ArticlePDF Available

Daphnia as an Emerging Epigenetic Model Organism


Abstract and Figures

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.
Content may be subject to copyright.
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.
[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. Zaagnini, “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,
[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,
[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 eects, 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.
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 dierentiation
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,
[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
eect 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 eects 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 eects 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,
[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. Zaagnini and B. Sabelli, “Karyologic observations on the
maturation of the summer and winter Eggs of Daphnia pulex
8 Genetics Research International
and Daphnia middendorana,” Chromosoma, vol. 36, no. 2,
pp. 193–203, 1972.
[44] K. K. Sanford, “The eect 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,
[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 tradeos, 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 eects 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.
... Under laboratory conditions, daphnids are maintained in their parthenogenetic state. During female parthenogenesis, recombination does not occur, and parthenogenetic offspring are genetically identical to their mother [22,23]. This reproductive trait makes Daphnia a model organism for studying epigenetic changes. ...
... This reproductive trait makes Daphnia a model organism for studying epigenetic changes. Environmentally controlled multifarious polyphenism, phenotypic alterations, and sex determination were observed in genetically identical daphnids [23]. Furthermore, various environmental stressors alter global and gene-specific DNA methylation levels in daphnids, and these epigenetic alterations can support phenotypic responses to chemical exposure [23,24]. ...
... Environmentally controlled multifarious polyphenism, phenotypic alterations, and sex determination were observed in genetically identical daphnids [23]. Furthermore, various environmental stressors alter global and gene-specific DNA methylation levels in daphnids, and these epigenetic alterations can support phenotypic responses to chemical exposure [23,24]. ...
Full-text available
The mixture of 5-chloro-2-methylisothiazol-3(2H)-one and 2-methylisothiazol-3(2H)-one, CMIT/MIT, is an isothiazolinone biocide that is consistently detected in aquatic environments because of its broad-spectrum usage in industrial fields. Despite concerns about ecotoxicological risks and possible multigenerational exposure, toxicological information on CMIT/MIT is very limited to human health and within-generational toxicity. Furthermore, epigenetic markers altered by chemical exposure can be transmitted over generations, but the role of these changes in phenotypic responses and toxicity with respect to trans- and multigenerational effects is poorly understood. In this study, the toxicity of CMIT/MIT on Daphnia magna was evaluated by measuring various endpoints (mortality, reproduction, body size, swimming behavior, and proteomic expression), and its trans- and multigenerational effects were investigated over four consecutive generations. The genotoxicity and epigenotoxicity of CMIT/MIT were examined using a comet assay and global DNA methylation measurements. The results show deleterious effects on various endpoints and differences in response patterns according to different exposure histories. Parental effects were transgenerational or recovered after exposure termination, while multigenerational exposure led to acclimatory/defensive responses. Changes in DNA damage were closely associated with altered reproduction in daphnids, but their possible relationship with global DNA methylation was not found. Overall, this study provides ecotoxicological information on CMIT/MIT relative to multifaceted endpoints and aids in understanding multigenerational phenomena under CMIT/MIT exposure. It also emphasizes the consideration of exposure duration and multigenerational observations in evaluating ecotoxicity and the risk management of isothiazolinone biocides.
... The changes in DNA methylation in D. magna can be induced by environmental factors such as metals, persistent organic pollutants, and pharmaceuticals (Athanasio et al. 2018;Kusari et al. 2017). Harris et al. (2012) reported that 5-azacytidine can inhibit DNA methylation by inhibiting DNA methyltransferase, thus reducing the total DNA methylation level in D. magna. Epigenetics may play a role in the regulation of Nrf2 transcriptional activity. ...
... It does not only serve as bait for predatory invertebrates and fish, but also feeds on algae to improve water quality (Tkaczyk et al. 2021). In addition, it has the advantages in ecotoxicological experiments and epigenetic studies because of its short life cycle, high sensitivity to poisons, parthenogenesis reproduction, and easy culture in the laboratory (Harris et al. 2012;Tatarazako and Oda 2007). In ecotoxicology investigation, D. magna is one of the most commonly used model organisms to evaluate the toxicity of various drugs, such as antibiotics, anticancer drugs, antidepressants and anti-inflammatory drugs (Tkaczyk et al. 2021). ...
Full-text available
Aspirin (acetylsalicylic acid, ASA), a widely used non-steroidal anti-inflammatory drug, was frequently detected in aquatic environments around the world. However, information on the potential toxic effects of aspirin on non-target aquatic invertebrates is limited. In the present study, we investigated the effects of ASA on the transcriptional expressions of antioxidant genes (Nrf2, Keap1, HO-1, GCLC, GPx, TRX, TrxR and Prx1) and DNA methylation genes (DNMT1, DNMT3 and TET2) in Daphnia magna (D. magna)for 24, 48 and 96 h and the changes of antioxidant enzymatic activity and GSH, MDA content for 48 h. The effects of ASA on the life traits of D. magna were also addressed via a 21-days chronic toxicity test. Results showed that the expressions of Nrf2 and its target genes (HO-1, GPx and TrxR, GCLC, TRX and Prx1) were induced to different degrees at 48 h and/or 96 h. The activity of antioxidant enzymes (SOD, CAT, GST and GPx) and MDA content increased but GSH content decreased, indicating that ASA caused oxidative stress in D. magna. ASA also changed the expression of DNA methylation genes, such as DNMT and TET2, in D. magna. We speculated that ASA may affect the antioxidant system responses through regulation of Nrf2/Keap1 signaling pathway, and/or through indirectly influencing DNA methylation levels by DNMT and TET gene expression, but the detailed mechanism needs further investigations. Chronic exposure to ASA for 21 days caused inhibitions on the growth, reproduction and behavior of D. magna (e.g., delaying days to the first brood and shortening the body length). In summary, ASA significantly affected the antioxidant responses of D. magna, and negatively disturbed its life traits in growth, development and reproduction.
... Some Cladocera species, such as Daphnia magna, Daphnia pulex and Daphnia cucullata, are often used as bioindicators of water pollution and a good model organism of freshwater ecology. Research from Poland suggests that the body size of Daphnia cucullata seems to be a useful indicator of the ecological status of lakes [5][6][7][8][9][10][11]. Daphnia has been considered to be a control organism in freshwater as a kind of convergence model with adaptive features in radically different habitats [12,13]. ...
Full-text available
We have used Daphnia cucullata (Crustacea: Cladocera) as a model organism for the first time in the four deepest Latvian lakes from the Boreal biogeographical region in order to find the genetic diversity of these populations. During the research, we detected the most appropriate microsatellite markers for future genetic studies of Daphnia cucullata populations of lakes Svente, Riča, Dridzis and Geran , imovas-Ilzas in the Boreal biogeographical region. Based on these microsatellite markers, we determined the genetic diversity of these populations. The loci Dgm105 and Dgm101 had the maximum number of alleles and the maximum number of private alleles. The specific locus Dgm105 had five private alleles (62% of all detected alleles), and locus Dgm101 had four private alleles (57% of all detected alleles) in these loci. We determined the observed heterozygosity (H obs) and the expected heterozygosity (H exp) level (via Hardy-Weinberg equilibrium), the number of polymorphic loci, the number of detected alleles in each analyzed microsatellite locus, the average number of alleles at the locus (N a), the average effective number of alleles at the locus (N e), the F ST of the population's genetic differentiation, the genetic distance (D) (following Nei) and the significance (χ 2-test) of differences between the levels of observed and expected heterozygosity. It was shown that Daphnia cucullata populations from lakes with a low number of zooplankton taxa (Riča and Geran , imovas-Ilzas) have a higher genetic diversity compared to lakes with a high number of zooplankton taxa (Dridzis and Svente). It was found that Daphnia cucullata populations from lakes Dridzis and Svente have the least genetic distance, and these populations form a single genetic group, as confirmed via clustering.
... They regulate bacterial and detrital quantity and they are an important component of the feed for juvenile fish, plankton-feeding fish and many other aquatic animals [1][2][3][4]. Some Cladocera species such as Daphnia magna, Daphnia pulex, Daphnia cucullata are often used as bioindicators of water pollution and a good model organism of freshwater ecology [5][6][7][8][9][10]. Daphnia has been considered to be a control organism in the freshwater as a kind of convergence model with the adaptive features in radically different habitats [11,12]. ...
Full-text available
We have used Daphnia cucullata as a model organism for the first time in the four deepest Latvian lakes from the Boreal biogeographical region lakes in order to find the genetic diversity of Daphnia cucullata populations. During the research, the most appropriate microsatellite markers for future genetic studies of Daphnia cucullata populations in Boreal biogeographical region lakes. It was the loci Dgm105 and Dgm101, in which the maximum number of alleles and the maximum number of private alleles were found. The Dgm105 locus had five private alleles (62% of all detected alleles), while the Dgm101 locus had four private alleles (57% of all detected alleles) in these loci. We was determined the observed heterozygosity (Hobs) and the expected heterozygosity (Hexp) level (by Hardy-Weinberg), the number of polymorphic loci, the number of detected alleles in each analyzed microsatellite locus, the average number of alleles at the locus (Na), the average effective number of alleles at the locus (Ne), FST of the population genetic differentiation, the genetic distance (D) (by Nei) and significance (χ2- test) of differences between the levels of observed and expected heterozygosity. It was shown that Daphnia cucullata populations from lakes with a low number of zooplankton taxa (Riča and Geraņimovas-Ilzas) have a higher genetic diversity compared to lakes with a high number of zooplankton taxa (Dridzis and Svente) in lakes. It was found, that Daphnia cucullata populations from lakes Dridzis and Svente have the least genetic distance and these populations form a single genetic group, as confirmed by clustering
... Under low stress, females reproduce asexually, and only females are produced. However, when high stress is introduced to the population, the epigenetic landscape of Daphnia, or heritable phenotype changes that do not alter DNA, shifts genetically female Daphnia into males to allow for sexual reproduction to occur (8). Sex determination occurs after the egg is produced and before development begins, allowing for the Daphnia to express its phenotype in response to environmental stress. ...
In the modern world people are often impacted by factors that control when they can sleep and for how long, which results in an altered photoperiod. Alterations in photoperiod can occur from a variety of factors, ranging from working night shifts to travelling and changing time zones, and therefore influence the daily lives of many people. The purpose of these experiments was to determine if alterations in photoperiod affect the stress response in Daphnia magna. We hypothesized that if Daphnia magna are exposed to alterations in photoperiod, then there will be an increased stress response. We kept two distinct Daphnia populations and exposed the experimental group to a shorter photoperiod of 12 hours instead of the traditional 24-hour photoperiod. During the testing period, we tracked possible stress responses, including mean heart rate, brood size and male-to-female ratio. There were statistically significant differences between the control and experimental groups for both heart rate and brood size. Specifically, the experimental group exhibited an increased heart rate and brood size relative to the control group. Within the experimental group, there was statistically significant variation in the brood size over time, with the peak brood size occurring on day three or four in the seven-day trial. Male-to-female ratio did not have a statistically significant response to the altered photoperiod. The results found support the hypothesis of increased stress when photoperiod is altered, which could also indicate that altered photoperiod may have an impact on people that constantly experience changes in their photoperiod.
... It leads to a further extrapolation from a small number of model species to a vast number of species with differences in their taxonomy, size, life story, physiology, and geographical range (Forbes and Forbes, 1993). Criteria to select species used in standardized bioassays could include, for example, their sensitivity to many xenobiotics (Religia et al., 2021); a parthenogenetic life cycle that becomes a valuable feature for transgenerational studies (Harris et al., 2012) as is the case of Daphnia magna; transparent embryos allowing developmental studies and a well-known genome sequence which facilitates gene expression studies as is the case of zebrafish (Danio rerio) (Howe et al., 2013). A wide distribution of the species is also a desirable feature for model species, as in the case of the algae Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) (Guiry, 2021), or their economic relevance, as in the case of rainbow trout (Oncorhynchus mykiss) (FAO, 2020). ...
The presence of pesticides in aquatic ecosystems is one of the most relevant stressors which biota usually face. Laboratory tests using model organisms for pesticides toxicity assessment are employed worldwide. The use of these species has been encouraged in the scientific community due to their advantageous features and their acceptation by regulatory and standardization organizations. However, non-model species as well as those belonging particular ecosystems could contribute in the laboratory-field toxicity extrapolation. In this context, this work aims on exploring the state of the ecotoxicological studies of pesticides in neotropical aquatic species, focusing on bioassays performed in Argentina over the last 20 years as a case of study. Furthermore, we analyzed the possible advantages and disadvantages of these studies, possible differential sensitivities among native and model species, and future challenges to be faced. The analysis of more than 150 publications allowed identify the chemical identity of tested compounds, organisms used for the bioassays, characteristics of the experimental designs, and the toxicity endpoints. Particularly, the studied cases showed that the tested chemicals are related to those most used in the agricultural activity in Argentina, the predilection for particular species in some taxonomic groups (e.g. amphibians), and the wide election of biochemical biomarkers in the studies. Regarding the sensitivity comparison between native and non-native species, the amount of data available indicates that there is not a clear difference beyond some particular cases. However, deeper understanding of toxic effects of pesticides on non-model species could help in a more comprehensive ecological risk assessment in different ecosystems.
... The significant change in neonate size in the F3 generation in the absence of direct or indirect contact with the contaminant suggests that benzotriazole exerts a transgenerational effect on the reproduction of D. magna. The F3 generation is considered the first truly unexposed generation because the F1 and F2 generations are directly or indirectly exposed to the substance as embryos and germline cells, respectively, when their F0 generation ancestors are exposed (Harris et al., 2012;Jeremias et al., 2018;Trijau et al., 2018). Maternal and transgenerational effects are underpinned by epigenetic mechanisms, and environmentally induced epigenetic modifications can influence fertility, offspring quality, and the health of the next generation (Soubry, 2018; Viluksela and Pohjanvirta, 2019). ...
Due to its widespread and intensive use as a corrosion inhibitor, benzotriazole is ubiquitously detected from a few parts per billion to several hundred parts per million in aquatic environments. The long-term toxicity of benzotriazole is unclear despite its low acute toxicity. Therefore, we investigated the transgenerational effects of benzotriazole at the genomic and individual levels using the freshwater zooplankton Daphnia magna. Maternal exposure to sublethal concentrations (15 and 30 mg/L) of benzotriazole exerted transgenerational effects on D. magna at the genomic and individual levels even in descendants that have never been exposed to benzotriazole. Significant alterations in the expression of Cyp, GST, Vtg1, and Hb and in neonate size were observed in the unexposed F3 generation, confirming the transgenerational effect of benzotriazole. Interestingly, detoxification related genes Cyp and GST were unaffected or downregulated in the exposed generation but upregulated in the following unexposed generations. Furthermore, continuous multigenerational exposure to an environmental concentration (4.3 μg/L) of benzotriazole also upregulated detoxification genes in decent generations but exerted no individual-level effects in subsequent generations.
... Another potential mechanism of evolved salt tolerance could be epigenetic changes (i.e., changes in gene activity without changing DNA sequence), some of which can be inherited across multiple generations (Ashe et al. 2021). Selection on the phenotypic variance caused by the epigenetic changes can lead to adaptive evolution (Shea et al. 2011;Ashe et al. 2021), which has been observed for Daphnia exposed to heavy metals (Harris et al. 2012). We were unable to determine whether adaptation to increased salinity in the isofemale lines in our study was the result of selection on existing variation or epigenetic changes. ...
Full-text available
Adaptation to one stressor can influence organismal responses to future environmental changes, either to the same or a novel stressor. But there is a lack of research on this topic, particularly in the context of freshwater habitats. Fluctuating salinity and heatwaves that are becoming more frequent and intensified are affecting freshwater ecosystems. We applied an experimental evolution approach to examine the influence of adaptation to elevated salinity on the population’s responses to subsequent salt and heat stress. We conducted lab experiments using Daphnia pulicaria cultured from individuals with previous 8-week exposure history to two salinity treatments (6.5 or 350 mg Cl−/L). Iso-female lines with or without prior exposure to elevated salinity were assayed along a salt gradient (18.5 to 1500 mg Cl−/L) or an acute heat gradient treatment (20 to 35 °C). Our results showed that Daphnia survival, fecundity, and body length growth rate declined with increased salt concentration, with survival and fecundity being most sensitive. The treatment group with previous salt exposure history had higher survival and fecundity than the naïve treatment group when treated with salt, without loss of fitness under low-salinity conditions. Daphnia survival and growth rates were reduced in temperatures higher than 30 °C. Despite the fact that the two stressors can induce similar defense mechanisms, previous exposure history to salt did not prevent Daphnia populations from experiencing reduced survival and growth rates under heated conditions. Our work demonstrates that organisms can rapidly adapt to a stressor that protects them from later exposure to increases in this stressor, without a trade-off in fitness under undisturbed conditions, but this evolved tolerance cannot protect them from all levels of this stressor or alleviate damage by a novel one.
Full-text available
Although its role in the functioning of aquatic systems is widely recognized, the contribution of freshwater metazooplankton (metazoan plankton) to ecosystem services (ES) is seldom considered. Here we aim at providing a first overview of how this group contributes to ecosystem services according to the Millennium Ecosystem Assessment framework. We show that although metazooplankton hardly generates any provisioning services, it provides crucial support to the generation of other services. Metazooplankton is important for fisheries because it forms an essential food item for the larval and juvenile stages of most freshwater fish and acts as a trophic link between phytoplankton and microbial communities and the fish community. Through its stoichiometric homeostasis and ability to feed on biochemically complementary food sources it may also act as a buffer against bottom-up effects of nutrient deficiencies in primary producers. Metazooplankton often has a crucial regulatory function by controlling phytoplankton growth and dissolved organic carbon, contributing to the quality of drinking and irrigation water supplies and of the underwater light climate. It provides attractive study material for didactic purposes and some taxa have served as model systems that have considerably aided progress in scientific disciplines such as ecology, evolutionary biology, ecotoxicology, environmental and biomedical sciences.
Full-text available
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.
Full-text available
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).