Figures and data
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 (week 1 to week 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 5 independent culture experiments, i.e. 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.
Parasites cultured in Human Serum grew in size unlike those cultured in Foetal Bovine Serum.
Bar plot representing area measurements of schistosomula developed in vitro and cultured in media complemented either in Foetal Bovine Serum (FBS) or Human Serum (HS) at indicated weeks after cercarial transformation. Bars indicate mean area (µm²) ± SEM. HS: Human Serum (light brown); FBS: Foetal Bovine Serum (blue). *: P-value < 0.05 in indicated pairwise comparisons.
Parasites developed in Human Serum readily digest red blood cells.
A. Bar Plot representing the percentage of Human Serum (HS)- or Foetal Bovine Serum (FBS)-cultured schistosomula with (BG+, light brown) or without (BG-, blue) black guts 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 one or two days later. Error Bars = SEM. B. Representative images of in vitro developed schistosomula cultured in FBS or HS one day after adding hRBC (+ RBC) and controls without RBC (- RBC). Scale bars: 100 µm.
Development of parasites in human serum 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 Human Serum (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 mean of EdU+ cells per worm. Statistical analysis was performed by Kruskal-Wallis test with Dunn multiple comparison post-hoc test, with P≤0.05 (*) considered significant. 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.
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-E, G), cyan (F, 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.
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: grey, CellMask Deep Red Plasma Membrane Stain: magenta. Scale bar: 150 µm (E), 50 µm (F).
