Comparative Study of Transcriptome Profiles of Mechanical- and Skin-Transformed Schistosoma mansoni Schistosomula
Berriman M (2013) Comparative Study of Transcriptome Profiles of Mechanical- and Skin-Transformed Schistosoma mansoni
Schistosomula. PLoS Negl Trop Dis 7(3): e2091. doi:10.1371/journal.pntd.0002091
Comparative Study of Transcriptome Profiles of Mechanical- and Skin-Transformed Schistosoma mansoni Schistosomula
Anna V. Protasio 0
David W. Dunne 0
Matthew Berriman 0
John Pius Dalton, McGill University, Canada
0 1 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom, 2 Department of Pathology, University of Cambridge , Cambridge , United Kingdom
Schistosome infection begins with the penetration of cercariae through healthy unbroken host skin. This process leads to the transformation of the free-living larvae into obligate parasites called schistosomula. This irreversible transformation, which occurs in as little as two hours, involves casting the cercaria tail and complete remodelling of the surface membrane. At this stage, parasites are vulnerable to host immune attack and oxidative stress. Consequently, the mechanisms by which the parasite recognises and swiftly adapts to the human host are still the subject of many studies, especially in the context of development of intervention strategies against schistosomiasis infection. Because obtaining enough material from in vivo infections is not always feasible for such studies, the transformation process is often mimicked in the laboratory by application of shear pressure to a cercarial sample resulting in mechanically transformed (MT) schistosomula. These parasites share remarkable morphological and biochemical similarity to the naturally transformed counterparts and have been considered a good proxy for parasites undergoing natural infection. Relying on this equivalency, MT schistosomula have been used almost exclusively in high-throughput studies of gene expression, identification of drug targets and identification of effective drugs against schistosomes. However, the transcriptional equivalency between skin-transformed (ST) and MT schistosomula has never been proven. In our approach to compare these two types of schistosomula preparations and to explore differences in gene expression triggered by the presence of a skin barrier, we performed RNAseq transcriptome profiling of ST and MT schistosomula at 24 hours post transformation. We report that these two very distinct schistosomula preparations differ only in the expression of 38 genes (out of ,11,000), providing convincing evidence to resolve the skin vs. mechanical long-lasting controversy.
Funding: This work was funded by the Wellcome Trust, through core support of the Wellcome Trust Sanger Institute (grant WT 098051). Additional support was
provided by a Wellcome Trust Programme Grant to DWD (WT 083931/Z/07/Z). 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.
Schistosomiasis is a parasitic disease caused by platyhelminths of
the genus Schistosoma. It has been estimated that ,200 million
people are infected and ,200,000 die due to schistosomiasis-related
pathologies . Without a vaccine, mechanisms of prophylaxis rely
primarily on reduction of the number of infected individuals
through mass-administration of the only available drug
praziquantel. However, the number of infected people has changed little over
the last decades . What is more, reduced susceptibility of
Schistosoma mansoni worms to praziquantel has been reported in the
field [2,3] and resistance to the drug can be induced under
experimental conditions [4,5], raising the possibility that a similar
situation could be also seen in the field. Consequently, the
development of new mechanisms of intervention is a priority.
In this context, it is important that the process of infection is well
characterised. The infectious agents for the human host, the
cercariae, are microscopic free-living larvae released by infected
fresh water snail hosts. Cercariae infect their mammalian host during
water contact by trespassing across the skin barrier. This process is
characterised by rapid morphologic, metabolic and physiological
changes  that results in obligate parasitic schistosomula in as
little as 2 hours . The most prominent aspects of this
transformation are the loss of the cercarial tail and a series of
changes in the parasites surface. During skin penetration, the
outermost layer in the parasites surface, the glycocalyx, gets thinner
by the action of secretions from the parasites own acetabular glands
, which are emptied during the process of transformation .
The remains of the glycocalyx are shed together with transient
microvilli structures that form and disappear during this
transformation process . At the same time, pre-packed multi-laminated
vesicles originating from the body of the parasite make their way to
the surface where they release their contents; these contribute to the
generation of the new double-bilayer membrane, characteristic of
the intra-mammalian stage of the parasite .
Increasing research on the schistosomulum stage required the
development of efficient, reproducible and rapid ways to generate
large quantities of biological material. Various effectors are known
to elicit the artificial transformation of cercariae into
schistosomula, for example, cell growth media at 37uC [15,16] or just low
Schistosomiasis is an endemic parasitic disease affecting
,200 million people in the most socioeconomically
deprived regions of the world. Human infection occurs
during water contact where free-living larvae called
cercariae penetrate host skin and become parasitic
organisms called schistosomula. This stage represents the
first encounter of the parasites with the host and is also
regarded as one of the most vulnerable stages of the
parasites life cycle. Therefore, schistosomula are the focus
of many studies, many of which look at changes in the
expression of genes as a way of understanding the process
of infection, identifying potential drug targets and vaccine
candidates. Because collecting enough parasitic material
from natural infections is not possible for certain types of
studies (for example, gene expression studies), a
mechanical transformation of the cercariae into schistosomula is
often used instead and assumed as a good proxy for the
natural transformation process. However, the equivalency
of gene expression profiles between naturally transformed
parasites and the mechanically transformed counterparts
has never been studied. In this report, we analyse
differences in gene expression patterns between these
two different parasite preparations and provide enough
data to resolve a long-lasting controversy.
osmolarity phosphate buffer saline solution  seem to be
enough to trigger the cercariae to schistosomula transformation.
The presence of certain skin lipids, yet is not essential , also
plays a role in the process of cercariae transformation and
penetration [18,19] probably by triggering the release of
acetabular glands contents . The most popular method for
obtaining artificially transformed schistosomula uses a mechanical
transformation (MT) protocol that includes some sort of shear
force (centrifugation , passages through an emulsifying
needle , or shaking ) applied to freshly shed cercariae
followed by separation of cercariae heads from tails (usually by
centrifugation in a density gradient) and posterior incubation of
the cercariae heads/schistosomula in culture media at 37uC.
Parasites obtained using this protocol show no major
morphological or biochemical differences with those recovered from natural
infections [10,15]; making the MT the method of choice for
obtaining large quantities of schistosomula.
However, at the level of the whole transcriptome, equivalency of
MT schistosomula to those obtained from natural infections has not
been established; even though these artificial parasite preparations
have been used almost exclusively in the identification of potential
vaccine proteins and in high-throughput studies of gene expression
, identification of drug targets  and screening of a
compound library . Artificial induction of stress or mechanical
damage may induce gene expression signals that are not responding
to the natural process of infection. Moreover, failure to induce
physiologically important transcription events, triggered by host-skin
specific signals, could lead to exploitable vulnerabilities being missed.
Our work presented here uses high throughput transcriptome
sequencing technology, known as RNA-seq , in combination
with the latest genome assembly available for S. mansoni  to
compare the profile of genes expressed in MT and ST schistosomula.
Materials and Methods
S. mansoni (NMRI strain of Puerto Rican origin) cercariae were
shed from infected Biomphalaria glabrata snails by exposing them to
the light for 1.5 hours. MT schistosomula were obtained using an
optimised version of the protocol used by Brink et al., .
Optimisation steps of the protocol were implemented in the tail
detachment step (shake cercariae vigorously for approximately
30 seconds in a vortex mixer before passing these through a 21G
syringe needle approximately 1315 times) and the separation of
heads/schistosomula and tails (by placing the heads plus tails
suspension on 10 ml of ice-cold 70% Percoll (Sigma, UK) and
90 mM NaCl solution in DMEM in 15 ml conical tubes) by
centrifugation at no more than 1000 g for 10 minutes at 4uC.
Skin-transformed (ST) schistosomula were obtained using a
modified version of the protocol published by Clegg et al., .
TO (Tuck Ordinary) mice (Harland, UK) were killed with an
overdose of anaesthetics followed by cervical dislocation according
to Home Office regulations. Hair was removed from the
abdominal and dorsal skin areas using clippers and skin was later
excided from the animal using dissecting scissors. Each animal
provided an area of skin of approximately 7.5 cm2; which was
divided into two halves. Gel-like dermal tissue was removed by
rubbing the skin gently (for approximately 5 minutes) with
sterilized gauze soaked in supplemented DMEM (Dulbeccos
Modified Eagles medium supplemented with 100 U/L penicillin,
0.1 mg/L streptomycin and 10 mM L-glutamine). The
transformation apparatus is presented in Figure 1A. The lower
compartment of the assembly was filled with supplemented
DMEM containing 2% fetal calf serum (FCS) and one half of
prepared skin was mounted covering the opening of the tube with
the dermal side facing downwards. The upper compartment was
placed above the lower compartment with a rubber O-ring in
between. All pieces were kept in place by holding both tubes with a
metal clip (Figure 1B). The skin surface was washed three times
with aquarium water and assemblies were checked for leaks. All
assemblies were placed in a water bath pre-warmed at 37uC; the
bottom compartment of the assembly was constantly kept at this
temperature (Figure 1C). Experiments were carried out in a room
with controlled temperature of 28uC. Approximately 12,000
14,000 freshly shed cercariae kept in aquarium water were placed
in each assembly and these were left in the water bath for 3 hours.
Schistosomula preparations were individually checked for
contamination with tails. Samples with more than 4% tails/cercariae
contamination were discarded.
MT and ST schistosomula preparations were placed in
individual tubes, washed 3 times in supplemented DMEM and
incubated at 37uC and 5% CO2 for a total of 24 hours in growth
media (supplemented DMEM, 10% FCS, 1% Hepes buffer).
Schistosomula preparations were observed under the microscope
using a Leica DM 1 L inverted microscope (Leica, Milton Keynes,
Bucks, UK) at 106 or 406. Video recordings were taken using a
Dino-Lite AM-423X camera and DinoXcope software (Version
1.7.3). The criterion used for evaluating parasites is the one used in
Mansour et al., .
After the incubation period was completed, parasites were
transferred to 15 ml conical tubes and centrifuged at 1,000 g for
5 minutes, supernatant was discarded and schistosomula were
suspended in 1 ml of TRIzol reagent (Invitrogen, UK) and stored
at 280uC until RNA extraction.
RNA extraction, library preparation and sequencing
Total RNA from parasite material was extracted using TRIzol
(Invitrogen, UK) according to manufacturer specifications. After
extraction, RNA quality was assessed using an Agilent RNA 6000
Nano - Bioanalyzer and quantified using a NanoDrop ND-1000
UV-Vis spectrophotometer. RNA-seq libraries were prepared as
previously described  and sequenced as 76-base paired reads
Figure 1. Diagram and photographs of assemblies used in skin-transformation of schistosomula. A Graphical representation of a
transformation assembly. B Photograph of one of the transformation assembly prior to use. C Three transformation assemblies in use during an
experiment. The lower compartment of the assembly is placed in a water bath with a constant temperature of 37uC while the upper compartment is
left at a room temperature (28uC).
using the Illumina Genome Analyzer IIx platform. Raw sequence
data were submitted to ArrayExpress (http://www.ebi.ac.uk/
arrayexpress/) under accession number E-MTAB-451.
RNA-seq reads alignment and gene expression analysis
RNA-seq reads were aligned to the latest S. mansoni reference
genome (version 5.0, ) using TopHat  (version 1.3.1) with
default parameters except for minimum and maximum intron
sizes which were set to 10 and 30,000 bp respectively. Other
parameters that were specified included the type of library
sequenced (set to standard cDNA Illumina library; library-type
fr-unstranded) and the mate pair distance (or insert size; -r option),
which was calculated individually for each library. Only uniquely
mapping reads were kept for further analyses. The number of
reads aligned to each transcript was calculated using BEDTools
 and used to calculate RPKM (reads per kilobase per million
of reads mapped) values  for each transcript. A threshold
RPKM value was calculated as described in Protasio et al., 
and transcripts with expression ,2 RPKM were removed from
the dataset resulting in the reduction of the total number of
transcripts from 11,778 to 9,291 (2,487 transcripts had negligible
expression in both samples). Differential expression of transcripts
was performed using EdgeR . P-values were adjusted for
multiple testing  and the threshold for significance set at
adjusted p-value#0.05. A complete list with fold change values
and associated adjusted p-values obtained from EdgeR are
provided in Supplementary Table S1.
RT-qPCR validation of differentially expressed genes
Relative expression of a subset of genes found differentially
expressed between the ST and MT schistosomula were assayed
using real time quantitative PCR (RT-qPCR). Primers for these
genes were designed using Primer3 software  and ordered
from Sigma, UK (primer sequences are available upon request).
First strand cDNA was synthesised from 1 ug of original total
RNA samples (MT2 and ST2 Table 1) using SuperscriptII
(Invitrogen, UK) according to manufacturers instruction. All
RTqPCR reactions were performed in a Mx3005P QPCR System
(Agilent Technologies) and using KAPA SYBR FAST qPCR Kit
(Kapa Biosystems). PCR efficiencies for each primer pair were
calculated using 10-fold dilutions of MT2 cDNA. Relative gene
expression for a given gene was quantified relative to the
expression of a reference gene (rRNA18S). Cycle thresholds (Ct)
for each reaction where obtained using the MxPRO QPCR
Software (Agilent Technologies) and used in the Pfaffl equation
 to calculate the fold change expression of a target gene
between samples. Fold change values reported are the mean of
four replicates. In order to compare RT-qPCR and RNA-seq
derived fold change values, RNA-seq standard deviation (SD) was
calculated using the method described by Busby et al., .
Metabolic activity of schistosomula
AlamarBlue incorporates a colour indicator of metabolic
activity of the mitochondrial function  and has previously
been used to assess the viability of schistosomula . In order to
assess metabolic activity of MT and ST parasites, 250
24-hoursold-schistosomula obtained from MT and ST methods were
incubated in AlamarBlue (Invitrogen, UK) for either 3 or 24 hours
prior to measurement. Eleven and 12 samples were assayed for
MT and ST respectively. Absorbance was measured at 570 nm
(with reference at 600 nm) using a microplate reader BioTek
PowerWave HT (BioTek Instruments Inc., Winooski, VT, USA);
data collection was performed using the software Gen5 (BioTek
Instruments Inc., Winooski, VT, USA). Raw absorbance data is
presented in Supplementary Table S2. Students t-test was used to
evaluate the significance between mean absorbance.
MT: mechanically transformed; ST: skin-transformed.
All animal procedures were performed in accordance with the
UK Animals (Scientific Procedures) Act 1986 and as authorised on
personal and project licences issued by the UK Home Office.
Optimization of transformation protocols
Both MT and ST transformation protocols were subjected to
optimization. Results comparing the standard and optimised MT
protocols are shown in Figure 2A and 2B respectively. For the
MT, optimised conditions resulted in increased numbers of
schistosomula and a lower percentage of damaged or non-viable
individuals. Reduction of the number of syringe passages resulted
in increased number of viable parasites while a higher percentage
of Percoll resulted in less contaminating tails (,1%). For the ST,
we found that schistosomula preparations virtually free from tail
contamination (,1% to 4%) could be obtained by placing no
more than 14,000 cercariae in the upper compartment of the
transformation apparatus. However, tail contamination was more
frequent in the ST preparations than in MT and samples
dedicated for RNA-seq libraries had to be carefully selected.
Contrary to schistosomula resulting from MT, non-viable parasites
were hardly ever observed in ST preparations. Under the light
microscope, schistosomula obtained from both protocols were
indistinguishable from each other (Supplementary Video S1 and
Video S2) and both schistosomula preparations progressed to later
stages in the life cycle (up to two weeks post transformation) when
cultured in vitro (data not shown). In terms of recovery, MT yields
,90% of the applied cercariae, while the ST yields only ,10%.
Differential gene expression between MT and ST
An overview of the sequencing and alignment results obtained
for the sequenced samples is presented in Table 1; where samples
1 and 2 (for both MT and ST) represent independent
schistosomula transformations (replicates). In the case of ST samples, and
due to the limited number of schistosomula obtained in each
experiment, approximately 3 experiments were pooled to provide
enough biological material.
Our dataset included 11,778 annotated transcripts and we
found that 9,291 showed expression above background in at least
one of the samples. Correlation analysis of the 24-hour old MT
and ST schistosomula transcriptome samples showed high values
for both Pearsons product and Spearmans rank coefficients (0.98
and 0.99, respectively; Figure 3A). Using the software package
EdgeR , we found only 38 differentially expressed transcripts
(adjusted p-value,0.05) between MT and ST schistosomula. Of
these, 28 transcripts showed higher relative expression in the ST
parasites (Table 2) while 10 transcripts showed higher relative
expression in the MT (Table 3). A graphical representation of
differentially expressed transcripts is shown in Figure 3B.
RT-qPCR validation was performed for 33 of the 38 genes
found differentially expressed between ST and MT schistosomula
(Figure 4). With 95% confidence interval, the fold change values
obtained from both methods overlapped in 14 cases
(Smp_029780, Smp_057860, Smp_124000, Smp_132670,
Smp_172770, Smp_211020, Smp_212760, Smp_900010,
Smp_900020, Smp_900030, Smp_900050, Smp_900070,
Smp_900080, Smp_900090). Moreover, fold change values
obtained from these two different methods are highly correlated
(Pearsons correlation 0.89, p-value 3.85E212) and only in 4 cases
(Smp_028850, Smp_067800, Smp_155320, Smp_001070) the
direction of the fold change disagrees between the two methods.
Genes more highly expressed in ST schistosomula
Table 2 shows a list of genes more highly expressed in
skintransformed parasites. We found that all 12 mitochondrial genes
(Smp_900000Smp_900110) are found in this list. In order to
investigate whether the higher expression of the mitochondrial
genes had any consequences on metabolic activity we used the
AlamarBlue (AB) assay. AB is a good indicator of mitochondrial
activity through the measurement of redox species generated by
the respiratory electron chain . Incubation of 24-hour old
schistosomula for 3 hours in AB showed no significant difference
between MT and ST parasites (Figure 5 blue boxplots).
Increasing the incubation time to 24 hours showed an incremental
increase in the absorbance (compared to blank wells) and a
significant difference (t-test, p-value,0.01) between MT and ST
schistosomula (Figure 5 green boxplots) suggesting that these two
populations of parasites have not only different rates of
mitochondrial metabolism but that ST parasites are more
metabolically active than their MT counterparts after 24 hours of
in vitro culture.
The remainder of genes found relatively more expressed in the
ST sample included examples that could be associated with the
infection process. Two of these genes, for instance, are involved in
calcium sensing (Smp_151600) or binding (Smp_132670),
functions that have been associated with schistosomula adaptation to
the mammalian host . It is possible that such mechanisms are
induced by contact with the host skin explaining the reduced
expression of such transcripts in MT parasites.
Genes more highly expressed in MT schistosomula
Proteases and proteases inhibitors are among the transcripts
that were more expressed in MT schistosomula. Proteases have a
recognised role in schistosomes; for example, adult worms use a set
of aspartic proteases called cathepsins for the purpose of feeding
 while cercariae use elastase and other proteases during the
process of skin invasion [45,46]. We found a gene encoding a
secreted serine protease from the trypsin family (Smp_002150),
with expression in MT parasites double that of ST parasites.
RNAseq data (Supplementary Table S3 in Ref. ) showed that the
expression of this gene is developmentally up regulated after the
transformation in schistosomula, showing an impressive 30-fold
increase between 3-hour and 24-hour post-transformation
Alongside the serine protease we found two protease inhibitors
that were also differentially expressed. Protease inhibitors can
neutralise the action of host- and/or parasite-derived proteases.
Smp_089670 encodes a 1,800 amino acids polypeptide with high
similarity to an alpha-macroglobulin. Macroglobulin-type
inhibitors entrap their target proteases limiting the range of substrates
they can act upon; hence, they have a regulatory role rather than
strictly inhibitory effect . Macroglobulins can also inhibit
coagulation , perhaps indicating that the secretion of the S.
mansoni alpha-macroglobulin functions as a facilitator of
schistosomula migration through the broken tissue/vessels during the
skin stage. The second protease inhibitor was a kunitz-type serine
protease inhibitor (Smp_147730). These types of inhibitors have
been postulated to have an important role in the host-parasite
interaction in Echinococcus granulosus infections . Interestingly,
both protease inhibitors were significantly up regulated during
transformation from cercariae to schistosomula (Supplementary
Table S3 in Ref. ) suggesting that their expression is
Two microexon genes are more expressed in MT
Microexons (,36 bp, in multiples of 3 bases) typically form a
small part of some genes in most eukaryotes  but for a few
genes in S. mansoni, microexons comprise the majority (,75%) of
the sequence length and these genes have therefore been termed
microexon genes (MEGs) [51,52]. Each MEG has the potential to
generate an enormous repertoire of splicing variants through exon
skipping because missed exons do not cause frame-shifts. The
particular gene structure of MEGs therefore provides an easy
mechanism to generate protein variation and seems to be both
time and tissue specific .
Two MEGs from two different families appeared more
expressed in MT compared to ST schistosomula at 24 hours
after transformation (Table 3). In the case of Smp_180340, a
MEG-2 member, RNA-seq coverage was poor and not specific to
the exons in both samples; probably indicating unprocessed
transcripts and was not considered for further analysis. For
Smp_124000, RNA-seq data from schistosomula samples agreed
with the current annotation of the gene (Figure 6). Intriguingly
however, the isoforms expressed in MT and ST differed from
each other, with three exons that were expressed in the ST being
absent from the MT schistosomula sample (Figure 6). Because the
RNA-seq experiments assayed the transcriptional status of large
numbers of parasites simultaneously, we can therefore rule out
at least in this example that exon skipping is simply a stochastic
Gene product description
Differentially expressed genes with adjusted p-value,0.05 are shown. Gene identification numbers with a prefix Smp_90 are those encoded in the mitochondrial
Gene product description
Differentially expressed genes with adjusted p-value,0.05 are shown.
Figure 4. RT-qPCR and RNA-seq fold change values are highly correlated. Bar plots and inset scatter plot show a comparison of RNA-seq
and RT-qPCR fold change values obtained for 33 differentially expressed genes. Fold change values are represented in the log2 scale and error bars
represent 95% confidence interval (fold change 61.96xSD). The inset scatter plot highlights the high correlation found between both methods
(Pearsons correlation = 0.89, p-value = 3.85E212). The solid lane represents the linear regression of this correlation; as a guide the x = y correlation is
also shown as a dashed line.
The process of cercarial invasion and early stages of
schistosomula migration are relevant for the development of intervention
strategies against schistosomiasis. The skin schistosomula stage
represents the first encounter of the parasite with the mammalian
host and it is regarded as a vulnerable stage for parasite killing
. Hence, schistosomula have been the target of many
studies that focus on both the adaptation of the parasite to its host
and the identification of drug targets and vaccine development.
The study of changes in gene expression across different stages
of host invasion can be used to investigate parasite adaptation to
the host. With one exception, where in vivo recovered and in vitro
cultured S. japonicum schistosomula were compared , all
highthroughput gene expression studies have used MT schistosomula
at different developmental stages by just prolonging the in vitro
incubation time [25,27,28,32,57]. Since MT schistosomula are
only proxies for natural infections, the differences between these
and more naturally transformed parasites needs to be established.
For instance, misleading artefactual parasite responses induced by
stress or damage need to be identified as well as potentially
important parasite responses that are only induced during the
rapid natural transformation of free-living cercariae into
obligatory parasitic schistosomula. Our work used the latest RNA-seq
technology to investigate the differences in the gene expression of
MT and ST schistosomula at 24-hour post-transformation. We
found that these samples differ only in the expression of 38
transcripts (out of 9,291 expressed transcripts; adjusted
pvalue,0.05). In order to validate our approach, we performed
RT-qPCR on 33 out of the 38 genes found differentially expressed
and found that, at least in this experiment, RNA-seq seems to over
estimate the fold change values of differentially expressed genes.
However, a high correlation value of 0.89 was found between the
two methods (similar values have been reported elsewhere )
suggesting that the RNA-seq is a valid and reliable method for
high-throughput identification of differentially expressed genes.
Increased transcript coverage (greater sequencing depth) as well as
the addition of more biological replicates may result in better
measurement by providing greater statistical power.
Transcripts encoded in the mitochondrial genome
(mitochondrial genes) were found more highly expressed in ST parasites
resulting in higher metabolic rates in ST parasites. Previously
Brink et al.,  suggested that ST parasites are a selection of the
most fit cercariae. We suggest that MT preparations may
contain a mixture of fit and less-fit parasites and therefore not all of
them are expected to develop at their maximum metabolic rate
resulting in an averaged reduced metabolic activity for the MT
MT parasites showed higher expression of a protease and two
protease inhibitors. Proteases have been linked to host tissue
invasion [59,60] and have been shown expressed in parasites
recovered from in vivo infections  as well as in purely MT
schistosomula when compared to cercariae . Therefore it is
not surprising that they are present in our schistosomula samples.
However, the unexpected finding is that these are more expressed
in the MT rather than in the ST sample. If proteases were linked
to the process of invasion, it would be expected that these be
triggered by the presence of components of the skin, elements that
are absent during the MT transformation. Further research will be
needed to understand what triggers the expression of these genes.
Two members of the microexon gene family were found
overexpressed in the MT parasites. What is more, the transcript
variants found here are different from the ones previously reported
confirming that different splice variants from the same loci are
expressed at different time points of the life cycle. Interestingly,
one of the variants expressed in 24 hours old schistosomula has a
different exon profile in the two preparations, suggesting that cues
from the environment might be triggering splicing variation. The
role and regulation of alternate splicing in MEG may become
clearer as the functions of microexon genes are further elucidated.
Due to the different treatments received by MT and ST
schistosomula, including the low temperature and mechanical
stress endured by MT schistosomula, we had hypothesised that
stress-associated transcripts (e.g. stress/apoptotic pathways) would
be differentially expressed. Surprisingly, we could not identify clear
markers of stress, possibly because we had optimised our MT
protocol to yield a minimum proportion of damaged parasites.
Other MT protocols involving, for example a greater number of
passages through a syringe-needle or a different source of
mechanical stress may give different results.
Since MT schistosomula are not exposed to skin lipids that are
known to play a role in transformation [18,19] and induce the
release of contents from the acetabular glands , we had
anticipated observing differences related to the presence of lipids
in the ST. However, we could not identify transcripts related to the
binding (fatty-acid binding proteins) or to the transport of fatty
acids and therefore conclude that the effect elicited by the presence
of skin lipids is independent from transcriptional regulation at that
time and is more likely related to machinery that the parasite may
already have in place prior to its encounter with host skin.
Previously, gene expression changes using in vivo recovered (IVS)
and mechanically transformed schistosomula (MTS) in S. japonicum
at 3 days after transformation has been published . The
authors showed that IVS parasites show higher expression of
transcripts encoding protaglandins, glutathione-S-transferase
Sm28GST, paramyosin, stress related proteins and transcripts
related to markers of anti-inflammatory and immunomodulatory
processes. In the case of MTS parasites, the authors report higher
expression of transcripts involved in glucose transport, fatty acids
transport and haemoglobin digestion. None of the genes found
differentially expressed in our analysis could be associated with the
functions described in the S. japonicum study; probably due to the
differences in the experimental design of both studies (i.e., time
points at which differential expression is assessed).
Our study represents a snapshot of the schistosomula
transcriptome after transformation. It is possible that a greater effect of
the different treatments applied to both populations might be seen
at shorter times after transformation. Ideally, a time-course
experiment comprising more than one time point comparison
between ST and MT schistosomula should have been performed.
Nevertheless, should differences in gene expression exist at earlier
time points, they disappear at 24 hours after transformation and
are unlikely to have consequences on the gene expression profile of
Finally, we emphasise that in view of the great differences in the
transformation processes analysed here, the number of genes
found differentially expressed between ST and MT at 24-hours
Figure 6. The microexon gene Smp_124000 is expressed as different isoforms in the ST and MT schistosomula. The exons or lack of
them that contribute to the new isoform are marked in dashed boxes while exons that are differentially expressed between the ST and MT
schistosomula are marked with a star (*).
post-transformation is unexpectedly small, suggesting that changes
in gene expression induced upon transformation might be
independent from the methodology employed to transformed
parasites, at least in the two methods here studied.
In this work, we provide further evidence that transformation
might be triggered by more robust signals, such as the change in
osmotic pressure  and/or temperature  between the water
and the host environments. We recommend that, except for the
reported genes, these samples should be considered as
transcriptionally equivalent. Our work contributes to the validation of gene
expression studies that have used MT schistosomula and provides
further evidence that the MT is a good proxy for natural
Table S1 Complete list of differentially expressed genes. Fold
change values, p-values and adjusted p-values are shown for each
transcript as reported by EdgeR .
We would like to thank Maureen Laidlaw and Frances Jones from the
Schistosomiasis Research Group (Department of Pathology, University of
Cambridge, UK) for their assistance during experimental procedures; Dr.
Avril Coghlan and Dr. James Cotton (Parasite Genomics, Wellcome Trust
Sanger Institute) for their help in RT-qPCR data analysis; Dr. Adam Reid
(Parasite Genomics, Wellcome Trust Sanger Institute) and Dr. Colin M.
Fitzsimmons (Department of Pathology, University of Cambridge, UK) for
critical reading of this manuscript.
Conceived and designed the experiments: AVP DWD MB. Performed the
experiments: AVP. Analyzed the data: AVP. Contributed reagents/
materials/analysis tools: DWD MB. Wrote the paper: AVP DWD MB.
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