In vitro sexual dimorphism establishment in schistosomes.

Schistosomes are parasitic flatworms that cause Schistosomiasis, a major neglected tropical disease that affects more than 250 million people worldwide. With two 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 only becomes apparent by adulthood within the mammalian host. However, the cellular and molecular mechanisms underlying the sexual differentiation of are poorly understood, partly due to intrinsic challenges in assessing parasite development in vivo. Therefore, robust and reproducible approaches for maintaining and developing parasites in vitro are needed to overcome these difficulties. To date, few studies have focused on protocols that allow cultured parasites to reach sexual dimorphic stages, and none have been reproduced, limiting the ability to understand the sexual biology of this major human parasite. Here, we refine a protocol for long-term culture of newly transformed cercariae that developed in vitro into sexually dimorphic forms. We assessed the effect of adding two different sera, foetal bovine serium (FBS) and human serum (HS), to culture medium supplemented with red blood cells. In contrast to FBS-culture parasites, those grown in HS digested red blood cells, a crucial step for long term parasite development. Furthermore, sexual dimorphism was clearly established in the HS-cultured parasites, albeit delayed, in contrast to most FBS-cultured parasites that did not progress beyond an early liver stage. Moreover, in EdU-pulse experiments, cells within HS-cultured parasites continuously proliferated, but markedly fewer proliferated in FBS-culture. By enabling reproducible parasite develoment in vitro, this protocol creates new opportunities for dissecting mechanisms that underly sexual dimorphim, as well as for screening in vitro for new interventions across the life cycle of these major human parasites.