Genetic Manipulation of Schistosoma haematobium, the
Gabriel Rinaldi1,2, Tunika I. Okatcha1,3, Anastas Popratiloff4, Mary A. Ayuk1, Sutas Suttiprapa1,
Victoria H. Mann1, Yung-san Liang5, Fred A. Lewis5, Alex Loukas6, Paul J. Brindley1*
1Department of Microbiology, Immunology and Tropical Medicine, School of Medicine and Health Sciences, The George Washington University Medical Center,
Washington, DC, United States of America, 2Departamento de Gene ´tica, Facultad de Medicina, Universidad de la Repu ´blica, (UDELAR), Montevideo, Uruguay,
3Department of Tropical Medicine, School of Public Health and Tropical Medicine, Tulane University, New Orleans, Louisiana, United States of America, 4Center for
Microscopy and Image Analysis, The George Washington University, Washington, DC, United States of America, 5Biomedical Research Institute, Rockville, Maryland,
United States of America, 6Queensland Tropical Health Alliance, James Cook University, Cairns, Queensland, Australia
Background: Minimal information on the genome and proteome of Schistosoma haematobium is available, in marked
contrast to the situation with the other major species of human schistosomes for which draft genome sequences have been
reported. Accordingly, little is known about functional genomics in S. haematobium, including the utility or not of RNA
interference techniques that, if available, promise to guide development of new interventions for schistosomiasis
Methods/Findings: Here we isolated and cultured developmental stages of S. haematobium, derived from experimentally
infected hamsters. Targeting different developmental stages, we investigated the utility of soaking and/or square wave
electroporation in order to transfect S. haematobium with nucleic acid reporters including Cy3-labeled small RNAs,
messenger RNA encoding firefly luciferase, and short interfering RNAs (siRNAs). Three hours after incubation of S.
haematobium eggs in 50 ng/ml Cy3-labeled siRNA, fluorescent foci were evident indicating that labeled siRNA had
penetrated into miracidia developing within the egg shell. Firefly luciferase activity was detected three hours after square
wave electroporation of the schistosome eggs and adult worms in 150 ng/ml of mRNA. RNA interference knockdown
(silencing) of reporter luciferase activity was seen following the introduction of dsRNA specific for luciferase mRNA in eggs,
schistosomules and mixed sex adults. Moreover, introduction of an endogenous gene-specific siRNA into adult
schistosomes silenced transcription of tetraspanin 2 (Sh-tsp-2), the apparent orthologue of the Schistosoma mansoni gene
Sm-tsp-2 which encodes the surface localized structural and signaling protein Sm-TSP-2. Together, knockdown of reporter
luciferase and Sh-tsp-2 indicated the presence of an intact RNAi pathway in S. haematobium. Also, we employed laser
scanning confocal microscopy to view the adult stages of S. haematobium.
Conclusions: These findings and approaches should facilitate analysis of gene function in S. haematobium, which in turn
could facilitate the characterization of prospective intervention targets for this neglected tropical disease pathogen.
Citation: Rinaldi G, Okatcha TI, Popratiloff A, Ayuk MA, Suttiprapa S, et al. (2011) Genetic Manipulation of Schistosoma haematobium, the Neglected
Schistosome. PLoS Negl Trop Dis 5(10): e1348. doi:10.1371/journal.pntd.0001348
Editor: Malcolm K. Jones, University of Queensland, Australia
Received June 17, 2011; Accepted August 22, 2011; Published October 11, 2011
Copyright: ? 2011 Rinaldi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Schistosoma haematobium infected hamster livers and intestines, and developmental stages including adults and cercariae of S. haematobium were
supplied under NIAID-NIH contract HHSN272201000005I. The authors acknowledge support from grant no. S10RR025565 (to AP) from the National Center for
Research Resources, NIH (http://www.ncrr.nih.gov/) for microscopical analysis. 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.
* E-mail: email@example.com
More people are infected with Schistosoma haematobium than with
the other schistosomes combined. Of .110 million cases of S.
haematobium infection in sub-Saharan Africa, 70 million are
associated with hematuria, 18 million with major bladder wall
pathology, and 10 million with hydronephrosis leading to severe
kidney disease [1,2,3]. In many patients, chronic inflammation in
response to S. haematobium ova leads to squamous cell carcinoma of
the bladder [4,5]. S. haematobium is classified as a Group 1
carcinogen by the World Health Organization’s International
Agency for Research on Cancer [6,7] although the cellular and/or
molecular mechanisms linking S. haematobium infection with cancer
formation have yet to be defined . One quarter to three
quarters of women infected with S. haematobium suffer from female
genital schistosomiasis (FGS) of the lower genital tract . FGS
results from deposition of the schistosome eggs in the uterus,
cervix, vagina and/or vulva, with ensuing host inflammatory
responses comprised of granulomas, fibrosis, and pathological
localized blood vessel formation . FGS increases susceptibility
to HIV/AIDS [10,11,12], and decreases female fertility .
Given the enormous numbers of people infected with S.
haematobium, and the pathogenesis of S. haematobium infection,
including its association with bladder cancer and HIV/AIDS,
www.plosntds.org1 October 2011 | Volume 5 | Issue 10 | e1348
there is a pressing need for new approaches to control including
the development of a vaccine to prevent infection with S.
haematobium. With regard to fundamental aspects of the host-
parasite relationship, research on S. haematobium is in its infancy
compared to S. mansoni and S. japonicum . There have been
massive recent advances in genomic, transcriptomic, and proteo-
mic datasets for both S. japonicum and S. mansoni [15,16,17]. There
now is an urgent need to establish similar datasets for S.
haematobium, and in addition to establish tools and approaches to
determine the function and importance of these schistosome genes
- including S. haematobium-specific genes . Here we cultured
several developmental stages of S. haematobium and applied several
functional genomics approaches to this species. We report that this
schistosome, like S. mansoni and S. japonicum, is amenable to
transformation with nucleic acid probes. Notably, the findings
indicated the presence of an intact, active RNA interference
pathway in S. haematobium, the neglected schistosome.
Materials and Methods
Eggs of an Egyptian strain of S. haematobium were isolated from
either small intestines, that had been thoroughly rinsed in 16PBS
to remove the gut contents, or livers of experimentally infected
Syrian golden hamsters  following a protocol optimized for
isolating eggs of S. mansoni from livers of mice . In brief, three
to five livers or two to three washed small intestines were chopped
finely with a scalpel blade, and then blended to a smooth
consistency in 50 ml of phosphate-buffered saline, pH 7.4 (PBS),
5 ml of 0.5% clostridial collagenase (Sigma) and 500 ml of
polymyxin B (Sigma). Digests were incubated with gentle shaking
at 37uC overnight, after which the contents were subjected to
centrifugation at 4006g for 5 min. The supernatant was removed
and the pellet resuspended in 50 ml PBS. This wash procedure
was repeated twice more, with the exception that after the final
centrifugation the pellet was resuspended into 25 ml of PBS. The
resuspended mixture from liver was passed sequentially through
250 and 150 mm sieves. No passes through sieves were performed
with the gut mixture. The liver mixture filtrate or the gut mixture
were centrifuged at 4006g for 5 min, the supernatant discarded
and the pellet resuspended in 3 ml of PBS. This was applied to a
column of Percoll, prepared by mixing 8 ml of Percoll (GE
Healthcare Bio-Science AB) with 32 ml of 0.25 M sucrose in a
50 ml tube. The tube was centrifuged at 8006g for 10 min. Liver
or intestinal cells and debris that remained on the top of the
Percoll were removed with a Pasteur pipette. The schistosome
eggs, which pelleted tightly at the bottom of the tube, were washed
three times with PBS and any residual host cells were removed by
discarding the supernatant. Further purification of eggs was
achieved by resuspension in 0.5 ml of PBS and application on to a
second Percoll column, prepared by mixing 2.5 ml of Percoll with
7.5 ml of 0.25 M sucrose in a 15 ml polypropylene tube. The eggs
were pelleted and then washed as before. Some eggs were snap
frozen and stored at 280uC until use for extraction of total RNA.
For other aliquots, the eggs were resuspended in 6 ml of complete
culture medium - Dulbecco’s modified Eagle’s medium (DMEM)
with 10% fetal bovine serum (FBS) and 100 U of penicillin and
streptomycin (Invitrogen, Carlsbad, CA), split into 2 ml aliquots in
a six-well plate and cultured at 37uC under 5% CO2.
S. haematobium schistosomula were obtained by mechanical
transformation of cercariae released from infected Bulinus truncatus
truncatus snails and cultured at 37uC in modified Basch’s medium
under 5% CO2in air as described for S. mansoni schistosomula
. Mixed sex adults of S. haematobium were obtained by portal
perfusion of infected hamsters followed by mesenteric vessel
dissection and manual removal of adult worms using forceps under
a magnification glass . The adults were rinsed several times in
PBS and cultured in complete culture medium.
Exposure of S. haematobium eggs to Cy3-siRNA
S. haematobium eggs were either electroporated and soaked in
non-coding Cy3-labeled siRNAs (Silencer Cy3-Labeled Negative
Control siRNA, Ambion, Austin, TX) at 50 ng/ml with conditions
as described . Briefly, eggs were washed in DMEM
supplemented with 200 U/ml penicillin G sulfate, 200 mg/ml
streptomycin sulfate, 500 ng/ml amphotericin B, 10 mM HEPES
(wash medium) and transfected in 100 ml of the same medium in
4 mm gap cuvettes with an ElectroSquarePorator ECM830 (BTX,
San Diego, CA) using a single square wave pulse of 125 volts of
20 milliseconds duration. After electroporation, eggs were washed
in PBS three times to remove the unincorporated Cy3-labeled
siRNA. Subsequently, eggs were transferred into complete
DMEM at 37uC for three hours. Other eggs were soaked for
three hours in Cy3-siRNA, then washed in PBS three times in
order to remove the unincorporated Cy3-labeled siRNAs. The
Cy3-siRNA exposed eggs, with or without electroporation, were
examined under bright and fluorescent light (below) using a Zeiss
Axio Observer A.1 inverted microscope fitted with a digital
camera (AxioCam ICc3, Zeiss). Manipulation of digital images
was undertaken with the AxioVision release 4.6.3 software (Zeiss).
These manipulations were limited to insertion of scale bars,
adjustments of brightness and contrast, cropping and the like;
image enhancement algorithms were applied in linear fashion
across the entire image and not to selected aspects.
Synthesis of mRNA, dsRNA, and siRNAs
To synthesize firefly luciferase mRNAs (mLuc), in vitro transcrip-
tions of capped RNAs from PCR DNA templates were accom-
plished using the mMachine T7 Ultra kit (Ambion) as described
[22,23]. Subsequently, RNAs were precipitated with ammonium
acetate, dissolved in nuclease-free water and quantified by
spectrophotometry (NanoDrop Technologies, Wilmington, DE).
The dsRNAs were generated by in vitro transcription using, as
templates, PCR products amplified with gene specific primers tailed
More people are infected with Schistosoma haematobium
than other major human schistosomes yet it has been less
studied because of difficulty in maintaining the life cycle in
the laboratory. S. haematobium might be considered the
‘neglected schistosome’ since minimal information on the
genome and proteome of S. haematobium is available, in
marked contrast to the other major schistosomes. In this
report we describe tools and protocols to investigate the
genome and genetics of this neglected schistosome. We
cultured developmental stages of S. haematobium, and
investigated the utility of introducing gene probes into the
parasites to silence two model genes. One of these, firefly
luciferase, was a reporter gene whereas the second was a
schistosome gene encoding a surface protein, termed Sh-
tsp-2. We observed that both genes could be silenced – a
phenomenon known as experimental RNA interference
(RNAi). These findings indicated that the genome of S.
haematobium will be amenable to genetic manipulation
investigations designed to determine the function and
importance of genes of this schistosome and to investigate
for novel anti-parasite treatments.
Genetic Manipulation of Schistosoma haematobium
www.plosntds.org2October 2011 | Volume 5 | Issue 10 | e1348
with the T7 promoter sequence. A luciferase dsRNA (dsLuc)
template encoding the full length 1,672 kb was amplified from the
pGL3-basic plasmid (Promega, Madison, WI), (F: 59TAATAC-
R: 59- TAATACGACTCACTATAGGGGCAACCGCTTCCC-
CGACTTCCTTA-39). The siRNAs were designed with the
assistance of the BLOCK-iTTMRNAi Designer Tool, https://
RNA of 19 nt in length named siShTSP 2 (59-GGA AUC CUG
UUU CAA AGA U-39), specific for residues 159–177 of the
extracellular loop 2 of S. haematobium tetraspanin 2 (Sh-tsp-2) and an
irrelevant siRNA (control) termed siScrambled, 59-GGA GUC
CCU UUA AAU AGA U-39, the sequence of which included the
sameresidues of siSh-tsp-2but in which the order of the residues had
been randomly mixed, were purchased from Invitrogen.
Transfection of developmental stages of S. haematobium
with mRNA and/or dsRNA
S. haematobium eggs were maintained for one day after isolation
from hamsters, then subjected to electroporation in the presence of
mLuc at 150 ng/ml . Briefly, ,2,000 eggs were subjected to
the square wave electroporation in 4 mm gap pathway cuvettes
(BTX) in 100 ml wash medium, as above. A group electroporated
in the absence of mLuc was included as a mock-treated control.
Thereafter the eggs were kept in culture for 3 or 20 hours,
harvested and stored at 280uC. For RNAi approaches, one group
of eggs was incubated with 30 mg of dsLuc, and other two groups
were incubated without dsLuc. After 10 min at 23uC, 15 mg of
mLuc was added to eggs in wash medium, except to a mock
control group, i.e. a group of eggs not treated with exogenous
nucleic acids. The eggs were subjected to square wave electropo-
ration (above), transferred to pre-warmed culture medium and
harvested three hours later.
Schistosomula of S. haematobium were removed from culture
three hours after cercarial transformation, washed and resus-
pended in 100 ml of wash medium containing 30 mg of dsLuc.
Two other groups of schistosomules were incubated in the absence
of dsLuc, in 4 mm gap cuvettes. After 10 min incubation at 23uC,
15 mg of mLuc was added to the wash medium in each group,
except to a mock control group. Thereafter the schistosomules
were subjected to square wave electroporation, 125 V, 20 ms,
transferred to prewarmed Basch’s medium and harvested three
We have recently determined that dicing adult schistosomes into
several fragments results in more reporter gene activity than in
similar numbers of intact worms . Accordingly, ,50 mixed sex
adults of S. haematobium were removed from culture 24 hours after
perfusion from hamsters, washed, diced into three or four
fragments using a sterile blade. Intact or fragmented S. haematobium
worms were placed into 4 mm gap pathway cuvettes in the
presence of 15 mg of mLuc resuspended in 100 ml of wash medium
and subjected to square wave electroporation, 125 V, 20 ms, one
pulse. After electroporation, the worms and fragments were
transferred into pre-warmed complete culture medium, incubated
at 37uC under 5% CO2in air, and harvested 3 hours later.
For RNAi approaches targeting the luciferase reporter gene, the
worms were diced into three or four fragments using a sterile
blade, washed three times in wash medium and transferred to
4 mm gap cuvettes containing 100 ml wash medium. One group of
diced adult worms was incubated with 30 mg of dsLuc, and the
other two were incubated in the absence of dsLuc. Following
incubation at 23uC for 10 min, 15 mg of mLuc was added to each
group, except to the mock control group after which the parasites
were subjected to a single pulse of square wave electroporation,
125 V, 20 ms. Subsequently, the diced worms were transferred to
complete medium and maintained in culture; the worm fragments
remained active (displaying movements) during the study.
For RNAi targeting an endogenous S. haematobium gene, intact
adult worms were electroporated in the presence of 10 mg of siSh-
tsp-2 or 10 mg of siScrambled in 100 ml (16.5 mM) of wash
medium. We targeted intact worms for this experiment, dealing
with silencing of an endogenous gene, with the aim of determining
whether a gross phenotype might accompany gene knockdown.
After electroporation, worms were transferred to complete
medium for 48 h, then stored at 280uC.
Developmental stages of S. haematobium were harvested three
hours after electroporation unless otherwise indicated, washed
three times with wash medium and stored as wet pellets at 280uC.
Luciferase activity in extracts of parasites was determined using
Promega’s luciferase assay reagent system and a tube luminometer
(Sirius, Berthold, Pforzheim, Germany) . In brief, pellets of
parasites were subjected to sonication (365 s bursts for schisto-
somula and adults and 565 s bursts for eggs, output cycle 4,
Misonix Sonicator 3000, Newtown, CT) in 300 ml 16CCLR lysis
buffer (Promega). The sonicate was clarified by centrifugation at
20,800 g, 15 min, 4uC and the supernatant, containing the soluble
fraction, analyzed for luciferase activity. Aliquots of 100 ml of
soluble fraction were injected into 100 ml luciferin at 23uC, mixed,
and relative light units (RLUs) determined 10 s later by the
luminometer. Replicate samples were measured, with results
presented as the average of the readings per mg of protein. The
protein concentration in the soluble fraction of the schistosome
extract was determined using the bicinchoninic acid assay (Pierce,
Rockford, IL). Recombinant luciferase (Promega) was included as
a positive control.
Gene expression analysis of Sh-tsp-2
Expression of Sh-tsp-2 mRNA was analyzed in adults of S.
haematobium harvested 48 hours after RNAi treatment. Total RNA
was extracted from the worms using the RNAqueusH-4PCR Kit
(Ambion).Any residualDNAremaining in the RNA wasremoved by
DNase digestion using TurboDNase (Ambion) and cDNA was
synthesized from 100 ng of total RNA using the iScript cDNA
Synthesis Kit (Bio-Rad, Hercules, CA). Primers and TaqMan probes
were designed with the assistance of Beacon Designer (Premier
2 and S. haematobium tropomyosin (ShTrop) (GenBank L76202.1)
genes, as follows: for ShTSP 2, forward primer: 59-GAT GCA TTA
AGA GAA TTC GTA A- 39; reverse primer: 59-TGG TGG AGT
GAC ATA ATC-39; probe: 59-/56-FAM/TGA AGA ATC AGC
ACC ACA GCA TTG/3IABlk_FQ/-39; for ShTrop, forward
primer: 59-ATC CGA GAT TTA ACA GAA C-39; reverse primer:
59-CGC TAA GAG CTT TGT ATC-39; probe: 59-/56-FAM/TTC
TCA GCC AGT AAG TCA TCT TCC AA/3IABlk_FQ/-
39.Quantitative PCRs were performed in triplicate, using 96-well
plates (Bio-Rad), with an initial denaturation step at 95uC for
3 minutesfollowedby40cyclesof30 secat95uCand30 secat50uC,
using a thermal cycler (iCycler, Bio-Rad) and a Bio-Rad iQ5 detector
to scan the plates in real time. Reactions were carried out in 20 ml
FastMix,UNG(Quanta Bioscience,Gaithersburg,MD).The relative
quantification assay 22DDCtmethod  was employed, using
ShTrop as the reference gene. Results were plotted as Sh-tsp-2 gene
expression level relative to the reference gene considering 1=Sh-tsp-2
relative expression level measured in the irrelevant control group.
Genetic Manipulation of Schistosoma haematobium
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Laser scanning confocal microscopical imaging
Adult flukes were fixed in 4% paraformaldehyde overnight,
rinsed with PBS, then incubated in propidium iodine (PI) diluted
1:1000 for one day. The PI-stained worms were placed on
polylysine coated 50 mm Petri dishes, covered with PBS, and
examined using a Carl Zeiss LSM 710 confocal system. This
system includes a Zeiss Axio Examiner Z1 upright microscope
equipped with a 206/1.0 water dipping objective lens, deploy-
ment of which seemed prudent for imaging entire schistosomes
since this objective does not require a coverslip (which markedly
diminishes spherical aberrations). Confocal images were captured
using a Qasar 32-channel spectral detector. Briefly, the worms
were simultaneously scanned with 488 and 561 nm laser lines
(multiline argon and diode laser, respectively), while the backward
light was registered in 102461024 lambda-stack images taken
simultaneously at a spectral resolution of 9.6 nm. Thus, for each
single optical section, 32 images were recorded covering the visible
spectrum from 423–721 nm, allowing the analysis of each of the
(1) reflected light, (2) autofluorescence, and (3) characteristic
emission at 617 nm from PI. To detect the reflected laser light, we
utilized a T80/R20 beamsplitter, which only partially attenuates
the laser lines in the backward direction. Confocal stacks for three-
dimensional (3D) rendering were taken at z-scaling of 1.7 mm,
which matched the pinhole opening (34 mm). Pixel resolution was
0.59 mm. After completion of the online acquisition, a linear
spectral unmixing protocol was applied to the lambda-stacks to
generate two three-channel confocal stacks. To generate reliable
spectral unmixed channels, various sites from the worm were
tested and representative for the 488-line reflection, autofluores-
cence and PI were selected and used as reference for unmixing.
Thus, the resulting images, encoded in three channels reflected
light, autofluorescence and PI signals from the nuclei. Unmixed,
confocal stacks were imported to Volocity (v.5.5, Perkin Elmer/
Improvision) for further three-dimensional rendering and analysis.
Male LVG hamsters were purchased from Charles River
(Wilmington, MA) and maintained in the Biomedical Research
Institute’s (BRI) animal facility, which is accredited by the
American Association for Accreditation of Laboratory Animal
Care (AAALAC; #000779), is a USDA registered animal facility
(51-R-0050), and has an Animal Welfare Assurance on file with
the National Institutes of Health, Office of Laboratory Animal
Welfare (OLAW), A3080-01. Maintenance of the hamsters,
exposure to S. haematobium cercariae, and subsequent harvesting
of tissues were approved by the BRI Institutional Animal Care and
Use Committee (protocol approval number 09-03). All procedures
employed were consistent with the Guide for the Care and Use of
Culture of developmental stages of Schistosoma
Given the scarcity of reports on in vitro culture techniques
focused on S. haematobium we adapted protocols from studies with
S. mansoni , to maintain some developmental stages in culture.
Thus, eggs isolated either from small intestines or livers of
hamsters, schistosomula mechanically transformed from cercariae
released by experimentally infected B. t. truncatus snails, and mixed
sex adults from portal perfusion and mesenteric vessel dissection of
hamsters were cultured in the indicated medium at 37uC, 5%
CO2. No differences in gross appearance were evident between the
eggs isolated from intestines (Figure 1A and B) or liver (Figure 1C
and D). The eggs were cultured in complete medium (Dulbecco’s
modified Eagle’s medium (DMEM) with 10% fetal bovine serum
(FBS) and 100 U of penicillin and streptomycin (Invitrogen,
Carlsbad, CA), for up to seven days (not shown). Mixed sex adults
were cultured in complete medium (above) for up to five days
(Figure 2A). Notably, at higher magnification, longitudinal
orientation of the eggs within the uterus of the female schistosome
was apparent (Figure 2B) which is in marked contrast to the
transverse disposition in utero of S. mansoni eggs, e.g. [26,27].
Schistosomula of S. haematobium, obtained by cercarial transfor-
mation as described above, were cultured in modified Basch’s
medium (Figure 2C and 2D).
Cy3-siRNA is effectively incorporated into eggs of S.
To investigate whether macromolecules could be introduced
into S. haematobium eggs, cultures of eggs were incubated in a Cy3-
siRNA (13.8 kDa) with or without concomitant square wave
electroporation. Three hours after exposure to Cy3-siRNA, eggs
were examined by fluorescence microscopy. Surprisingly, strong
fluorescence including foci of intense fluorescence was revealed in
Cy3-siRNA soaked eggs in contrast to those subjected to
electroporation (Figure 3 and S1). More than 80% of the treated
eggs emitted fluorescence as revealed at low magnification (Figure
S1). These results indicated that it is possible to introduce Cy3-
siRNA into S. haematobium eggs by simple soaking and that
electroporation was not essential for this reporter probe.
(However, with some stages, electroporation is more efficient: it
mobilizes dsRNA or mRNA into the target worms quickly, which
is advantageous when working with RNAs that are labile.)
Although the structure of the S. haematobium eggshell is not well
described, pores are present in the eggshell of S. mansoni eggs – the
eggshell has been described as cribriform .
Reporter firefly luciferase is active in S. haematobium
To ascertain if transgene mRNAs could penetrate schistosome
eggs and be translated into an active protein, we electroporated
cultured eggs in the presence of firefly luciferase mRNA (mLuc)
(512 kDa). More specifically, 48 hours after isolation 1,500–2,500
eggs were subjected to electroporation in the presence of 150 ng/
ml of mLuc, and collected three and 20 hours later. Luciferase
activity was detected in the mLuc electroporated group compared
with untreated control at 3 h, and even higher luciferase activity
was measured in eggs harvested at 20 h after electroporation
(Figure 4A). (A signal of ,100–150 RLUs/sec/mg was measured
in the mock control group, which represents the background
baseline of this assay.) We electroporated intact and fragmented
adult worms in the presence of 15 mg of luciferase mRNA, and
measured the luciferase activity 3 hours later. Several fold (,3.5
times) more activity was detected in fragmented than intact worms
(Figure 4B), in like fashion to S. mansoni . Collectively, these
findings indicated that square wave electroporation efficiently
delivered exogenous nucleic acids into the eggs and adults of S.
haematobium and that reporter luciferase was functionally translated
from this exogenous mRNA.
dsRNA silences reporter luciferase mRNA in S.
We have reported that it is feasible to knock down an exogenous
reporter transgene by dsRNA in order to detect an active RNAi
pathway in flukes [23,29]. Given that S. haematobium can be
productively transformed with mRNA by square wave electropo-
ration, we proceeded to investigate silencing of expression of the
Genetic Manipulation of Schistosoma haematobium
www.plosntds.org4October 2011 | Volume 5 | Issue 10 | e1348
exogenous reporter transcript (mLuc). About 2,000 eggs were
removed from culture four days after isolation, washed and
subjected to electroporation in the presence of both mLuc and
dsLuc (mLuc+dsLuc group). Control eggs electroporated in the
absence of exogenous RNAs (mock control) and positive control
eggs electroporated in the presence of mLuc (mLuc group) were
included (Figure 5A). Reduced luciferase activity was evident in
the mLuc+dsLuc group in comparison with the mLuc group, even
though the luciferase activity in terms of absolute RLUs/sec/mg
measured in eggs at three hours after electroporation was relatively
low in comparison to the other developmental stages (Figure 5B,
left panel). (It appears to be more difficult to introduce mRNA into
eggs than other developmental stages, likely because of the
presence of the eggshell.) The experiment with eggs was repeated
three times; knock down was apparent in two of the three trials.
Fragmented adults were also examined; .75% knockdown of
luciferase was observed (Figure 5B, center panel). The experiment
was repeated; knock down was obvious on each occasion.
Furthermore, three hour old schistosomula were co-transfected
with messenger RNA encoding luciferase (mLuc) and dsRNA
targeting the luciferase transcript (dsLuc) by electroporation
(mLuc+dsLuc group), along with controls (experimental design
shown in Figure 5A). At 3 h after electroporation, luciferase
activity of 14,080 RLU/sec/mg was evident in lysates of the
positive control schistosomules transfected with mLuc. By contrast,
luciferase activity in the schistosomules exposed to both dsLuc and
mLuc was significantly lower, 6073 RLUs/sec/mg, representing
43% of the positive mLuc control (Figure 5B, right panel). In
review, a similar trend was apparent in each of these develop-
mental stages: it is feasible to knock down the reporter luciferase
gene in eggs, schistosomules and adults of S. haematobium.
Suppression of an endogenous gene in adults of S.
In addition to reporter luciferase, we introduced siRNA specific
for Sh-tsp-2, an orthologue of a S. mansoni membrane protein
critical for tegument formation, the tetraspanin Sm-TSP-2 
into intact S. haematobium mixed sex adults. At 48 hours after
electroporation of siRNAs – siSh-tsp-2 and a control siRNA, we
observed a significant knock-down of levels of the Sh-tsp-2
transcript (Figure 6). This experiment was repeated three times;
knock down was seen on each occasion, and on two of these three
occasions the knock-down was .75%. Notably, no gross
phenotypic differences among the adult worms were evident by
light microscopy (not shown).
Confocal micrographs highlight characteristic
morphology of S. haematobium
In addition to the images of cultured stages of S. haematobium,
adult worms were fixed in 4% paraformaldehyde and stained with
PI. Spectral confocal microscopy was used to image the entire
volume of the paraformaldehyde fixed male and female worms at
high resolution (Figure 7A). We employed A T80/R20 beams-
plitter to image the flukes, using backward scattered laser light.
The reflected light is registered on the lambda stack as a dual-peak
at the wavelength of the laser used for excitation. In this case, the
Figure 1. Eggs of Schistosoma haematobium. These schistosome eggs were obtained from experimentally infected hamsters, and thereafter were
maintained in culture. Eggs were recovered from small intestines (panels A and B) or liver (panels C and D). Scale bars, 100 mm.
Genetic Manipulation of Schistosoma haematobium
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488 nm laser line produced a large reflection response, from which
images of the surface of the schistosomes were assembled
(Figure 7B and F). The approach also recorded in consistent and
reproducible manner, autofluorescence deriving from the gut and,
dramatically, eggs in utero (Figure 7D and E). The autofluorescence
registered on the lambda stack displayed a broad spectrum - peak
,560 nm, range 500–650 nm (overlapping with numerous widely
employed dyes and fluorescent proteins). The signals from nuclei
stained with PI (Figure 7C and G) registered as a spectral curve
(peak 617 nm) that partially overlapped with the red-shifted slope
of the autofluorescence. Thus, we could select discrete sites on
worms representing reflected light, autofluorescence and PI
fluorescence that served as references for linear unmixing .
The three-channel confocal stacks, derived after linear unmixing,
comprised channels representing the reflection, autofluorescence
and PI signal at high signal to noise ratio. Figures 7E and 7H show
three-dimensional images assembled from the merged reflected
light, autofluorescence and the PI fluorescence signals.
Using S. haematobium eggs from livers and intestines of
experimentally infected hamsters, adult worms perfused from the
hamsters and cercariae from B. t. truncatus snails, and using similar
approaches to those for S. mansoni , we were able to culture
eggs, schistosomules, and adults of S. haematobium and to subject
these developmental stages to genetic manipulation. We trans-
formed eggs of S. haematobium with a small nucleic acid probe, Cy3-
siRNA. Experience with the other two major schistosomes has
revealed that the schistosome egg represents an attractive
developmental stage at which to target transgenes because it is
readily obtained from experimentally-infected rodents or naturally
infected people, is easily maintained in vitro, has a high ratio of
germ to somatic cells and contains a miracidium that can be
employed to infect snails to propagate the life cycle [21,32,33].
Furthermore, from the clinical perspective, the egg represents the
major source of pathogenesis in human schistosomiasis haemato-
bia. We observed that exogenous macromolecules penetrate into
cultured eggs, and we speculate that small macromolecules such as
Cy3-Silencer siRNA (13.8 kDa) enter eggs through the pores that
likely anastomose throughout the eggshell and which provide
access from sub-shell envelope and the developing miracidium to
the exterior, in like fashion to the egg of S. mansoni [29,34,35].
Others and we have described the utility of firefly luciferase as a
transgene probe in S. mansoni and the liver fluke Fasciola hepatica
[21,22,23,29,36]. We have also reported the utility of luciferase as
a model target to identify the presence of an active RNA
interference pathway in less well studied helminth parasites,
especially where genome sequences are unavailable . Using
this strategy, we now present findings that indicate for the first
time the presence of an intact RNAi pathway in S. haematobium. In
each of three developmental stages investigated – eggs, schisto-
somula, and mixed sex adults, co-introduction of dsRNA spanning
Figure 2. Adults and micrograph showing the characteristic longitudinal disposition of the eggs along the body of the
schistosomules of Schistosoma haematobium. Panel A: micrograph illustrating a population of mixed sex adults obtained by portal perfusion
from infected hamsters and maintained in culture. Panel B: high magnification micrograph showing the characteristic longitudinal disposition of the
eggs along the body of the female. Panel C: images of cercariae released from infected snails. Panel D: images of representative schistosomules in
culture 3 hours after cercarial transformation. Scale bars, 500 mm (A) and 100 mm (B, C and D).
Genetic Manipulation of Schistosoma haematobium
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the transcript of firefly luciferase and of mRNA encoding firefly
luciferase resulted in robust knockdown of the exogenous mRNA.
Luminometric measurement of luciferase activity provided a direct
demonstration of gene silencing at the protein level.
In S. mansoni, comparative studies indicate that efficiency of
RNAi efficiency following electroporation is superior to passive
soaking . Here we employed square wave electroporation to
introduce the dsRNA and luciferase mRNA into developmental
stages of S. haematobium. Eggs, schistosomula and adults of S.
haematobium were amenable to transfection with foreign nucleic
acids using this technique. Given that these stages tolerated the
electro-transfection conditions well, we anticipate that this
technique can be optimized for genetic analysis and genomic
manipulation of S. haematobium. Whereas soaking performed better
than electroporation alone for eggs of S. haematobium, it will be
worthwhile to employ electroporation followed by soaking of the
transfected eggs, a combination that is superior to soaking alone in
eggs of S. mansoni .
Although little is known about the protein encoding genes of S.
haematobium, we obtained the sequence of Sh-tsp-2, an apparent
orthologue of Sm-tsp-2 which encodes a lead vaccine antigen for
schistosomiasis mansoni . By targeting the sequence encoding
the extracellular loop 2 domain of this protein with a 19 nt
siRNA, we observed strong knockdown of the Sh-tsp-2 transcript
in adult worms. Thorough studies targeting this gene are
warranted given its performance as a vaccine antigen for S.
mansoni infection and because of the integral role that Sm-TSP-2
plays in development, maturation or stability of the tegument
Figure 3. Labeled short interfering RNA enters cultured eggs of Schistosoma haematobium. Representative images of schistosome eggs
3 hours after soaking in Cy3-siRNA; panel A: no Cy3-siRNA treatment control, bright field; panel B: no Cy3-siRNA treatment control, fluorescence field,
panel C: soaked eggs in medium containing 50 ng/ml of Cy3-siRNA, bright field, panel D: soaked eggs in medium containing 50 ng/ml of Cy3-siRNA,
fluorescence field. Scale bar, 50 mm.
Figure 4. Luciferase activity measured in Schistosoma haemato-
bium. Panel A: S. haematobium eggs transfected with 150 ng/ml of
firefly luciferase mRNA. Detection of luciferase activity in mock control
(mock) and in mLuc treated eggs, measured three (3 h) and 20 (20 h)
hours after electroporation. Panel B: Luciferase activity measured in
extracts of adult worms 3 h after electroporation, (mock) adult worms
treated with no molecule, (intact) intact worms treated with 150 ng/ml
mRNA, and (fragmented) worms diced into three or more pieces and
treated with 150 ng/ml mRNA.
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We deployed laser scanning confocal microscopy to view the
adult stage of S. haematobium. In addition to facilitating views of the
entire worms ($1 cm in length), the approach circumvents
barriers to reliable fluorescence imaging of schistosomes, including
the notion of autofluorescence of schistosome eggs, e.g. . The
provenance of signals from the eggs (and gut) aside, the
phenomenon deserves deeper exploration since it has the potential
to chart predictable anatomical landmarks of the schistosome that
can facilitate microanalysis of schistosome organs and tissues.
Future characterization of native fluorescence signals of schisto-
somes can be expected to be of interest. Unlike fixed worms,
evaluation of the living worms critically depends on minimizing
invasive approaches. Also, fluorescence labels suitable for living
cells generally cause some perturbation of normal functions. Thus,
a library of spectrally distinctive signals – including the signal from
eggs reported here - can be expected to facilitate microscopic
imaging of viable schistosomes. Collectively, spectral confocal
imaging provided the technological capacity to document eggs in
utero of this neglected schistosome by extracting their emission of
autofluorescence. Also, we imaged adult S. haematobium worms by
staining tegumental nuclei with propidium iodide, which allowed
the assembly of the three dimensional structure of the blood fluke.
The spectral confocal microscopy approaches allowed differenti-
ation of a fluorochrome from natural signals, e.g. PI versus
autofluorescence, and portends its likely utility for monitoring
reporter genes such as green fluorescent protein in transgenic
In conclusion, this is the first report of genetic manipulation of S.
haematobium. The procedures described here are expected to find
application in determining the importance of S. haematobium genes.
Figure 5. Suppression of exogenous luciferase activity in transfected eggs, chopped/diced adults and schistosomules of
Schistosoma haematobium. Panel A: schematic representation of the experimental designs. Panel B: Luciferase activity measured in the indicated
groups three hours after electroporation.
Figure 6. Silencing of the gene encoding the tetraspanin 2
antigen of Schistosoma haematobium. Quantitative RT-PCR analysis
of the mRNAs from adult S. haematobium worms at 48 h after
transfection by electroporation with siRNA specific for Sh-tsp-2. .80%
silencing of the Sh-tsp-2 (siShTSP2) evident when compared to the
control group treated with siRNA scrambled control (siScrambled). Sh-
tsp-2 expression was normalized to a control mRNA encoding
Genetic Manipulation of Schistosoma haematobium
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Whereas few sequences are yet available, there is now increasing
interest in sequencing the S. haematobium genome. Tools and
procedures for genetic and genomic manipulation of S. haematobium
will soon be needed to determine the importance of prospective
new gene targets for development of novel interventions.
hours after exposure to Cy3-siRNA. Panels A and B: control
without Cy3-siRNA, bright (A) and fluorescence (B) fields; panels
C and D: soaked eggs in medium containing 50 ng/ml of Cy3-
siRNA, bright (C) and fluorescence (D) fields. Panels E and F:
control electroporated eggs without Cy3-siRNA, bright (E) and
Representative micrographs at low magnifi-
6) of Schistosoma haematobium eggs at three
fluorescence (F) fields, panels G and H: eggs electroporated in the
presence of 50 ng/ml of Cy3-siRNA, bright (G) and fluorescence
(H) fields. Scale bar, 200 mm.
We thank Dr. Peter J. Hotez for helpful discussions and advice.
Conceived and designed the experiments: GR SS AP VHM AL PJB.
Performed the experiments: GR TIO AP MAA SS. Analyzed the data: GR
AP SS VHM AL PJB. Contributed reagents/materials/analysis tools: GR
AP SS AL VHM Y-sL FAL PJB. Wrote the paper: GR AP FAL AL PJB.
Confocal microscopy and imaging: AP SS GR.
Figure 7. Representative three dimensional (3D) renderings from laser scanning confocal images of adult forms of Schistosoma
haematobium. Panel A: 3D rendering from the female (left) and male (right) S. haematobium. Images were captured with 56objective as tile-scan to
cover the entire worm in addition to z-stacks. Propidium iodide (PI) was used to label the nuclei (red). B–E: High power (206/1.0) 3D rendering from a
female S. haematobium capturing the anterior of the worm. Three channels were extracted after applying a linear spectral unmixing algorithm to a
lambda stack confocal images. B, The surface of the female S. haematobium visualized using reflected light scattering from the 488 nm laser line. C, PI
–labeling, D, ‘autofluorescence’ from the schistosome eggs, E, merge of B–D, showing the structure of the anterior of the worm, with a semi-
transparent visualization. In D and E, arrows indicate location of schistosome eggs. F–H, 3D rendering from the anterior of a male worm visualized
with a similar approach used in B–E. F, reflected light channel, G, PI channel, H, merge of F and G using semi-transparent visualization. Scale bars,
500 mm (A), 400 mm (B–E), 200 mm (F–H).
Genetic Manipulation of Schistosoma haematobium
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