- Email: [email protected]
PL10-like genes in the ovary of Schistosoma mansoni
Journal Pre-proof Functional analysis of vasa/PL10-like genes in the ovary of Schistosoma mansoni Danielle E. Skinner (Conceptualization) (Methodology) (Validation) (Formal analysis) (Investigation) (Visualization)Writing – original draft)Writing – review and editing), Anastas Popratiloff (Methodology) (Validation) (Formal analysis) (Investigation) (Resources) (Visualization)Writing – original draft), Yousef N. Alrefaei (Visualization)Writing – original draft)Writing – review and editing), Victoria H. Mann (Supervision) (Resources), Gabriel Rinaldi (Conceptualization) (Supervision) (Resources) (Funding acquisition)Writing – original draft)Writing – review and editing), Paul J. Brindley (Conceptualization) (Supervision) (Resources) (Methodology) (Formal analysis) (Visualization) (Funding acquisition)Writing – original draft)Writing – review and editing)
PII:
S0166-6851(19)30149-5
DOI:
https://doi.org/10.1016/j.molbiopara.2020.111259
Reference:
MOLBIO 111259
To appear in:
Molecular & Biochemical Parasitology
Received Date:
30 September 2019
Revised Date:
23 December 2019
Accepted Date:
13 January 2020
Please cite this article as: Skinner DE, Popratiloff A, Alrefaei YN, Mann VH, Rinaldi G, Brindley PJ, Functional analysis of vasa/PL10-like genes in the ovary of Schistosoma mansoni, Molecular and amp; Biochemical Parasitology (2020), doi: https://doi.org/10.1016/j.molbiopara.2020.111259
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
Functional analysis of vasa/PL10-like genes in the ovary of Schistosoma mansoni
Danielle E. Skinner1, 2, Anastas Popratiloff3, Yousef N. Alrefaei1, 4, Victoria H. Mann1, Gabriel Rinaldi1, 5, *, #, Paul J. Brindley1,*, #
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1. Department of Microbiology, Immunology & Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, The George Washington University, Washington, DC 20037 USA.
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2. Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego 9500 Gilman Dr, La Jolla, CA, 92093, USA.
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3. Center for Microscopy and Image Analysis, The George Washington University, Washington, D.C., 20037, USA.
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4. Department of Medical Laboratory Technology, The Public Authority of Applied Education and Training, Shuwaikh, Kuwait.
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5. Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, UK.
These authors contributed equally.
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*Correspondence:
Gabriel Rinaldi, Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK; email, [email protected]
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Paul J. Brindley, Department of Microbiology, Immunology & Tropical Medicine, School of Medicine & Health Sciences, The George Washington University, 2300 I Street NW, Washington, DC 20037 USA; email, [email protected]
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Graphical abstrct
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A novel confocal-microscopy approach for ovary volume estimation and autofluorescence control was employed to evaluate the effect of knocking down vasa-like genes in Schistosoma mansoni.
Highlights A novel confocal microscopy-based approach to precisely estimate the volume of the female Schistosoma mansoni ovary while controlling for tissue autofluorescence was developed Gene silencing of S. mansoni vasa-like gene 1 was associated with a reduction in the ovary volume in treated female worms, but not with egg number or morphology alterations A lower number of dividing cells in immature ovary of the RNAi-treated parasites compared to controls suggested a key role of S. mansoni vasa-like gene 1 in germline homeostasis
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Abstract The RNA helicase Vasa plays a pivotal role in the development of the germ line. To decipher the functional roles of vasa/PL10-like genes in the human blood fluke Schistosoma mansoni, we performed RNA interference followed by the analysis of the ovary in the adult female. Doubled stranded RNA targeting the schistosome vasa-like gene Smvlg1 reduced the volume of the ovary. Changes in morphology of the ovary were analysed using carmine red-staining of the parasites followed by a novel confocal laser scanning microscopy (CLSM)-based approach to control for natural autofluorescence in female schistosome tissues. The reduction in the ovary volume may have been promoted by the loss of germ cells. By contrast, significant differences were not apparent in the number of eggs produced or hatching rate of eggs laid by the female schistosomes transfected with Smvlg-specific dsRNA. The findings suggested a role for S. mansoni vasa/PL10-like gene -1 in germ cell development within the schistosome ovary that might impact in the pathogenesis and disease transmission by this neglected tropical disease pathogen.
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Keywords. Schistosoma mansoni; vasa/PL10-like genes; Functional Genomic tools; RNAi; germline; confocal microscopy
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1. Introduction
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The catalogue of species of flatworms for which there are draft sequences of the entire genome is expanding now at pace: four tapeworm genomes were reported – the human hydatid pathogens Echinococcus multilocularis and E. granulosus, the causative agent of neurocysticercosis Taenia solium, and model tapeworm of rodents, Hymenolepis microstoma [1]. In addition, since 2009 draft genomes for the main blood fluke species are available which have been regularly curated for the last decade. This information follows on the reports of the genome sequences of the three major schistosome species of humans, Schistosoma haematobium, S. japonicum and S. mansoni [2-7]. More recently, 81 genomes of parasitic and non-parasitic worms have been broadly compared, identifying key gene families involved in immune responses, parasite migration and infection establishment [8]. Several genes revealed in these studies need to be functionally addressed employing genetic approaches under ongoing development. Noteworthy advances in functional genomic tools and transgenesis for schistosomes have been made, including CRISPRCas-based genome editing in S. mansoni and the liver fluke Opisthorchis viverrini [9, 10]. However, progress still lags behind model species such as the fruit fly, Caenorhabditis elegans, zebrafish, and the laboratory mouse.
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It is usually informative to utilize data on gene expression and genomics from related taxa to identify orthologues/ paralogues within the species of interest. For schistosomes, in addition to the higher metazoans listed above, these informative taxa include planarians (which are freeliving, ‘capable-of-regeneration’, turbellarians) such as Schmidtea mediterranea, Macrostomum lignano, and Dugesia japonica. The analyses revealed that the schistosome vasa-like genes Smvlg1, Smvlg2, and Smvlg3, and several other vasa-like genes of platyhelminths likely are not bona fide orthologues of vasa, or orthologues of PL10, a DEAD-box helicase closely related to Vasa [11]. Moreover, close scrutiny of four cyclophyllidean tapeworm genomes failed to locate an orthologue of vasa [1], which supports the hypothesis that vasa was lost during the evolutionary history of the Trematoda and Cestoda [12]. In addition, parasitic flatworms do not possess other post transcriptional regulators such as Piwi and Group 4 tudor proteins that, like Vasa, are associated with germline development and maintenance of somatic stem cells [1, 13]. These findings indicate that parasitic flatworms may have evolved an independent and/ or modified mechanism for germline and somatic stem cell development and maintenance [1, 1215].
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Functional roles of Vasa helicases are poorly understood in species of the Lophotrochozoa and little is known about the function of vasa in flatworms. Nonetheless, several functional analyses have focused on vasa-like genes in platyhelminths, including the monogenean Neobenedenia girellae, the free-living planarian Dugesia japonica [16, 17], and the larval and adult developmental stages of S. mansoni [14] and S. japonicum [18], respectively. RNA interference (RNAi) studies with N. girellae vasa-like genes (Ngvlgs) revealed that the knockdown of Ngvlg1 and Ngvlg2 (related to the Smvlg1 and Smvlg2, respectively) leads to a partial and complete loss of germ cells in the ovary and testis [16]. In addition, the hatching rates of eggs laid by the dsRNA-treated worms were decreased compared to controls [16]. These findings indicate that Ngvlg1 and Ngvlg2 play critical roles in the formation of germ cells and fertilization of the eggs and/or development of the eggs [16]. Functional studies with D. japonica vasa-like genes (DjvlgA and DjvlgB) were performed by exposure of planarians to X-irradiation; the expression of these genes was investigated as well as the regenerative capacity of the flatworm [17]. DjvlgA 4
and DjvlgB are expressed in the germ cells and DjvlgA appears to play a role in the totipotency of the neoblasts [17]. In N. girellae and D. japonica, vasa-like genes appear to play a role in the differentiation and maintenance of germ cells and in D. japonica to play a role in the differentiation and maintenance of the neoblasts. A vasa-like gene from S. mansoni sporocysts was silenced using Smvlg3-specific siRNAs, revealing a critical role in germline proliferation and maintenance [14]. In S. japonicum, the vasa homologue SjVasa3, (91% amino acid identity to Smvlg3) plays a key role in development of the reproductive tract [18].
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Key details provided by these studies notwithstanding, the vasa-like genes of platyhelminths remain poorly understood. Therefore, here we investigated functional aspects of S. mansoni vasa-like genes that we have previously reported [11] by RNAi following approaches already reported [19-21]. In recent years there have been advances in the development of molecular tools for schistosomes and RNAi has become the benchmark approach for functional gene analyses (reviewed in [21, 22]). Herein, the functional analysis was undertaken by the delivery of dsRNAs against the three vasa-like genes in S. mansoni by electroporation to adult females, followed by analysis of the morphology and volume of the ovary. Although, significant knock down was not apparent for Smvlg-2 and -3, the findings indicated that Smvlg-1 gene might be involved in the development and maintenance of the reproductive system of the female schistosome. 2. Materials and Methods
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2.1 Schistosomes
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Mice infected with the NMRI (Puerto Rican) strain of S. mansoni were supplied by the Biomedical Research Institute (BRI) Rockville, Maryland. Maintenance of the mice infected with S. mansoni at GWU was approved by the GWU Institutional Animal Care and Use Committee of the IACUC of The George Washington University. All procedures employed were consistent with the Guide for the Care and Use of Laboratory Animals. Adult worms were recovered after portal perfusion of mice at seven weeks after infection [23]. Thereafter, the adult worms were transferred to a 50 ml conical tube and washed several times with 37C 1phosphate-buffered saline, pH 7.4 (1PBS) supplemented with 2% penicillin, streptomycin, fungizone (2% PSF) by gravity wash. The adult worms were transferred to schistosomule wash medium (DMEM supplemented with 10 mM HEPES and 2% PSF) and cultured at 37C, under 5% CO2 until treatment with double stranded RNA.
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2.2 Synthesis of dsRNA
Double stranded (ds) RNA template was prepared by PCR with the pCR4.0 TOPO TA vector containing the S. mansoni vasa-like gene 1 (Smvlg1) cDNA (JQ619869, Smp_033710) as template, and Smvlg1 specific primers tailed with the T7 promoter sequence at the 5’ termini. The primer sequences were (T7 promoter sequence italicized): dsSmvlg1 forward primer, 5′TAA TAC GAC TCA CTA TAG GGC AAA CGG TTC AGA TGG TGG TGG TGC C-3′ and dsSmvlg1 reverse primer, 5′- TAA TAC GAC TCA CTA TAG GGG CGC TTA TAG AAT CAC CAG GAC CTT GC -3′, spanning coding DNA positions 62-736. The irrelevant control firefly luciferase dsRNA (dsLuc) template was generated using the pGL3-basic plasmid (Promega, Madison, WI; GenBank accession U47295.2) and primers as follows: dsLuc forward primer, 5’5
TAA TAC GAC TCA CTA TAG GGG TGC CAG AGT CCT TCG ATA G-3’ and dsLuc reverse primer, 5’-TAA TAC GAC TCA CTA TAG GG CAA CTT TAC CGA CCG CGC C-3’, spanning coding DNA positions 563-1097.
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The dsRNAs were synthesized and purified using the MEGAscript RNAi kit according to the manufacturer’s instructions (Ambion, Austin, TX). The dsRNAs were precipitated by adding one volume of 5 M ammonium acetate, 2.5 volumes of 95% ethanol, and stored at -20C for 24 h. Thereafter, precipitated dsRNAs were recovered by centrifugation at 4C, 15,000 g for 30 min. The pellets washed with -20C 70% EtOH and then pelleted by centrifugation at 4, 14,500 rpm for 30 min. The dsRNAs were resuspended in schistosomule wash medium (DMEM supplemented with 10 mM HEPES and 2% PSF - 10,000 units/ml of penicillin, 10,000 µg/ml of streptomycin, and 25 µg/ml of Amphotericin B or Fungicide). The integrity of the dsRNAs was verified by non-denaturing 1% agarose gel electrophoresis, and concentration and purity determined with a spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington DE). dsRNAs were also synthesized for Smvlg2 and Smvlg3 (Supplementary Methods). 2.3 Delivery of dsRNA
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Immediately after the portal perfusion (above), adult female and male schistosomes were separated, and the females dispensed into 4 mm gap cuvettes (BTX, San Diego, CA), with 20 females per cuvette with 200 l schistosomule wash medium, three cuvettes total. The cuvettes were incubated for 10 min at room temperature (RT) with 15 g dsSmvlg1 or 15 g dsLuc, the latter serving as irrelevant dsRNA control. The third cuvette did not receive dsRNA was included as a mock-treatment control. Thereafter, the cuvettes were subjected to square wave electroporation, one pulse of 125 V, 20 ms in duration, using the ElectroSquarePorator model ECM830 (BTX, San Diego, CA). Immediately after, one ml of pre-warmed (37C) modified Basch’s medium was added to the cuvettes [23, 24]. The female worms were transferred to 6well tissue culture plates, the volume of Basch’s medium increased to 5 ml, and the plate incubated at 37C under 5% CO2. Forty eight hours later the Basch’s medium was changed. At 4 days in culture, the Basch’s medium was replaced and fresh 15 g of dsRNAs was added to the wells. The female worms were harvested at day 8 for subsequent RNA and phenotype analyses (below). The experiment was performed in duplicate. In addition, the RNAi experiment was performed in triplicate with five treatment groups (dsSmvlg1, dsSmvlg2, dsSmvlg3, dsLuc, and mock-treatment control) and harvested four days later (Supplementary Methods). The number of eggs laid by females across the experimental groups and controls, and hatching rate also were recorded. 2.4 Gene expression analysis The adult females were harvested 4 or 8 days after the transfection with dsRNA. Total RNA was extracted from a subpopulation (five female worms) of each treatment group using the RNAqueous®-4PCR kit (Ambion, Austin TX). Residual DNA contaminating the RNA was removed by digestion with RNase-free DNase I (TurboDNase, Ambion) at 37°C for 60 min. cDNAs were synthesized using the iScript™ cDNA synthesis kit (Bio-Rad, Hercules CA) using 150 ng of total RNA. Relative expression of genes of interest was analyzed by quantitative RTqPCR. TaqMan probes and qPCR primers were designed with the assistance of Beacon 6
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Designer (Premier Biosoft International, Palo Alto, CA): Smvlg1 forward primer, 5′-ACG ACT ATA ATG AGA ATA ATC TTG-3′; Smvlg1 reverse primer, 5′-CCA AAC TTT ATG TGC CTC-3′; Smvlg1 probe, 5′-/56-FAM/GTT CAG ATG GTG GTG GT/3IABlk_FQ/-3′. TaqMan probe and primers targeting the S. mansoni reference gene, glyceraldehyde 3-phosphate dehydrogenase (SmGAPDH) (GenBank accession M92359, Smp_056970), were: SmGAPDH forward primer, 5’-TGT GAA AGA GAT CCA GCA AAC-3’; SmGAPDH reverse primer, 5’GAT ATT ACC TGA GCT TTA TCA ATG G-3’; and SmGAPDH probe, 5’-/56-FAM/AAG ACT CCA GTA GAC TCA ACG ACA T/3IABlk_FQ/-3’. Quantitative PCRs were performed in triplicates, and reactions were carried out in volumes of 20 µl with primer-probe sets and Perfecta qPCR FastMix, UNG (Quanta Bioscience, Gaithersburg, MD). The qPCR conditions included an initial denaturation at 95°C for 3 min followed by 40 cycles of 30 s at 95°C and 30 s at 55°C, performed in a thermal cycler (iCycler, Bio-Rad) and a Bio-Rad iQ5 detector to scan the plate in real time. Relative quantification was calculated following the 2−ΔΔC method [25]. SmGapdh was employed as the reference and the mock-treatment control as calibrator sample. T
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2.5 Carmine staining of adult female schistosomes
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The adult females were fixed for at least 24 h with AFA (85% EtOH, 37% formalin commercial grade, glacial acetic acid, 85:10:5) at -20C. Excess fixative was removed by washing worms with 70% EtOH for 5 min at RT. The adult females were stained with hydrochloric carmine for at least 30 min at RT [26, 27]. Hydrochloric carmine was prepared by mixing 1 g of carmine (alum lake carminic acid, Acros Organic, 1390-65-4) with 500 l concentrated HCl (>12 M), 500 l H2O, volume brought up to 20 ml with 90% ethanol. The mixture was heated to ~80C for about 20-30 minutes until the carmine dissolved. The solution was cooled to ambient temperature, filtered, and stored in the dark at 4C. The excess, was differentiated in acidic 70% EtOH (1 ml HCl, 1M per 10 ml of 70% EtOH) for 1 to 2 min at RT. Thereafter, worms were dehydrated through a graded ethanol series at RT: 70%, 90%, and 100% each performed once for 5 min at RT, then cleared in a xylene series: 50:50; xylene:100% ethanol for one wash for 5 min, and 100% xylene for three washes for 5 min each at RT. The female worms were mounted on glass slides with Permount (Fisher Scientific, USA) before confocal microscopy. 2.6 Confocal laser scanning microscopy
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Confocal laser scanning microscopy (CLSM) images were captured on a Carl Zeiss LSM 710 confocal system. Confocal images were captured with a Plan-Apochromat 20x/0.8 objective lens, deployment of which was considered to be suitable for imaging the entire ovary of schistosomes. Confocal images were also captured with an Alpha Plan Apochromat 100x/1.46 objective lens with oil immersion, deployment of which was prudent for imaging cells within the immature ovary. The female worms were excited with 488 nm argon laser and 561 nm diode-pumped laser. Images were acquired using the linear spectral unmixing approach. The rationale for this approach over the conventional bandwidth emission filtering was that schistosomes exhibit substantial auto-fluorescence that interfered with the measurement of the carmine-specific fluorescence. The key difference was that instead of capturing the emission in a relatively large range of the spectrum, i.e., 560-620 nm, which contains convolved auto-fluorescence and carmine fluorescence, the instrument utilizes a 32-channel spectral photomultiplier that is capable of capturing simultaneously spectral data sets in bands of 9.6 nm. This was possible 7
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2.7 Quantification of mitotic cells in the immature ovary
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since the emission deriving from the sample is spectrally separated by a diffraction grating and sent to the 32-channel photomultiplier. Since the 32-channels are aligned along the visible spectrum, they can capture simultaneously a set of 32 images that are known as lambda stacks or spectral image sets. Further, analyses of the emission was done from these image sets pixel by pixel, revealing specific emission curves present in the sample. Linear spectral unmixing, utilized herein refers to application of spectral disconsolation algorithm that relies on the linear additive properties of the fluorescence signal. Therefore, if there are two emission curves present in the sample with at least 10% difference, linear spectral unmixing could be used to generate two intensity modulated channels representing the areas of the image expressing the two emissions. The final product was a two-channel image that resembled a standard confocal micrograph, but the way it was generated was fundamentally different as explained. Confocal zstacks were collected from the top to the bottom of the worms at the levels of the ovaries, ensuring that the entire organ is within the 3D image set. Z-steps were optimized according to the software optimal calculations. The resulting images, encoded two channels — autofluorescence and carmine stain signal. Unmixed, confocal stacks were imported to Volocity (v.6.2.1, Perkin Elmer/Improvision) for three-dimensional rendering and analysis of ovary volume.
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In order to quantify the percentage of cells undergoing mitosis in the immature ovary of Smvlg1-silenced parasites or controls, cell numbers in each of 31 high power (100x/1.46 objective) images were estimated, in a blinded-to-operator fashion, counting an average of 45 cells per image with the assistance of Image J (version 1.52p). Cells displaying clear metaphasic, anaphasic, or telophasic chromosomes with no obvious nuclear membrane were counted as ‘dividing cells’. By contrast, cells where the nuclear membrane appeared to be intact, with or without an evident nucleolus, were considered to be non-dividing. The raw data collected in Microsoft Excel (version 16.16.16) were exported to RStudio (Version 1.2.5001) to generate the boxplots of percentage of dividing cells to total cells, and for statistical analysis. 2.8 Statistical analysis
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Levels of statistical significance among and between the experimental groups were determined using Analysis of Variance (ANOVA) and Student’s t-test for pairwise comparisons. P-values of ≤ 0.05 were considered to be statistically significant. Plots were generated with the assistance of GraphPad (Prism 8, version 8.2.1) (San Diego, CA) and RStudio (Version 1.2.5001) (Boston, MA). 3. Results
3.1 Significant knockdown of S. mansoni vasa-like-gene 1 Smvlg1-specific dsRNA was synthesized spanning the amino-terminus region of the transcript avoiding the highly conserved core motifs characteristic of the putative DEAD-box RNA helicases, of which S. mansoni encodes 35 based on a search on v7 genome using Biomart with Pfam ID PF00270 and InterPro ID IPR011545 criteria in Worm Base Parasite, version 14 8
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(August 2019) (Supplementary Tables S1 and S2) (https://parasite.wormbase.org/index.html) [1, 11]. Firefly luciferase-specific dsRNA was included as irrelevant-dsRNA control, and female worms electroporated under the same conditions but with no dsRNA molecules served as mocktreatment control. To investigate the RNAi effects at the molecular level, transcript levels of Smvlg1 were analyzed in the three independent treatment groups by qRT-PCR for each experiment. Eight days after treatment, double stranded RNA interference (dsRNA)-mediated post-transcriptional gene silencing of Smvlg1 led to a robust reduction (68%) in transcript levels compared to the mock-treatment control and the control worms treated with dsRNA for luciferase (Figure 1). Transcript levels of Smvlg1, Smvlg2, and Smvlg3 were also analyzed in three independent experiments in which females were harvested four days after treatment with dsSmvlg1, dsSmvlg2, dsSmvlg3, dsLuc, or without dsRNAs. The dsSmvlg1 treatment group was the only one of the three dsSmvlg treatment groups that delivered a consistent and significant knockdown of its target in at least three independent replicates (Supplementary Figure S1). Even though gene silencing was attempted in male adult worms targeting these genes, knock down was not apparent or unsuccessful for all three vasa-like genes. In addition, we had previously identified and characterized these genes in the female schistosome [11]. Therefore, we focused here on the vasa-like genes in the female worm.
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3.2 A novel approach to control for natural tissue autofluorescence in the female schistosome
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RNAi-treated female schistosomes were stained with carmine red-stain and imaged by confocal laser scanning microscopy (CLSM). The ovary of the female worms was imaged by spectral confocal microscopy with a 488 nm laser line and the volume estimated as described above. In brief, discrete sites on the worms were selected representing the autofluorescence and the fluorescence of the carmine stain and served as emission references for linear unmixing [28]. Autofluorescence registered on the lambda stack displayed a broad spectrum with a peak at ~600 nm, and the carmine signal peaked at ~635 nm. The two-channel confocal stacks, derived after linear unmixing, comprised channels representing the autofluorescence and carmine signal (Figure 2A). 3.3 Silencing to S. mansoni vasa-like-gene 1 decreases the ovarian volume
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To specifically investigate the effects of the vasa-like gene silencing on the ovary volumes a thorough quantitative analysis was performed. The confocal 3D image sets were rendered in three-dimensions by importing into the Volocity 3D imaging software. Volocity was used to establish a measurement protocol to locate the significant intensities of the carmine signals specific to the ovary and render the volume measurements in µm3 (Figure 2B). This approach used intensity threshold of the carmine channel to create a specific volume of the ovary and output its volume. No discernable gross morphological abnormalities were evident within the ovaries of dsRNA-treated or control parasites (Figure 3A). However, in two independent experiments harvested at 8 days, significantly smaller ovary volumes were observed in females treated with dsSmvlg1 compared to the ovary volumes of the mock-treatment control (Figure 3B). Overall, a trend was apparent where reduced ovary volumes were more likely in the females transfected with dsSmvlg1 than in the controls (Figure 3C, Supplementary Tables S3 and S4). A similar trend was observed in the three independent experiments harvested at 4 days 9
(Supplementary Figure S2). Remarkably, no significant differences in number of laid eggs or hatching rate were seen among the experimental and control groups (not shown). 3.4 Smvlg1 may be involved in germinal cell proliferation
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To analyze the underlying mechanism for the volume regression of ovaries in the dsSmvlg1treated females, cellular proliferation was investigated. In the anterior region of the schistosome ovary, i.e. immature ovary, oogonia undergo mitosis and meiosis leading to small poorly differentiated immature oocytes, whereas the posterior region of the ovary, i.e., mature ovary, consists of large well-differentiated mature primary oocytes [29-33]. CLSM x100 magnification images were captured of the immature region of the ovaries from the carmine red-stained wholemounts of the S. mansoni females. Images were taken of ovaries with the largest and the smallest volumes from both the controls and the dsSmvlg1-treated females. In 100 images, cells at rest (i.e., visible nucleolus, no mitotic figures) can be distinguished from actively dividing cells (i.e., visible mitotic figures) (Figure 4A-C). Accordingly, the percentage of dividing cells was estimated for each of the three groups of parasites. A significant trend towards a lower percentage of dividing cells in the parasites transfected with dsRNA against Smvlg-1 compared to controls was evident (Figure 4D). The latter might suggest a functional role of Smvlg-1 in the control of cell proliferation in the immature lobe of the schistosome ovary.
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4. Discussion
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In the current study RNAi-induced suppression of S. mansoni vasa-like genes (Smvlg) [11] was accomplished by delivering dsRNA into cultured worms by square wave electroporation. The knockdown of the vasa-like gene 1 (Smvlg1) was consistent across four experiments where the females were harvested at four days, 38-60% (mean=54%) knockdown in Smvlg1 transcript levels was observed in each replicate. In the two experiments where the females were harvested at eight days, transcript levels of Smvlg1 were reduced by 59-78% (mean=68%). Given that the levels of knockdown achieved for Smvlg2 and Smvlg3 were not consistent across all the experiments, we decided to focus on the functional role of Smvlg1. Remarkably, the silencing of Smvlg1 was associated with a reduced volume of the ovary in female worms. These changes were detected using a novel approach that employs carmine red-staining of the parasite followed by confocal laser scanning microscopy (CLSM) to disentangle the carmine-specific signal from the native autofluorescence. The reduction in ovary volume after the treatment with Smvlg1specific dsRNA might have followed the loss of germ cells, suggesting that Smvlg1 may be involved in germ cell homeostasis in the schistosome ovary. However, further studies are needed to prove this hypothesis. Unexpectedly, no significant differences were seen in the number or hatching rate of eggs laid by female schistosomes transfected with Smvlg-specific dsRNAs compared to control parasites. When vasa-like genes were silenced in S. japonicum, the authors reported not only evident morphological alterations in both testis and ovaries, but also a reduction in the number of eggs in female uterus and in vitro laid eggs [18]. In the monogenean N. girellae, Ohashi and colleagues also showed that vasa-like genes were expressed in the germline, and when silenced a partial or complete loss of germ cells that led to a reduction in the hatching rate of eggs was evident [16]. The unexpected absence of a reduction in the number of eggs in Smvlg1-silenced parasites might be explained by functional redundancy of the vasa-like genes, in particular Smvlg2 and Smvlg3 that were not silenced in our approach. Similar outcomes 10
were observed in the free-living flatworm Macrostomum lignano where not only a functional redundancy of vasa and vasa-like genes has been described, but also difficulties in achieving substantial silencing for some of the vasa genes, probably due to long half-life of the mRNA and/ or the protein, were discussed [34]. Accordingly, we cannot rule out the presence of similar phenomena in our study. This will be investigated in future studies.
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Analyses by CLSM revealed no discernable morphological abnormalities within the ovaries of the dsRNA-treated schistosomes compared to controls in both the three independent experiments harvested at 4 days and the two independent experiments harvested at 8 days. The schistosome ovaries from the treatment groups harvested at 8 days post-treatment were more regressed reproductively than the schistosome ovaries from the treatment groups harvested at 4 days, which would be expected from female schistosomes separated from their male partners and cultured in vitro for 8 days as previously described [29]. In addition, we cannot rule out that this well-described regression of the reproductive tissue in worms cultured in vitro might have been exacerbated by the electroporation itself. Recently, a novel tissue culture media complemented with ascorbic acid and cholesterol was reported to maintain the reproductive organs and vitellaria of female schistosomes longer in culture [35]. Future experiments employing this tissue culture media would avoid the confounding effect of the ovary regression due to the in vitro culture conditions. Moreover, in vivo RNAi by delivering dsRNA against S. japonicum genes into experimentally-infected mice has been recently shown to be successful to induce a gene silencing of >79.4% in male and >91.5% in female compared to control parasites [36]. This novel approach provides a more natural environment to induce germline gene silencing allowing the detection of more reliable and maybe more reproducible phenotypes, including alterations in the reproductive system and number of eggs trapped in the host liver.
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Many genes, which are specifically expressed in germ cells and somatic stem cells of higher metazoans, have been lost during the evolution of parasitic flatworms, including schistosomes [1, 13-15, 37]. In particular, Vasa and PL10, two closely related proteins in the ATP-dependent DEAD-box helicase superfamily are not present in schistosomes although they occur widely in species from bacteria to humans [38]. Nonetheless, we reported previously that three vasa-like genes (Smvlg1, Smvlg2, and Smvlg3) evolved in S. mansoni that form a parasitic flatwormspecific clade discrete from the PL10 clade [11, 12]. This flatworm-specific clade includes vasalike genes from other flatworms including N. girellae, D. japonica, and S. polychroa [12, 16, 17, 39]. Whereas the phylogenetic analysis revealed that these DEAD-box helicases were not orthologues of either vasa or PL10, transcripts of the vasa-like genes are expressed in the schistosome ovary in a germline-specific manner and are abundantly expressed in intrasnail developmental stages [14]. Moreover, among the schistosome developmental stages, eggs laid in vitro by worms in culture within 12 hours after their collection from the mouse display the highest levels of vasa-like gene expression [11]. Other reports showed that vasa-like genes exhibit germline-specific expression [14, 16, 17, 39]. Future studies on the vasa-like genes and related genes of schistosomes using functional genomics approaches such as genome editing [9, 10] and single-cell transcriptomic [15, 40] are expected to contribute to the understanding of the germ line development, pathogenesis, and disease transmission.
CRediT author statement. 11
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Danielle E. Skinner: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Visualization, Writing – Original Draft, Review and Editing Anastas Popratiloff: Methodology, Validation, Formal analysis, Investigation, Resources, Visualization, Writing – Original Draft Yousef N. Alrefaei: Visualization, Writing – Original Draft, Review and Editing Victoria H. Mann: Supervision, Resources Paul J. Brindley: Conceptualization, Supervision, Resources, Funding acquisition, Writing – Original Draft, Review and Editing Gabriel Rinaldi: Conceptualization, Supervision, Resources, Methodology, Formal analysis, Visualization, Funding acquisition, Writing – Original Draft, Review and Editing Acknowledgements
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We thank Anna Protasio for inisghtful, helpful comments and informative discussion. These studies were supported by NIH-NIAID awards R01AI072773 (PJB) and R21AI109532 (GR), and the Wellcome Trust Strategic Award number 107475/Z/15/Z; the content is solely the responsibility of the authors and does not necessarily represent the official views of the NIAID, the NIH or the Wellcome Trust. Mice infected with S. mansoni were provided by the NIAID Schistosomiasis Resource Center for distribution through BEI Resources, NIH-NIAID Contract HHSN272201000005I. References
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Figure legends Figure 1. Knockdown of vasa-like gene 1 in females of S. mansoni at eight days posttransfection. Panel A. Timeline for RNAi analysis. Groups of 20 female worms, previously separated from male worms, were subjected to electroporation in the absence of dsRNA (8d mock control), in the presence of 15 µg of Luc-specific dsRNA (8d dsLuc; irrelevant dsRNA control), or Smvlg1-specific dsRNA (8d dsSmvlg1). At day 4, an additional 15 µg of indicated dsRNAs were added to the culture medium, and the worms harvested at day 8. Panel B. Relative gene expression levels measured by RT-qPCR; Smvlg1 transcript levels were normalized to those of SmGAPDH. The Smvlg1 transcript level was determined in two independent experiments with the 8d mock, 8d dsLuc, and 8d dsSmvlg1 treatment groups. The bars represent the means ± standard deviations. Student’s t-test: *p ≤ 0.05 and **p ≤ 0.01. Lower table displays the transcript levels normalized to SmGAPDH (100% in the calibrator sample, i.e. mock control) for each treatment group from the two independent experiments. Exp1, experiment 1; Exp2, experiment 2; SD, standard deviation.
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Figure 2. Estimation of the ovary volume and tissue autofluorescence. Panel A. High power (20x/0.8 objective) images of S. mansoni female excited with 488 nm laser line. Two channels were extracted after applying a linear spectral unmixing algorithm to the lambda stack confocal images (see Methods). S. mansoni female visualized with the autofluorescence channel (grey, 1), with the carmine stain signal channel (red, 2), and merged channels (3). Scale bars, 50 μm. Panel B. High power (20x/0.8) images of S. mansoni females excited with 488 nm laser line. Two channels were extracted after applying a linear spectral unmixing algorithm to the lambda stack confocal images (see Methods). Three dimensional (3D) renderings and ovary volume analysis of the stack confocal images were performed with the 3D imaging software Volocity (v6.2.1, PerkinElmer/Improvisation). 1) 3D rendering of S. mansoni female visualized with the 15
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autofluorescence channel (grey). Overt threshold intensities of the carmine stain signals specific to the female ovary displayed in blue. 2) 3D rendering of S. mansoni female visualized with the carmine stain channel (red). 3) Merged images shown in 1 and 2. Overt threshold intensities of the carmine stain specific to the female ovary displayed in blue. 4) Overt threshold intensities of the carmine specific to the female ovary in blue visualized without the autofluorescence and carmine stain channels. Scale bar, 100 μm.
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Figure 3. RNAi-based knockdown of vasa-like gene 1 led to a reduction in the ovary volume. Panel A. Representative images of ovaries 4 days post-treatment in the indicated groups. Scale bar: 50 m. MO, mature ovary; IO, immature ovary. A single slide from a z-stack capture did not reveal evident gross morphological changes. Therefore, a precise protocol to estimate the ovary volumes was developed. Panel B. Violin plots summarizing the data from two independent experiments (top and bottom). Volume of ovaries are displayed for the indicated groups of parasites 8 days post-RNAi treatment. * p 0.0.5. The violin plots are basically box plots, plus the addition of the probability density of the data at different values, i.e. the wider the violin plot in a particular region, the higher the data point density in that region. Panel C. Frequency plots showing volumes of dsRNA-treated S. mansoni females harvested at day eight. Female schistosomes electroporated without dsRNA (8d mock, no treatment control), or with 15 μg of control luciferase gene-specific dsRNA (8d dsLuc, irrelevant dsRNA control), or Smvlg1specific dsRNA (8d dsSmvlg1) are indicated in the inset. Ovary volume measurements (μm3) from each of the indicated group were represented in Log10 and graphed by percent frequency of the Log10 values. Data from two independent experiments are displayed in the left and right plots.
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Figure 4. Representative rendering from laser scanning confocal images of the anterior lobe containing immature oogonia (IO) in adult Schistosoma mansoni female worms. High power (100x/1.46 objective) images captured of S. mansoni females with 488 nm laser line. Two channels were extracted after applying a linear spectral unmixing algorithm to the lambda stack confocal images (see Methods). Panel A: Control females four days after electroporation without dsRNA (4d mock). Panel B: Control females four days after electroporation with luciferase-targeted dsRNA (4d dsLuc) Panel C: Females four days after electroporation with Smvlg1 dsRNA (4d dsSmvlg1). Cells at rest (i.e., visible nucleolus, no mitotic figures), and actively dividing cells (i.e., visible mitotic figures) are indicated with light blue or yellow arrows, respectively. Scale bars, 10 m. Panel D: Box plots indicating the percentage of dividing cells in the mock control, dsRNA luciferase treatment (dsLuc) or dsRNA Smvlg-1 treatment (dsSmvlg1). The mean of dividing cells differed significantly between groups (One-way ANOVA, F=5.9544, df=2, p = 0.007).
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PII:
S0166-6851(19)30149-5
DOI:
https://doi.org/10.1016/j.molbiopara.2020.111259
Reference:
MOLBIO 111259
To appear in:
Molecular & Biochemical Parasitology
Received Date:
30 September 2019
Revised Date:
23 December 2019
Accepted Date:
13 January 2020
Please cite this article as: Skinner DE, Popratiloff A, Alrefaei YN, Mann VH, Rinaldi G, Brindley PJ, Functional analysis of vasa/PL10-like genes in the ovary of Schistosoma mansoni, Molecular and amp; Biochemical Parasitology (2020), doi: https://doi.org/10.1016/j.molbiopara.2020.111259
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
Functional analysis of vasa/PL10-like genes in the ovary of Schistosoma mansoni
Danielle E. Skinner1, 2, Anastas Popratiloff3, Yousef N. Alrefaei1, 4, Victoria H. Mann1, Gabriel Rinaldi1, 5, *, #, Paul J. Brindley1,*, #
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1. Department of Microbiology, Immunology & Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, The George Washington University, Washington, DC 20037 USA.
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2. Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego 9500 Gilman Dr, La Jolla, CA, 92093, USA.
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3. Center for Microscopy and Image Analysis, The George Washington University, Washington, D.C., 20037, USA.
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4. Department of Medical Laboratory Technology, The Public Authority of Applied Education and Training, Shuwaikh, Kuwait.
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5. Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, UK.
These authors contributed equally.
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*Correspondence:
Gabriel Rinaldi, Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK; email, [email protected]
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Paul J. Brindley, Department of Microbiology, Immunology & Tropical Medicine, School of Medicine & Health Sciences, The George Washington University, 2300 I Street NW, Washington, DC 20037 USA; email, [email protected]
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Graphical abstrct
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A novel confocal-microscopy approach for ovary volume estimation and autofluorescence control was employed to evaluate the effect of knocking down vasa-like genes in Schistosoma mansoni.
Highlights A novel confocal microscopy-based approach to precisely estimate the volume of the female Schistosoma mansoni ovary while controlling for tissue autofluorescence was developed Gene silencing of S. mansoni vasa-like gene 1 was associated with a reduction in the ovary volume in treated female worms, but not with egg number or morphology alterations A lower number of dividing cells in immature ovary of the RNAi-treated parasites compared to controls suggested a key role of S. mansoni vasa-like gene 1 in germline homeostasis
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Abstract The RNA helicase Vasa plays a pivotal role in the development of the germ line. To decipher the functional roles of vasa/PL10-like genes in the human blood fluke Schistosoma mansoni, we performed RNA interference followed by the analysis of the ovary in the adult female. Doubled stranded RNA targeting the schistosome vasa-like gene Smvlg1 reduced the volume of the ovary. Changes in morphology of the ovary were analysed using carmine red-staining of the parasites followed by a novel confocal laser scanning microscopy (CLSM)-based approach to control for natural autofluorescence in female schistosome tissues. The reduction in the ovary volume may have been promoted by the loss of germ cells. By contrast, significant differences were not apparent in the number of eggs produced or hatching rate of eggs laid by the female schistosomes transfected with Smvlg-specific dsRNA. The findings suggested a role for S. mansoni vasa/PL10-like gene -1 in germ cell development within the schistosome ovary that might impact in the pathogenesis and disease transmission by this neglected tropical disease pathogen.
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Keywords. Schistosoma mansoni; vasa/PL10-like genes; Functional Genomic tools; RNAi; germline; confocal microscopy
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1. Introduction
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The catalogue of species of flatworms for which there are draft sequences of the entire genome is expanding now at pace: four tapeworm genomes were reported – the human hydatid pathogens Echinococcus multilocularis and E. granulosus, the causative agent of neurocysticercosis Taenia solium, and model tapeworm of rodents, Hymenolepis microstoma [1]. In addition, since 2009 draft genomes for the main blood fluke species are available which have been regularly curated for the last decade. This information follows on the reports of the genome sequences of the three major schistosome species of humans, Schistosoma haematobium, S. japonicum and S. mansoni [2-7]. More recently, 81 genomes of parasitic and non-parasitic worms have been broadly compared, identifying key gene families involved in immune responses, parasite migration and infection establishment [8]. Several genes revealed in these studies need to be functionally addressed employing genetic approaches under ongoing development. Noteworthy advances in functional genomic tools and transgenesis for schistosomes have been made, including CRISPRCas-based genome editing in S. mansoni and the liver fluke Opisthorchis viverrini [9, 10]. However, progress still lags behind model species such as the fruit fly, Caenorhabditis elegans, zebrafish, and the laboratory mouse.
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It is usually informative to utilize data on gene expression and genomics from related taxa to identify orthologues/ paralogues within the species of interest. For schistosomes, in addition to the higher metazoans listed above, these informative taxa include planarians (which are freeliving, ‘capable-of-regeneration’, turbellarians) such as Schmidtea mediterranea, Macrostomum lignano, and Dugesia japonica. The analyses revealed that the schistosome vasa-like genes Smvlg1, Smvlg2, and Smvlg3, and several other vasa-like genes of platyhelminths likely are not bona fide orthologues of vasa, or orthologues of PL10, a DEAD-box helicase closely related to Vasa [11]. Moreover, close scrutiny of four cyclophyllidean tapeworm genomes failed to locate an orthologue of vasa [1], which supports the hypothesis that vasa was lost during the evolutionary history of the Trematoda and Cestoda [12]. In addition, parasitic flatworms do not possess other post transcriptional regulators such as Piwi and Group 4 tudor proteins that, like Vasa, are associated with germline development and maintenance of somatic stem cells [1, 13]. These findings indicate that parasitic flatworms may have evolved an independent and/ or modified mechanism for germline and somatic stem cell development and maintenance [1, 1215].
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Functional roles of Vasa helicases are poorly understood in species of the Lophotrochozoa and little is known about the function of vasa in flatworms. Nonetheless, several functional analyses have focused on vasa-like genes in platyhelminths, including the monogenean Neobenedenia girellae, the free-living planarian Dugesia japonica [16, 17], and the larval and adult developmental stages of S. mansoni [14] and S. japonicum [18], respectively. RNA interference (RNAi) studies with N. girellae vasa-like genes (Ngvlgs) revealed that the knockdown of Ngvlg1 and Ngvlg2 (related to the Smvlg1 and Smvlg2, respectively) leads to a partial and complete loss of germ cells in the ovary and testis [16]. In addition, the hatching rates of eggs laid by the dsRNA-treated worms were decreased compared to controls [16]. These findings indicate that Ngvlg1 and Ngvlg2 play critical roles in the formation of germ cells and fertilization of the eggs and/or development of the eggs [16]. Functional studies with D. japonica vasa-like genes (DjvlgA and DjvlgB) were performed by exposure of planarians to X-irradiation; the expression of these genes was investigated as well as the regenerative capacity of the flatworm [17]. DjvlgA 4
and DjvlgB are expressed in the germ cells and DjvlgA appears to play a role in the totipotency of the neoblasts [17]. In N. girellae and D. japonica, vasa-like genes appear to play a role in the differentiation and maintenance of germ cells and in D. japonica to play a role in the differentiation and maintenance of the neoblasts. A vasa-like gene from S. mansoni sporocysts was silenced using Smvlg3-specific siRNAs, revealing a critical role in germline proliferation and maintenance [14]. In S. japonicum, the vasa homologue SjVasa3, (91% amino acid identity to Smvlg3) plays a key role in development of the reproductive tract [18].
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Key details provided by these studies notwithstanding, the vasa-like genes of platyhelminths remain poorly understood. Therefore, here we investigated functional aspects of S. mansoni vasa-like genes that we have previously reported [11] by RNAi following approaches already reported [19-21]. In recent years there have been advances in the development of molecular tools for schistosomes and RNAi has become the benchmark approach for functional gene analyses (reviewed in [21, 22]). Herein, the functional analysis was undertaken by the delivery of dsRNAs against the three vasa-like genes in S. mansoni by electroporation to adult females, followed by analysis of the morphology and volume of the ovary. Although, significant knock down was not apparent for Smvlg-2 and -3, the findings indicated that Smvlg-1 gene might be involved in the development and maintenance of the reproductive system of the female schistosome. 2. Materials and Methods
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2.1 Schistosomes
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Mice infected with the NMRI (Puerto Rican) strain of S. mansoni were supplied by the Biomedical Research Institute (BRI) Rockville, Maryland. Maintenance of the mice infected with S. mansoni at GWU was approved by the GWU Institutional Animal Care and Use Committee of the IACUC of The George Washington University. All procedures employed were consistent with the Guide for the Care and Use of Laboratory Animals. Adult worms were recovered after portal perfusion of mice at seven weeks after infection [23]. Thereafter, the adult worms were transferred to a 50 ml conical tube and washed several times with 37C 1phosphate-buffered saline, pH 7.4 (1PBS) supplemented with 2% penicillin, streptomycin, fungizone (2% PSF) by gravity wash. The adult worms were transferred to schistosomule wash medium (DMEM supplemented with 10 mM HEPES and 2% PSF) and cultured at 37C, under 5% CO2 until treatment with double stranded RNA.
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2.2 Synthesis of dsRNA
Double stranded (ds) RNA template was prepared by PCR with the pCR4.0 TOPO TA vector containing the S. mansoni vasa-like gene 1 (Smvlg1) cDNA (JQ619869, Smp_033710) as template, and Smvlg1 specific primers tailed with the T7 promoter sequence at the 5’ termini. The primer sequences were (T7 promoter sequence italicized): dsSmvlg1 forward primer, 5′TAA TAC GAC TCA CTA TAG GGC AAA CGG TTC AGA TGG TGG TGG TGC C-3′ and dsSmvlg1 reverse primer, 5′- TAA TAC GAC TCA CTA TAG GGG CGC TTA TAG AAT CAC CAG GAC CTT GC -3′, spanning coding DNA positions 62-736. The irrelevant control firefly luciferase dsRNA (dsLuc) template was generated using the pGL3-basic plasmid (Promega, Madison, WI; GenBank accession U47295.2) and primers as follows: dsLuc forward primer, 5’5
TAA TAC GAC TCA CTA TAG GGG TGC CAG AGT CCT TCG ATA G-3’ and dsLuc reverse primer, 5’-TAA TAC GAC TCA CTA TAG GG CAA CTT TAC CGA CCG CGC C-3’, spanning coding DNA positions 563-1097.
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The dsRNAs were synthesized and purified using the MEGAscript RNAi kit according to the manufacturer’s instructions (Ambion, Austin, TX). The dsRNAs were precipitated by adding one volume of 5 M ammonium acetate, 2.5 volumes of 95% ethanol, and stored at -20C for 24 h. Thereafter, precipitated dsRNAs were recovered by centrifugation at 4C, 15,000 g for 30 min. The pellets washed with -20C 70% EtOH and then pelleted by centrifugation at 4, 14,500 rpm for 30 min. The dsRNAs were resuspended in schistosomule wash medium (DMEM supplemented with 10 mM HEPES and 2% PSF - 10,000 units/ml of penicillin, 10,000 µg/ml of streptomycin, and 25 µg/ml of Amphotericin B or Fungicide). The integrity of the dsRNAs was verified by non-denaturing 1% agarose gel electrophoresis, and concentration and purity determined with a spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington DE). dsRNAs were also synthesized for Smvlg2 and Smvlg3 (Supplementary Methods). 2.3 Delivery of dsRNA
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Immediately after the portal perfusion (above), adult female and male schistosomes were separated, and the females dispensed into 4 mm gap cuvettes (BTX, San Diego, CA), with 20 females per cuvette with 200 l schistosomule wash medium, three cuvettes total. The cuvettes were incubated for 10 min at room temperature (RT) with 15 g dsSmvlg1 or 15 g dsLuc, the latter serving as irrelevant dsRNA control. The third cuvette did not receive dsRNA was included as a mock-treatment control. Thereafter, the cuvettes were subjected to square wave electroporation, one pulse of 125 V, 20 ms in duration, using the ElectroSquarePorator model ECM830 (BTX, San Diego, CA). Immediately after, one ml of pre-warmed (37C) modified Basch’s medium was added to the cuvettes [23, 24]. The female worms were transferred to 6well tissue culture plates, the volume of Basch’s medium increased to 5 ml, and the plate incubated at 37C under 5% CO2. Forty eight hours later the Basch’s medium was changed. At 4 days in culture, the Basch’s medium was replaced and fresh 15 g of dsRNAs was added to the wells. The female worms were harvested at day 8 for subsequent RNA and phenotype analyses (below). The experiment was performed in duplicate. In addition, the RNAi experiment was performed in triplicate with five treatment groups (dsSmvlg1, dsSmvlg2, dsSmvlg3, dsLuc, and mock-treatment control) and harvested four days later (Supplementary Methods). The number of eggs laid by females across the experimental groups and controls, and hatching rate also were recorded. 2.4 Gene expression analysis The adult females were harvested 4 or 8 days after the transfection with dsRNA. Total RNA was extracted from a subpopulation (five female worms) of each treatment group using the RNAqueous®-4PCR kit (Ambion, Austin TX). Residual DNA contaminating the RNA was removed by digestion with RNase-free DNase I (TurboDNase, Ambion) at 37°C for 60 min. cDNAs were synthesized using the iScript™ cDNA synthesis kit (Bio-Rad, Hercules CA) using 150 ng of total RNA. Relative expression of genes of interest was analyzed by quantitative RTqPCR. TaqMan probes and qPCR primers were designed with the assistance of Beacon 6
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Designer (Premier Biosoft International, Palo Alto, CA): Smvlg1 forward primer, 5′-ACG ACT ATA ATG AGA ATA ATC TTG-3′; Smvlg1 reverse primer, 5′-CCA AAC TTT ATG TGC CTC-3′; Smvlg1 probe, 5′-/56-FAM/GTT CAG ATG GTG GTG GT/3IABlk_FQ/-3′. TaqMan probe and primers targeting the S. mansoni reference gene, glyceraldehyde 3-phosphate dehydrogenase (SmGAPDH) (GenBank accession M92359, Smp_056970), were: SmGAPDH forward primer, 5’-TGT GAA AGA GAT CCA GCA AAC-3’; SmGAPDH reverse primer, 5’GAT ATT ACC TGA GCT TTA TCA ATG G-3’; and SmGAPDH probe, 5’-/56-FAM/AAG ACT CCA GTA GAC TCA ACG ACA T/3IABlk_FQ/-3’. Quantitative PCRs were performed in triplicates, and reactions were carried out in volumes of 20 µl with primer-probe sets and Perfecta qPCR FastMix, UNG (Quanta Bioscience, Gaithersburg, MD). The qPCR conditions included an initial denaturation at 95°C for 3 min followed by 40 cycles of 30 s at 95°C and 30 s at 55°C, performed in a thermal cycler (iCycler, Bio-Rad) and a Bio-Rad iQ5 detector to scan the plate in real time. Relative quantification was calculated following the 2−ΔΔC method [25]. SmGapdh was employed as the reference and the mock-treatment control as calibrator sample. T
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2.5 Carmine staining of adult female schistosomes
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The adult females were fixed for at least 24 h with AFA (85% EtOH, 37% formalin commercial grade, glacial acetic acid, 85:10:5) at -20C. Excess fixative was removed by washing worms with 70% EtOH for 5 min at RT. The adult females were stained with hydrochloric carmine for at least 30 min at RT [26, 27]. Hydrochloric carmine was prepared by mixing 1 g of carmine (alum lake carminic acid, Acros Organic, 1390-65-4) with 500 l concentrated HCl (>12 M), 500 l H2O, volume brought up to 20 ml with 90% ethanol. The mixture was heated to ~80C for about 20-30 minutes until the carmine dissolved. The solution was cooled to ambient temperature, filtered, and stored in the dark at 4C. The excess, was differentiated in acidic 70% EtOH (1 ml HCl, 1M per 10 ml of 70% EtOH) for 1 to 2 min at RT. Thereafter, worms were dehydrated through a graded ethanol series at RT: 70%, 90%, and 100% each performed once for 5 min at RT, then cleared in a xylene series: 50:50; xylene:100% ethanol for one wash for 5 min, and 100% xylene for three washes for 5 min each at RT. The female worms were mounted on glass slides with Permount (Fisher Scientific, USA) before confocal microscopy. 2.6 Confocal laser scanning microscopy
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Confocal laser scanning microscopy (CLSM) images were captured on a Carl Zeiss LSM 710 confocal system. Confocal images were captured with a Plan-Apochromat 20x/0.8 objective lens, deployment of which was considered to be suitable for imaging the entire ovary of schistosomes. Confocal images were also captured with an Alpha Plan Apochromat 100x/1.46 objective lens with oil immersion, deployment of which was prudent for imaging cells within the immature ovary. The female worms were excited with 488 nm argon laser and 561 nm diode-pumped laser. Images were acquired using the linear spectral unmixing approach. The rationale for this approach over the conventional bandwidth emission filtering was that schistosomes exhibit substantial auto-fluorescence that interfered with the measurement of the carmine-specific fluorescence. The key difference was that instead of capturing the emission in a relatively large range of the spectrum, i.e., 560-620 nm, which contains convolved auto-fluorescence and carmine fluorescence, the instrument utilizes a 32-channel spectral photomultiplier that is capable of capturing simultaneously spectral data sets in bands of 9.6 nm. This was possible 7
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2.7 Quantification of mitotic cells in the immature ovary
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since the emission deriving from the sample is spectrally separated by a diffraction grating and sent to the 32-channel photomultiplier. Since the 32-channels are aligned along the visible spectrum, they can capture simultaneously a set of 32 images that are known as lambda stacks or spectral image sets. Further, analyses of the emission was done from these image sets pixel by pixel, revealing specific emission curves present in the sample. Linear spectral unmixing, utilized herein refers to application of spectral disconsolation algorithm that relies on the linear additive properties of the fluorescence signal. Therefore, if there are two emission curves present in the sample with at least 10% difference, linear spectral unmixing could be used to generate two intensity modulated channels representing the areas of the image expressing the two emissions. The final product was a two-channel image that resembled a standard confocal micrograph, but the way it was generated was fundamentally different as explained. Confocal zstacks were collected from the top to the bottom of the worms at the levels of the ovaries, ensuring that the entire organ is within the 3D image set. Z-steps were optimized according to the software optimal calculations. The resulting images, encoded two channels — autofluorescence and carmine stain signal. Unmixed, confocal stacks were imported to Volocity (v.6.2.1, Perkin Elmer/Improvision) for three-dimensional rendering and analysis of ovary volume.
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In order to quantify the percentage of cells undergoing mitosis in the immature ovary of Smvlg1-silenced parasites or controls, cell numbers in each of 31 high power (100x/1.46 objective) images were estimated, in a blinded-to-operator fashion, counting an average of 45 cells per image with the assistance of Image J (version 1.52p). Cells displaying clear metaphasic, anaphasic, or telophasic chromosomes with no obvious nuclear membrane were counted as ‘dividing cells’. By contrast, cells where the nuclear membrane appeared to be intact, with or without an evident nucleolus, were considered to be non-dividing. The raw data collected in Microsoft Excel (version 16.16.16) were exported to RStudio (Version 1.2.5001) to generate the boxplots of percentage of dividing cells to total cells, and for statistical analysis. 2.8 Statistical analysis
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Levels of statistical significance among and between the experimental groups were determined using Analysis of Variance (ANOVA) and Student’s t-test for pairwise comparisons. P-values of ≤ 0.05 were considered to be statistically significant. Plots were generated with the assistance of GraphPad (Prism 8, version 8.2.1) (San Diego, CA) and RStudio (Version 1.2.5001) (Boston, MA). 3. Results
3.1 Significant knockdown of S. mansoni vasa-like-gene 1 Smvlg1-specific dsRNA was synthesized spanning the amino-terminus region of the transcript avoiding the highly conserved core motifs characteristic of the putative DEAD-box RNA helicases, of which S. mansoni encodes 35 based on a search on v7 genome using Biomart with Pfam ID PF00270 and InterPro ID IPR011545 criteria in Worm Base Parasite, version 14 8
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(August 2019) (Supplementary Tables S1 and S2) (https://parasite.wormbase.org/index.html) [1, 11]. Firefly luciferase-specific dsRNA was included as irrelevant-dsRNA control, and female worms electroporated under the same conditions but with no dsRNA molecules served as mocktreatment control. To investigate the RNAi effects at the molecular level, transcript levels of Smvlg1 were analyzed in the three independent treatment groups by qRT-PCR for each experiment. Eight days after treatment, double stranded RNA interference (dsRNA)-mediated post-transcriptional gene silencing of Smvlg1 led to a robust reduction (68%) in transcript levels compared to the mock-treatment control and the control worms treated with dsRNA for luciferase (Figure 1). Transcript levels of Smvlg1, Smvlg2, and Smvlg3 were also analyzed in three independent experiments in which females were harvested four days after treatment with dsSmvlg1, dsSmvlg2, dsSmvlg3, dsLuc, or without dsRNAs. The dsSmvlg1 treatment group was the only one of the three dsSmvlg treatment groups that delivered a consistent and significant knockdown of its target in at least three independent replicates (Supplementary Figure S1). Even though gene silencing was attempted in male adult worms targeting these genes, knock down was not apparent or unsuccessful for all three vasa-like genes. In addition, we had previously identified and characterized these genes in the female schistosome [11]. Therefore, we focused here on the vasa-like genes in the female worm.
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3.2 A novel approach to control for natural tissue autofluorescence in the female schistosome
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RNAi-treated female schistosomes were stained with carmine red-stain and imaged by confocal laser scanning microscopy (CLSM). The ovary of the female worms was imaged by spectral confocal microscopy with a 488 nm laser line and the volume estimated as described above. In brief, discrete sites on the worms were selected representing the autofluorescence and the fluorescence of the carmine stain and served as emission references for linear unmixing [28]. Autofluorescence registered on the lambda stack displayed a broad spectrum with a peak at ~600 nm, and the carmine signal peaked at ~635 nm. The two-channel confocal stacks, derived after linear unmixing, comprised channels representing the autofluorescence and carmine signal (Figure 2A). 3.3 Silencing to S. mansoni vasa-like-gene 1 decreases the ovarian volume
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To specifically investigate the effects of the vasa-like gene silencing on the ovary volumes a thorough quantitative analysis was performed. The confocal 3D image sets were rendered in three-dimensions by importing into the Volocity 3D imaging software. Volocity was used to establish a measurement protocol to locate the significant intensities of the carmine signals specific to the ovary and render the volume measurements in µm3 (Figure 2B). This approach used intensity threshold of the carmine channel to create a specific volume of the ovary and output its volume. No discernable gross morphological abnormalities were evident within the ovaries of dsRNA-treated or control parasites (Figure 3A). However, in two independent experiments harvested at 8 days, significantly smaller ovary volumes were observed in females treated with dsSmvlg1 compared to the ovary volumes of the mock-treatment control (Figure 3B). Overall, a trend was apparent where reduced ovary volumes were more likely in the females transfected with dsSmvlg1 than in the controls (Figure 3C, Supplementary Tables S3 and S4). A similar trend was observed in the three independent experiments harvested at 4 days 9
(Supplementary Figure S2). Remarkably, no significant differences in number of laid eggs or hatching rate were seen among the experimental and control groups (not shown). 3.4 Smvlg1 may be involved in germinal cell proliferation
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To analyze the underlying mechanism for the volume regression of ovaries in the dsSmvlg1treated females, cellular proliferation was investigated. In the anterior region of the schistosome ovary, i.e. immature ovary, oogonia undergo mitosis and meiosis leading to small poorly differentiated immature oocytes, whereas the posterior region of the ovary, i.e., mature ovary, consists of large well-differentiated mature primary oocytes [29-33]. CLSM x100 magnification images were captured of the immature region of the ovaries from the carmine red-stained wholemounts of the S. mansoni females. Images were taken of ovaries with the largest and the smallest volumes from both the controls and the dsSmvlg1-treated females. In 100 images, cells at rest (i.e., visible nucleolus, no mitotic figures) can be distinguished from actively dividing cells (i.e., visible mitotic figures) (Figure 4A-C). Accordingly, the percentage of dividing cells was estimated for each of the three groups of parasites. A significant trend towards a lower percentage of dividing cells in the parasites transfected with dsRNA against Smvlg-1 compared to controls was evident (Figure 4D). The latter might suggest a functional role of Smvlg-1 in the control of cell proliferation in the immature lobe of the schistosome ovary.
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4. Discussion
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In the current study RNAi-induced suppression of S. mansoni vasa-like genes (Smvlg) [11] was accomplished by delivering dsRNA into cultured worms by square wave electroporation. The knockdown of the vasa-like gene 1 (Smvlg1) was consistent across four experiments where the females were harvested at four days, 38-60% (mean=54%) knockdown in Smvlg1 transcript levels was observed in each replicate. In the two experiments where the females were harvested at eight days, transcript levels of Smvlg1 were reduced by 59-78% (mean=68%). Given that the levels of knockdown achieved for Smvlg2 and Smvlg3 were not consistent across all the experiments, we decided to focus on the functional role of Smvlg1. Remarkably, the silencing of Smvlg1 was associated with a reduced volume of the ovary in female worms. These changes were detected using a novel approach that employs carmine red-staining of the parasite followed by confocal laser scanning microscopy (CLSM) to disentangle the carmine-specific signal from the native autofluorescence. The reduction in ovary volume after the treatment with Smvlg1specific dsRNA might have followed the loss of germ cells, suggesting that Smvlg1 may be involved in germ cell homeostasis in the schistosome ovary. However, further studies are needed to prove this hypothesis. Unexpectedly, no significant differences were seen in the number or hatching rate of eggs laid by female schistosomes transfected with Smvlg-specific dsRNAs compared to control parasites. When vasa-like genes were silenced in S. japonicum, the authors reported not only evident morphological alterations in both testis and ovaries, but also a reduction in the number of eggs in female uterus and in vitro laid eggs [18]. In the monogenean N. girellae, Ohashi and colleagues also showed that vasa-like genes were expressed in the germline, and when silenced a partial or complete loss of germ cells that led to a reduction in the hatching rate of eggs was evident [16]. The unexpected absence of a reduction in the number of eggs in Smvlg1-silenced parasites might be explained by functional redundancy of the vasa-like genes, in particular Smvlg2 and Smvlg3 that were not silenced in our approach. Similar outcomes 10
were observed in the free-living flatworm Macrostomum lignano where not only a functional redundancy of vasa and vasa-like genes has been described, but also difficulties in achieving substantial silencing for some of the vasa genes, probably due to long half-life of the mRNA and/ or the protein, were discussed [34]. Accordingly, we cannot rule out the presence of similar phenomena in our study. This will be investigated in future studies.
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Analyses by CLSM revealed no discernable morphological abnormalities within the ovaries of the dsRNA-treated schistosomes compared to controls in both the three independent experiments harvested at 4 days and the two independent experiments harvested at 8 days. The schistosome ovaries from the treatment groups harvested at 8 days post-treatment were more regressed reproductively than the schistosome ovaries from the treatment groups harvested at 4 days, which would be expected from female schistosomes separated from their male partners and cultured in vitro for 8 days as previously described [29]. In addition, we cannot rule out that this well-described regression of the reproductive tissue in worms cultured in vitro might have been exacerbated by the electroporation itself. Recently, a novel tissue culture media complemented with ascorbic acid and cholesterol was reported to maintain the reproductive organs and vitellaria of female schistosomes longer in culture [35]. Future experiments employing this tissue culture media would avoid the confounding effect of the ovary regression due to the in vitro culture conditions. Moreover, in vivo RNAi by delivering dsRNA against S. japonicum genes into experimentally-infected mice has been recently shown to be successful to induce a gene silencing of >79.4% in male and >91.5% in female compared to control parasites [36]. This novel approach provides a more natural environment to induce germline gene silencing allowing the detection of more reliable and maybe more reproducible phenotypes, including alterations in the reproductive system and number of eggs trapped in the host liver.
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Many genes, which are specifically expressed in germ cells and somatic stem cells of higher metazoans, have been lost during the evolution of parasitic flatworms, including schistosomes [1, 13-15, 37]. In particular, Vasa and PL10, two closely related proteins in the ATP-dependent DEAD-box helicase superfamily are not present in schistosomes although they occur widely in species from bacteria to humans [38]. Nonetheless, we reported previously that three vasa-like genes (Smvlg1, Smvlg2, and Smvlg3) evolved in S. mansoni that form a parasitic flatwormspecific clade discrete from the PL10 clade [11, 12]. This flatworm-specific clade includes vasalike genes from other flatworms including N. girellae, D. japonica, and S. polychroa [12, 16, 17, 39]. Whereas the phylogenetic analysis revealed that these DEAD-box helicases were not orthologues of either vasa or PL10, transcripts of the vasa-like genes are expressed in the schistosome ovary in a germline-specific manner and are abundantly expressed in intrasnail developmental stages [14]. Moreover, among the schistosome developmental stages, eggs laid in vitro by worms in culture within 12 hours after their collection from the mouse display the highest levels of vasa-like gene expression [11]. Other reports showed that vasa-like genes exhibit germline-specific expression [14, 16, 17, 39]. Future studies on the vasa-like genes and related genes of schistosomes using functional genomics approaches such as genome editing [9, 10] and single-cell transcriptomic [15, 40] are expected to contribute to the understanding of the germ line development, pathogenesis, and disease transmission.
CRediT author statement. 11
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Danielle E. Skinner: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Visualization, Writing – Original Draft, Review and Editing Anastas Popratiloff: Methodology, Validation, Formal analysis, Investigation, Resources, Visualization, Writing – Original Draft Yousef N. Alrefaei: Visualization, Writing – Original Draft, Review and Editing Victoria H. Mann: Supervision, Resources Paul J. Brindley: Conceptualization, Supervision, Resources, Funding acquisition, Writing – Original Draft, Review and Editing Gabriel Rinaldi: Conceptualization, Supervision, Resources, Methodology, Formal analysis, Visualization, Funding acquisition, Writing – Original Draft, Review and Editing Acknowledgements
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We thank Anna Protasio for inisghtful, helpful comments and informative discussion. These studies were supported by NIH-NIAID awards R01AI072773 (PJB) and R21AI109532 (GR), and the Wellcome Trust Strategic Award number 107475/Z/15/Z; the content is solely the responsibility of the authors and does not necessarily represent the official views of the NIAID, the NIH or the Wellcome Trust. Mice infected with S. mansoni were provided by the NIAID Schistosomiasis Resource Center for distribution through BEI Resources, NIH-NIAID Contract HHSN272201000005I. References
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Figure legends Figure 1. Knockdown of vasa-like gene 1 in females of S. mansoni at eight days posttransfection. Panel A. Timeline for RNAi analysis. Groups of 20 female worms, previously separated from male worms, were subjected to electroporation in the absence of dsRNA (8d mock control), in the presence of 15 µg of Luc-specific dsRNA (8d dsLuc; irrelevant dsRNA control), or Smvlg1-specific dsRNA (8d dsSmvlg1). At day 4, an additional 15 µg of indicated dsRNAs were added to the culture medium, and the worms harvested at day 8. Panel B. Relative gene expression levels measured by RT-qPCR; Smvlg1 transcript levels were normalized to those of SmGAPDH. The Smvlg1 transcript level was determined in two independent experiments with the 8d mock, 8d dsLuc, and 8d dsSmvlg1 treatment groups. The bars represent the means ± standard deviations. Student’s t-test: *p ≤ 0.05 and **p ≤ 0.01. Lower table displays the transcript levels normalized to SmGAPDH (100% in the calibrator sample, i.e. mock control) for each treatment group from the two independent experiments. Exp1, experiment 1; Exp2, experiment 2; SD, standard deviation.
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Figure 2. Estimation of the ovary volume and tissue autofluorescence. Panel A. High power (20x/0.8 objective) images of S. mansoni female excited with 488 nm laser line. Two channels were extracted after applying a linear spectral unmixing algorithm to the lambda stack confocal images (see Methods). S. mansoni female visualized with the autofluorescence channel (grey, 1), with the carmine stain signal channel (red, 2), and merged channels (3). Scale bars, 50 μm. Panel B. High power (20x/0.8) images of S. mansoni females excited with 488 nm laser line. Two channels were extracted after applying a linear spectral unmixing algorithm to the lambda stack confocal images (see Methods). Three dimensional (3D) renderings and ovary volume analysis of the stack confocal images were performed with the 3D imaging software Volocity (v6.2.1, PerkinElmer/Improvisation). 1) 3D rendering of S. mansoni female visualized with the 15
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autofluorescence channel (grey). Overt threshold intensities of the carmine stain signals specific to the female ovary displayed in blue. 2) 3D rendering of S. mansoni female visualized with the carmine stain channel (red). 3) Merged images shown in 1 and 2. Overt threshold intensities of the carmine stain specific to the female ovary displayed in blue. 4) Overt threshold intensities of the carmine specific to the female ovary in blue visualized without the autofluorescence and carmine stain channels. Scale bar, 100 μm.
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Figure 3. RNAi-based knockdown of vasa-like gene 1 led to a reduction in the ovary volume. Panel A. Representative images of ovaries 4 days post-treatment in the indicated groups. Scale bar: 50 m. MO, mature ovary; IO, immature ovary. A single slide from a z-stack capture did not reveal evident gross morphological changes. Therefore, a precise protocol to estimate the ovary volumes was developed. Panel B. Violin plots summarizing the data from two independent experiments (top and bottom). Volume of ovaries are displayed for the indicated groups of parasites 8 days post-RNAi treatment. * p 0.0.5. The violin plots are basically box plots, plus the addition of the probability density of the data at different values, i.e. the wider the violin plot in a particular region, the higher the data point density in that region. Panel C. Frequency plots showing volumes of dsRNA-treated S. mansoni females harvested at day eight. Female schistosomes electroporated without dsRNA (8d mock, no treatment control), or with 15 μg of control luciferase gene-specific dsRNA (8d dsLuc, irrelevant dsRNA control), or Smvlg1specific dsRNA (8d dsSmvlg1) are indicated in the inset. Ovary volume measurements (μm3) from each of the indicated group were represented in Log10 and graphed by percent frequency of the Log10 values. Data from two independent experiments are displayed in the left and right plots.
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Figure 4. Representative rendering from laser scanning confocal images of the anterior lobe containing immature oogonia (IO) in adult Schistosoma mansoni female worms. High power (100x/1.46 objective) images captured of S. mansoni females with 488 nm laser line. Two channels were extracted after applying a linear spectral unmixing algorithm to the lambda stack confocal images (see Methods). Panel A: Control females four days after electroporation without dsRNA (4d mock). Panel B: Control females four days after electroporation with luciferase-targeted dsRNA (4d dsLuc) Panel C: Females four days after electroporation with Smvlg1 dsRNA (4d dsSmvlg1). Cells at rest (i.e., visible nucleolus, no mitotic figures), and actively dividing cells (i.e., visible mitotic figures) are indicated with light blue or yellow arrows, respectively. Scale bars, 10 m. Panel D: Box plots indicating the percentage of dividing cells in the mock control, dsRNA luciferase treatment (dsLuc) or dsRNA Smvlg-1 treatment (dsSmvlg1). The mean of dividing cells differed significantly between groups (One-way ANOVA, F=5.9544, df=2, p = 0.007).
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