In vitro sexual dimorphism establishment in schistosomes
- Department of Biology, University of Oxford, United Kingdom
- Department of Life Sciences, Aberystwyth University, United Kingdom
- Wellcome Sanger Institute, United Kingdom
- School of Infection and Immunity, College of Medical, Veterinary & Life Sciences, University of Glasgow, United Kingdom
Figures
Figure 1 with 3 supplements
Dimorphic female and male schistosomes entirely developed in vitro from cercariae.
(A) Schematic representation of the collection and mechanical transformation of cercariae into schistosomula for long-term in vitro culture. (B) Morphological scoring of cultured S. mansoni schistosomula at the indicated time points after in vitro transformation (weeks 1–10) for worms in long-term culture (LTC) medium supplemented either with human serum (HS - left) or foetal bovine serum (FBS - right). Heatmap columns represent five distinct morphological categories, and rows indicate five independent culture experiments, that is parasites obtained from different batches of infected snails. Heatmap colours represent the percentage of worms for each replicate in each morphological category. Middle panel: representative images of in vitro schistosomula cultured in either HS or FBS as indicated. Scale bars: 100 µm. Category 1, early schistosomula; category 2, lung schistosomula; category 3, early liver schistosomula; category 4, late liver schistosomula; category 5, dimorphic schistosomula. A detailed description of the developmental categories and representative images are provided in Figure 1—figure supplement 1.
Figure 1—figure supplement 1
Developmental stage scoring.
Summary of morphological attributes for the five different categories of development of worms cultivated in vitro (HS), as well as representative bright field images of each of these morphological categories. Arrows in categories 2 and 4 indicate elongated lung schistosomula, and the fusion of the two caeca posterior to the ventral sucker in the late liver schistosomula, respectively. Scale bar: 100 µm.
Figure 1—figure supplement 2
Representative FBS-cultured parasites by week 10 in culture; most of the worms are dead (red arrows) and the few still alive are lung schistosomula (green arrows).
Scale bar: 100 µm.
Figure 1—figure supplement 3
Dimoprhic worms developed in vitro.
(A) Representative pictures of in vitro developed parasites by day ~80 in culture, with male or female worms as indicated. Scale bar: 500 µm (B) Representative results of the PCR-based sex genotyping using primers to amplify a fragment of the control actin gene in both male and female (upper band), and primers to amplify a W-specific region only in female worms (lower band). A non-template control, and male and female positive controls were included as indicated. Assigned sexes for each of the groups of clonal cercariae emitted by individual snails infected with single miracidia.
Figure 2
Parasites cultured in human serum (HS) grew in size unlike those cultured in foetal bovine serum (FBS).
Bar plot representing area measurements of schistosomula developed in vitro and cultured in media complemented either in FBS or HS at indicated weeks after cercarial transformation. Bars indicate mean area (µm²) ± SEM. HS (light brown); FBS (blue). *: p-value <0.01 in indicated pairwise comparisons (Supplementary file 1F).
Figure 3
Parasites developed in human serum (HS) readily digest RBCs.
(A) Bar Plot representing the percentage of HS- or foetal bovine serum (FBS)-cultured schistosomula with (BG+, light brown bar) or without (BG-, blue bar) black guts (BG) due to the presence of intestinal hemozoin. Washed human red blood cells (hRBCs) were added into the media at day 13 post-transformation and images captured 1 or 2 days later. Error bars = SEM. (Statistical analyses in Supplementary file 1F). (B) Representative images of in vitro developed schistosomula cultured in FBS or HS 1 day after adding hRBC (+RBC) and controls without RBC (- RBC). Scale bars: 100 µm.
Figure 4 with 1 supplement
Development of parasites in human serum (HS) may be driven by stem cell proliferation.
(A) Violin plots showing the number of EdU + cells per worm at indicated time points (2, 8, and 15 days post-cercarial transformation) in parasites cultured either in foetal bovine serum (FBS, blue) or HS (light brown). Human red blood cells (hRBCs) were added in the culture at day 13 post-cercarial transformation. The small black dots indicate individual worms, and the big black point indicates the median of EdU + cells per worm. All worms showing ⪰ 60 EdU+ cells were counted and clustered together in the group named ‘60 EdU + cells’. Hence, the data were treated as ordinal, and statistical analysis performed by Kruskal–Wallis test with Dunn multiple comparison post-hoc test, with p≤0.05 (*) considered significant (Supplementary file 1E and F). (B) Representative images of parasites displaying Edu + cells at each indicated time point and culture condition. Edu + cells and nuclei were labelled with Alexa fluor 488 (green) and DAPI (white/grey), respectively. Scale bars: 50 µm or 75 µm as indicated.
Figure 4—figure supplement 1
Representative confocal high-magnification images of individual parasites displaying Edu + cells at each indicated time point and culture condition.
Edu + cells and nuclei were labelled with Alexa fluor 488 (green) and DAPI (white/grey), respectively. Scale bars: 50 µm.
Figure 5 with 1 supplement
In vitro cultured schistosomes display sexual dimorphism and developing reproductive systems.
Representative confocal microscopy images of in vitro developed male (A–D) and female (E–H) at day 60 in culture. Worms shown in panels G and H are different individuals. CellMask Green Actin Tracking Stain: green, DAPI: grey (A–H) . C, cirrus; T, testis; OS, oral sucker; VS, ventral sucker; GC, gynaecophoric canal; G, gut; GP, genital pore; U, uterus; O, ovary; Oo, ootype; OV, oviduc; S, sperm. Scale bar: 50 µm.
Figure 5—figure supplement 1
Representative bright-light pictures parasites cultured in HS supplemented medium for more than 100 days, indicating male and female worms.
(A-F) Parasites cultured for 103 days. (G, H) Parasites cultured for 145 days. Scale bar: 100 µm.
Figure 6
In vitro cultured schistosomes are capable of pairing.
(A, B) Representative bright-field images of pairs of schistosomes developed entirely in vitro after 80 (A) and 150 (B) days of culture in culture medium supplemented with HS. Scale bars: 500 µm (A), 100 µm (B). (C, D) Representative bright-field images of a worm pairs between ex vivo-collected males and in vitro-developed females (C) and ex vivo-collected females and in vitro-developed males (D) within 24 hours after placing the worms in the same well to facilitate pairing. Scale bars: 100 µm, (E, F) Confocal microscopy image of a schistosome pair (ex vivo-collected male and in vitro-developed female) in copula (E), and magnification of the ovarian area, highlighting maturing oocytes (F). CellMask Green Actin Tracking Stain: green, DAPI: cyan , CellMask Deep Red Plasma Membrane Stain: magenta. Scale bar: 150 µm (E), 50 µm (F).
Author response image 1
Videos
Video 1
Representative Z-stack of EdU + cells after 2 days of in vitro culture with human serum.
EdU + cells and nuclei were labelled with Alexa fluor 488 (green) and DAPI (grey), respectively. Scale bars: 50 µm.
Video 2
Representative Z-stack of EdU + cells after 2 days of in vitro culture with foetal bovine serum.
EdU + cells and nuclei were labelled with Alexa fluor 488 (green) and DAPI (grey), respectively. Scale bars: 50 µm.
Video 3
Representative Z-stack of an in vitro developed male worm.
CellMask Green Actin Tracking Stain (green), and DAPI-stained nuclei (cyan). Scale bar: 25 µm.
Video 4
Representative Z-stack of an in vitro developed female worm.
CellMask Green Actin Tracking Stain (green) and DAPI-stained nuclei (cyan). Scale bar: 10 µm.
Video 5
Representative video of in vitro developed females and males in copula at ~80 days in culture.
Video 6
Representative video of in vivo developed male and in vitro developed female in copula.
Video 7
Representative video of in vivo developed female and in vitro developed male in copula.
Video 8
Z-stack of a schistosome pair (in vivo-developed male and in vitro-developed female) in copula.
CellMask Green Actin Tracking Stain: green, DAPI: grey, CellMask Deep Red Plasma Membrane Stain purple. Scale bar: 25 µm.
Video 9
Same Z-stack of a schistosome pair as Video 8 (in vivo-developed male and in vitro-developed female) in copula with higher magnification of the ovarian area, highlighting maturing oocytes.
CellMask Green Actin Tracking Stain: green, DAPI: grey, CellMask Deep Red Plasma Membrane Stain purple. Scale bars: 50 µm.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (S. mansoni) | S. mansoni NMRI strain | Wellcome Sanger Institute (WSI) and Aberystwyth University (AU) | ||
| Biological sample (Biomphalaria glabrata) | Bi. glabrata | Geyer et al., 2017 | ||
| Sequence-based reagent | W1a – forward | Grevelding, 1999 | PCR primers | 5′-CAACACAGTGAAATTCTTCC-3′ |
| Sequence-based reagent | W1b – reverse | Grevelding, 1999 | PCR primers | 5′-GAATTCACCACTCGACATTC-3′ |
| Sequence-based reagent | Actin – forward | Rinaldi et al., 2009 | PCR primers | 5′-CAG TGT TCC CTT CCA TCG TT-3′ |
| Sequence-based reagent | Actin – reverse | Rinaldi et al., 2009 | PCR primers | 5′-GGA CAG GGT GTT CTT CTG GA-3′ |
| Chemical compound, drug | 5-Ethynyl-2'-deoxyuridine (EdU) | Cambridge bioscience | 61135-33-9 | |
| Chemical compound, drug | Azide fluor 488 | Sigma-Aldrich | 760765–1 MG | |
| Chemical compound, drug | Fluoromount-G Mounting Medium, with DAPI | Invitrogen | 00-4959-52 | |
| Chemical compound, drug | CellMaskTM Green Actin Tracking Stain | Invitrogen | 17163269 | |
| Chemical compound, drug | CellMaskTM Deep Red Plasma Membrane Stain | Invitrogen | C10046 | |
| Chemical compound, drug | NucBlue Live ReadyProbes Reagent | Invitrogen | R37606 | |
| Chemical compound, drug | Lactalbumin hydrolysate | Merk Life Sciences | Cat. 61300–500 G | |
| Chemical compound, drug | Hypoxanthine | Merk Life Sciences | Cat. H9636-1G | |
| Chemical compound, drug | Serotonin | Merk Life Sciences | Cat. H9523 | |
| Chemical compound, drug | Hydrocortisone | Merk Life Sciences | Cat. H0888-1G | |
| Chemical compound, drug | Triiodothyronine | Merk Life Sciences | Cat. T6397-100 mg | |
| Chemical compound, drug | MEM vitamins | Merk Life Sciences | Cat. M6895-100 ml | |
| Chemical compound, drug | Schneider’s insect media | Merk Life Sciences | Cat. 50146–500 ml | |
| Chemical compound, drug | Hepes | Merk Life Sciences | Cat. H0887-100 ml | |
| Chemical compound, drug | Heat-inactivated human serum | NHSBT serum | Cat. NC02 | |
| Chemical compound, drug | Heat foetal bovine serum | Thermo Fisher Scientific | Cat. 11550356 | |
| Chemical compound, drug | Antibiotic antimycotic solution | Thermo Fisher Scientific | Cat. 15140–122 | |
| Chemical compound, drug | Insulin solution | Merk Life Sciences | Cat. I9278-5 ml | |
| Chemical compound, drug | L-glutamine | Thermo Fisher Scientific | Cat. 11539876 | |
| Chemical compound, drug | Human red blood cells | NHSBT-NCI | Cat. NC15 | |
| Software, algorithm | CVAT (Computer Vision Annotation Tool) online server | https://www.cvat.ai/ |
Table 1
Long-term culture medium (LTC medium) composition.
| Reagent name | Final concentration | Reagent source |
|---|---|---|
| DMEM, high glucose, sodium pyruvate | Thermo Fisher Scientific (Cat. 13476146) | |
| Lactalbumin hydrolysate | 1 mg/ml | Merk Life Sciences (Cat. 61300–500 G) |
| Hypoxanthine | 500 nM | Merk Life Sciences (Cat. H9636-1G) |
| Serotonin | 1 μM | Merk Life Sciences (Cat. H9523) |
| Hydrocortisone | 1 μM | Merk Life Sciences (Cat. H0888-1G) |
| Triiodothyronine | 200 nM | Merk Life Sciences (Cat. T6397-100MG) |
| MEM vitamins | 1X | Merk Life Sciences (Cat. M6895-100mL) |
| Schneider’s insect media | 10% | Merk Life Sciences (Cat. 50146–500 mL) |
| Hepes | 20 mM | Merk Life Sciences (Cat. H0887-100mL) |
| Heat-inactivated human serum Foetal bovine serum | 20% | HS: NHSBT serum (Cat. NC02) FBS: Fisher Scientific (Cat. 11550356) |
| Antibiotic antimycotic solution | 2X | Thermo Fisher Scientific (Cat. 15140–122) |
| Insulin solution | 8 μg/ml | Merk Life Sciences (Cat. I9278-5mL) |
| L-glutamine | 2 mM | Thermo Fisher Scientific (Cat. 11539876) |
| Human red blood cells | 0.02% v/v | NHSBT-NCI (Cat. NC15) |
Additional files
-
Supplementary file 1
Supplementary tables.
Table A. Raw counts of parasites within each developmental stage category. Each row corresponds to a picture of parasites in culture medium containing FBS or HS. Each column corresponds to the raw parasite counts at indicated stage development (categories 0–5), time in culture (Time in days - D), and experimental condition. Table B. Primers used in PCR for sexing parasites. Table C. Area raw measurements of developing worms. Table D. Percentage of parasites with either black positive (hemozoin) or black negative (no hemozoin) intestine. BG: Black gut. Table E. Raw counting of EdU positive cells per parasite for indicated experimental group, replicate and experiment in long format. The worms were classified by group (column C) and replicate (column D), using the following code: E (‘early’), M (‘medium’) and L (‘late’), corresponding to days 2, 8 and 15, respectively. R and W correspond to conditions with (R) or without (W) human red blood cells, and HS and FBS to culture medium employed. Table F. Summary of all statistical tests employed in this study. 1. Statistical tests of parasite mortality and the raw data table used for this test. 2. Statistical tests for worm size comparisons (correspond to Figure 2). 3. Statistical tests for worm black gut comparisons (correspond to Figure 3). BG: Black gut. 4. Statistical tests for EdU positive cells comparisons (correspond to Figure 4). Replicate code: E, M and L correspond to day 2, 8 and 15 respectively; R and W correspond to the presence (R) or absence (W) of RBCs added 13 days after transformation.
- https://cdn.elifesciences.org/articles/111066/elife-111066-supp1-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/111066/elife-111066-mdarchecklist1-v1.docx
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In vitro sexual dimorphism establishment in schistosomes
eLife 15:RP111066.
https://doi.org/10.7554/eLife.111066.3
