Frequency of TUNEL positive cells in specific tissues of female <i>S. mansoni</i>.

<p>Frequency of TUNEL positive cells in specific tissues of female <i>S. mansoni</i>.</p

Paired and separated females shrink and lose vitellarial mass when placed into tissue culture.

<p>Adult schistosomes were placed in culture paired with male worms, or as singled females, and followed over time. A) Changes in cross sectional area of females over time in culture, and compared to freshly recovered paired females (ex-vivo) and virgin females from single sex infections. The slope of decline in size is slightly steeper for un-paired females; although the difference is statistically significant, it is so small that it is unlikely to be biologically significant. Data were log transformed for analysis, but not display. Linear regression analysis revealed a significant (P<.005) difference in the slopes, but there was no significant difference between the size of paired and un-paired females at any time point when compared independently using t-tests. Each data point represents a single female worm. B) H&E stained sections of a freshly recovered female parasite (Ex-vivo) and females that had been maintained as paired or singled worms in vitro for 7 days. The location of the vitellarial tissue (v) and gut (g) in the paired ex-vivo female are shown for reference. C) Total DNA was measured relative to a standard curve with known quantities of DNA, and normalized to the ex vivo control (day 0). Data was log transformed for analysis. Linear regression analysis revealed significant (P<0.01) difference in the slopes of DNA loss between paired and singled (un-paired) females, and t-test showed significant difference between paired and un-paired females at day 7 (P<0.001) and day 11 (P<0.01). Day 11 DNA levels in singled females that were re-paired with males at day 7 were no different from those in females that remained singled through this time.</p

Involution of the vitellarium is associated with significant apoptosis within the vitelline cell population.

<p>Adult females were recovered from mixed sex infections and placed in culture as paired or singled, un-paired worms for 1 h in the presence of BrdU, or cultured for 24 h, with BrdU added at the 23^rd hour. Worms were fixed, sectioned and stained with TUNEL and DAPI (A) or with anti-BrdU and DAPI (C). The ratios of double positive cells to DAPI single positive cells within the vitellaria were enumerated microscopically. TUNEL-positive cells were extremely rare in worms examined within 1 h of recovery. However, all females examined exhibited a significant increase in apoptotic vitelline cell death within 24 h (A). In contrast we measured a significant reduction in the frequency of proliferating cells over this same time period (C) (p<0.01 in paired and in singled worms, as assessed by Student's t-test). B) Casapse-3 activity was monitored from the cell lysates of ex-vivo, paired and unpaired female worms. Results are given as an optical density of 405 nm normalized by parasite total protein concentration (**, <i>p</i><0.01; *, <i>p</i><0.05, as assessed by Student's t-test).</p

Females stop laying eggs and expressing genes encoding egg protein when placed into tissue culture.

<p>Adult females from mixed sex infections were placed in culture with or without male parasites (Four female parasites per well of a 6 well plate). A) Total numbers of eggs per well produced during each 24 h period were counted. Results are pooled from 3 individual experiments. The rate of decline of egg production is significantly faster in un-paired females. Data were log transformed (Y = log(Y+1)) for analysis. Linear regression analysis revealed a highly significant (P<0.0001) difference in the slopes of egg production decline. A 2-way-ANOVA showed a significant interaction of pairing and time on egg production, and Bonferroni post-test determined significant effects of pairing on egg production at days 3 through 6. B) Eggs produced during each day of culture were photographed. C) Real-time RT-PCR analyses of p14 transcripts. Expression of S. mansoni alpha-tubulin was used as the endogenous control to calculate relative expression levels, and expression levels were normalized to ex vivo females. Data were log transformed for analysis. Expression of P14 declined significantly over time in culture in both paired and un-paired females. Linear regression analysis revealed a highly significant (P<0.0001) difference in the slopes of p14 expression decline between paired and un-paired females. Independent t-tests performed at each time point showed dramatic and highly significant differences in expression of p14 at days 7 and 11 between paired and un-paired females (P<0.0001). Females that were un-paired for 7 days in vitro, then re-paired for 4 days showed a trend of higher P14 expression at day 11 than females who were un-paired for the full 11 days. This suggests that re-pairing with the male may be sufficient to prevent further decline of eggshell protein expression. There were no significant changes in alpha-tubulin expression over the time period of the experiment. D) Real time RT-PCR analyis of p14 transcripts in virgin females from single sex infections following culture in vitro paired or un-paired with male parasites. Only worms that were actually paired for the 11day duration were considered paired. Expression of alpha-tubulin was used as the endogenous control to calculate relative expression levels, and expression levels were normalized to the ex vivo females. Expression of P14 increased significantly (P<0.01) in single sex females that were paired for 11 days in vitro. Furthermore, the already low expression of P14 in virgine females from single sex infections was further reduced upon culturing in the absence of males (P<0.05). Thus, the difference in P14 expression levels at day 11 in females that are paired vs un-paired is highly significant (P<0.0001). Data were log transformed for analysis. T-tests were done to compare expression between samples. E) Changes in cross sectional area of virgin females from single sex infections over time in culture with or without males. No significant differences in size over time with or without males were noted. Each data point represents a single female worm.</p

Viteline cells are proliferating in paired females and in unpaired females from single sex infections.

<p>In contrast, apoptosis is occurring only in the vitellarial tissues of single females. Mice infected with mixed sex or female-only parasites were injected with BrdU 24 h prior to sacrifice. Recovered parasites were fixed, sectioned and stained with TUNEL and DAPI (A) or with anti-BrdU and DAPI (B). A) TUNEL positive cells were more frequent in the vitellaria of females from single sex infections (P<0.0001) compared to ex vivo females from a mixed sex infection. Data was log transformed for analysis. Significance was determined by t-tests assuming unequal variances. TUNEL positive cells occurred almost exclusively in the vitellaria (data not shown). DAPI (blue), TUNEL (Red). Autofluorescence in the FITC channel is shown in grey for tissue context. 40× magnification. G = gut. B) The frequency of BrdU positive cells in the vitellaria of females from mixed sex infections and single sex infections were not significantly different. DAPI (blue), BrdU (red). Autofluorescence in the FITC channel is shown in grey for tissue context. 20× magnification.</p

Loss of ACSL and ACAD function inhibits egg production.

<p><b>A.</b> Average basal OCR of fecund females incubated without (Ctrl) or with Triascin C (TC) over the first 30 min ex vivo. <b>B.</b> Numbers of eggs produced in 24 h per female parasite in the absence or presence of Triascin C. Average basal OCR (<b>C & F</b>), numbers of eggs produced in 72 h per female (<b>D & G</b>) and quantitation of Oil-Red-O staining (<b>E</b>) and measurement of FAO activity (<b>H</b>) 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002996#ppat.1002996.s002" target="_blank">Fig. S2</a>.</p

Parasitic and free-living adults from a stable transgenic line of <i>Strongyloides ratti</i> express GFP in the body wall-specific manner expected for the <i>Ss-act-2</i>prom::<i>gfp</i> transcriptional reporter.

<p>Typical patterns of GFP expression in parasitic female, free-living male and free-living female <i>S. ratti</i> expressing the integrated reporter transgene encoded in pPV356. (<b>A, B</b>) DIC and fluorescence images, respectively, of a parasitic female <i>S. ratti</i> from integrated line PV2. Note expression predominating in the body wall up to the level of the esophageal/intestinal boundary (<b>ei</b>). Position of the head is indicated (<b>h</b>). (<b>C, D</b>) DIC and fluorescence images, respectively, of a free-living female <i>S. ratti</i> from integrated line PV2. Note uniform expression throughout the body wall with additional loci of expression in the vulva (<b>v</b>) and rectum (<b>re</b>). (<b>E, F</b>) DIC and fluorescence images, respectively, of a free-living male <i>S. ratti</i> from transgenic line PV2. Note uniform expression in the body wall with additional expression in the cloaca (<b>cl</b>). (<b>G, H</b>) DIC and fluorescence images, respectively, of a non-transformed free-living male <i>S. ratti</i>. The fluorescence image in panel H was exposed for a period≥exposure times in panels B, D and F. Scale bar = 200 µm in all panels.</p

Vector constructs used to establish stable transgenic lines of <i>Strongyloides ratti</i> incorporate elements of the <i>piggyBac</i> transposon system.

<p><b>A</b>) Donor vector pPV254 incorporating a fluorescent reporter gene in which expression of GFP is driven by the <i>Ss-act-2</i> promoter and terminated by the <i>Ss era-1</i> 3′ UTR. The reporter transgene in pPV254 is flanked by the inverted terminal repeats (ITR) plus internal sequences common to <i>piggyBac</i> transposable elements. <b>B</b>) Donor vector pPV356, which is like pPV254 in all respects except that the coding sequence is flanked by the gypsy retroviral insulator sequences from <i>Drosophila</i>. <b>C</b>) Helper vector pPV402 in which expression of the <i>piggyBac</i> transposase gene is driven by <i>Ss-rps-21</i> promoter and terminated by the <i>Ss-era-1</i> 3′ UTR. <b>D</b>) Plasmid pPV257 for in vitro transcription of mRNA encoding the <i>piggyBac</i> transposase under the T7 promoter. In lieu of helper vector pPV402, this mRNA was capped, tailed and co-injected with donor vector pPV356 in Experiment 2.</p

Analysis of relative transgene copy number via qRT-PCR for stable transgenic lines of <i>S. ratti</i> transformed with the <i>piggyBac</i> transposon system.

a<p>Calculated from three technical replicates within the assay.</p>b<p>Calculated from three replicate assays.</p>c<p>Differences between mean relative copy number for stable lines not significant by one-way ANOVA (P>0.05).</p>d<p>Wildtype <i>S. ratti</i>.</p

Stable transgenic lines of <i>S. ratti</i> are established by microinjection of nucleic acid constructs followed alternating rounds of host and culture passage, selecting on expression of a fluorescent reporter gene product.

<p><b>A</b>) Diagram of major steps in isolation of stable transgenic lines of <i>S. ratti</i>. Initial gene transfer into parental (P0) free-living females is by gonadal microinjection of donor vectors alone or in combination with helper vector or capped transposase encoding mRNA. F1 larvae are derived in culture and screened for GFP expression. Transgene expressing individuals are reared to infective L3 (L3i) and inoculated into rats to establish as parasitic females. F2 progeny released in the feces of these rats are screened for transgene expression and positive larvae reared in culture to free-living males and females and allowed to mate. F3 progeny arising from these crosses are reared to L3i in culture in inoculated into rats. F4 progeny arising in the feces are screened for reporter transgene expression and used for further alternating rounds of culture and host passage with continued selection on GFP. The timeline indicates the intervals in days following microinjection of parental worms in which each generation is isolated up to the F5 when transmission and expression are generally stabilized. <b>B</b>) Frequency of transgene expressing progeny by generation during selection of stable transgenic lines of <i>S. ratti</i>. Data are mean (± one standard deviation) percentages of reporter transgene-expressing individuals in each generation of alternating host and culture passage with selection on GFP (see panel A). Means are derived from three independently established lines (PV2, PV3 and PV4). Sample sizes (<b>n</b>) indicated below the abscissa are totals of worms examined from all three lines. Statistics: in panel B, 2-way ANOVA, performed using Prism ver. 5.0c (GraphPad Software, Inc., La Jolla, California, USA), revealed a highly significant effect due to generation (P<0.0001) but no significant effect (P = 0.2) due to donor plasmid.</p