Daphnia as an Emerging Epigenetic Model Organism

Abstract

Daphnia offer a variety of benefits for the study of epigenetics. Daphnia's parthenogenetic life cycle allows the study of epigenetic effects in the absence of confounding genetic differences. Sex determination and sexual reproduction are epigenetically determined as are several other well-studied alternate phenotypes that arise in response to environmental stressors. Additionally, there is a large body of ecological literature available, recently complemented by the genome sequence of one species and transgenic technology. DNA methylation has been shown to be altered in response to toxicants and heavy metals, although investigation of other epigenetic mechanisms is only beginning. More thorough studies on DNA methylation as well as investigation of histone modifications and RNAi in sex determination and predator-induced defenses using this ecologically and evolutionarily important organism will contribute to our understanding of epigenetics.

Full text

Hindawi Publishing Corporation
Genetics Research International
Volume 2012, Article ID 147892, 8 pages
doi:10.1155/2012/147892
Review A rticle
Daphnia as an Emerging Epigenetic Model Organism
Kami D. M. Harris, Nicholas J. Bartlett, and Vett K. Lloyd
Department of Biology, Mount Allison University, 63B York Street, Sackville, NB, Canada E4L 1G7
Correspondence should be addressed to Vett K. Lloyd, vlloyd@mta.ca
Received 23 August 2011; Accepted 25 October 2011
Academic Editor: Jennifer Brisson
Copyright © 2012 Kami D. M. Harris et al. This is an open acce ss article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Daphnia offer a variety of benefits for the study of epigenetics. Daphnia’s parthenogenetic life cycle allows the study of epigenetic
effects in the absence of confounding genetic differences. Sex determination and sexual reproduction are epigenetically determined
as are several other well-studied alternate phenotypes that arise in response to environmental stressors. Additionally, there is
a large body of ecological literature available, recently complemented by the genome sequence of one species and transgenic
technology. DNA methylation has been shown to be altered in response to toxicants and heavy metals, although investigation
of other epigenetic mechanisms is only beginning. More thorough studies on DNA methylation as well as investigation of histone
modifications and RNAi in sex determination and predator-induced defenses using this ecologically and evolutionarily important
organism w ill contribute to our understanding of epigenetics.
1. Introduction
The unusual life cycle of the freshwater microcrustacean,
Daphnia, has been studied for more than 150 years [1]. Most
species are cyclic parthenogens able to produce two types of
eggs, diploid parthenogenetic eggs or haploid sexual egg s,
in response to environmental cues [2, 3]. Sex determination
is likewise environmentally controlled; males are produced
in response to suitable environmental cues [3]. Additionally,
Daphnia exhibit a range of spectacular polyphenisms, phe-
notypic alternations including helmet and neckteeth forma-
tion, in response to predators [4, 5]. This makes Daphnia
an excellent candidate for studying environmental influences
on epigenetic developmental programs. Most importantly
in the context of epigenetics, clonal lines are genetically
identical yet consist of phenotypically divergent individuals.
This offers a unique opportunity to separate genetic and
epigenetic influences on the phenotype, an invaluable asset
when studying epigenetics. The attractiveness of Daphnia as
a potential epigenetic model organism is further enhanced by
the fact that they are easy and inexpensive to maintain and
have a rapid life cycle. As a primary consumer and a food
source for invertebrates and fish [6], there is an extensive
body of literature on their ecological role, de velopment, and
the evolution of parthenogenesis. Thus, Daphnia is an ec-
ologically important organism well-studied in the con-
text of evolution, ecology, ecotoxicology, predator-induced
polyphenisms, and genomics [7, 8]andoffers unparalleled
opportunities to study epigenetics in these biologically im-
portant processes.
Epigenetics is the study of mitotically or meiotically her-
itable changes in phenotypes that occur without changes in
the DNA sequence [9]. Altered gene expression can be caused
by DNA methylation, histone modifications, and RNA in-
terference as well as other, less well-studied, epigenetic mech-
anisms such as variant histones, nucleosome phasing, higher-
order chromatin structures, and nuclear localization [4, 9].
DNA methylation, p erformed by either de novo or main-
tenance DNA methyltransferases, has been associated with
transcriptional regulation, chromosome inactivation, and
transposable element regulation, among other funct ions
[10]. Although DNA methylation is found in a wide variety
of eukaryotes, the amount of methylation and its org ani-
zation within the genome differ dramatically between species
and developmental stages [4]. DNA methylation interacts
with other epigenetic processes [11]. Modifications to the
amino- or carboxyl-terminal histone tails affect the inter-
actions of histones with DNA, other histones, and other

2 Genetics Research International
chromatin-associated proteins [12]. These modifications
are performed by specialized enzymes and include acety-
lation, ubiquitination, sumoylation, phosphorylation, and
methylation, all of which can alter gene expression [12].
DNA methyltransferases and histone modifying enzymes can
recruit each other by way of a mutual attraction to the
modifications imposed by the other [11]. DNA methylation
and histone modifications also interact with the RNA
silencing system [13]. The RNA silencing system operates
through the production of small noncoding RNA molecules
(ncRNA) and is referred to as RNA interference (RNAi).
Small RNAs, microRNA (miRNA) and short interfering RNA
(siRNA) are excised from larger double-stranded molecules
can form RNA-induced silencing complexes (RISC) that
target complementary nucleic acid sequences and recruit or
activate DNA methyltransferases and histone modifying en-
zymes [14].
Epigenetic marks are modified by external environmental
factors such as nutrition and exposure to chemicals, as well
as developmental cues [15]; these epigenetic alterations can
enhance the cell and org anism’s ability to respond to its
environment and thrive [16]. DNA methylation, histone
modifications, and RNAi are all mitotically transmissible.
Additionally, as epigenetic changes can be adaptive, selec-
tion for meiotic transmission might be expected to allow
epigenetic information to be passed between generations [4].
Such transgenerational inheritance has been documented in
Arabidopsis [17], mice [18], Drosophila [19], and humans
[20, 21] and is postulated in Daphnia [16]. However, identi-
fication of transgenerational effects can be problematic when
the embryo undergoes development in the mother’s body, as
is the case in Daphnia. In such situations, maternal exposure
to environmental factors could affect the offspring either
by retention of maternal epigenetic states in the germ line
cells that give rise to the embryo, a true transgenerational
effect, or more simply by exposure of the somatic cells of the
embryo while it is in the mother. To resolve this ambiguity,
the persistence of the trait needs to be monitored in the F3
and subsequent generations, those which were not exposed
as either the embryos that produce the F1 or the embryonic
germ line that produce the F2.
Spurred by the use of Daphnia as a subject of ecological
and developmental research, numerous techniques have been
developed that can equally enhance its use in epigenetic stud-
ies. Conventional cytological methods have been employed
[22] and more recently these have been extended to include
fluorescence in situ hybridization (FISH) [ 23]. This could
allow examination of higher-order chromatin structures that
have been associated with the epigenetic status of genome
regionsinotheranimals[24]. Recently Daphnia pulex was
the first crustacean to have its genome sequenced, revealing
the largest number of genes yet found in a single organism,
yet present in a remarkably compact genome [25]. The large
numberofgenesisduetoaveryhighrateoftandemgene
duplication events, and approximately 30% of the genes
are unique to Daphnia [25]. The availability of the genome
sequence allows for the development of microarrays for
genome-wide transcriptional studies [26]. Daphnia embryos
are transparent and can develop independently of the
mother, and as a result embryogenesis of Daphnia has been
well documented [2,
27, 28]. With the genomic sequence
available, conventional embryology can be extended to look
at specific gene products. Methods for in situ immuno-
hybridization and immunohistology have been developed
so the tissue- and developmental-specific localization of
RNAs and proteins can be examined [29]. In the context
of epigenetics, this approach could be used to detect devel-
opmental and tissue-specific histone modifications. While
there are no immortalized cell lines currently available for
Daphnia, methods for primary culture have been developed
[30]. These cells are viable for at least one week and can
be transformed to study the role of overexpression of en-
dogenous or foreign genes [30]. More recently, Kato et al.
[31, 32] showed that it is possible to insert double-stranded
RNA to reduce the expression of genes by RNAi-based gene
knockdowns. The same technique can be used to over-
express selected genes [33]. Knockdown of specific genes
encoding for DNA methyltransferases, histone modifying
enzymes, and their interacting proteins would allow for
an assessment of the role of DNA methylation, histone
modification, and related epigenetic processes correlated
with the well-defined phenot ypes that arise from epigenetic
alterations.
2. The
Daphnia
Life Cycle and Epigenetic
Phenotypic Variation
2.1. The Daphnia Life Cycle. Most Daphnia can reproduce
either asexually or sexually, depending on environmental
cues. In both cases, eggs are produced by stem cells in the
ovary [2]. In sexual eggs, meiosis is conventional and the
haploid oocytes are fertilized. Parthenogenetic oocytes un-
dergo only the equational meiotic division and so remain
diploid and embryogenesis occurs without fertilization. Early
embryogenesis commences as the egg matures on route to the
brood pouch. Sexually produced eggs are typically produced
in pairs, arrest in the blastula stage in the brood pouch, and
the carapace overlying the brood pouch is modified into a
tough, desiccation-resistant structure called the ephippium,
which allows the eggs to survive harsh environmental con-
ditions [2]. Parthenogenetic eggs complete embryogenesis in
the brood pouch and are released as miniature versions of the
adult [2]. Once hatched, the neonates typically undergo four
to six larval instars, depending on species, before reaching
reproductive maturity (Figure 1)[7, 38].
2.2. Epigenetic Regulation of the Life Cycle. Epigenetic
changes in gene expression can modify an organism’s pheno-
type and these changes are particularly obvious when there
are no genetic differences between individuals of any one
strain. Sensitivity of the epigenome to environmental cues
occurs at different stages of the Daphnia life cycle. In general,
the embryonic stages appear important for establishing the
epigenetic states of genes involved in phenotypic variation,
whereas exposure to environmental cues in the postembry-
onic larval stages is important for maintaining the epigenetic
state (Figure 1).

Genetics Research International 3
Sex determinism
Kairomone sensitive period
for establishment of
helmets and neckteeth
Initiation of neckteeth
development
Kairomone sensitive
period for
maintenance of
helmets and neckteeth
4–6 instars
Daphnia life cycle
Brood pouch
Adult
Meiosis occurs
in ovaries
Parthenogenetic
versus sexual
Embryogenesis
Figure 1: The Daphnia life cycle. The life cycle is shown with the stages at which the epigenome is sensitive to the environmental inputs that
regulates sexual reproduction, sex determination, helmets, and neckteeth (indicated in red).
The production of sexual versus asexual eggs is environ-
mentally cued by environmental factors such as photoperiod,
temperature, food abundance, and crowding [3]. In sexual
eggs meiosis is conventional whereas asexual partheno-
genetic eggs arise from an abortive first meiotic division,
resulting in diploid eggs able to initiate development in the
absence of fertilization [2]. In parthenogenetic eggs the first
division is abortive; however, many of the same meiotic genes
are expressed in parthenogenetic as in sexual reproduction
[39] and bivalents are produced [40]. Nevertheless, genes
suppressing recombination are overrepresented in the Daph-
nia genome relative to those promoting recombination [39],
chiasmata are not observed [40] and genetic evidence of
recombination has not been observed [41]. Thus, barring
rare conversion, mitotic recombination or mutation events
[42] parthenogenetic progeny are genetically identical. Since
the ovary can simultaneously contain parthenogenetic and
sexual eggs [2], the cues must act during the first meiotic
division, as the oocytes form (Figure 1). How these environ-
mental signals are interpreted and the molecular mechanism
by which meiosis is regulated, remains unknown. The pro-
duction of males is triggered by similar environmental cues
as sexual egg production [3]; however, the control of male
sex determination is independent of the regulation of female
meiosis [2, 43]. Males are produced in either mixed or,
more typically, all-male broods [3, 44, 45] and at least in
some species can emerge from fertilized sexual ephippial eggs
[46]. Despite obv ious morphological differences—males
being smaller, having testes, modified appendages, and
carapace—all parthenogenetic offspring , male or female,
and their mothers, are genetically identical. The mechanism
of sex determination is thus clearly environmental and
epigenetic. As juvenile hormone analogs induce males even
in the absence of environmental cues, this suggests envi-
ronmental cues are transduced by the endocrine system
[33, 45]. Based on the production of intersexes in D. magna
and D. longispina, induced by altered temperature or inter-
mediate hormone concentrations, respectively, the determi-
native events in sex determination have been shown to act
in oocyte maturation before the first embryonic division
[44, 45]. Interestingly, Sanford [44] shows that intersex
progeny are produced in broods long after the mother has
been moved from the inducing conditions. This underscores
the epigenetic nature of sex determination and might rep-
resent an example of transgenerational inheritance, but
could equally reflect the early developmental action of the
sex determination process. Many genes show differential
expression between males and females [47], including the
core sex-determination gene, doublesex, that is expressed at
higher levels during embryogenesis in males than in females
[33]. This suggests that, in Daphnia, environmental sex de-
termination arose by imposing environmentally mediated

4 Genetics Research International
regulation on the conserved doublesex genetic sex determi-
nation pathway. Identification of differences in the epigenetic
status of the doublesex gene in males and females would fur-
ther our understanding of environmental sex determination
and the role of epigenetics in such a key aspect of the life
cycle.
3. Epigenetic Regulation of Helmet Formation
Predators are an important aspect of an organism’s envi-
ronment, and various predator-induced defenses, such as
helmets, have been well documented in Daphnia [16]. Hel-
mets are cranial extensions of the exoskeleton that have been
shown to decrease the daphnids’ chances of predation [48].
Helmet growth is induced by kairomones, w hich are aquatic
chemicals released by predators [48]. Circulating kairomones
can double the relative helmet size in some daphnids [49].
Agrawal et al. [ 16] have shown that kairomones induce
helmet growth in Daphnia cucullata both in the genera-
tion exposed to the kairomones and in their nonexposed
progeny (Ta ble 1). Newly hatched animals were exposed
to kairomone-containing water, or control non-kairomone
water and the size of helmets were monitored. Additionally,
females were exposed and successive broods of their progeny
were monitored for helmet production to detect transgener-
ational effects.
Exposure of neonates to kairomones induced helmet
formation and removal of kairomones reduced helmet size
[16]. This shows that kairomones act directly during early
larval stages to promote helmet growth. Interestingly, when
mothers were exposed, helmets were present in their neonate
progeny, even if the progeny were not exposed [16]. Helmet
formation in the neonates foll owing only maternal exposure,
could arise either from a transgenerational effect, transmis-
sion of the altered maternal epigenome to the F1 progeny via
the oocyte, or, as the embryos are brooded in the mother,
sensitivity of the embryonic somatic cells to kairomones.
The latter is suggested by the fact that final helmet size is
diminished in successive broods, which would have been
younger, with fewer somatic cells, at the time of exposure,
and that the F2 was not strongly influenced by grand-
maternal exposure [16]. This finding also implies that late
embryonic stages are more sensitive than earlier ones.
The effect of kairomone exposure on helmet size was
cumulative; the largest helmets were obtained when b oth the
mother and the neonates were exposed [16]. This additive
effect indicates that both stages are sensitive. The possibility
of different epigenetic events contributing to cuticular
growth during embryonic and larval stages is suggested by
similar studies on neckteeth formation (see below) [48].
Growth of the helmet is accomplished by mitotic division of
diploid epidermal cells, thought to be triggered by sig-
nals from adjacent polyploid epidermal cells [50]. It is
possible that kairomone exposure during late embryonic
stages induces cell fate changes producing more polyploid
cells whereas kairomone exposure during the larval stages
increases the mitogenic activity of these polyploid cells.
4. Epigenetic Regulation of
Neckteeth Formation
Another common predator-induced defense is exhibited by
several species, including Daphnia pulex. In the presence
of kairomones produced by Chaoborus (phantom midge)
larvae, Daphnia pulex produces structures known as neck-
teeth, small protrusions on the neck region accompanied by
a strengthened carapace [48, 51]. Daphnids that have these
outgrowths have a higher predator escape rate, presumably
due to the thickened exoskeleton that makes handling and
consumption more difficult [48]. Development of the neck-
teeth begins in the first larval instar and growth continues
until the third instar [52]. Withdrawal of the predatory cue
at the first, second, or third instar reduces the number of
neckteeth at successive instars [52]. Thus, the maintenance
of epigenetic mar ks on the genes controlling the growth of
neckteeth requires kairomone exposure in the larval stages
[52]. However, Miyakawa et al. [51] were able to show
that production of neckteeth involves at least two additional
critical stages in late embryonic development. Few or no
neckteeth form when kairomones are absent during embryo-
genesis, even if kairomones are present during the postem-
bryonic larval stages [50]. Thus, as for helmet formation,
embryonic exposure appears to be required to establish cell
fates, while larval exposure is required to maintain and
express the phenotype. Differential Display 1 (DD1)isagene
identified as having altered expression in the embryonic stage
in kairomone-exposed daphnids [51]. It is proposed that
DD1 plays a role in kairomone reception and/or cell fate
determination that establishes the epigenetic state of target
genes leading to the formation of neckteeth [51]. Multiple
endocrine and morphogenetic genes, such as Hox3, exd,
JHAMT, Met, InR, IRS-1, DD1, DD2, and DD3, were shown
to be upregulated in the exposed postembryonic larvae [51].
The Hox gene upregulated in kairomone-exposed daphnids
encodes a transcription factor associated with chromatin
[53]. The exd and met gene products can similarly act as
transcr iption factors and potentially alter the epigenetic
status of downstream genes [54, 55]. Thus, the upregulation
of these genes supports the conclusion that the maintenance
and growth of neckteeth production is a result of epigenetic
changes.
5. Epigenetic Regulation of Growth
In much the same way that external environmental cues
such as kairomones can affect the development of helmets
and neckteeth, environmental toxicants can affect the body
length and growth in Daphnia magna [34]. Again, as the ani-
mals are all genetically identical, differences between exposed
and nonexposed animals must be epigenetic. Among many
others, 5-azacytidine, genistein, biochanin A, vinclozolin,
and zinc, all of which can alter DNA methylation, were
shown to have an effect on body length (Ta ble 1)[34, 56].
This growth effect, however, was transient as it was only
seen in 7-day-old animals but not adults [34]. Additionally,
zinc exposure significantly reduced body length of 6-day-old
animals in the untreated F1 generation [56]. This finding

Genetics Research International 5
Table 1: Epigenetic assay systems.
Assay system Species F0 treatment F0 effects F1 F2 Reference
Helmets
D. cucullata Kairomones n.d. Increase Increase [16]
D. cucullata Kairomones Increase n.d. n.d. [29]
D. lumholtzi Kairomones Increase n.d. n.d. [29]
D. ambigua Kairomones Increase n.d. n.d. [29]
Neckteeth D. pulex Kairomones Increase n.d. n.d. [29]
Growth D. magna
5-azacytidine
Decrease
(day 7 only)
Decrease
Decrease
(day 7 only)
[34]
Genistein Decrease n.s. n.s. [34]
Vinclozolin Decrease n.s. n.s. [34]
Zinc
Decrease
(day 6 only)
Decrease
(day 6 only)
n.s. [35]
Reproduction D. magna
5-azacytidine Decrease Decrease n.s. [34]
Genistein Decrease n.s. n.s. [34]
Vinclozolin n.s. n.s. n.s. [34]
Zinc Decrease n.s. n.s. [36]
Global DNA
methylation
D. magna
Zinc n.s. Decrease Increase [37]
5-azacytidine Decrease Decrease Decrease [34]
Genistein n.s. n.s. n.s. [34]
Vinclozolin Decrease n.s. Decrease [34]
Data summarized here is for a treated F0 generation with subsequent generations untreated. n.s. denotes nonsignificant results. n.d. denotes that those trials
were not done.
might be an indication of a transgenerationally heritable
effect but as it did not persist to the F2 generation, it is more
likely the result of embryonic exposure (Ta ble 1).
6. Epigenetic Regulation of Fertility
Fertility was also shown to be affected by chemical treatment.
While vinclozolin exposure had no significant effect, 5-
azacytidine, 5-aza-2

-deoxycytidine, genistein, biochanin A,
and cadmium all reduced reproduction in surviving females,
in comparison to nonexposed females (Ta ble 1)[34, 57].
Zinc exposure was found to have complex effec ts; exposure
decreased reproductive success in the F0, but not in the
subsequent F1 and F2 generations when these were raised
in control medium (Tabl e 1)[36]. When animals were con-
tinuously exposed to zinc, reproduction was reduced in
the F0 and F1 but not the F2 [36]. These results were
interpreted as an acclimation effect [36], which would be
interesting; however, this conclusion would be strengthened
by results from a larger number of reproducing females and
corroborating molecular data.
The effects of chemical exposure occurred in genetically
identical individuals and in some cases were heritable be-
tween generations, suggesting that the phenotypic variability
is epigenetic. This possibility is reinforced by the fact that
some of these chemicals have been shown to alter DNA
methylation [34].
7. Epigenetic Mechanisms—DNA Methylation
The role of epigenetic mechanisms such as DNA methyla-
tion, histone modification, and RNAi in normal Daphnia
development and the epigenetic adaptations described above
is still in its infancy. Vandegehuchte et al. [57] were the
first to determine that D. magna is capable of methylating
DNA. They found genes homologous to the three main
human DNA methyltransferases and confirmed that DNA
methylation occurred. Through the use of ultraperformance
liquid chromatography (UPLC) and microarrays, Vandege-
huchte et al. [34] examined the DNA methylation and
transcription levels, respectively, in D. magna exposed to
various chemicals. They measured direct effects on methy-
lation in the exposed generation as well as the effects in
subsequent generations (Ta ble 1). Global or localized DNA
methylation levels were found to be affected by 5-azacytidine,
vinclozolin, genistein, and zinc but were not affected by 5-
aza-2

-deoxycytidine, biochanin A, and cadmium [34, 57].
5-azacytidine is known to hinder DNA methylation in
humans by inhibiting DNA methyltransferases and, consist-
ent with this, D. magna treated with 5-azacytidine showed a
decrease in global DNA methylation [ 34, 58]. Interestingly,
the untreated offspring of 5-azacytidine exposed daphnid
mothers also showed decreased methylation when compared
to nonexposed daphnids of the same generation (Ta ble 1).
Vandegehuchte et al. [34] interpreted this as a transgenera-
tional effect; however, the F1 were exposed to the toxicant as
embryos, a time shown to be sensitive to epigenetic pertur-
bations in many animals [20, 59, 60] including Daphnia
[51] so these results are more likely due to exposure of the
F1 as embryos rather than a true transgenerational effect.
Conclusive evidence for a transgenerational effect would be
the persistence of the effect into nonexposed generations be-
yond the F2, a result not observed in this series of experi-
ments. The sensitivity of this experiment and confirmation

6 Genetics Research International
of any transgener ational effectswouldbeenhancedbyexam-
ination of gene-specific epigenetic alterations as opposed
to global DNA methylation levels, and monitoring changes
persisting to the F3 and subsequent generations.
In comparison to nonexposed daphnids, when the F0
was exposed to zinc, there was decreased methylation of the
F0 and F1 generations followed by a significant increase in
the F2 generation (Tabl e 1 )[37]. Vandegehuchte et al. [37]
attributed the increase in the third generation to acclimation.
While possible, this explanation cannot be confirmed until
the study is repeated with a larger sample size. Additionally,
as age affects DNA methylation levels in Daphnia [37] the
age of the daphnids would have to be tightly controlled.
Treatment with vinclozolin showed a significant decrease in
DNA methylation in D. magna in the F0 and F1 exposed gen-
erations; however, the F2 showed a nonsignificant increase
in overall methylation levels [34]. This implies that while the
fungicide v inclozolin does alter DNA methylation, evidence
for a transgenerational effect is still lacking. Unusual results
were seen with genistein treatment. In mammals, genistein
causes global DNA hypomethylation [61]butinD. magna it
yielded hypermethylated DNA [34]. This confounding result
could be attributed to differences in genomic organization
between mammals and daphnids, the possibility exists that
the sequences that are hypermethylated in the much larger
daphnid genome do not exist in humans.
The microarray platform used for these studies was origi-
nally designed for investigation of developmentally regulated
genes and allowed monitoring of only a subset of those
genes, so it was not ideal for global transcription assessment
[36]. Until the D. magna genome is ful ly sequenced and a
more complete microarray can be employed, it would be
preferable to monitor specific genes or to use a species with
a fully sequenced genome, such as D. pulex. Additionally,
bisulfite sequencing, methylated DNA immunoprecipitation
(meDip), or DNA methylation sensitive restriction enzyme
digests, which allow monitoring of the methylation status
of individual genes would be more biologically informative.
Candidate genes include those that are involved in repro-
duction and growth since brood size and body length is
affected by toxicant exposure in D. magna [34, 36, 56, 57],
sex determination [47], as well as those involved in helmet
and neckteeth formation [16, 51].
8. Conclusion
Daphnia have the potential to be invaluable animals for
epigenetic study. They are already well-studied in the context
of their important ecological and evolutionary roles, as well
as being readily available and inexpensive to maintain. The
ability of Daphnia to produce clones parthenogenetically
allows for the elimination of genetic var iability, a valuable re-
source in the study of epigenetics. Obvious phenotypic assay
systems such as sexual reproduction, helmets, neckteeth,
growth, and fertility allow correlations to be made between
such phenotypic responses and the epigenetic changes that
accompany them (Tabl e 1 ). Further, potential tr ansgener-
ational effects in the production of polyphenisms, inter-
sex individuals, and other epigenetically determined states
remain to be explored [44, 45]. There are also many classical
and molecular tools available for use in studying epigenetics
in Daphnia.
The next steps in establishing Daphnia as an epigenetic
model organism will be to determine the genetic and
epigenetic mechanisms responsible for the establishment and
maintenance of phenotypic responses to the environment
such as sexual reproduction, helmets, and neckteeth. It is also
essential to extend the research on epigenetic mechanisms
to include histone modifications, RNAi, and further define
the baseline levels and changes in DNA methylation in re-
sponse to environmental stimuli throughout development.
Documenting the epigenetic differences between sexual and
asexual Daphnia and stressed and unstressed individuals
would prove a fruitful area of research with important
implications for evolutionary and developmental biology.
Authors’ Contribution
All authors contributed to the writing of this review. Kami D.
M. Harris and Nicholas J. Bartlett contributed equally.
Acknowledgments
This paper was supported by a Natural Sciences and Engi-
neering Research Council grant to V. K. Lloyd T he authors
would like to thank M. J. Beaton and the anonymous review-
ers for discussion and comments on this paper.
References
[1] J. Lubbock, “An account of the two methods of reproduction
in Daphnia, and of the structure of ephippium,” Philosophical
Transactions of the Royal Society, vol. 57, pp. 79–100, 1857.
[2] F. Zaffagnini, “Reproduction in Daphnia,” in Daphnia,R.H.
Peters and R. De Bernardi, Eds., vol. 45 of Memorie dellIstituto
Italiano di Idrobiologia, pp. 245–284, 1987.
[3] O. T. Kleiven, P. Larsson, and A. Hobaek, “Sexual reproduction
in Daphnia magna requires three stimuli,” Oikos, vol. 65, no. 2,
pp. 197–206, 1992.
[4] M. B. Vandegehuchte and C.R. Janssen, “Epigenetics and
its implications for ecotoxicology,” Ecotoxicology, vol. 20, pp.
607–624, 2011.
[5] L. J. Weider and J. Pijanowska, “Plasticity of Daphnia life
histories in response to chemical cues from predators,” Oikos,
vol. 67, no. 3, pp. 385–392, 1993.
[6] J. K. Colbourne, P. D. N. Hebert, and D. J. Taylor, “Evolu-
tionar y origins of phenotypic diversity in Daphnia,” in
Molecular Evolution and Adaptive Radiation, T. J. Givnish and
K. J. Sytsma, Eds., pp. 163–188, Cambridge University Press,
Cambridge, UK, 1997.
[7] A. Stollewerk, “The water flea Daphnia—a “new” model sys-
tem for ecology and evolution?” Journal of Biology, vol. 9, no.
2, article 21, 2010.
[8] D. Ebert, Ecology, Epidemiology, and Evolution of Parasitism in
Daphnia, National Library of Medicine (US), National Center
for Biotechnology Information, Bethesda, Md, USA, 2005,
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db
=Books/.
[9] R. Jaenisch and A. Bird, “Epigenetic regulation of gene expres-
sion: how the genome integrates intrinsic and environmental
signals,” Nature Genetics, vol. 33, pp. 245–254, 2003.

Genetics Research International 7
[10] K. F. Santos, T. N. Mazzola, and H. F. Carvalho, “The prima
donna of epigenetics: the regulation of gene expression by
DNA methylation,” Brazilian Journal of Medical and Biological
Research, vol. 38, no. 10, pp. 1531–1541, 2005.
[11] F. Fuks, “DNA methylation and histone modifications: team-
ing up to silence genes,” Current Opinion in Genetics and
Development, vol. 15, no. 5, pp. 490–495, 2005.
[12] A. Lennartsson and K. Ekwall, “Histone modification patterns
and epigenetic codes,” Biochimica et Biophysica Acta, vol. 1790,
no. 9, pp. 863–868, 2009.
[13] Z. Lippman and R. Martienssen, “The role of RNA interfer-
ence in heterochromatic silencing,” Nature, vol. 431, no. 7006,
pp. 364–370, 2004.
[14] R. W. Carthew and E. J. Sontheimer, “Origins and Mechanisms
of miRNAs and siRNAs,” Cell, vol. 136, no. 4, pp. 642–655,
2009.
[15] S. M. Reamon-Buettner, V. Mutschler, and J. Borlak, “The next
innovation cycle in toxicogenomics: environmental epigenet-
ics,” Mutation Research, vol. 659, no. 1-2, pp. 158–165, 2008.
[16] A. A. Agrawal, C. Laforsch, and R. Tollrian, “Transgenerational
induction of defences in animals and plants,” Nature, vol. 401,
no. 6748, pp. 60–63, 1999.
[17] F. Johannes, E. Porcher, F. K. Teixeira et al., “Assessing the
impact of transgenerational epigenetic variation on complex
traits,” PLoS Genetics, vol. 5, no. 6, Article ID e1000530, 2009.
[18] J. E. Cropley, C. M. Suter, K. B. Beckman, and D. I. K. Martin,
“Germ-line epigenetic modification of the murine Avy allele
by nutritional supplementation,” Proceedings of the National
Academy of Sciences of the United States of America, vol. 103,
no. 46, pp. 17308–17312, 2006.
[19] Y. Xing, S. Shi, L. Le, C. A. Lee, L. Silver- Morse, and W. X.
Li, “Evidence for transgenerational transmission of epigenetic
tumor susceptibility in Drosophila,” PLoS Genetics, vol. 3, no.
9, pp. 1598–1606, 2007.
[20] N. A. Youngson and E. Whitelaw, “Transgenerational epige-
netic effects,” Annual Review of Genomics and Human Gene tics,
vol. 9, pp. 233–257, 2008.
[21] D. K. Morgan and E. Whitelaw, “The case for transgeneration-
al epigenetic inheritance in humans,” Mammalian Genome,
vol. 19, no. 6, pp. 394–397, 2008.
[22] Y. Ojima, “A cytological study on the development and matu-
ration of the parthenogenetic and sezual eggs of Daphnia pulex
(Crustacea-Cladocera),” Kwansei Gakuin Unic Ann Studies,
vol. 6, pp. 123–176, 1958.
[23] D. Tsuchiya, B. D. Eads, and M. E. Zolan, “Methods for
meiotic chromosome preparation, immunofluorescence, and
fluorescence in situ hybridization in Daphnia pulex,” Methods
in Molecular Biology, vol. 558, pp. 235–249, 2009.
[24] T. Cremer and C. Cremer, “Chromosome terri tories, nuclear
architecture and gene regulation in mammalian cells,” Nature
Reviews Genetics, vol. 2, no. 4, pp. 292–301, 2001.
[25] J. K. Colbourne, M. E. Pfrender, D. Gilbert et al., “The
ecoresponsive genome of Daphnia pulex,” Science, vol. 331, no.
6017, pp. 555–561, 2011.
[26]A.Soetaert,K.vanderVen,L.N.Moens,T.Vandenbrouck,
P.vanRemortel,andW.M.DeCoen,“Daphnia magna
and ecotoxicogenomics: gene expression profiles of the anti-
ecdysteroidal fungicide fenar imol using energy-, molting- and
life stage-related cDNA libraries,” Chemosphere, vol. 67, no. 1,
pp. 60–71, 2007.
[27] V. Obreshkove and A.W. Fraser, “Growth and differentiation
of Daphnia magna eggs in vitro,” Biological Bulletin , vol. 78,
pp. 428–436, 1940.
[28] C. Laforsch and R. Tollrian, “Embryological aspects of
inducible morphological defenses in Daphnia,” Journal of
Morphology, vol. 262, no. 3, pp. 701–707, 2004.
[29] K. Sagawa, H. Yamagata, and Y. Shiga, “Exploring embryonic
germ line development in the water flea, Daphnia magna,by
zinc-finger-containing VASA as a marker,” Gene Expression
Patterns, vol. 5, no. 5, pp. 669–678, 2005.
[30] C. D. Robinson, S. Lourido, S. P. Whelan, J. L. Dudycha,
M. Lynch, and S. Isern, “Viral transgenesis of embryonic cell
cultures from the freshwater microcrustacean Daphnia,” Jour-
nal of Experimental Zoology, vol. 305, no. 1, pp. 62–67, 2006.
[31] Y. Kato, K. Kobayashi, H. Watanabe, and T. Iguchi, “Intro-
duction of foreign DNA into the water flea, Daphnia magna,
by electroporation,” Ecotoxicology, vol. 19, no. 3, pp. 589–592,
2010.
[32] Y. Kato, Y. Shiga, K. Kobayashi et al., “Development of an RNA
interference method in the cladoceran crustacean Daphnia
magna,” Development Genes and Evolution, vol. 220, no. 11-12,
pp. 337–345, 2011.
[33] Y. Kato, K. Kobayashi, H. Watanabe, and T. Iguchi, “Envi-
ronmental sex determination in the branchiopod crustacean
Daphnia magna: deep conservation of a Doublesex gene in the
sex-determining pathway,” PLoS Genetics, vol. 7, no. 3, Article
ID e1001345, 2011.
[34] M. B. Vandegehuchte, F. Lemi
`
ere, L. Vanhaecke, W. Vanden
Berghe, and C. R. Janssen, “Direct and transgenerational
impact on Daphnia magna of chemicals with a known
effect on DNA methylation,” Comparative Biochemistry and
Physiology, vol. 151, no. 3, pp. 278–285, 2010.
[35] M. B. Vandegehuchte, D. De Coninck, T. Vandenbrouck, W.
M. De Coen, and C. R. Janssen, “Gene transcription profiles,
global DNA methylation and potential transgenerational
epigenetic effects related to Zn exposure history in Daphnia
magna,” Environmental Pollution, vol. 158, no. 10, pp. 3323–
3329, 2010.
[36] M. B. Vandegehuchte, T. Vandenbrouck, D. D. Coninck, W. M.
De Coen, and C. R. Janssen, “Can metal stress induce trans-
ferable changes in gene transcription in Daphnia magna?”
Aquatic Toxicology, vol. 97, no. 3, pp. 188–195, 2010.
[37] M. B. Vandegehuchte, F. Lemi
`
ere, and C. R. Janssen, “Quan-
titative DNA-methylation in Daphnia magna and effects of
multigeneration Zn exposure,” Comparative Biochemistry and
Physiology, vol. 150, no. 3, pp. 343–348, 2009.
[38] S. T. Threlkeld, “Daphnia life history strategies and resource
allocation patterns,” in Daphnia,R.H.PetersandR.De
Bernardi, Eds., vol. 45 of
Memorie dellIstituto Italiano di
Idrobiologia, pp. 353–366, 1987.
[39] A. M. Schurko, J. M. Logsdon, and B. D. Eads, “Meiosis genes
in Daphnia pulex and the role of parthenogenesis in genome
evolution,” BMC Evolutionary Biology, vol. 9, no. 1, article 78,
2009.
[40] C. Hiruta, C. Nishida, and S. Tochinai, “Abortive meiosis in the
oogenesis of parthenogenetic Daphnia pulex,” Chromosome
Research, vol. 18, no. 7, pp. 833–849, 2010.
[41] P. D. Hebert and R . D. Ward, “Inheritance during partheno-
genesis in Daphnia magna,” Genetics, vol. 71, no. 4, pp. 639–
642, 1972.
[42] A. R. Omilian, M. E. A. Cristescu, J. L. Dudycha, and M. Lynch,
“Ameiotic recombination in asexual lineages of Daphnia,”
Proceedings of the National Academy of Sciences of the United
States of A merica, vol. 103, no. 49, pp. 18638–18643, 2006.
[43] F. Zaffagnini and B. Sabelli, “Karyologic observations on the
maturation of the summer and winter Eggs of Daphnia pulex

8 Genetics Research International
and Daphnia middendorffiana,” Chromosoma, vol. 36, no. 2,
pp. 193–203, 1972.
[44] K. K. Sanford, “The effect of temperature on the intersex
character of Daphnia longispina,” Physiological Zoology, vol.
20, pp. 325–332, 1947.
[45] A. W. Olmstead and G. A. LeBlanc, “The environmental-
endocrine basis of gynandromorphism (intersex) in a crus-
tacean,” International Journal of Biological Sciences, vol. 3, no.
2, pp. 77–84, 2007.
[46] S. S. Schwartz and P. D. N. Hebert, “Daphniopsis ephemeralis
sp.n. ( Cladocera, Daphniidae): a new genus for North
America,” The Canadian Journal of Zoology, vol. 63, no. 11, pp.
2689–2693, 1985.
[47] B. D. Eads, J. K. Colbourne, E. Bohuski, and J. Andrews,
“Profiling sex-biased gene expression during parthenogenetic
reproduction in Daphnia pulex,” BMC Genomics, vol. 8, article
464, 2007.
[48] R. Tollrian and S. T. Dodson, “Inducible defenses in Cladocera:
constraints, costs, and multipredator environments,” in The
Ecology and Evolution of Inducible Defenses, R. Tollrian and
C. D. Harvell, Eds., pp. 177–202, Princeton University Press,
Princeton, NJ, USA, 1999.
[49] R. Tollrian, “Predator-induced helmet formation in Daphnia
cucullata (Sars),” Archiv fur Hydrobiologie, vol. 119, pp. 191–
196, 1990.
[50] M. J. Beaton and P. D. N. Hebert, “Patterns of DNA synthesis
and mitotic activity during the intermoult of Daphnia,”
Journal of Ex p erimental Zoology, vol. 268, no. 5, pp. 400–409,
1994.
[51] H. Miyakawa, M. Imai, N. Sugimoto et al., “Gene up-
regulation in response to predator kairomones in the water
flea, Daphnia pulex,” BMC Developmental Biology, vol. 10,
article 45, 2010.
[52] M. Imai, Y. Naraki, S. Tochinai, and T. Miura, “Elaborate
regulations of the predator-induced polyphenism in the water
flea Daphnia pulex: Kairomone-sensitive periods and life-
history tradeoffs,” Journal of Experimental Zoology, vol. 311,
no. 10, pp. 788–795, 2009.
[53] D. Lemons and W. McGinnis, “Genomic evolution of hox gene
clusters,” Science, vol. 313, no. 5795, pp. 1918–1922, 2006.
[54] C. Rauskolb, M. Peifer, and E. Wieschaus, “extradenticle, a
regulator of homeotic gene activ ity, is a homolog of the
homeobox-containing human proto-oncogene pbx1,” Cell,
vol. 74, no. 6, pp. 1101–1112, 1993.
[55] K. Miura, M. Oda, S. Makita, and Y. Chinzei, “Characteri-
zation of the Drosophila Methoprene-tolerant gene product:
Juvenile hormone binding and ligand-dependent gene regula-
tion,” FEBS Journal, vol. 272, no. 5, pp. 1169–1178, 2005.
[56] M. B. Vandegehuchte, T. Vandenbrouck, D. De Coninck, W.
M. De Coen, and C. R. Janssen, “Gene transcription and
higher-level effects of multigenerational Zn exposure in Daph-
nia magna,” Chemosphere, vol. 80, no. 9, pp. 1014–1020, 2010.
[57] M. B. Vandegehuchte, T. Kyndt, B. Vanholme, A. Haegeman,
G. Gheysen, and C. R. Janssen, “Occurrence of DNA methyla-
tion in Daphnia magna and influence of multigeneration Cd
exposure,” Environment International, vol. 35, no. 4, pp. 700–
706, 2009.
[58] A. Baccarelli and V. Bollati, “Epigenetics and environmental
chemicals,” Current Opinion in Pediatrics, vol. 21, no. 2, pp.
243–251, 2009.
[59] D. C. Dolinoy, D. Huang, and R. L. Jirtle, “Maternal nutri-
ent supplementation counteracts bisphenol A-induced DNA
hypomethylation in early development,” Proceedings of the
National Academy of Sc iences of the United States of America,
vol. 104, no. 32, pp. 13056–13061, 2007.
[60] S. Feng, S. E. Jacobsen, and W. Reik, “Epigenetic reprogram-
ming in plant and animal development,” Science, vol. 330, no.
6004, pp. 622–627, 2010.
[61] M. Z. Fang, D. Chen, Y. Sun, Z. Jin, J. K. Christman, and
C. S. Yang, “Reversal of hypermethylation and reactivation of
p16INK4a, RARβ, and MGMT genes by genistein and other
isoflavones from soy,” Clinical Cancer Research, vol. 11, no. 19
I, pp. 7033–7041, 2005.