification of DNA methylation in fish genomes
and/or broad-scale taxonomic comparisons (Bird and Taggart, 1980;
Kato, 1996; Vanyushin et al., 1970, 1973). In addition, an early compar-
ative study using medaka (Oryzias latipes) and the Atlantic Killifish
(Fundulus heteroclitus) examined levels of methylation of an introduced
transgene (Winn et al., 1995), and demonstrated that this transgenic
DNA was methylated. More recent studies have focussed on changes
in DNA methylation in response to environmental factors or during
development.
4.1. Environmental contaminants
Some of the earliest studies of changes in DNA methylation associat-
ed with environmental factors in marine fishes examined the effect of
environmental toxins (Aniagu et al., 2008; Timme-Laragy et al., 2005;
Wang et al., 2009), and toxicology has continued to be an important
focus of DNA methylation studies in fishes (Pierron et al., 2014). These
studies span a range of organisms, pollutants, and methods. The first
toxicological study of DNA methylation in a marine fish used single-
locus bisulfite sequencing to examine DNA methylation in the cyp1a
promoter of the Atlantic Killifish (F. heteroclitus) from creosote contam-
inated and reference sites, and found no detectable DNA methylation in
the cyp1a promoter region (Timme-Laragy et al., 2005). This study illus-
trates one of the risks of using candidate approaches to studying DNA
methylation. These authors focussed on the proximal promoter region
of an important candidate gene in the detoxification pathway for organ-
ic contaminants. The promoter of this gene was known to be CpG-rich,
and thus contained many potential methylation sites. However, cyp1a
 D.C.H. Metzger, P.M. Schulte / Marine Genomics xxx (2016) xxx–xxx
is expressed at detectable levels under control conditions in killifish tis-
sues, and is induced by contaminant exposure (Meyer et al., 2003),
which suggests that it is an actively transcribed gene under a variety
of environmental conditions. CpG rich regions (CpG islands) located
immediately upstream of the transcription start sites of actively tran-
scribed genes typically have low levels of methylation, and may not un-
dergo dynamic changes in DNA methylation, which may not make them
ideal candidate regions to explore without some a priori indication of
differential methylation.
Several studies have demonstrated that whole-genome DNA
methylation levels change in response to toxicant exposure in ways
that can be detected using low-resolution approaches (Aniagu et al.,
2008; Mirbahai et al., 2011; Pierron et al., 2014; Wang et al., 2009). In
Threespine Stickleback (Gasterosteus aculeatus) there were significant
changes in whole-genome DNA methylation levels in gonad tissue fol-
lowing environmental exposure to estradiol (E2) as detected by HPLC,
but no changes in response to hexa-bromo-cyclododecane (HBCD)
exposure in liver (Aniagu et al., 2008). HPLC-based methods were
also used to examine the effects of contaminant exposure in a rock-
fish (Sebastiscus marmoratus). In this case, there were significant
concentration-dependent changes in whole-genome DNA methyla-
tion levels in liver tissue following environmental exposure to tribu-
tyltin and triphenyltin in Wang et al. (2009). Results from these
studies illustrate both the potential and the limitations of low-
resolution DNA methylation assays in that important DNA methyla-
tion changes could be occurring at targeted locations that would not
be detected at the whole-genome level, making negative results diffi-
cult to interpret.
Studies using low-resolution whole-genome assays followed by
intermediate-resolution methods and targeted or whole-genome se-
quencing to detect differentially methylated loci have yielded additional
insights into the effects of environmental toxicants on the epigenome.
For example, exposure of European Eel (Anguilla anguilla) to environ-
mental cadmium resulted in a dose-dependent increase in whole-
genome methylation levels using a low-resolution ELISA method
(Pierron et al., 2014). These authors then identified multiple genes
that were differentially methylated using MS-AFLP. Similarly, tumori-
genesis has been shown to have an epigenomic component in the Com-
mon Dab (Limanda limanda) (Mirbahai et al., 2011). These authors first
used an HPLC-based approach to detect a reduction in whole-genome
methylation levels in tumor tissues. They then used MeDIP-seq to spe-
cifically identify differentially methylated genomic regions at single
base-pair resolution, which were then correlated with changes in gene
expression. Many of the differentially methylated and expressed genes
were in pathways known to be associated with tumorigenesis in other
organisms.
4.2. Temperature
Temperature has profound effects on the biology of fishes, and there
have been a few studies investigating its effects on the epigenome of
marine fishes (Campos et al., 2013; Shao et al., 2014; Varriale and
Bernardi, 2006). A particularly intriguing study (Varriale and Bernardi,
2006) found that whole-genome methylation levels (as assessed using
RP-HPLC) were inversely correlated with temperature, with the ge-
nomes of fishes from cooler habitats exhibiting higher levels of
whole-genome DNA methylation compared to fish from warmer
habitats. Several subsequent studies have also identified tempera-
ture as an important factor that may modify methylation levels in
marine fishes (Campos et al., 2013; Shao et al., 2014). For example,
temperature has been shown to affect methylation levels in the pro-
moter of the myogenin gene in Senegalese Sole (Solea senegalensis), as
detected using candidate-gene bisulfite sequencing (Campos et al.,
2013). These changes were correlated with changes in myogenin
expression and muscle cellularity, clearly demonstrating the linkages
between changes in DNA methylation and important organismal
Please cite this article as: Metzger, D.C.H., Schulte, P.M., Epigenomics in marine fishes, Mar. Genomics (2016), http://dx.doi.org/10.1016/
j.margen.2016.01.004
7
phenotypes. These results also illustrate the important point that al-
though proximal promoter regions tend to be hypomethylated relative
to other regions of the genome, changes in methylation status in the
promoter at specific loci can be important.
4.3. Development and growth
It has been well documented in mammalian systems that whole-
genome DNA methylation patterns undergo dramatic changes during
development and cellular differentiation. As briefly summarized
above, much of what is known about changes in methylation patterns
during development is derived from work in zebrafish (e.g. Jiang et al.,
2013; Potok et al., 2013). However, two studies have examined changes
in DNA methylation with development in marine fishes. For example,
early maturation in Atlantic Salmon (Salmo salar) is associated with
changes in DNA methylation patterns as detected using MS-AFLP
(Morán and Pérez-Figueroa, 2011). Observed differences DNA methyla-
tion associated with early maturation were consistent across two popu-
lations in testis tissue, whereas methylation differences in the brain
associated with early maturation were observed in only one of two pop-
ulations studied. In contrast, no differences in DNA methylation pat-
terns associated with early maturation were observed in liver tissue.
The role of DNA methylation in metamorphosis has been explored in
Sea Lamprey (Petromyzon marinus). Larval lampreys (ammocoetes) are
filter feeders that burrow into streambeds. During the larval stage, lam-
preys go through a series of metamorphoses until the final transforma-
tion into a tissue-feeding adult that migrates to the marine environment
(Manzon et al., 2015). There are substantial differences in whole-
genome methylation levels in the muscle tissue of larval and adult lam-
preys as detected by MS-AFLP (Covelo-Soto et al., 2015a, 2015b). These
authors then sequenced eleven of the differentially methylated bands,
and were able to identify six of these bands, including several hox
genes, which are known to be involved in metamorphosis.
Changes in DNA methylation may also play a role in the regulation of
growth during development. A study conducted by Si et al. (2015) in
Half-Smooth Tongue Sole (Cynoglossus semilaevis) used bisulfite treat-
ment followed by sequencing of a specific locus to examine the effect
of genotype on methylation level and gene expression of pituitary ade-
nylate cyclase activating polypeptide (pacap). In this study, the authors
examine three pacap genotypes that are known to differ in growth and
show that DNA methylation levels and gene expression vary significant-
ly between the three pacap alleles suggesting that there may be an epi-
genetic component to genotype-mediated differences in growth rates.
Changes in DNA methylation may also play a role in establishing var-
iation in life-history traits. For example, a recent study (Baerwald et al.,
2015) used RRBS to examine variation in methylation patterns associat-
ed with variation in the timing of smoltification and the propensity to
migrate in Rainbow Trout (Oncorhynchus mykiss). This study took ad-
vantage of the existence of double-haploid clonal lines of resident and
anadromous Rainbow Trout to produce double-haploid F2 siblings
with varying genomic combinations of parental migratory and resident
DNA. These F2s were used to identify fifty-seven differentially methylat-
ed regions that correlated with smoltification, including multiple genes
in pathways associated with circadian rhythms. Over half of the differ-
entially methylated regions were in or near CpG islands associated
with proximal promoters, suggesting that they could be functionally
important and affect gene expression. These data demonstrate the
power of RRBS for the detection of differential methylation patterns as-
sociated with ecologically important traits in marine fishes.
4.4. Sex determination
Fishes provide an excellent model for the study of sex-determination
mechanisms, as they exhibit a wide range of sex-determination strategies,
including both genetic sex-determination and environmental sex deter-
mination (Devlin and Nagahama, 2002). While the mechanisms of sex
8 D.C.H. Metzger, P.M. Schulte / Marine Genomics xxx (2016) xxx–xxx
determination are beyond the scope of this review, the studies conducted
to date suggest an important role of epigenetics in sex-determination in
several species.
The first study to associate changes in DNA methylation patterns
with sex determination in a marine fish was conducted in the
European Sea Bass (Dicentrarchus labrax). In this study Navarro-
Martín et al. (2011) used a targeted bisulfite sequencing approach
to show that the methylation patterns of CpG locations in the pro-
moter of the cyp19a gene are hypermethylated in male gonad tissue
compared to female gonad tissue. cyp19a is an aromatase gene that
converts androgen into estrogen. Hypermethylation in male gonad
tissue is therefore consistent with reduced expression of cyp19a
and a reduction in the synthesis of estrogen in male gonad tissue. Inter-
estingly, exposure of European Sea Bass larvae to elevated temperatures
resulted in increased methylation of the cyp19a promoter and a mascu-
linization of female gonad tissue.
The role of epigenetics in sex determination has also been investigat-
ed in the Half-Smooth Tongue Sole (C. semilaevis), which undergoes
environmentally induced changes in sex ratios following exposure to
elevated temperatures during development. This species has a ZZ/ZW
sex determination system in which it has been previously demonstrated
that male-specific expression of the Z-linked dmrt1 gene is associated
with male gonad development (Chen et al., 2014). Shao et al. (2014)
used WGBS to determine whole-genome DNA methylation patterns in
male, female, and pseudo-male gonads. They found that genes associated
with sex-determination pathways are differentially methylated between
male and female gonad tissue. Specifically, the authors discovered that
the dmrt1 gene has high levels of DNA methylation in female gonads,
whereas ZZ males and ZW pseudo-males were hypomethylated at the
dmrt1 locus suggesting that demethylation of dmrt1 is necessary for
male gonad development. Interestingly, although ZW pseudo-males can
be produced by exposing ZW females to high environmental tempera-
tures, pseudo-males can also give rise to ZW pseudo-male offspring in
the absence of a high-temperature environmental cue (Chen et al.,
2014). This suggests that there may be a transgenerational epigenetic
mechanism of sex determination in this species.
A study by Wen et al. (2014) measured DNA methylation and gene
expression levels of both cyp19a and dmrt1 in Japanese Flounder
(Paralichthys olivaceus). They found that high levels of dmrt1 expression
in male gonad tissue are correlated with low levels of DNA methylation
in the dmrt1 promoter, whereas low levels of expression are correlated
with high levels of DNA methylation. In contrast, the promoter of
cyp19a was hypermethylated in testes compared to ovaries, which
corresponded to lower levels of cyp19a mRNA in testis tissue compared
to ovaries. Subsequent studies in Japanese Flounder have observed sim-
ilar patterns in which DNA methylation patterns of cyp19a1a and foxl2
are inversely correlated with mRNA expression levels during ovarian
development (Si et al., 2016). Taken together, these studies demon-
strate that the methylation of the dmrt1 and cyp19a genes is important
for sex determination in a variety of fish species.
4.5. Morphological variation
There has been considerable interest in the potential role of epige-
netic mechanisms in causing intra-specific morphological variation
(Bonasio, 2015). There is a single study examining this question in a
species with marine populations: the Threespine Stickleback (Smith
et al., 2015).The Threespine Stickleback species complex contains ma-
rine, anadromous, and freshwater ecotypes that differ in many morpho-
logical and physiological characteristics, including variation in the
extent of bony lateral plates (Bell and Foster, 1994). Smith et al.
(2015) used RRBS to characterize whole-genome DNA methylation pat-
terns in muscle tissue from low plated and high plated stickleback
morphotypes from a single population. Using this method, a variety of
differentially methylated regions were identified, some of which were
associated with genes involved in biological processes that have
Please cite this article as: Metzger, D.C.H., Schulte, P.M., Epigenomics in marine fishes, Mar. Genomics (2016), http://dx.doi.org/10.1016/
j.margen.2016.01.004
previously been shown to differ between morphotypes. Because these
analyses were performed on wild-caught individuals, it is not possible
to determine whether the observed differences in methylation were a
cause or consequence of the morphological differences between the
ecotypes or whether they may have been caused by environment-
dependent effects.
4.6. Aquaculture
Artificial triploidization is commonly used in aquaculture settings to
alter the growth and fertility of fishes (Piferrer et al., 2009). Using MS-
AFLP, Covelo-Soto et al. (2015a) measured whole genome DNA methyl-
ation levels in the brain, gill, heart, liver, kidney and muscle tissue of
diploid and triploid Brown Trout, (Salmo trutta L.) to test whether
changes to whole-genome DNA methylation levels are important for
the maintenance of polyploidy in fish. Although they did not find signif-
icant differences between any of the diploid and triploid tissues at the
whole genome level there were intriguing patterns at a small number
of individual loci. Thus, it is possible that compensatory epigenetic
mechanisms may still play a role in the proper genomic function of
polyploidy in fish but that these changes could not be reliably detected
at the level of resolution provided by MS-AFLP.
Exposure to differing environments during early life has been
shown to cause persistent changes in DNA methylation patterns in
mammals (Bird, 2002). These observations have motivated interest
in examining whether the effects of hatchery rearing on perfor-
mance could be due to epigenetic effects (Taylor et al., 2010). No differ-
ences in MS-AFLP banding patterns were detected between returning
wild and hatchery-reared adult steelhead Trout (O. mykiss). However,
MS-AFLP is unable to resolve quantitative differences in the percent
DNA methylation at any given site. Thus, it is impossible to rule out
the possibility differences in DNA methylation between wild and
hatchery-reared fish from these data.
The role of changes in DNA methylation has also been examined in
hatchery-reared Brown Trout (S. trutta L.) (Morán et al., 2013). Using
MS-AFLP, these authors observed substantial differences in DNA
methylation patterns in the gill between fish held in fresh water and
fish acclimated to seawater for 10–12 days. These data clearly show
that environmental salinity can have a profound effect on DNA methyl-
ation in fish. In addition, these authors examined the role of the salt con-
tent of the diet in altering DNA methylation levels, as high-salt diets
have been shown to be important in the transformation of the gill to
saltwater phenotype (Perry et al., 2006). There were transient changes
in DNA methylation in the gills of fish fed a high-salt diet that made
their methylation patterns somewhat more similar to fish held in sea-
water than those of fish in freshwater that were fed the control diet
(in at least on one of the two principle component axes). These data
suggest that diet-induced epigenetic modifications may be important
for the transformation of the gill during salinity acclimation.
Geay et al. (2012) used a candidate-locus approach to examine
the effects of diets differing in their levels of highly unsaturated
fatty acids (HUFA) on methylation levels in the promoter of the
Delta(6) desaturase (fads2) gene in European Sea Bass (D. labrax). The
promoter of the fads2 gene was methylated at very low levels, and
there was no significant change in DNA methylation with diet. This il-
lustrates the general principle that the DNA methylation status of the
promoters of highly expressed genes tends to be maintained at consti-
tutively low levels. However, the expression of the fads2 gene was ob-
served to increase in Sea Bass fed a low HUFA diet, illustrating that
changes in gene expression can occur in the absence of changes in
methylation at the proximal promoter of a gene.
5. The future of epigenomics research in marine fishes
The studies presented here clearly demonstrate the potential for
epigenomic research in marine fishes. The continued development of
 D.C.H. Metzger, P.M. Schulte / Marine Genomics xxx (2016) xxx–xxx
methods that can be applied to non-model systems is making address-
ing these epigenomic questions increasingly tractable. As sequencing
technologies continue to develop and the cost of whole genome sequenc-
ing decreases, we expect that high-resolution epigenomic studies in ma-
rine fishes will become increasingly common. Below we provide some
concluding remarks regarding the utility of fish as model systems for
epigenomic research, methodological considerations regarding applying
epigenomic techniques in marine fishes, and some particularly promising
areas of research where epigenomic methods could be applied.
5.1. Fish as model systems for epigenomics
Fish have many useful characteristics that make them attractive
model systems for addressing questions in evolutionary biology, embry-
ology, and physiology (Cossins and Crawford, 2005; Powers, 1989).
Many of these characteristics also make fish particularly suitable
model systems for epigenomic research (Kamstra et al., 2015). For
example, one of the key questions in epigenomics is to understand the
transgenerational effects of environmental factors on organismal phe-
notypes, and whether and how methylation patterns can be transmitted
across generations. In mammals, an environmentally-induced pheno-
type must be maintained at least to the F3 generation to convincingly
demonstrate a true epigenetic effect. This is because exposure of the
gestating female (F0) simultaneously exposes the developing embryo
(F1) and the developing germ line of the embryo (F2). In contrast, in
fish only the mother (FO) and her eggs (F1) are exposed. Thus, in fish
it is possible to conclusively demonstrate an epigenetic effect by the
F2 generation. This, in combination with the rapid generation time of
many species of fish makes them a compelling model for demonstrating
transgenerational effects. Another key question in epigenomics is the
role of early developmental environment on the phenotype of the
offspring and the mechanisms underlying developmental plasticity. Be-
cause many fishes develop externally, it is relatively straightforward to
manipulate the environment during development, especially in com-
parison to the challenges of these approaches in mammals. In addition,
many species have large clutch sizes, which allows for full-sib compar-
isons by using a split-clutch design in which multiple members of the
same family are exposed to different treatments. Similarly, a split clutch
design can be used to generate multiple half-sib families, which could
help to disentangle paternal and maternal effects.
In addition, many of the unique biological features of fish make them
an interesting model of the investigation of the role of epigenomics in a
variety of fundamental biological processes. For example, fish demon-
strate a wide range of sex-determining mechanisms, including both ge-
netic and environmental sex-determination, but the underlying
mechanisms remain poorly understood (Devlin and Nagahama, 2002).
Recent studies suggest that there may be an epigenomic component to
sex-determination in some species (Navarro-Martín et al., 2011; Shao
et al., 2014), highlighting the potential for research in this area. Similarly,
polyploidization is common in fish, and induction of triploidy is routinely
used in aquaculture. Thus fish provide a vertebrate model for the study of
the role of epigenomics in dosage compensation in polyploids (Pala et al.,
2008).
5.2. Unique features of epigenomics in fishes
Although fish and mammals share many common features of their
methylation patterns, which are distinct from the patterns in other
major taxonomic groups such as the invertebrates, fungi and plants
(Zemach et al., 2010), there are also fundamental differences in methyla-
tion patterns and mechanisms between fish and mammals (Goll and
Halpern, 2011). For example, genomic imprinting is thought to be absent
in fish, and they lack the dnmt3L gene that is involved in mammalian im-
printing. However, recent data suggest that sex-specific methylation may
occur in fish (Jiang et al., 2013; Potok et al., 2013; Shao et al., 2014), but
the mechanisms involved remain unknown. Similarly, fish and mammals
Please cite this article as: Metzger, D.C.H., Schulte, P.M., Epigenomics in marine fishes, Mar. Genomics (2016), http://dx.doi.org/10.1016/
j.margen.2016.01.004
9
appear to differ in the extent to which methylation patterns are erased
early in development, with current studies suggesting that fish retain a
higher proportion of methylated sites (Jiang et al., 2013; Potok et al.,
2013). In mammals, the high degree of erasure of methylated sites has
been used to argue against the role of DNA methylation in transmitting
transgenerational epigenetic information. This difference highlights the
potential importance of pursuing studies of transgenerational epigenet-
ic inheritance of DNA methylation patterns in fish. Fish genomes also
contain additional DNA methyltransferase genes not present in the ge-
nomes of other organisms. Whether these genes facilitate a unique
functional role of DNA methylation, and the evolution of this gene fam-
ily in fish remains to be studied.
5.3. Methodological considerations
Throughout this review we have highlighted the particular strengths
and weaknesses associated with the various techniques that can be used
to measure DNA methylation in marine fishes. Low-resolution tech-
niques such as ELISA-based detection of whole-genome methylation
levels are easy to apply and can be useful for the identification of large-
scale changes in methylation levels. However, most questions require
higher-resolution approaches. Although MS-AFLP provides only inter-
mediate resolution, this technique can be fairly easily applied in any lab-
oratory that is equipped for molecular biology, and access to a reference
genome and sophisticated bioinformatics expertise is not required. As a
result, MS-AFLP can be an appropriate choice for preliminary studies or
for laboratories that are beginning to address epigenomic questions.
MS-AFLP can be particularly useful for screening to identify candidate
differentially methylated loci for further examination.
The current gold-standard for characterizing DNA methylation levels
at high resolution is the application of bisulfite sequencing, which can
be applied to single candidate loci, subsets of the whole genome (via
RRBS), or as WGBS. Bisulfite sequencing of candidate loci is cost-
effective and straightforward, but is critically dependent on the appropri-
ate choice of candidate gene. This can be difficult in the absence of prior
information suggesting that a genomic region is likely to be differentially
methylated. Application of MS-AFLP to identify promising candidates
followed by bisulfite sequencing may provide a good balance of the
strengths of these two techniques. In contrast, WGBS provides complete
information about the methylation status of every cytosine in the ge-
nome, but this technique remains prohibitively expensive for many
studies. RRBS provides a good alternative that captures a large subset
of the methylated regions of the genome at a more reasonable cost.
Similarly, the recently developed non-bisulfite-dependent method of
EpiRADseq represents another approach to increase the tractability of
high-resolution epigenomic techniques. The development of these
reduced-representation sequencing techniques opens exciting new
possibilities for studies of epigenomics in marine fishes.
One key challenge for epigenomic studies of DNA methylation is that
these studies examine correlations between changes in methylation
patterns and phenotypic changes, which does not directly establish cau-
sation. To demonstrate that changes in DNA methylation levels are
causing the observed changes in phenotype requires direct functional
assays such as mutagenesis of the putatively important methylated
sites. New technologies such as the CRISPR/Cas9 endonuclease system
(Hsu et al., 2014) should allow these causal links to be established.
This technique has been proposed to allow targeted genetic manipula-
tion of essentially any species and has already been successfully applied
in a marine fish species (Aluru et al., 2015).
5.4. Research on epigenomics in aquaculture
Studies of diet (Geay et al., 2012; Morán et al., 2013), the effects of
hatchery rearing (Taylor et al., 2010), triploidization (Covelo-Soto et al.,
2015a, 2015b), and early maturation (Morán and Pérez-Figueroa, 2011)
demonstrate the enormous potential for the application of epigenomics
10 D.C.H. Metzger, P.M. Schulte / Marine Genomics xxx (2016) xxx–xxx
in aquaculture. Many other aspects of aquaculture practice may have
epigenomic effects including disease exposure, stress, density, water
quality, none of which have yet been investigated. Determining whether
exposure to such conditions has persistent effects the phenotype of
cultured fishes could have important implications for improving the
yield or efficiency of aquaculture operations.
5.5. Research in environmental biology and evolution
Epigenomic changes have the potential to play an important role in
the response of marine fishes to environmental change, and may influ-
ence adaptation to new environments (Franks and Hoffmann, 2012).
These questions are of increasing importance in the context of global
environmental change and other human impacts on the marine envi-
ronment. For example, as discussed above, multiple studies have con-
vincingly shown that the DNA methylation patterns of marine fishes
change following exposure to environmental toxicants, highlighting
an underappreciated impact of environmental toxicants on organismal
biology. However, many of the studies to date have utilized low-
resolution methods, and thus the specific biological processes and path-
ways that are being affected remain unknown. Continued investigation
into the environmental effects on DNA methylation using higher resolu-
tion methods and assessment of the persistent consequences of these
changes is urgently needed to fully appreciate and predict the effects
of toxicant exposure and other environmental changes at the individual,
population, and ecological levels. A long-term goal of research efforts in
this area would be to establish DNA methylation-based assays as an
indicator of the effects of anthropogenic activities, and incorporate
this information into the risk-assessment process (Ray et al., 2014).
Multiple studies have shown that epigenetic mechanisms may be
important in the colonization of new environments and the process of
adaptation to environmental change (e.g. Schrey et al., 2012). For exam-
ple, variation in DNA methylation patterns could be an important source
of inter-individual and inter-population variation on which natural
selection could act (Massicotte et al., 2011). At the present time these
questions have not been addressed in any marine fish species, but
there is substantial scope for experimentation, and addressing these
questions may be particularly important as we attempt to predict the
responses of commercially important marine fishes to climate change
(Franks and Hoffmann, 2012).
6. Conclusions
The studies outlined above clearly demonstrate the pervasive effects
of the environment on the epigenomes of marine fishes, and indicate
the potential for ongoing epigenomic research to provide an increased
understanding of the responses of fishes to environmental change.
The integration of the various “omics” approaches, including tran-
scriptomics, proteomics, genomics, and epigenomics holds particular
promise in linking genotype, phenotype and environment and dissect-
ing the mechanisms underlying genotype by environment interactions.
Studies of these phenomena in marine fishes will provide new insights
that will increase our understanding of epigenomic phenomena in nat-
ural environments.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.margen.2016.01.004.
Acknowledgments
This work was supported through a Natural Sciences and Engineer-
ing Research Council of Canada (NSERC) Discovery Grant to PMS and a
University of British Columbia PhD Fellowship to DCHM. Thanks to the
participants in the symposium “Genome-Powered Perspectives in
Integrative Physiology and Evolutionary Biology” for their insightful
discussions and to the Society for Experimental Biology for supporting
this session.
Please cite this article as: Metzger, D.C.H., Schulte, P.M., Epigenomics in marine fishes, Mar. Genomics (2016), http://dx.doi.org/10.1016/
j.margen.2016.01.004
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