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Daphnia as an Emerging Epigenetic Model Organism


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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.
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Hindawi Publishing Corporation
Genetics Research International
Volume 2012, Article ID 147892, 8 pages
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,
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
Daphnia oer a variety of benefits for the study of epigenetics. Daphnia’s parthenogenetic life cycle allows the study of epigenetic
eects in the absence of confounding genetic dierences. 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 oers 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]andoers 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 dier dramatically between species
and developmental stages [4]. DNA methylation interacts
with other epigenetic processes [11]. Modifications to the
amino- or carboxyl-terminal histone tails aect 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 eects 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 aect the ospring either
by retention of maternal epigenetic states in the germ line
cells that give rise to the embryo, a true transgenerational
eect, 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
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
2. The
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 organisms pheno-
type and these changes are particularly obvious when there
are no genetic dierences between individuals of any one
strain. Sensitivity of the epigenome to environmental cues
occurs at dierent 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
Kairomone sensitive
period for
maintenance of
helmets and neckteeth
4–6 instars
Daphnia life cycle
Brood pouch
Meiosis occurs
in ovaries
versus sexual
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 dierences—males
being smaller, having testes, modified appendages, and
carapace—all parthenogenetic ospring , 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 dierential
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 dierences 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
3. Epigenetic Regulation of Helmet Formation
Predators are an important aspect of an organisms 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 eects.
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 eect, 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 eect 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
eect indicates that both stages are sensitive. The possibility
of dierent 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 dicult [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. Dierential 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
5. Epigenetic Regulation of Growth
In much the same way that external environmental cues
such as kairomones can aect the development of helmets
and neckteeth, environmental toxicants can aect the body
length and growth in Daphnia magna [34]. Again, as the ani-
mals are all genetically identical, dierences 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 eect on body length (Ta ble 1)[34, 56].
This growth eect, 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 eects F1 F2 Reference
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
(day 7 only)
(day 7 only)
Genistein Decrease n.s. n.s. [34]
Vinclozolin Decrease n.s. n.s. [34]
(day 6 only)
(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
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
eect 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 aected by chemical treatment.
While vinclozolin exposure had no significant eect, 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 eec 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 eect [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 eects 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 eects on methy-
lation in the exposed generation as well as the eects in
subsequent generations (Ta ble 1). Global or localized DNA
methylation levels were found to be aected by 5-azacytidine,
vinclozolin, genistein, and zinc but were not aected by 5-
-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 ospring 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 eect; 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 eect.
Conclusive evidence for a transgenerational eect would be
the persistence of the eect 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 eectswouldbeenhancedbyexam-
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 aects 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 eect 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 dierences 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
aected 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 eects 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 dierences 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.
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.
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... Additionally, somatic growth inhibition of D. magna caused by BP-3 exposure in the F0 generation was recovered in the F3 generation (Fig. 4a). However, the reproduction of D. magna was still significantly decreased (p < 0.05) compared to that of the control (Fig. 4b), indicating a transgenerational effect (Harris et al., 2012;Shaw et al., 2017;De Liguoro et al., 2019). Several studies have reported the transgenerational effects of toxic chemicals on crustaceans. ...
... However, this hypothesis can explain only multigenerational effects in the F0-F2 generation, but not in the F3 generation. The transgenerational effect observed in this study can be induced by epigenetic DNA methylation (Harris et al., 2012;Yu et al., 2021). However, there were no significant differences (p > 0.05) in global DNA methylation among all treatments and generations (Fig. 5). ...
Maternal exposure to microplastics (MPs) plays an important role in the fitness of unexposed progeny. In this study, the transgenerational effects of polyethylene MP fragments (17.35 ± 5.50 µm) containing benzophenone‐3 (BP-3; 2.85 ± 0.16% w/w) on chronic toxicity (21 d) in Daphnia magna were investigated across four generations. Only D. magna in the F0 generation was exposed to MP fragments, MP/BP-3 fragments, and BP-3 leachate to identify the transgenerational effect in the F3 generation. The mortality of D. magna induced by MP and MP/BP-3 fragments was recovered in the F3 generation, but somatic growth and reproduction significantly decreased compared to the control. Additionally, reproduction of D. magna exposed to BP-3 leachate significantly decreased in the F3 generation. These findings confirmed the transgenerational effects of MP fragment and BP-3 additive on D. magna. Particularly, the adverse effect on D. magna reproduction seemed to be cumulative across four generations for MP/BP-3 fragments, while it was an acclimation trend for BP-3 leachate. However, there was no significant difference in global DNA methylation in D. magna across four generations, thus requiring a gene-specific DNA methylation study to identify different epigenetic transgenerational inheritance.
... Daphnia magna (D. magna) species are environmental biomarker models widely used to study and comprehend the ecotoxicology effects of different inorganic and organic materials (e.g., nanoparticles and pharmaceuticals) on different biological levels, such as the cellular, reproductive, and molecular levels [1,2], the last one often applied to understand genome behavior. From the evolutionary point of view, changes, adaptations, and phenotypic variations are fundamental features to be modeled and studied during an ecotoxicology analysis due to its important location in the trophic chain when nanomaterials are used for water cleaning processes of real effluents [3][4][5]. For instance, recent advances and developments in several nanohybrids for the removal of toxic metals and other organic hazardous materials suggest their final spreading into water bodies, soils, various effluents, and the construction industry [6][7][8][9][10], a condition that requires tests of their ecotoxicological effects. ...
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A ternary nanocomposite made of nanomaghemite, nanoanatase, and graphene oxide has been successfully synthesized using an inorganic coprecipitation approach, and it has been systematically investigated by X-ray diffraction, transmission electron microscopy, and different spectrocopic techniques (electron energy loss, µ-Raman, and 57Fe Mössbauer) after interaction with an effluent containing Daphnia magna individuals. Specifically, the influence of the nanocomposite over the Daphnia magna carapace, administered in two doses (0.5 mg mL−1 and 1 mg mL−1), has been characterized using µ-Raman spectroscopy before and after laser burning protocols, producing information about the physicochemical interaction with the biomarker. The thermal stability of the nanocomposite was found to be equal to 500 °C, where the nanoanatase and the nanomaghemite phases have respectively conserved their structural identities. The magnetic properties of the nanomaghemite have also been kept unchanged even after the high-temperature experiments and exposure to Daphnia magna. In particular, the size, texture, and structural and morphological properties of the ternary nanocomposite have not shown any significant physicochemical modifications after magnetic decantation recuperation. A significant result is that the graphene oxide reduction was kept even after the ecotoxicological assays. These sets of observations are based on the fact that while the UV-Vis spectrum has confirmed the graphene oxide reduction with a localized peak at 260 nm, the 300-K and 15-K 57Fe Mössbauer spectra have only revealed the presence of stoichiometric maghemite, i.e., the two well-defined static magnetic sextets often found in the bulk ferrimagnetic counterpart phase. The Mössbauer results have also agreed with the trivalent-like valence state of Fe ions, as also suggested by electron energy loss spectroscopy data. Thus, the ternary nanocomposite does not substantially affect the Daphnia magna, and it can be easily recovered using an ordinary magnetic decantation protocol due to the ferrimagnetic-like character of the nanomaghemite phase. Consequently, it shows remarkable physicochemical properties for further reuse, such as cleaning by polluted effluents, at least where Daphnia magna species are present.
... The LPO levels for the low concentration series (Fig. 4m) under the different MP and APFO co-exposures tended to be comparable (p > 0.1), with the exception of aged PS + APFO at 1:4 where the intestinal damage was correspondingly more severe (Fig. 4j). This again supports that the combined toxicity of MPs and APFO at low exposure concentrations could pose environmental risks by disturbing the important roles of zooplankton in aquatic ecosystems (Harris et al., 2012). ...
Microplastics (MPs) have worldwide accumulated in aquatic environments and coexisted with various water contaminants including perfluorinated compounds (PFCs) that are frequently detected. The adverse effects of individual MPs or PFCs on aquatic organisms have been extensively reported; however, the combined toxicity of MPs and PFCs remains unknown. This study evaluated the combined toxicity of MPs [pristine and aged polystyrene (PS)] and a PFC [ammonium perfluorooctanoate (APFO)] to Daphnia magna under different concentration ratios by three classic methods: toxicity unit, additive index, and mixed toxicity index. The adsorption kinetics of APFO on MPs, aggregation of MPs in exposure medium, MP gut fullness of daphnids, intestinal histology, and lipid peroxidation were analyzed to reveal the mechanism underlying the combined toxicity. Our results showed that the combined toxic modes varied with the concentration ratios of MPs to APFO (antagonism at 4:1 and 1:4, synergism at 3:1, 1:2, and 1:3, and partial addition/antagonism at 2:1 and 1:1 for pristine PS + APFO; antagonism at all ratios except partial addition/antagonism at 3:1 and 1:3 for aged PS + APFO), which could be attributed to the alteration of MP aggregation and thus MP gut fullness in the daphnids. The combined toxicity was further confirmed to occur in the daphnid's gut, which was reflected in physiological and biochemical responses mediated by intestinal blockage. Observable intestinal damages under co-exposures at μg•L⁻¹ levels indicated the risks from future long-term exposure to MPs and PFCs in aquatic environments. This work demonstrates the necessity of assessing combined toxicity with different concentration ratios and provides new insights into the potential risks of MPs in aquatic environments.
... In addition to its role as a keystone species, there are several others reasons why Daphnia is particularly useful to study epigenetic inheritance. Individuals frequently reproduce clonally, which makes it possible to study epigenetic inheritance without the confounding effects of genetic variation (Duki c et al., 2019;Harris et al., 2012). Furthermore, Daphnia inhabit waters with seasonal environmental variation, spanning periods of multiple asexual generations, a situation that should favor incomplete epigenetic resetting (McNamara et al., 2016;Rivoire and Leibler, 2014;Uller et al., 2015). ...
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Transgenerational inheritance of environmentally-induced epigenetic marks can have significant impacts on eco-evolutionary dynamics, but the phenomenon remains controversial in ecological model systems. We used whole-genome bisulfite sequencing of individual water fleas (Daphnia magna) to assess whether environmentally-induced DNA methylation is transgenerationally inherited. Genetically identical females were exposed to one of three natural stressors, or a de-methylating drug, and their offspring were propagated clonally for four generations under control conditions. We identified between 70 and 225 differentially methylated CpG positions (DMPs) in F1 individuals whose mothers were exposed to a natural stressor. Roughly half of these environmentally-induced DMPs persisted until generation F4. In contrast, treatment with the drug demonstrated that pervasive hypo-methylation upon exposure is reset almost completely after one generation. These results suggest that environmentally-induced DNA methylation is non-random and stably inherited across generations in Daphnia, making epigenetic inheritance a putative factor in the eco-evolutionary dynamics of fresh-water communities.
... The approach to generating environmentally driven phenotypes mediated through epigenetic mechanisms has been considered during the two main periods in the life cycle of farmed animals: (1) embryonic development and early life stages and (2) adult and broodstock. It has been demonstrated that the establishment of epigenetic states of genes related to phenotypic variation occurs mainly during these stages, and exposure to altered environmental conditions during these stages is important for the maintenance of the epigenetic state (Fig. 18.2; Burggren and Blank, 2009; a u t h o r e p r i n t Harris et al., 2012). Many studies, in species ranging from salmonids to oysters and (brine) shrimp, have provided evidence that epigenetic mechanisms are associated with commercially important traits such as metabolism, growth, sex determination, fecundity and behavior (for details, see reviews in Granda et al., 2018Norouzitallab et al., 2019Roy et al., 2020). ...
The global demand for high-quality animal protein is expected to increase considerably with the rise in the human population. It is predicted that farmed crustacean shrimps could contribute significantly to this increasing demand, and eventually to the world´s future food protein security and nutrition. Diseases, however, impose a major yield-limiting effect on the farmed production of high-value shrimps, causing devastating socioeconomic impacts. To avoid disease-mediated production losses there has been a continuous effort to develop effective and sustainable health management and disease prevention strategies. Lately, epigenetics has attracted considerable attention because there is now compelling evidence indicating that epigenetics alterations are involved in the induction of disease resistance in aquaculture animals, including shrimps, both within and across generations. This chapter brings this information together and discusses the potential of this new concept in the design of new-generation health management and disease preventional strategies in farmed shrimp.
... Daphnia species have long been used as sentinel species to indicate water quality and ecosystem health in freshwater systems (Shaw et al. 2008). But they are now also increasingly used as epigenetic model systems because epigenetic effects can easily be disentangled from genetic effects (Ebert 2011;Harris et al. 2012). This derives from the fact that Daphnia species normally reproduce via parthenogenesis, resulting in clonal lineages that allow phenotypic comparison of genetically identical individuals across a range of different environments. ...
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It has been hypothesized that the effects of pollutants on phenotypes can be passed to subsequent generations through epigenetic inheritance, affecting populations long after the removal of a pollutant. But there is still little evidence that pollutants can induce persistent epigenetic effects in animals. Here, we show that low doses of commonly used pollutants induce genome‐wide differences in cytosine methylation in the freshwater crustacean Daphnia pulex. Uniclonal populations were either continually exposed to pollutants or switched to clean water, and methylation was compared to control populations that did not experience pollutant exposure. Although some direct changes to methylation were only present in the continually exposed populations, others were present in both the continually exposed and switched to clean water treatments, suggesting that these modifications had persisted for 7 months (>15 generations). We also identified modifications that were only present in the populations that had switched to clean water, indicating a long‐term legacy of pollutant exposure distinct from the persistent effects. Pollutant‐induced differential methylation tended to occur at sites that were highly methylated in controls. Modifications that were observed in both continually and switched treatments were highly methylated in controls and showed reduced methylation in the treatments. On the other hand, modifications found just in the switched treatment tended to have lower levels of methylation in the controls and showed increase methylation in the switched treatment. In a second experiment, we confirmed that sublethal doses of the same pollutants generate effects on life histories for at least three generations following the removal of the pollutant. Our results demonstrate that even low doses of pollutants can induce transgenerational epigenetic effects that are stably transmitted over many generations. Persistent effects are likely to influence phenotypic development, which could contribute to the rapid adaptation, or extinction, of populations confronted by anthropogenic stressors.
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Water fleas are an important lower invertebrate model that are usually used for ecotoxicity studies. Contrary to mammals, the heart of a water flea has a single chamber, which is relatively big in size and with fast-beating properties. Previous cardiac chamber volume measurement methods are primarily based on ImageJ manual counting at systolic and diastolic phases which suffer from low efficiency, high variation, and tedious operation. This study provides an automated and robust pipeline for cardiac chamber size estimation by a deep learning approach. Image segmentation analysis was performed using U-Net and Mask RCNN convolutional networks on several different species of water fleas such as Moina sp., Daphnia magna, and Daphnia pulex. The results show that Mask RCNN performs better than U-Net at the segmentation of water fleas’ heart chamber in every parameter tested. The predictive model generated by Mask RCNN was further analyzed with the Cv2.fitEllipse function in OpenCV to perform a cardiac physiology assessment of Daphnia magna after challenging with the herbicide of Roundup. Significant increase in normalized stroke volume, cardiac output, and the shortening fraction was observed after Roundup exposure which suggests the possibility of heart chamber alteration after roundup exposure. Overall, the predictive Mask RCNN model established in this study provides a convenient and robust approach for cardiac chamber size and cardiac physiology measurement in water fleas for the first time. This innovative tool can offer many benefits to other research using water fleas for ecotoxicity studies.
Purpose: To analyze the results of direct and transgenerational effects of radio frequency electromagnetic fields (RF-EMF) on the model organism of crustaceans Daphnia magna. Materials and methods: D. magna were chronically exposed at 900 GHz EMF with an energy flux density (EFD) of about 1 mW/cm2 in the juvenile and pubertal periods of their ontogenesis. The cytotoxicity of exposure as well as survival, fertility and teratogenic effect of directly exposed daphnids and their progeny across three generations were analyzed. Results and conclusions: The results of our study show that exposure of RF-EMF at juvenile period can significantly affect the fertility and size of irradiated daphnids and their offspring of the first generation. The decrease in fertility may be associated with a cytotoxic effect on the cells of irradiated animals. The reduction in the size of the terminal spine and the body of individuals is an indicator of the negative impact of radiation on the protective strategy of the crustacean population. The reproductive process is restored by the second generation. The results of our study provide further insights into the possible mechanisms underlying the in vivo effects of RF-EMF.
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Daphnids are small crustaceans ubiquitous in fresh water; they have been a subject of study in ecology, evolution, and environmental sciences for decades. To understand data accumulated in daphnid biology at the molecular level, expressed sequence tags and a genome sequence have been determined. However, these discoveries lead to the problem of how to understand the functions of newly discovered genes. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a useful tool to achieve specific gene silencing in nontransformable species. Hence, we established a technique to inject exogenous materials into ovulated eggs and developed a dsRNA-based RNAi method for Daphnia magna. Eggs were collected just after ovulation and injected with dsRNA specific to the Distal-less (Dll) gene, which functions in appendage development in invertebrates and vertebrates. We found that the dsRNA successfully triggered the degradation of Dll mRNAs, which induced the truncation of the second antenna in a dose-dependent manner. This effect was sequence specific in that: (1) an unrelated dsRNA did not induce any morphological abnormalities and (2) two non-overlapping Dll dsRNAs generated the same phenotype. This is the first report of an RNAi technique in D. magna and, together with the emerging genome sequences, will be useful for advancing knowledge of the molecular biology of daphnids.
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Sex-determining mechanisms are diverse among animal lineages and can be broadly divided into two major categories: genetic and environmental. In contrast to genetic sex determination (GSD), little is known about the molecular mechanisms underlying environmental sex determination (ESD). The Doublesex (Dsx) genes play an important role in controlling sexual dimorphism in genetic sex-determining organisms such as nematodes, insects, and vertebrates. Here we report the identification of two Dsx genes from Daphnia magna, a freshwater branchiopod crustacean that parthenogenetically produces males in response to environmental cues. One of these genes, designated DapmaDsx1, is responsible for the male trait development when expressed during environmental sex determination. The domain organization of DapmaDsx1 was similar to that of Dsx from insects, which are thought to be the sister group of branchiopod crustaceans. Intriguingly, the molecular basis for sexually dimorphic expression of DapmaDsx1 is different from that of insects. Rather than being regulated sex-specifically at the level of pre–mRNA splicing in the coding region, DapmaDsx1 exhibits sexually dimorphic differences in the abundance of its transcripts. During embryogenesis, expression of DapmaDsx1 was increased only in males and its transcripts were primarily detected in male-specific structures. Knock-down of DapmaDsx1 in male embryos resulted in the production of female traits including ovarian maturation, whereas ectopic expression of DapmaDsx1 in female embryos resulted in the development of male-like phenotypes. Expression patterns of another D. magna Dsx gene, DapmaDsx2, were similar to those of DapmaDsx1, but silencing and overexpression of this gene did not induce any clear phenotypic changes. These results establish DapmaDsx1 as a key regulator of the male phenotype. Our findings reveal how ESD is implemented by selective expression of a fundamental genetic component that is functionally conserved in animals using GSD. We infer that there is an ancient, previously unidentified link between genetic and environmental sex determination.
Defining ParasitesAlthough parasites have traditionally been defined by a combination of conceptual and taxonomic features, I use an entirely conceptual definition here. I consider a parasite to be any small organism (including viruses) that lives in close association with a host organism and for which it seems reasonable to assume that the host carries some cost. These costs may be clearly visible, in the form of reduced fecundity or survival, but may in some cases be subtle. For example, reduced sexual attractiveness (leading to reduced mating success) or reduced competitive ability may not be very visible. I devote an entire chapter to discussing the fitness costs caused by parasites. This conceptual definition of a parasite includes members of various taxa, such as viruses, bacteria, fungi, and protozoa, but also includes functional categories (not taxonomically defined), such as pathogens and helminths. In contrast to typical predators, parasites do not always kill their hosts, and if they do, it may take a considerable amount of time, during which the parasite may be transmitted to other hosts, and the host remains in the community competing with other organisms for space, food, and mating partners.In the literature on Cladocera and more specifically on Daphnia, parasites are often distinguished from epibionts. Whereas the former are usually endoparasites, i.e., located within the body of the host, the latter are located on the body surface and may therefore be labeled as ectoparasites. In the main part of this book, I concentrate on endoparasites and exclude epibionts. However, this is not to say that epibionts are not parasites or are not important. In fact, I believe that most epibionts fulfill the definition of parasites used here, because they are often closely associated with their hosts and cause harm to their hosts. This harm may not be directly visible, but there are certainly increased costs for swimming, which may have consequences for other fitness components, such as fecundity, survival, competition, and mate finding (Threlkeld et al. 1993). It has also been suggested that epibiontic filter feeders compete with their hosts for food (Kankaala and Eloranta 1987). On the other hand, it has been suggested that under certain conditions, high loads of algal epibionts may provide additional food for the host and thus result in a net benefit (Barea-Arco et al. 2001). However, this form of a food supplementation is certainly not the typical effect of epibionts. I do not include epibionts in this book, because I feel that there is less need to discuss the epidemiology of this functional group than for endoparasites. However, I will refer to them whenever it might further our understanding of Daphnia–parasite interactions.
The factors inducing sexual eggs, as well as the offspring sex ratio preceding sexual egg formation, were investigated in a cyclic parthenogen, Daphnia magna (Crustacea, Cladocera). Laboratory experiments were conducted on individual animals living in flow-through chambers, making it possible to separate the effects of two density-dependent factors: food limitation and a chemically mediated cue. These factors were studied under a short-day photoperiod and in permanent light. The simultaneous actions of an inductive photoperiod, food limitation and chemically mediated crowding, were all needed to induce sexual egg formation. When only two stimuli were present, the offspring sex ratio was 0.50 or lower, and no sexual eggs were produced. By contrast, environmental conditions inducing sexual eggs also effected strongly male-biased asexual offspring, with an average sex ratio up to 0.8 (even 1.0 in individual females).