Sex-Biased Expression of MicroRNAs in Schistosoma mansoni
Citation: Marco A, Kozomara A, Hui JHL, Emery AM, Rollinson D, et al. (
Sex-Biased Expression of MicroRNAs in Schistosoma mansoni
Antonio Marco 0 1
Ana Kozomara 0 1
Jerome H. L. Hui 0 1
Aidan M. Emery 0 1
David Rollinson 0 1
Sam Griffiths- Jones 0 1
Matthew Ronshaugen 0 1
Malcolm K. Jones, University of Queensland, Australia
0 Current address: School of Life Sciences, The Chinese University of Hong Kong , Shatin, N.T., Hong Kong , China
1 1 Faculty of Life Sciences , Michael Smith Building, Oxford Road , University of Manchester , Manchester , United Kingdom , 2 School of Biological Sciences, University of Essex , Colchester , United Kingdom , 3 Wolfson Wellcome Biomedical Laboratories , Department of Life Sciences , Natural History Museum, London , United Kingdom
Schistosomiasis is an important neglected tropical disease caused by digenean helminth parasites of the genus Schistosoma. Schistosomes are unusual in that they are dioecious and the adult worms live in the blood system. MicroRNAs play crucial roles during gene regulation and are likely to be important in sex differentiation in dioecious species. Here we characterize 112 microRNAs from adult Schistosoma mansoni individuals, including 84 novel microRNA families, and investigate the expression pattern in different sexes. By deep sequencing, we measured the relative expression levels of conserved and newly identified microRNAs between male and female samples. We observed that 13 microRNAs exhibited sex-biased expression, 10 of which are more abundant in females than in males. Sex chromosomes showed a paucity of female-biased genes, as predicted by theoretical evolutionary models. We propose that the recent emergence of separate sexes in Schistosoma had an effect on the chromosomal distribution and evolution of microRNAs, and that microRNAs are likely to participate in the sex differentiation/maintenance process.
Funding: This work was supported by the Wellcome Trust Institutional Strategic Fund (097820/Z/11/Z) and the Biotechnology and Biological Sciences Research
Council (BB/G011346/1 and BB/H017801/1). JHLH was supported by a Faculty of Life Sciences, University of Manchester Career Development Award. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Human schistosomiasis is a neglected tropical disease (NTD)
caused by blood flukes of the genus Schistosoma. Schistosomiasis is
estimated to affect over 200 million people in developing tropical
and subtropical countries, with over 90% of cases being confined
to Africa [1,2]. Schistosoma mansoni is primarily responsible for
intestinal and hepatic schistosomiasis in Africa, the Arabian
peninsula, parts of South America and the Caribbean Islands .
Unlike most other flatworms (phylum Platyhelminthes), Schistosoma
species are dioecious; that is, they have two differentiated sexes.
The emergence of sexual dimorphism in these species is believed
to be associated with adaptation to warm-blooded vertebrates
from a hermaphrodite ancestor in cold-blooded vertebrates .
Schistosoma mansoni has seven pairs of autosomes and one pair of
sexual chromosomes with a ZW system, i.e., females are the
heterogametic sex . Like other species with a ZW-based system
of sex determination, there is no apparent global dosage
compensation in females .
The origin of Schistosoma sexuality has attracted much attention
[4,79]. Moreover, as the eggs laid by the female worms are
primarily responsible for the pathology associated with
schistosomiasis, the mechanisms associated with pairing and egg-laying,
including expression of sex specific genes are of great interest. In
the last years, different groups have characterized, using genomic
and proteomic approaches, gene products with differential
expression between males and females in Schistosoma species [10
13]. Since both sexes are necessary for the colonization of the host,
sex-biased genes are potential targets for the infection control of
MicroRNAs are short endogenous RNA molecules that regulate
gene expression by targeting mature mRNA transcripts . This
mechanism of post-transcriptional regulation is conserved in
animals and is likely to be involved in all aspects of cellular
function . The microRNA content of Schistosoma japonicum,
which affects large endemic areas around the river Yangtze in
China, has been studied in detail by several groups . In
addition, the previous characterization of microRNAs from other
non dioecious species of flatworms, such as Echinococcus granulosus
and the non-parasitic Schmidtea mediterranea [22,23], provides a
background against which to identify Schistosoma-specific
microRNAs with a potential role in sexual development and
Current knowledge of S. mansoni microRNAs is limited and
mostly based on computational predictions [24,25]. Moreover, S.
mansoni provides an excellent model to study the evolution and
function of sex-biased microRNAs. Here we use deep sequencing of
RNA libraries to explore the microRNA content of S. mansoni,
identify microRNAs specific to the schistosomes, and study the
potential impact of sex-biased microRNAs in sexual differentiation.
Schistosomiasis is the second most common disease
caused by a parasite, affecting over 200 million people.
The parasites involved are flatworms of the genus
Schistosoma. Unlike most non-parasitic flatworms,
Schistosoma species have separate sexes, and the emergence of
sex has been associated with the development of a
parasitic lifestyle. The identification of gene products that
are expressed in a sex-biased fashion permits the study of
the origin of sexual dimorphism and, in the case of the
schistosomes, the evolution of a human parasite. Here we
investigated the differential expression of microRNAs in
male and female individuals of the species Schistosoma
mansoni. MicroRNAs are crucial gene regulators. We
observed that many new microRNAs emerged in the
evolutionary lineage leading to the schistosomes.
However, many sex-biased microRNAs were present in the
hermaphrodite ancestor of the flatworms, and therefore
acquired sex-biased expression later on. Our results
suggest that changes in microRNA expression patterns
were associated with the emergence of separate sexes in
The microRNAs of Schistosoma mansoni
We have used small RNA deep sequencing to identify a total of
112 microRNAs in adult S. mansoni (Table 1, Supplementary Files
S1 and S2). Valid microRNA candidates were required to have
reads mapping to both arms of the precursor sequences
(representing mature microRNA and microRNA* sequences)
except for those with a previously validated homolog (see Materials
and Methods). Our microRNA annotation procedure was
intentionally conservative: we may not have detected some bona
fide microRNAs, but our predictions are of high confidence.
De Souza Gomes et al.  computationally identified 42
microRNA loci in S. mansoni, significantly expanding the previous
set of 6 microRNAs [16,24]. We confirmed 20 of these (Table 1),
all conserved in other species. We failed to detect the remaining
23. All but two of the unconfirmed microRNAs were not
conserved in other flatworms. A second recent work characterized
211 novel microRNAs in S. mansoni by cloning of small RNA
sequences from adults and schistosomulas . However, the
majority of the candidate microRNAs map to many positions in
the genome and only a few are reported to be within putative
precursor hairpin structures. Indeed, only two of the reported 211
microRNAs were confirmed in our analyses (mir-71a and let-7).
We identify 92 microRNAs not previously annotated in S. mansoni,
eight with obvious homologs in other species (Table 1). Amongst
these, we show that the deeply-conserved mir-124 locus produces
microRNAs from both genomic strands in S. mansoni
(Supplementary File S2). The remaining 84 novel predictions had no
detectable similarity with any known microRNA.
To characterize the microRNAs conserved in the parasitic
Schistosoma genus, we compared our sequenced microRNAs with
those already described for S. japonicum [1719,21]. We found that
26 out of our 112 microRNAs were conserved between these two
species (Figure 1). In order to determine how many of those 26 are
specific to the Schistosoma lineage, we searched the genomes of the
flatworms Schmidtea mediterranea  and Dugesia japonica  for
homologous sequences. Strikingly, all known microRNAs
conserved between Schistosoma species were also conserved in other
flatworms. We also detected a set of S. japonicum homologs for 12 of
our newly identified S. mansoni sequences that were not conserved
in other platyhelminthes (Figures 1A and B). Hence, these 12
microRNAs are the first instances of schistosome-specific
Homology searches in other animals showed that three
microRNAs are specific to platyhelminthes: mir-755, mir-2162
and mir-8451 (Figure 1A). Thirteen microRNAs are
protostomespecific and the remaining 10 are conserved across the animal
kingdom (Figure 1A). A total of 71 microRNAs identified in this
study are not detected in any other species, and are therefore likely
to be S. mansoni specific.
Sex-biased expression of microRNAs in S.mansoni
To explore potential sex-biased expression, we compared the
relative expression levels of the 112 microRNAs in males and
females (Figure 2A). We found 13 microRNAs that are
differentially expressed between males and females. A significant excess
 show increased expression in females (Figure 2A, Table 2).
We further quantified the relative expression level of all 3
malebiased and 4 of the female-biased microRNAs by real-time PCR
(see Materials and Methods). The observed fold changes in our
qPCR experiments were consistent with those observed in our
RNAseq analysis (Table 3), although the two least biased
microRNAs by RNAseq show small and non-significant changes
We next evaluated whether microRNA loci on the sex
chromosomes are biased towards differential expression between
sexes. The current assembly of the S. mansoni genome does not
differentiate between Z and W chromosomes. At this stage, we
cannot therefore evaluate the two sexual chromosomes separately.
In Figure 2B, we plot the relative enrichment of
sex-chromosomelinked microRNAs for male and female-biased expression. We
observed that the sex chromosome has fewer female-biased
microRNAs (,3-fold change) than expected by chance, although
the difference is only marginally significant (p = 0.10). The data
therefore indicate that microRNA genes with female sex-biased
expression may have a tendency to move out of sex chromosomes
The mir-71/mir-2 microRNA cluster is highly conserved in
invertebrates, and it has been shown that this cluster is duplicated
in Platyhelminthes [25,28]. Interestingly, one of the clusters
(mir71/2a/2b/2e) is on the sex chromosomes while the other
(mir71b/2f/2d/2c) is on chromosome 5 [25,29]. This has led some
authors to postulate that the mir-71/mir-2 clusters may be
involved in sexual maturation in Schistosoma . Our analysis
reveals that all microRNAs in the autosomal cluster have
femalebiased expression, while the sex chromosome cluster does not
show any bias (Figure 2A). Interestingly, the two clusters emerged
by a duplication in the ancestral lineage leading to Schistosoma, and
the multiple copies in other Platyhelminthes [29,30] came from
independent duplication events (Supplementary File S4). This
example may shed some light on how sex chromosomes evolved in
dioecious species (see Discussion).
Although the computational prediction of microRNAs has been
useful to understand the biology of small RNAs in S. mansoni ,
sequencing is required to validate the existence of these
microRNAs as well as for detecting new sequences. Our work
has confirmed the existence of 20 of the microRNAs predicted by
de Souza Gomes et al.  and we have expanded the S. mansoni
microRNA set to 112 loci. We specifically detect microRNAs
expressed in sexually mature adults, and microRNAs specifically
SO: AB SOLiD sequencing; MS: Illumina MiSeq sequencing.
expressed in other developmental stages (such as schistosomulas)
may have escaped our analysis. The use of deep sequencing also
permits the characterization of microRNAs produced from both
strands of the same locus, and we have identified sense and
antisense microRNA production from the mir-124 locus.
However, there is no evidence that this microRNA is also bidirectionally
transcribed in other species. Indeed, bidirectionally transcribed
microRNAs are rare and poorly conserved; only two cases of
conserved bidirectional microRNAs are known in protostomes:
iab-4 and mir-307 .
We observe an excess of microRNAs that exhibit female-biased
expression (Figure 2A). This is in agreement with the overall
female-bias observed for protein-coding genes in both S. mansoni
 and S. japonicum , although a recent expression analysis in
S. japonicum showed no gender bias . Recently, a work in the
parasitic nematode Ascaris suum showed that microRNAs are
differentially expressed between males and females . Although
the differences were small and the targeting properties of male and
female microRNAs similar, they reported a tendency of male
microRNA to target extracellular proteins . Another work in
the bird Taeniopygia guttata (zebra finch), suggests that the
malebiased expressed microRNA mir-2954 specifically target genes in
the Z chromosome, and may be involved in sexual dimorphism in
song behavior . Together, these papers and our work point to
a general mechanism of microRNA-modulation of sex-specific
A recent work shows that some microRNAs are specifically
expressed in Schistosoma japonicum eggs . In that work, the
authors also measured the microRNA expression levels in males
and females. We reanalyse their data and find that two out of
our three male-biased microRNAs (mir-1 and mir-61) have a
consistent bias in Schistosoma japonicum, while the third is not
present in their dataset. Also, five of our female-biased
microRNAs also showed a female bias in their work (mir-71b,
mir-2c, mir-2d, bantam and mir-31). These findings further
validate our results and show that the sex-biased pattern of
microRNA expression is evolutionarily conserved between these
1Log Fold Change after Upper Quartile normalization of Illumina MiSeq read counts.
2False discovery rate (q-value) for a dispersion of 0.2 according to edgeR manual (see Methods).
Sex-biased gene expression affects the genetic composition of
chromosomes, since selection has different effects on sex-biased
genes depending on whether they are located on sex chromosomes
or on autosomes (reviewed in ). Likewise, sex chromosomes
have distinctive evolutionary patterns, which affect the genes
encoded within . We may therefore expect to see signatures of
chromosome evolution in sex-biased microRNAs. Indeed, we
observed that microRNAs that are female sex-biased are depleted
in the sexual chromosomes (Figure 2B). This may indicate a loss of
sex-biased genes at the sex chromosomes. One interesting example
is the female-biased mir-71b/2f/2c/2d autosomal microRNA
cluster, which has a paralogous copy in the sex chromosome with
no biased expression. If a microRNA gene that is selectively
advantageous for females becomes part of a sex chromosome (by
sexualisation of the chromosome, or otherwise), selection over this
gene will be less efficient in the heterogametic sex (females in our
case), generating a conflict between expression pattern and
chromosomal location. Duplication of a gene into an autosome
1Log Fold Change for the differences between Ct values for target and control
2P-values based on t-test of Ct value differences for three technical replicates.
has been recently proposed as a way to escape such conflict
(reviewed in ), and the mir-71/mir-2 cluster duplication
appears to be an example of this. Although a more comprehensive
analysis of duplicated microRNAs with sex-biased expression is
required to confirm this, our analysis shows that sex-biased
expressed microRNAs have an impact in shaping the genome
The study of microRNAs in the schistosomes is of both
evolutionary and biomedical interest. First, the recent evolution of
a sexual reproductive system from a hermaphrodite ancestor can
give clues about how sexuality emerged in other species. Our data
are consistent with the acquisition of sex-biased expression of
conserved microRNAs soon after the species become dioecious.
Second, the characterization of Schistosoma-specific microRNAs
may provide new targets for infection control. In this work, we
characterize for the first time 12 microRNAs conserved between S.
mansoni and S. japonicum but not in other platyhelminthes (nor in
other animals). Two of these sequences also showed female-biased
expression (mir-8437 and mir-8447). However, some important
questions remain to be answered: Are microRNAs conserved in
the two studied schistosomes also conserved in other Schistosoma
species or in other trematodes? Is sex-biased expression of
microRNAs associated with sex-biased expression of their targets?
Do other dioecious flatworms have sex-biased expression of
microRNAs? The genomic sequencing of more platyhelminthes
and characterization of their microRNAs will help us to answer
Materials and Methods
Small RNA extraction, library construction and
For the detection of expressed microRNAs, female mice (BKW
strain) were infected with Belo-Horizonte strain Schistosoma mansoni
parasites by paddling in water containing 200 cercariae. Seven
weeks after infection, adult schistosomes were collected from the
mice post-mortem by hepatic portal perfusion. RNA was extracted
from adult schistosome samples with the miRVana miRNA
isolation kit (Ambion). We used two sequencing technologies, AB
SOLiD and Illumina MiSeq, to sequence S. mansoni small RNA
libraries. The extremely deep coverage provided by SOLiD
sequencing provides high sensitivity for the discovery of novel
microRNAs. We further used Illumina MiSeq sequencing of
gender-specific libraries to compare the expression level of
microRNAs between males and females. Library construction
was performed as previous described  using the SOLiD Small
RNA Expression Kit (Ambion). SOLiD sequencing was performed
at the Center for Genomic Research at the University of
Liverpool. We obtained a total of 124,341,126 SOLiD sequence
reads from two libraries. For the gender-specific differential
expression of microRNAs, we prepared RNA libraries with the
miRVana kit from separate male and female samples (provided by
Andrew MacDonald and Rinku Rajan at the University of
Edinburgh), and prepared libraries for Illumina MiSeq sequencing
according to the manufacturers instructions. MiSeq samples were
sequenced in the Genomics Core Facility at the University of
Manchester. High-throughput datasets were deposited in Gene
Expression Omnibus (GEO) at NCBI (accession number:
MicroRNA detection and annotation
Sequencing reads from male and female libraries were
separately mapped to the S. mansoni reference genome (assembly
5.1 available at http://www.genedb.org/Homepage/Smansoni;
[38,39]) with Bowtie 0.12 using the sequential trimming strategy
implemented in SeqTrimMap 1.0  allowing 2 mismatches.
Sequences mapping to potential rRNAs or tRNAs were first
removed. Putative tRNAs were predicted in the genome sequence
with tRNAscan-SE using default parameters  and ribosomal
RNAs (rRNAs) were extracted from the SILVA database (http://
www.arb-silva.de/, release 108). Mapped reads with a length of
1925 nucleotides, matching five or fewer positions in the genome
(a total of 63,771,124 sequence reads), were used to detect
microRNAs as previously described [37,40]. We further used
BLAST  to search microRNA candidates against the S. mansoni
genome and discarded those with more than 5 hits (E-value
,e10, 80% query coverage) to remove potential repetitive elements:
44 candidate sequences did not pass this filter. MicroRNA
candidates were manually inspected. Potential homologs of known
microRNAs (detected by BLASTN against all hairpin sequences
from miRBase version 17 ) with reads mapped from our
datasets but which did not pass our criteria were also retained.
Homolog of our microRNA candidates were predicted in the
genome sequences of S. japonicum (version 2), S. mediterranea (v. 3.1),
Caenorhabditis elegans (v. 7.1), Tribolium castaneum (v. 3.0), Drosophila
melanogaster (v. 5.1), Homo sapiens (v. 37.1) and Gallus gallus (v. 2.1),
with parameters: 2W 4, 2r +2, 2q 23. Only sequences with a
predicted hairpin structure and conserving at least one mature
sequence were considered as putative homologs.
Differential expression analyses
We mapped reads produced from MiSeq sequencing reactions
to our annotated S. mansoni microRNAs and discarded all reads
that map to more than one microRNA locus. Read counts were
transformed with the upper quartile normalization using the
edgeR package , following the suggestions in . Other
normalization procedures (TMM, LOWESS and no
normalization) did not change the results (Supplementary File S3). Fold
changes in expression levels are given in logarithms in base 2. We
consider a dispersion of expression between experiments of 0.2
and a false discovery rate of 10%. With these parameters,
microRNAs showing a two-fold difference in their expression
levels are considered to be sex-biased, as routinely suggested
[46,47]. The expression level in males and females of seven
microRNAs (mir-1b, mir-61, mir-281, mir-36b, mir-71b, bantam
and mir-8437) were further validated by quantitative PCR. We
used custom made TaqMan assays manufactured by Life
Technologies. Fluorescent quantification was done in a Chromo
4 qPCR system (BioRad) for a log fluorescent threshold of 0.05,
using mir-36a (the microRNA showing the least bias in the MiSeq
experiments) as the non-sex-biased control microRNA. For each
amplification, we performed three technical replicates to estimate
the significance of the observed differences.
Laboratory animal use was within a designated facility regulated
under the terms of the UK Animals (Scientific Procedures) Act,
1986, complying with all requirements therein. The experiments
involving mice in this study were approved by the Natural History
Museum Ethical Review Process and work was carried out under
Home Office project licence 70/6834.
File S1 Sequences, chromosomal location and number of reads
for all Schistosoma mansoni microRNAs characterized in this study.
Structural features of Schistosoma mansoni microRNA
File S3 Differential expression analysis between male and
female Schistosoma mansoni specimens for different normalization
File S4 Phylogenetic relationships between mir-71 sequences in
Schistosoma mansoni, S. japonicum and Schmidtea mediterranea.
We thank Andrew MacDonald and Rinku Rajan for supplying the worm
samples for sex-specific differential expression and Maria Ninova for
technical assistance with the TaqMan assays. We are also grateful to
three anonymous reviewers for helpful comments on the manuscript and
to Matt Berriman for discussion on the Schistosoma mansoni genome
Conceived and designed the experiments: AM SGJ MR. Performed the
experiments: AM AK JHLH AME. Analyzed the data: AM AK MR SGJ.
Contributed reagents/materials/analysis tools: AME DR. Wrote the
paper: AM SGJ MR.
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