Functional analyses of male-competence factors to unravel their role during the exceptional sex interplay in Schistosoma mansoni

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Schistosomes cause schistosomiasis, an infectious disease with worldwide meaning. A phenomenon of schistosomes, which are worm parasites, is the dependence of the development of the reproductive organs of the female on a continuous pairing contact with the male. Basis for this gender-interaction are molecular communication processes. Their consequences have been well studied in females, for which some knowledge exists. In males, however, knowledge about these processes is scarce, despite its relevance for basic research. Pairing is the prerequisite for egg production and thus essential for life-cycle maintenance. Furthermore, eggs are responsible for the clinical-pathological consequences of schistosomiasis, culminating in liver fibrosis. Based on transcriptomics with pairing-experienced males (bM) and pairing-unexperienced ones (sM), which were performed in previous funding periods, we have obtained a first comprehensive overview on the complexity of processes that are influence by pairing in males. Besides muscle and metabolism functions, stem cell-associated and particularly neural functions appeared to be important. Therefore, we focused on G protein-coupled receptors (GPCRs) and neuropeptides (Npps). Following a new description of all GPCRs (GPCRome), we analyzed their expression patterns on the basis of our transcriptomics data. We found many GPCRs to be more abundantly transcribed in bM, sM, and/or unpaired females compared to paired ones. This applied also to Npp-coding genes, which we identified by bioinformatics, and which we cloned. Next, we successfully established the MALAR yeast-2-hybrid system to identify Npp-GPCR interaction, and thus to “de-orphanize” GPCRs. We plan to confirm these interactions by a Fluorescence Resonance Energy Transfer (FRET) assays. By whole mount in situ hybridisation, we localised gpcr and npp transcripts, and Npps also by mass spectrometry imaging. First functional studies of Npp-GPCR partners showed interesting phenotypes such as deficits in egg production. For GPCR 9, which is testis-specific and pairing-dependently expressed, we discovered a role in spermatogenesis. We plan to extend these characterizations to all GPCRs, which exhibit this male-dominated expression profile. This allows a deeper insight into the biological relevance of these GPCRs and their ligands as well as their potential role(s) as male competence (MC) factors. Functional analyses of genes involved in dopamine synthesis, which are male-specific and pairing-dependently transcribed, confirmed their meaning for gonad differentiation in paired females. This is the first proof for the existence of MC factors. According to a meta-analysis, these genes start expression at the schistosomula stage. This motivates us to start KD of these genes also in this life stage using a new in vitro culture system, which allows rearing fully differentiated adult worms starting with schistosomula.