Single-cell atlas of the first intra-mammalian developmental stage of the human parasite Schistosoma mansoni

Abstract

Over 250 million people suffer from schistosomiasis, a tropical disease caused by parasitic flatworms known as schistosomes. Humans become infected by free-swimming, water-borne larvae, which penetrate the skin. The earliest intra-mammalian stage, called the schistosomulum, undergoes a series of developmental transitions. These changes are critical for the parasite to adapt to its new environment as it navigates through host tissues to reach its niche, where it will grow to reproductive maturity. Unravelling the mechanisms that drive intra-mammalian development requires knowledge of the spatial organisation and transcriptional dynamics of different cell types that comprise the schistomulum body. To fill these important knowledge gaps, we perform single-cell RNA sequencing on two-day old schistosomula of Schistosoma mansoni. We identify likely gene expression profiles for muscle, nervous system, tegument, oesophageal gland, parenchymal/primordial gut cells, and stem cells. In addition, we validate cell markers for all these clusters by in situ hybridisation in schistosomula and adult parasites. Taken together, this study provides a comprehensive cell-type atlas for the early intra-mammalian stage of this devastating metazoan parasite.

Fig. 1. Identification of 13 transcriptionally distinct cell types in schistosomula.

a Experimental scheme describing the sources of the parasite material, single-cell analysis and validation pipeline. Approximately 5000 schistosomula per experiment were dissociated, followed by enrichment of fluorescein diacetate (FDA+) live cells using fluorescence-activated cell sorting (FACS). Cells were loaded according to the 10X Chromium single-cell 3’ protocol. Clustering was carried out to identify distinct populations and population-specific markers. Validation of population-specific markers was performed by in situ hybridisation (ISH). b Uniform Manifold Approximation and Projection (UMAP) representation of 3226 schistosomulum single cells. Cell clusters  are coloured and distinctively labelled. c Gene-expression profiles of population markers identified for each of the cell clusters. The colours represent the level of expression from dark red (high expression) to light red (low expression). The sizes of the circles represent the percentages of cells in those clusters that expressed a specific gene. The colour bars under gene IDs represent the clusters in (b).

Fig. 2. Muscle cells express positional information underlying parasite development.

a Expression profiles of cell markers that are specific or enriched in the muscle clusters. Genes shown in red were validated by ISH. b FISH of Smp_161510. Smp_161510-expressing cells are found in dorsal and ventral sides along the midline. vs: ventral sucker. Single confocal sections shown for each image. c Double FISH of Smp_161510 and a pan-muscle marker troponin (Smp_018250) in the head region of the adult worm. A: anterior; P: posterior. Maximum intensity projection (MIP) is shown. d FISH of wnt-2 (Smp_167140) in 2-day old schistosomula, MIP. e Whole-mount in situ hybridisation (WISH) of wnt in the head region of the adult worm. The whole adult worm image is shown in Supplementary Fig. 4a. A: anterior; P: posterior. f Double FISH of wnt-2 (Smp_167140) and troponin (Smp_018250) in the head region of the adult worm, MIP. g FISH of rhodopsin GPCR (Smp_153210). Left: MIP; right: single magnified confocal sections of the dotted box. h Double FISH of myoD (Smp_167400) and troponin (Smp_018250) in adult soma, MIP. i, j Spatial distribution of actin-2 (Smp_307020) throughout the body of the parasite. i schistosomulum, MIP; j adult male, MIP. k Schematic that summarises the muscle cell types in 2-day old schistosomula. Marker genes identified in the current study are indicated in red. V: ventral; D: dorsal. The numbers of ISH experiments performed for each gene are listed in ‘Methods’ and Supplementary Data 7.

Fig. 3. Two distinct populations of tegumental cells in schistosomula.

a Expression profiles of cell marker genes that are specific to or enriched in the tegument clusters. Genes validated by ISH are marked in red. b annexin B2+ cells have taken up the fluorescent dextran. Yellow arrowheads indicate double-positive cells. Single confocal sections are shown. c Double FISH of Tegument 1 markers annexin B2 (Smp_077720) and Smp_022450. The majority of the cells show co-localisation (white signal). Left: MIP; right: zoomed in confocal sections. vs: ventral sucker; gc: germinal cell cluster. d Double FISH of Tegument 1 markers Smp_022450 and meg3 (Smp_138070). The majority of the cells show co-localisation (white signal). e Double FISH of Tegument 1 marker (Smp_022450) with ccdc74 (Smp_030010), MIP. The majority of cells show co-localisation (white signal), while a subset of cells in the neck region of the worm show single positive cells for Tegument 2 markers. f, g Double FISH of Tegument 2 marker mboat (Smp_169040) and f Tegument 1 marker annexin B2 (Smp_077720) and g oesophageal gland marker meg4 (Smp_307220), single confocal sections. Green arrowhead: mboat+ cells; white arrowhead: double-positive cells. h Schematic that summarises the tegument cell populations in 2-day old schistosomula. Marker genes identified in the current study are indicated in red. All previously reported genes are shown in black. The numbers of ISH experiments performed for each gene are listed in ‘Methods’ and Supplementary Data 7.

Fig. 4. Identification of schistosome parenchymal and primordial gut cells.

a Expression profiles of cell marker genes that are specific or enriched in the parenchymal clusters. Genes validated by ISH are marked in red. b Double FISH of parenchymal cathepsin B (Smp_141610) with a known marker of differentiated gut, cathepsin B’ (Smp_103610), MIP. No expression of parenchymal cathepsin B is observed in the primordial gut. gc: germinal cell cluster. c WISH of parenchymal cathepsin B in adult males. d–f FISH of parenchymal cathepsin B in different regions of adult worms: d testes lobes, e gut, and f vitellaria. White arrows indicate positive cells. Single confocal sections shown. g, h lap (Smp_030000) is expressed in both parenchyma and in the g gut primordia as well as h adult gut, shown by double FISH with the gut cathepsin B (Smp_103610). i Schematic that summarises the parenchymal cell populations in 2-day schistosomula. Marker genes identified in the current study are indicated in red. All previously reported genes are shown in black. The numbers of ISH experiments performed for each gene are listed in ‘Methods’ and Supplementary Data 7.

Fig. 5. A single cluster of stem cells in 2-day old schistosomula.

a FISH of h2a (Smp_086860) shows ~5 stem cells located at distinct locations—1 medial cell (M) and 2 lateral cells on each side (1L and 2L, 1R, and 2R; L: left; R: right), MIP. b Expression profiles of cell marker genes that are specific or enriched in the stem/germinal cell cluster. Genes validated by ISH are marked in red. c FISH of cam (Smp_032950) shows a similar localisation pattern as h2a, with some worms with a few more cam+ cells in the medial region as well as in the germinal cell cluster region, MIP. gc: germinal cluster. d Double FISH of cam (Smp_032950) and a previously validated schistosome stem cell marker h2b (Smp_108390), MIP. e WISH of cam (Smp_032950) in adult parasites shows enriched expression in the gonads including testis, ovary, and vitellarium, as well as in the mid-animal body region. f Double FISH of cam and h2b in adult soma. A single confocal section is shown. White arrows indicate co-localisation of two genes and magenta arrowheads indicate cells expressing only cam. g Schematic that summarises the stem and germinal cell populations in 2-day old schistosomula. Marker genes identified in the current study are indicated in red. All previously reported genes are shown in black. Genes that are enriched in this cluster but have not been directly shown by ISH are shown in grey. The numbers of ISH experiments performed for each gene are listed in ‘Methods’ and Supplementary Data 7.

Fig. 6. Heterogeneity in cells of schistosomula nervous system.

a Expression profiles of cell marker genes that are specific or enriched in the neuronal clusters. Genes validated by ISH are marked in red. b Cephalic ganglia marked by sWGA lectin shows co-localisation with 7b2 (Smp_073270)(white arrow), MIP. c WISH of 7b2 (Smp_073270) in adult male. d, e FISH of 7b2 (Smp_073270) in d cephalic ganglia and e body region of adult worms, single confocal sections. f Double FISH of 7b2 (Smp_073270) and Smp_203580 shows that six cells that are Smp_203580+ do not co-localise with 7b2+ cells. g Double FISH of Smp_203580 with Sm-kk7 (Smp_194830). All Smp_203580+ cells co-localise with Sm-kk7, MIP. h gnai (Smp_246100) FISH shows expression in a few cells along the anterior-posterior axis of the somule, MIP. i Double FISH of gnai and 7b2 shows some co-localisation in the nerve tracts in an adult male. Single confocal sections are shown. j Schematic that summarises the neuronal cell populations in two-day schistosomula. Marker genes identified in the current study are indicated in red. All previously reported genes are shown in black. The numbers of ISH experiments performed for each gene are listed in Methods and Supplementary Data 7.

Fig. 7. Gene-expression patterns of stem cells and neurons conserved between S. mansoni and Schmidtea mediterranea.

Random forest classifier used to assign cells from schistosomulum clusters into categories based on a Schmidtea mediterranea scRNAseq dataset. The colours and size of the circles represent the proportion of cells from each cluster (y-axis) that matches each S. mediterranea category label (x-axis). Only categories that received a maximum vote by a margin of >16% of trees during the prediction are included. Cells that did not fit any classification were classed as ‘not assigned’.