Fatty Acid Oxidation Is Essential for Egg Production by
the Parasitic Flatworm Schistosoma mansoni
Stanley Ching-Cheng Huang1,2, Tori C. Freitas2, Eyal Amiel2, Bart Everts1,2, Erika L. Pearce1,2,
James B. Lok3, Edward J. Pearce1,2*
1Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America, 2Trudeau Institute, Saranac Lake,
New York, United States of America, 3Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, United States of
Schistosomes, parasitic flatworms that cause the neglected tropical disease schistosomiasis, have been considered to have
an entirely carbohydrate based metabolism, with glycolysis playing a dominant role in the adult parasites. However, we
have discovered a close link between mitochondrial oxygen consumption by female schistosomes and their ability to
produce eggs. We show that oxygen consumption rates (OCR) and egg production are significantly diminished by
pharmacologic inhibition of carnitine palmitoyl transferase 1 (CPT1), which catalyzes a rate limiting step in fatty acid b-
oxidation (FAO) and by genetic loss of function of acyl CoA synthetase, which complexes with CPT1 and activates long chain
FA for use in FAO, and of acyl CoA dehydrogenase, which catalyzes the first step in FAO within mitochondria. Declines in
OCR and egg production correlate with changes in a network of lipid droplets within cells in a specialized reproductive
organ, the vitellarium. Our data point to the importance of regulated lipid stores and FAO for the compartmentalized
process of egg production in schistosomes.
Citation: Huang SC-C, Freitas TC, Amiel E, Everts B, Pearce EL, et al. (2012) Fatty Acid Oxidation Is Essential for Egg Production by the Parasitic Flatworm
Schistosoma mansoni. PLoS Pathog 8(10): e1002996. doi:10.1371/journal.ppat.1002996
Editor: David L. Williams, Rush University Medical Center, United States of America
Received April 21, 2012; Accepted September 13, 2012; Published October 25, 2012
Copyright: ? 2012 Huang 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: The work was supported by NIH-NIAID (http://www.niaid.nih.gov)grants AI075266 to EJP and AI082548 to JBL and EJP. Schistosome life stages were
provided by BRI through NIH-NIAID contract No. HHSN272201000005I. 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: firstname.lastname@example.org
Infection with helminth parasites of the genus Schistosoma causes
chronic and debilitating disease in over 200 million people
worldwide [1,2]. Adult S. mansoni worms live within the portal
vasculature, producing eggs (200–300/day/female) that are
intended to pass into the intestinal lumen for release into the
environment to allow transmission of the infection . However,
many eggs are carried by the blood flow to the liver, where they
become trapped in sinusoids and elicit strong Th2 cell mediated
immunopathology, which is the cause of disease manifestations
. Since egg production is key for both transmission and
pathogenesis, studying reproductive biology in schistosomes could
lead to new methods for preventing or treating disease .
Adult schistosomes exhibit sexual dimorphism, a trait that is
unusual among parasitic trematodes, and display a fascinating
codependency: the female resides in a groove (the gynecophoric
canal) on the ventral side of the male and is dependent on ongoing
physical pairing, but not sperm transfer , for proper sexual
development [5–11]. Virgin adult female schistosomes, from
female-only infections, are developmentally stunted compared to
fecund females from mixed-sex infections and are unable to lay
eggs [11,12]. Furthermore, egg-laying females that are physically
separated from their partners and surgically implanted into a host
in the absence of male worms cease egg production and regress
reproductively to an immature state. Interestingly, regression is
reversible because normal reproductive activity is resumed when
separated females are re-paired with males [11,13,14]. Regression
is largely the result of involution of the vitellarium, a proliferative
tissue that occupies the posterior two thirds of the female and
produces cells that surround the ovum and provide proteins for
eggshell formation and nutrients for the developing embryo .
There have been numerous suggestions that male parasites
promote female maturation by ‘‘providing’’ nutrients . The
fact that starvation in planaria (free living flatworms) can lead to
reversible tissue involution  is consistent with the possibility
that loss of vitelline cells is the end result of nutritional deprivation
in female parasites. Glucose is considered to be the key
macronutrient required by adult schistosomes to meet their
bioenergetics needs [17,18], but there is a lack of clarity in the
literature regarding the relative extent to which Warburg
metabolism (the homolactic fermentation of glucose in the
presence of oxygen) versus mitochondrial oxidative phosphoryla-
tion (OXPHOS) are important in these organisms [17,19,20].
Nevertheless, fecund adult females gradually stop ovipositing in
vitro even when glucose and oxygen are not limiting , and
under anaerobic conditions egg production ceases immediately
despite the fact that the worms remain viable for extended periods
. These findings led us to consider the possibility that worms
are able to survive using Warburg metabolism, but require
substrates other than glucose for oxidative metabolic pathways
critical for egg production. Despite the general view that there is
PLOS Pathogens | www.plospathogens.org1October 2012 | Volume 8 | Issue 10 | e1002996
no appreciable lipid catabolism in helminth parasites , the
genes encoding the enzymes of the b-oxidation pathway, through
which fatty acids (FA) are catabolized into the TCA cycle, are
conserved in schistosomes . Moreover, greater than 40% of
the lipid in adult schistosomes is in the form of triacylglyceride
(TG), usually considered an energy store for b-oxidation , and
FA are able to promote egg production and egg viability in vitro
. We therefore decided to ask whether adult female
schistosomes use FA oxidation (FAO) for egg production.
Fecund female schistosomes use OXPHOS
The b-oxidation pathway allows FA to be used as fuel for the
TCA cycle, which in turn generates substrates for the electron
transport chain to make ATP via OXPHOS. To examine whether
this process occurs in adult female schistosomes, we used
extracellular flux analysis to compare mitochondrial oxygen
consumption rates (OCR, ) in individual fecund and virgin
female schistosomes immediately ex vivo (Fig. S1). OCR in female
schistosomes declined in the presence of oligomycin, and
antimycin-A plus rotenone (Fig. 1A), indicating that it is largely
a function of mitochondrial OXPHOS (Fig. S1). Baseline OCR
(Fig. 1A,B), and mitochondrial spare respiratory capacity (SRC,
Fig. 1A,C) , were significantly higher in fecund vs. virgin
females (P,0.01). SRC is the difference between OCR at basal
state and after addition of FCCP (Fig. S1), and reflects the extra
mitochondrial capacity available to produce energy under
conditions of increased work or stress and is an important
determinant of long-term cellular survival and function [26,27].
Since the sizes of fecund and virgin adult females differ , the
SRC measurement also provides an internally controlled indica-
tion that there are significant qualitative differences in mitochon-
drial respiration between fecund and virgin worms.
OXPHOS is essential for egg production
Previous work showed that female schistosomes require oxygen
to produce eggs . To assess whether these findings reflect a
dependence on OXPHOS, we cultured fecund female worms for
24 h in oligomycin, antimycin A or rotenone, all of which inhibit
mitochondrial OCR (Fig. 1), and measured egg production and
worm viability; these inhibitors had a significant (p,0.01 in each
case) negative effect on egg production (Fig. 2A), but little adverse
effect on worm viability over this time period (Fig. 2B). Moreover,
when the inhibitors were washed out after 24 h, egg production
resumed at normal levels over the ensuing 24 h (Fig. 2C). These
data indicate that female worms can survive independently of
mitochondrial respiration, but absolutely require this process in
order to produce eggs.
Cpt1 activity regulates oxygen consumption and egg
Carnitine palmitoyl transferase 1 (Cpt1) catalyzes the initial rate
limiting step in FAO in which FA are transferred from the cytosol
into the mitochondria . To determine whether OXPHOS
depends on FAO we incubated fecund female worms with the
Cpt1 inhibitor etomoxir [29,30] immediately ex vivo and
measured OCR. We found that etomoxir caused a significant
decline in basal OCR (Fig. 3A; S2), without affecting basal
extracellular acidification, an indicator of glycolysis (data not
shown). Since OXPHOS is essential for egg production (Fig. 2), we
reasoned that if FAO is a significant source of substrates for the
TCA cycle and therefore for OXPHOS, then inhibition of FAO
should have a deleterious effect on egg output. To examine this we
recovered fecund female worms from infected mice and measured
the effect of etomoxir on egg production over 24 h in culture.
Under these conditions, etomoxir completely suppressed egg
production (Fig. 3B), although worms remained viable. These data
implicate FAO in egg production by female schistosomes.
Fecund female schistosomes have extensive lipid
The understanding of how FA are utilized by cells is evolving
rapidly. The current view is that FA are converted into TG and
stored in cytoplasmic lipid droplets, from which they are released
in a regulated fashion by lipolysis  to be used as energy
substrates in FAO, or as ligands for nuclear receptors. It has been
reported that schistosomes possess considerable TG stores when
recovered from mice , but the function and location of these
stores remains enigmatic . To examine this we stained female
worms immediately ex vivo with Oil-Red-O, which binds to
neutral TG and was recently authenticated as a true lipid stain in
the free-living helminth Caenorhabditis elegans . The results were
striking, revealing that fecund female parasites possess an extensive
lipid droplet network. This network was evident microscopically,
and by measuring extracted dye spectrophotometrically (Fig. 3C).
In contrast, virgin females had significantly lower lipid reserves
(Fig. 3C). Moreover, the intensity of Oil-Red-O staining declined
markedly over time as fecund worms were maintained in tissue
culture for 3 or 13 days (Fig. 3C). Previous reports have
commented on the presence of lipid droplets within mature (Stage
4) vitelline cells . Although we do note have proof that all of
the droplets that we have visualized using Oil-Red-O staining and
confocal microscopy are within the vitellarium, their location is
anatomically consistent with the majority of them being associated
with this organ. The lack of Oil-Red-O staining in virgin worms,
and in fecund females after culture, is consistent with the failure of
the vitellarial lineage to produce Stage 4 cells under these
The decline in lipid reserves in vitro is of interest since it occurs
with a similar kinetic to the decline in egg production by cultured
Schistosomes are parasitic worms that are the cause of the
Neglected Tropical Disease schistosomiasis. Female schis-
tosomes mated with males produce eggs, which either
pass out of the host’s body for transmission of the
infection, or become trapped in host tissues, where they
induce inflammation that contributes to disease symp-
toms. It has been assumed that egg production is a
bioenergetically-demanding process fuelled by glucose
metabolism. However, we have discovered that egg
production is blocked by inhibition of fatty acid oxidation
(FAO), the process through which FA are utilized within
mitochondria to fuel the tricarboxylic acid cycle and
thereby produce substrates for ATP synthesis through
oxidative phosphorylation. Consistent with a role for FAO
in egg production, fecund females have extensive fat
stores, in the form of lipid droplets, whereas virgin adult
females have little or no fat reserves. Moreover, fecund
females placed into tissue culture exhaust their fat reserves
and cease to be able to produce eggs. Since schistosomes
cannot produce their own FA, our data point to the
acquisition of FA from the host as a key process necessary
for egg production. Our findings point to the importance
of regulated lipid stores and FAO for egg production by
Fat Catabolism Supports Flatworm Reproduction
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worms . We reasoned that this could reflect a causal link
between lipid droplet exhaustion and the cessation of FAO under
these conditions. To explore this, we used real time flux analysis to
measure FAO activity and mitochondrial OCR in fecund females
immediately ex vivo and in vitro. We found that cumulative levels
of palmitate oxidation and basal OCR declined significantly in
vitro (Fig. 3D and 3E), and that as anticipated this was paralleled,
between days 3 and 13, by a significant decline in egg production
(Fig. 3F). To formally examine whether there is a link between
FAO and lipid droplet depletion in vitro, we recovered fecund
females from infected hosts and cultured them with etomoxir for
24 h and used Oil-Red-O staining to quantify lipid droplets. We
found that etomoxir significantly inhibited depletion of lipid
reserves in these worms (Fig. 3G).
FA catabolism is essential for egg production
FA liberated from lipid droplets by lipolysis are activated and
shuttled into mitochondriaforFAObyacyl-CoA synthetase(ACSL)
[33,34]. We reasoned that if FA are essential for OXPHOS and egg
parameters by preventing the use of FA resulting from lipolysis. We
examined this using chemical inhibitors and RNAi. First, we tested
the effect of the fungal metabolite Triacsin C, which is a potent
inhibitor of most mammalian ACSLs [35,36]. We recovered fecund
female parasites from their hosts and immediately assessed the effect
of Triacsin C on basal OCR and egg production. We found that
Triacsin C inhibited OCR (Fig. 4A) and blocked egg production
entirely (Fig. 4B). We used RNAi to substantiate the importance of
ACSL in OXPHOS and egg production. Immediately after
explantation, and prior to assessing OCR and egg production,
fecund females were electroporated with siRNAs against SmACSL,
or control siRNAs, . Using this approach, SmACSL expression
was significantly attenuated within 72 h (Fig. S2A). Concomitant
with reduced expression of SmACSL there were significant declines
in OCR (Fig. 4C) and egg production (Fig. 4D). Moreover,
SmACSL-siRNA resulted in greater retention of lipid reserves over
3 days in culture (Fig. 4E), which was also apparent to some extent
in Triascin-C treated worms (Fig. S2B).
Figure 1. Fecund female schistosomes have high mitochondrial OCR. A. OCR of Fecund female parasites recovered from mixed sex
infections, and Virgin adult females recovered from single sex infections were measured in real time by extracellular flux analysis, at basal
(immediately ex-vivo) and following the addition of oligimycin, FCCP and rotenone (Rot) and antimycin A (Ant) at the times indicated. B. Average
basal OCR readings of Fecund and Virgin females over the first 30 minutes ex-vivo. C. Spare respiratory capacity of Fecund and Virgin females,
calculated as shown in Fig. S1. Data are means plus SEM of readings from 4–5 individual female worms per experiment. Data are representative of at
least 3 individual experiments. See also Fig. S1.
Figure 2. OXPHOS is necessary for egg production. A. Eggs produced per fecund female during the first 24 h ex vivo, in the absence (Ctrl) or
presence of oligomycin (Olig), antimycin A (Ant) or rotenone (Rot). B. Survival of females, compared to untreated cultured worms, during the same
period and conditionS as described in A. C. Egg production between 24 h and 48 h in vitro following the washing out of inhibitors that were present
during the first 24 h ex vivo. Data are means plus SEM of readings from 10 individual female worms per experiment. Data are representative of at
least 3 individual experiments.
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The initial step in mitochondrial b-oxidation is catalyzed by
acyl-CoA dehydrogenase (ACAD). We targeted schistosome
ACAD using siRNAs; this approach resulted in reduced ACAD
mRNA, (Fig. S3C), decreased mitochondrial OCR (Fig. 4F), and
decreased egg production (Fig. 4G). Furthermore, the stimulation
of FAO by added palmitate was significantly impaired by this
siRNA treatment (Fig. 4H).
Taken together, these data support a role for the mobilization of
lipid droplet reserves for FAO in female schistosomes, and the use
of this pathway to support egg production.
Schistosomes cannot synthesize their own FA , but they can
take up lipids and convert them into TG [23,38]. Therefore we
propose that in vivo, TG in lipid droplets are continuously
catabolized for FAO and replenished through the uptake of FA
from the environment. We hypothesize that FAO is essential for
the differentiation and/or survival of Stage 4 vitelline cells. In this
model, the reduced OCR and SRC of virgin vs. fecund females
are due to the absence of mature vitellocytes that normally are
committed to FAO and OXPHOS. Our data indicate that, in
vitro, lipid stores are used but not replenished, thereby accounting
for the loss of Oil-Red-O staining and declines in OCR as TG
reserves are depleted in cultured parasites. Our data fit with the
view that reproductive maturation and regression are closely
linked to nutritional status in female schistosomes , and point
to FA as a key nutritional requirement for this process.
How male parasites help females to acquire FA remains to be
determined. Schistosomes eat blood, and it has been proposed that
male worms physically assist females in this process. However, we
Figure 3. Schistosomes use FA from lipid droplets for FAO to produce eggs. A. Average basal OCR of fecund females incubated without
(Ctrl) or with etomoxir (ETO) over the first 30 min ex vivo. See also Fig. S2. B. Numbers of eggs produced in 24 h per female parasite in the absence or
presence of etomoxir. C Oil-Red-O stained fecund females immediately ex vivo or at day 3 or day 13 of culture (red=Oil Red O; blue=Hoescht;
green=phalloidin) and quantitation of Oil-Red-O staining of females, as indicated. Images are optical sections through longitudinal axes. Scale
bar=50 mm. D. Palmitate induced mitochondrial FAO (% basal OCR) of fecund females ex vivo and after 13 days in culture. E. Average basal OCR of
fecund females ex vivo and after 3 or 13 days in culture (black bars) and numbers of eggs produced within the 24 h period immediately ex vivo or in
the 24 h period prior to day 3 or day 13 of culture (pink circles). F. Quantitation of Oil-Red-O staining of fecund females cultured without or with
etomoxir for 24 h. Data are means plus SEM of readings from 5–6 individual female worms per experiment. Data are representative of at least 3
individual experiments. ns=not significant.
Fat Catabolism Supports Flatworm Reproduction
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have been unable to show any positive effect in the assays
described herein of adding red blood cells to cultures of
schistosomes, regardless of whether males are present or not (data
not shown). Since glucose is an essential nutrient for schistosomes
(Krautz-Peterson et al. 2010, and data not shown), it is possible
that virgin females are subsisting largely on glucose absorbed
directly from the blood through tegumental surface transporters
[39,40]. A plausible explanation for the observation that females
cease egg production in vitro, even when male worms are present,
is that certain FA present in vivo are missing in the media that
have been routinely used to culture schistosomes. Possibilities
include short chain FA, which are present in high concentrations
in portal blood, and which interestingly are depleted in plasma
samples from schistosome-infected mice [41,42], and stearic acid,
which when complexed with bovine serum albumin is able to
replace fetal calf serum in a defined medium that is able to support
short term egg production by cultured schistosomes .
It has been assumed that FAO does not occur in schistosomes, and
that glucose is the key substrate for energy generation. However, the
data presented here indicate that schistosomes use FAO specifically
for the compartmentalized process of egg production. A rolefor FAO
FA in reproduction in insects and mammals [43,44]. It will be
important to identify the FA that support egg production and to
understand the specific mechanism by which male schistosomes assist
females in acquiring these nutrients. Unraveling the metabolic
requirements for reproduction in schistosomes may enable develop-
ment of enhanced tissue culture systems that will support continuous
egg production in vitro. This, in turn, would greatly facilitate the
application of emerging tools for transgenesis in these important
parasites . Moreover, it is conceivable that a greater understand-
ing of the metabolic processes that support schistosome egg
production may offer new opportunities to simultaneously prevent
transmission and disease development.
Figure 4. Loss of ACSL and ACAD function inhibits egg production. A. Average basal OCR of fecund females incubated without (Ctrl) or with
Triascin C (TC) over the first 30 min ex vivo. B. Numbers of eggs produced in 24 h per female parasite in the absence or presence of Triascin C.
Average basal OCR (C & F), numbers of eggs produced in 72 h per female (D & G) and quantitation of Oil-Red-O staining (E) and measurement of
FAO activity (H) in control fecund females, and in fecund females electroporated with SmASCL-siRNA (siASCL) or SmACAD-siRNA (siACAD), or with
control siRNA (-ve siRNA). Data are means plus SEM of readings from 5–6 individual female worms per experiment. Data are representative of at least
2 individual experiments. See also Fig. S2.
Fat Catabolism Supports Flatworm Reproduction
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Materials and Methods
This study was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. The
protocol was approved by the Institutional Animal Care and Use
Committee of Washington University School of Medicine (Animal
Welfare Assurance Number: A-3381-01).
Parasites and animals
Seven to eight wk old adult Schistosoma mansoni (NMRI strain)
were recovered from infected C57BL/6 female mice (Jackson
Laboratory). Parasites were cultured in RPMI containing 10%
fetal calf serum (FCS) (both from GIBCO), 2% antibiotic/
antimycotic, 1% HEPES, 10 mM glucose, 2 mM L-glutamine
and 1 mM sodium pyruvate (all from Sigma) at 37uC in 95%air/
5%CO2. Medium was replaced every 3 days. Eggs produced every
24 h were counted using a microscope.
Real-time measurements of OCR and extracellular acidification
were made using an XF-24 Extracellular Flux Analyzer (Seahorse
Bioscience). Worms were plated in XF-24 Islet Capture Micro-
plates (one worm per well) and analyzed in non-buffered RPMI
1640, 25 mM glucose, 10% FCS, 100 U/mL penicillin/strepto-
mycin, 2 mM L-glutamine and 1 mM sodium pyruvate under
basal conditions or in the presence of oligomycin (3 mM), fluoro-
carbonyl cyanide phenylhydrazone (FCCP, 4.5 mM), rotenone
(0.3 mM), antimycin A (3 mM), etomoxir (200 mM) (all from Sigma)
or Triacsin C (10 mM, Enzo Life Sciences). For FAO assay, real-
time oxidation rates of palmitate in worms was assessed by
extracellular flux analysis as described above. Basal OCR rates
were measured prior to 2 h treatment with palmitate (200 mM)
with fatty acid free bovine serum albumin (BSA), or with fatty acid
free BSA (0.17 mM) alone (Seahorse Bioscience).
Treatment of adult S. mansoni with small interfering RNA
siRNA targeting acyl-CoA synthetase (ACSL; GI: 256090263 and
GI: 238666949)and acyl-CoA
353231171 and GI: 256070604) were designed and synthesized by
Ambion, Applied Biosystems (Silencer Select Custom Designed siRNA;
http://www5.appliedbiosystems.com/tools/sirna/). siACSL: sense-
CUGUAUGCat. siACAD: sense-GGAAUCAAAUGAUAUCUUA-
tt; antisense-UAAGAUAUCAUUUGAUUCCat. Silencer Negative
control siRNA#1, which is not matched to any sequence in the
parasite genome, was also provided by the manufacturer and used as a
control. siRNA (10 mM) was delivered by electroporation .
Oil-Red-O staining and quantification
Parasites were fixed in 4% paraformaldehyde (Electron
Microscopy Sciences) diluted in PBSTx (PBS, 0.3% Triton X-
100) for 1 h, , dehydrated in 60% isopropanol for 15 min,
stained with Oil-Red-O (Sigma) overnight , washed in PBSTx
4 times and stained with phallotoxin-Alex Fluor 488 (Invitrogen)
confocal microscope and a PL APO CS 206NA=0.70 objective in
the format of 204862048. To quantify Oil-Red-O staining, dye was
eluted in 100% isopropanol for 30 min and absorbance of the eluate
vs. 100% isopropanol at 490 nm was measured .
The significance of observed differences was assessed using
function. Related to Fig. 1. The XF-24 Extracellular Flux Analyzer,
(Seahorse) was used to measure OCR as a basal rate, and after the
addition of Oligomycin (an inhibitor of the mitochondrial ATP
synthase, FCCP (to uncouple ATP synthesis from the electron
transport chain, ETC), or Antimycin A and Rotenone (to block
complex I and III of the ETC, respectively), as indicated. Resulting
changes in OCR indicate the amount of oxygen consumed for
mitochondrial ATP production, the maximal mitochondrial respira-
the amount of oxygen that is consumed by non-mitochondrial
processes when the ETC is inhibited. The SRC (spare respiratory
capacity) is the difference between maximal and basal OCRs.
Fundamental parameters of mitochondrial
of fecund females incubated without (0) or with etomoxir (ETO) at
different concentrations, over the first 30 min ex vivo. Data are
means plus SEM of readings from 5–6 individual female worms
Dose response to etomoxir. Average basal OCR
inhibition of, SmACSL and SmACAD. Related to Fig. 4.
Treatment of fecund parasites immediately ex-vivo with siRNA
specific for SmACSL (A) or SmACAD (C) led to a 50%–60%
reduction in encoding mRNAs after 72 h. Data points are means
plus SEM of readings from 5–6 individual female worms per
experiment. For real time RT-PCR, RNA was extracted using
RNeasy (Qiagen), contaminating genomic DNA was removed
using Turbo DNA-free endonuclease (Ambion) and cDNA was
synthesized using SuperScript II reverse transcriptase (Invitrogen),
and oligo dT. RT-minus controls were performed to confirm
absence of genomic DNA (data not shown). SmACSL transcripts
were quantified relative to a-tubulin using Applied Biosystems’
7500 real-time PCR system and SYBR green PCR Master Mix
(Applied Biosystems), and the 22DDCtmethod. Dissociation curves
were generated for each real-time RT-PCR to verify the
amplification of only one product. SmACSL primers were:
forward 59-TATGCCTCTGCCCAACTCTC-39 and reverse 59-
CACGTACGGGAAGTGCTAAA-39. SmACAD primers were:
forward 59-GCTGTCACACCACCTTGTCC-39 and reverse 59-
TCCAGATTGACTTGGCCTCT-39. a-Tubulin (GI: 8355916)
primers were: forward 59-TAGAGCGTCCAACCTACACAA-39
Quantitation of Oil-Red-O staining of fecund females cultured
without (Ctrl) or with Triacsin C (TC) for 24 h.
RNAi-mediated knockdown of, and targeted
The authors would like to thank Jonathan Curtis and Dr. Rianne van der
Windt and for their insightful suggestions and comments, and Mike Tighe
for help with microscopy.
Conceived and designed the experiments: SC-CH EA BE EJP. Performed
the experiments: SC-CH TCF EJP. Analyzed the data: SC-CH EA BE
ELP JBL EJP. Wrote the paper: SC-CH EJP.
Fat Catabolism Supports Flatworm Reproduction
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PLOS Pathogens | www.plospathogens.org7 October 2012 | Volume 8 | Issue 10 | e1002996