Authors

Discussion

Schistosomes are parasitic flatworms that cause Schistosomiasis, a major Neglected Tropical Disease (NTD) that affects more than 250 million people worldwide. With distinct sexes, a heterogametic female (ZW) and a homogametic male (ZZ), schistosomes are an exception among flatworms, which are largely hermaphroditic. Sexual dimorphism in schistosomes becomes most apparent by adulthood within the mammalian host. The cellular and molecular mechanisms underlying the sexual differentiation of these parasites are poorly understood, partly due to intrinsic challenges in assessing their development in vivo. Therefore, robust and reproducible approaches for maintaining and developing parasites in vitro are needed to overcome these difficulties. However, to date, only a few studies have focused on protocols that allow cultured parasites to reach sexually dimorphic stages, and none have been reproduced, limiting the ability to understand their unique sexual biology. Here, we refine a protocol for long-term culture of newly transformed cercariae that developed into sexually dimorphic forms in vitro. We assessed the effect of two different sera, Foetal Bovine Serum (FBS) and Human Serum (HS), added to the culture medium supplemented with human red blood cells. We found that in contrast to FBS-cultured worms, HS-cultured parasites digested red blood cells, a crucial step for long term parasite development. Additionally, while most FBS-cultured parasites did not progress beyond an early liver stage, sexual dimorphism was clearly established in the HS-cultured worms, albeit delayed compared to in vivo development. Moreover, EdU pulse-chase experiments revealed a continuous proliferation of cells over time in HS-cultured parasites, while a significantly lower number of proliferating cells were detected in FBS-cultured worms. Phenotypic differences between worms in cultured in the two sera were mirrored by significant changes at the single-cell transcriptomic level. Whereas all predicted cell clusters were present in HS-developed parasites, the worms cultured in FBS were strikingly depleted of key stem cell populations required for somatic and germ line development in the mammalian host. By coupling phenotype analysis of cultured parasites with single cell transcriptome profiling, this study represents a proof-of-principle for single cell phenotyping in schistosomes. Moreover, the protocol described in this study paves the way to study parasite development in vitro, positively impacting the principles of the3Rs (Replacement, Reduction and Refinement) for animal research, and allowing for in-depth studies of sexual dimorphism establishment as well as in vitro screening for novel control strategies across the life cycle of these major human parasites.