Dynamic single-cell transcriptomics reveals lsamp-guided neural network formation in male S. japonicum driving female reproduction

Dynamic single-cell transcriptomics reveals lsamp-guided neural network formation in male S. japonicum driving female reproduction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dynamic single-cell transcriptomics reveals lsamp-guided neural network formation in male S. japonicum driving female reproduction Wei Hu, Yanmin You, Shaoyun Cheng, Xu Chen, Xi Chen, Cun Yi, Mingqi Cai, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6983189/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jan, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Sexual maturation and egg production in schistosomes require sustained male-female pairing, yet the cellular dynamics underlying this process remain poorly understood. Here, we generated a high-resolution single-cell transcriptomic atlas of 104,671 cells from male and female Schistosoma japonicum across four developmental stages, identifying 76 distinct cell populations and mapping lineage trajectories in reproductive and neural systems. Notably, we discovered three male-specific neuron subtypes, including delta-3 neurons located in the gynecophoric canal, and identified lsamp as a critical gene co-expressed with nrps in delta-3 neurons. Loss of lsamp impaired axonal growth and disrupted the male ventral nerve network, reducing the targeted delivery of pheromone BATT precursors to delta-3 neurons, thereby hindering female reproductive maturation. This work maps schistosome male-female interactions at single-cell resolution, revealing a male ventral nerve network that facilitates intersex communication and promotes female reproductive development, offering the new strategies to disrupt parasite reproduction and combat schistosomiasis. Biological sciences/Microbiology/Parasitology/Parasite biology Biological sciences/Molecular biology/Transcriptomics Biological sciences/Microbiology/Pathogens Schistosomes single-cell RNA sequencing male-female interaction reproductive development ventral neural fibers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Schistosomiasis is a prevalent and devastating neglected tropical disease caused by parasitic flatworms called Schistosoma spp., which affects over 250 million people in disadvantaged areas 1 . Currently, no vaccine is available, and treatment relies on a single drug (praziquantel) 2 , 3 . Therefore, new therapeutic and preventive strategies are urgently needed. Eggs produced by mature female schistosomes drive the pathology and transmission of this disease. In a definitive mammalian host, one mature female parasite could produce hundreds to thousands of eggs daily that are deposited in the host tissues, e.g ., the liver, where they induce the local granuloma and further lead to tissue fibrosis 4 , 5 . Eggs excluded with feces or urine hatch in the water environment, and then the released miracidia invade the intermediate snail host before spreading. Therefore, developing strategies for obstructing the egg production of these parasites will conduce to the control of this disease. Schistosomes have a unique reproductive feature. The pairing with the male worm initiates the sexual development of a female worm and maintains her mature state 6 . Unlike females, the male worm can reach sexual maturity independently 7 . Continued investigations have been carried out to uncover the underlying mechanism of this male-induced female reproduction since the first report of the phenomenon in S. japonicum nearly a century ago 8 . Recently, a dipeptide pheromone (β-alanyl-tryptamine) synthesized and secreted by male worms has been identified in S. mansoni that stimulates female sexual development 9 . The discovery of this neuron-derived key molecular that can be transmitted from males to females highlights the importance of understanding the complex network during male-female interplay. The direct physical connection between males and females occurs at their late juvenile stage when the male worm clasps the female through the gynecophoric canal to form a worm pair 10 . Thereafter, the coupled male and female continue to grow and turn to be sexually mature. Besides the extension of body length, a series of previous studies have observed dramatic gene expression changes in males and females upon pairing 10 – 13 . These changes are not only restricted to the sexual organs but also somatic tissues, such as neurons and muscles. Additionally, labor division is more apparent between the two genders at their adult stage, which is represented by the distinct sex-associated gene expression patterns 10 , 14 – 17 . Recently, researchers have revealed that the cell types of S. mansoni expanded along with the growth and development of the parasite in the mammalian hosts 18 – 20 . Notably, single-cell RNA sequencing showed distinguished cell clusters between the adult male and female and between the virgin female and mature female in S. mansoni 21 , which highly indicates a dynamic sex-related cellular differentiation during the male-female interaction. However, the whole picture of the molecular changes of males and females during their interaction at the single-cell level is still lacking. In this study, we profiled the dynamic single-cell atlas of male and female S. japonicum across 4 time points throughout the reproductive development during their interplay. We sequenced 104, 671 single-cell transcriptomes and characterized 76 molecularly distinct cell populations. Along with the male-female interaction, the cell clusters of the reproductive and nervous systems undergo the most dramatic changes in both sexes. RNA velocity analysis revealed the detailed lineage of cells related to the reproductive development in both genders. We analyzed and predicted gene expression patterns and key regulatory factors involved in the maturation of male and female gametes. Furthermore, we noticed a complex heterogeneity and sex-biased characteristics of the nervous system during the maturation of the parasite. Notably, we identified a lsamp gene expressed within a male-specific neuron population, which triggers female reproductive development by fostering axon growth within the male gynecophoric canal in S. japonicum . Taken together, our datasets provide the comprehensive single-cell atlas of male and female S. japonicum across their maturation and reveal the key regulator of a male-specific neuron that is required for the stimulus of female sexual development, which will further prompt the innovation of strategies against this human parasite. Results Dynamic single-cell transcriptomes of male and female S. japonicum during their interplay S. japonicum primarily undergoes pairing and sexual maturation between 14–26 dpi (days post-infection) 10 . To characterize the dynamic single-cell atlas of S. japonicum throughout the female-male pairing and subsequential maturation process, we collected worms at four-time points (14, 18, 22, 26 dpi) from the mice model ( Fig. 1a ). Whole worms were separated by sex at each stage, except for those from 14 dpi (as gender could not be distinguished morphologically at this stage). Subsequently, we dissociated these samples and isolated live single cells using fluorescence-activated cell sorting (FACS). Utilizing a 10× Genomics chromium controller, we generated 7 single-cell RNA-seq (scRNA-seq) libraries ( Fig. 1a, and Supplementary Data 1 ). Through the analysis of these libraries using Cell Ranger, we obtained a total of 110,204 cells ( Supplementary Data 1 ). After excluding cells with more than 30% mitochondrial reads or fewer than 500 detected genes, the final count was 104, 671 cells, which were further clustered into 76 distinct cell clusters ( Supplementary Fig. 1, and Supplementary Data 1 ). Furthermore, we annotated the identities of these cell clusters by referring to cell markers defined in the single-cell sequencing of S. mansoni 18 , 19 , 21 ( Supplementary Data 2 ). In total, the S. japonicum single-cell atlas comprised 33 transcriptionally distinct neuron cell clusters, 14 neoblast clusters, 9 tegument cell clusters, 5 muscle cell clusters, 5 parenchyma clusters, 3 germ stem cell (GSC) clusters, 1 vitellocyte cluster, 1 male gamete cluster, 1 Mehlis' gland cell cluster, 1 gut cell cluster, 1 flame cell cluster, 1 esophageal gland cell cluster, and 1 unknown cluster ( Supplementary Fig. 1 ). Our single-cell atlas provided detailed lineage information for the parasite’s specific tissues. Interestingly, our data unexpectedly revealed many schistosome neural stem cells and neural precursor cells for the first time. In comparison, the single-cell atlas of S. mansoni depicts neural cells as scattered clusters lacking apparent lineage connections 21 . Based on their marked heterogeneity, we classified the neural cells into five categories: neural stem cells, neural precursor cells, kk7 + neurons, nonciliated neurons, and ciliated neurons ( Fig. 1b) . The tegument cells are divided into tegument progenitor, tegument progeny 1, tegument progeny 2, syncytial 1, and syncytial 2 ( Fig. 1b ). In addition to the well-defined cell markers in previous studies on S. mansoni 21 , we further identified the marker genes as shown in Supplementary Fig. 2 and Supplementary Data 3–4 through WISH (whole-mount in situ hybridization), including new markers such as Sjc-0003010 and Sjc-0008393 for GSCs (germline stem cells) ( Supplementary Fig. 3a ), Sjc-0001566 ( ccdc ) for male gametes ( Supplementary Fig. 3c ), Sjc-0004922 ( Tubulin alpha-1 chain ) for neoblasts ( Supplementary Fig. 3e ), Sjc-0008047 ( klf11 ) for neural stem cells ( Supplementary Fig. 3f ), Sjc-0005931 ( acss2 ) and Sjc-0007590 ( pcdh7 ) for neural precursor cells ( Supplementary Fig. 3g ), Sjc-0002588 ( slc6a5 ) for parenchyma cells ( Supplementary Fig. 3i ), Sjc-0001890 ( sgf1 ) for esophageal gland cells ( Supplementary Fig. 3k ), Sjc-0008416 ( cpb ) and Sjc-0000095 ( cacnb1 ) for muscle cells ( Supplementary Fig. 3m ), Sjc-0008478 and Sjc-0000277 ( Slc7a14 ) for syncytial 2 of tegument cells ( Supplementary Fig. 3n ). We first analyzed the distribution of cells in each sample, which allowed us to identify developmental differences at the cellular level between male and female worms at various stages ( Supplementary Fig. 4 ). The most significant differences observed between male and female worms were primarily related to cell types associated with the reproductive system. As the worms mature, the number of GSCs and vitellocyte cells in females gradually increases, while the number of male gametes rises in males ( Supplementary Fig. 4 ). To gain a general view of the changes in different cell clusters in male and female worms during their pairing, we analyzed the dynamics of the proportion for each cell type. In both sexes, neoblasts and neuronal cells individually accounted for ~ 20% of the total cells, representing the two largest cell populations in S. japonicum ( Fig. 1c, and Supplementary Data 5 ). The proportion of neural stem cells and precursor cells decreased after 14 dpi, while the populations of differentiated neural cells ( e.g ., nonciliated neurons and ciliated neurons) dramatically expanded ( Fig. 1c, and Supplementary Data 5 ). This finding indicates that neural progenitor cells are active during the juvenile stage, but gradually degenerate in the adulthood. Interestingly, the proportion of mature neurons in male worms continues to rise, whereas in female worms, it initially increases before eventually declining ( Fig. 1c, Supplementary Fig. 5, and Supplementary Data 5 ). In addition, the proportion of the reproduction-related cell types gradually increased throughout the sexual maturation process in each sex as expected, such as germline stem cells, female vitellocytes, and male gametes ( Fig. 1c, Supplementary Fig. 5, and Supplementary Data 5) . Additionally, the proportions of parenchyma and flame cells were enlarged in females and males along with their development ( Fig. 1c, Supplementary Fig. 5, and Supplementary Data 5 ). However, the percentage of muscle cells declined sharply in both genders after 14 dpi, especially in male worms ( Fig. 1c, Supplementary Fig. 5, and Supplementary Data 5 ). For neoblasts, a clear decrease was observed in male parasites across all four-time points. In females, however, the decrease was evident from 14 to 18 dpi, followed by an expansion as female development progressed. We further examined the tegument, which includes well-defined cell types from the progenitors to the progenies. Unlike the neuronal populations, the proportion of tegument progenitors remained stable even as the proportion of their progenitor cells and differentiated syncytial cells increased for both branches (Fig. 1c, Supplementary Fig. 5, and Supplementary Data 5) . This differing pattern of changes may be attributed to the tegument requiring renewal: as the interface between the host and parasite, the tegument needs rapid turnover to replace damaged or aging tissues, thereby maintaining the activity of progenitor cells. To validate the dynamic changes in cell populations along the male-female pairing indicated by our single-cell atlas, we performed WISH using the germline cell marker eled 18 . As shown in Fig. 1d , the expression region of eled expanded significantly in both female and male sexual organs from 18 to 26 dpi, which was consistent with the single-cell analysis data. Detailed lineage analysis of cells related to the reproductive development in both genders The schistosome germ cells originate from a subset of stem cells at the asexual stage and eventually separate from somatic cell lineage at the juvenile stage 18 . Upon pairing, the committed GSCs in both sexes proliferate and differentiate into mature gametes. To demonstrate the cellular dynamics of the parasite's reproductive system and the relationship between germline and somatic lineages, we first subset the cells defined as stem cells (neoblasts) and those present in the reproductive organs, including GSCs, male gametes, female gametes, vitellocytes, and Mehlis' glands, from both female and male samples (18, 22, and 26 dpi), and performed sub-cluster analysis. Then, we identified 17 clusters with representative markers for each subtype based on well-defined markers from previous studies ( Supplementary Fig. 6a, and Supplementary Data 2) , such as the neoblast marker nanos-2 ( Sjc-0005962 ) 22 , the vitellocyte S1 cell maker vf1 ( Sjc-0000108 ) 23 , the germline stem cell marker nanos-1 ( Sjc-0006622 ) and eled ( Sjc-0009220 ) 24 ( Supplementary Fig. 6b-c) . We also validated newly identified tissue-specific markers using WISH, including Sjc-0000843 ( ribc2 , RIB43A-like with coiled-coils protein 2 ) for male gametes, Sjc-0003040 for female gametes, esg-2a ( Sjc-0006798 ) for vitelline cells, and Sjc-0000795 for Mehlis' gland ( Fig. 2a, and Supplementary Fig. 6b-c ). These markers can effectively characterize the developmental status of each organ. Interestingly, female gametes were also observed in male worms, albeit in relatively low numbers ( Supplementary Fig. 6a, c-d ). To confirm the presence of female gamete cells in males, we performed WISH using the female gamete marker gene Sjc-0004512 ( clec, C-type lectin domain-containing protein ) and observed a signal in the testes of male worms ( Supplementary Fig. 6d ). By performing RNA velocity analysis, we inferred the possible developmental trajectories from germline stem cells to mature gametes, separately for female and male worms ( Fig. 2b ). To further investigate the differences in the germline lineage, we further performed RNA velocity analysis on the GSCs, GSC progenies, and the gametes of males and females. It revealed the cell trajectory originating from the GSCs population into mature gametes, in both sexes ( Fig. 2c-d) . Subsequently, we analyzed the expression patterns of genes involved in the differentiation of male or female gametes, including sex-specific highly expressed genes and highly expressed genes shared by both sexes ( Supplementary Fig. 7a, and Supplementary Data 6–8 ). We found that male and female gametes shared 156 genes in their lineages that could be further clustered into five modules ( Supplementary Fig. 7b, and Supplementary Data 6 ). Most of these genes, represented in C2-C5, showed similar expression trends in both genders. Interestingly, genes in the C1 module exhibited opposite expression patterns during the maturation of male and female gametes. Highly expressed genes in female gametogenesis also could be clustered into five modules ( Supplementary Fig. 7c, and Supplementary Data 7 ): The C1 module shows a gradual downregulation in expression as gametes mature, such as Sjc-0003683 and Sjc-0006860 ( hist1h1a ); The C2 module exhibits an initial slight downregulation followed by a rapid upregulation during gamete maturation, such as Sjc-0003078 and Sjc-0009371 ( nars , Asparagine-tRNA ligase, cytoplasmic ); The C3 and C4 modules maintain a constant expression level in the early stages, followed by a rapid upregulation in the mid to late stages, such as Sjc-0003040, Sjc-0004512 ( clec), Sjc-0004939 ( erc2 , ERC protein 2 ) and Sjc-0000072 ; The C5 module showed a positive correlation with gamete development, before stabilizing at a constant expression level during the later stages, as seen with Sjc-0008219 ( hormad1 , horma domain-containing protein 1 ) and Sjc-0001311. Highly expressed genes in male gametogenesis can also be clustered into five modules ( Supplementary Fig. 7d, and Supplementary Data 8 ): The gene expression trends in modules C1 to C4 are similar, showing a negative regulatory relationship with the gamete maturation, including Sjc-0003673 ( gpd1 , Glycerol-3-phosphate dehydrogenase ) and Sjc-0004527 ( eef1b , Elongation factor 1-beta ), while the C5 module exhibits a positive regulation, as exemplified by Sjc-0002056 ( zinc finger CCCH domain-containing protein 31 ) and Sjc-0000843 ( ribc2 ). The above results demonstrate that the development of male and female gametes is a complex process regulated by multiple gene modules. Further functional validation is required to explore the mechanisms of common and specific genes related to female or male gametogenesis. Vitellaria is the largest reproductive organ in adult female schistosomes that occupies two-thirds of the body's length. We found that both vitellaria S1 cells and GSCs express a certain level of eled ( Sjc-0009220 ) ( Supplementary Fig. 6b-c ), suggesting a potential shared origin from a population of eled + stem cells, as previously reported 18 . We analyzed the developmental relationship between the vitellaria and the female germline cells through RNA velocity. However, we did not find any connection between S1 vitellaria cells and GSCs in Fig. 2b , as the two cell clusters were spatially distant. Vitellaria maturation is crucial for egg production in schistosomes. Previous studies have shown that vitellaria development can be divided into stages S1-S4, all of which express vf1 ( Sjc-0000108 ) 23 . In the single-cell transcriptomic analysis of S. mansoni , the vitellaria development process was further categorized into five stages: S1, S1 progeny, early vitellocytes, late vitellocytes, and mature vitellocytes 21 . However, the detailed differentiation trajectory of vitellaria cells has not been fully elucidated at the single-cell level. In our study, we classified the vitellaria development process into four stages based on clustering results: S1 vitellocytes, S1 vitellocyte progeny, early vitellocytes (early vi), and late vitellocytes (late vi). Using RNA velocity, we inferred the developmental trajectory of vitellaria, revealing that S1 vitellocyte cells ( vf1 + esg-2a − ) gradually differentiate into S1 vitellocyte progenies ( vf1 + esg-2a + ), which subsequently give rise to early vitellocytes and late vitellocytes (Fig. 2b, and Supplementary Fig. 8) . This trajectory aligns with previous findings and provides new insights into the molecular mechanisms underlying vitellaria maturation. Further functional studies on key markers such as vf1 and esg-2a will enhance our understanding of their roles in vitellaria development and their potential as therapeutic targets. Mehlis' gland, a specialized organ unique to trematodes and cestodes, has been long speculated to play an essential role in eggshell formation and the release of eggshell granules 25 , 26 . However, research on its function in schistosomes remains limited. In our study, we observed that Mehlis' gland cells emerge earlier than vitelline cells, appearing in large numbers by 18 dpi, while vitelline cells do not proliferate in large numbers until 22 dpi ( Supplementary Fig. 9a-b ). These findings were further validated through WISH experiments using the marker gene Sjc-0000795 for Mehlis' gland, the marker gene esg-2a for vitellaria and the marker gene Sjc-0000278 ( rreb1 ) for both vitellaria and Mehlis' gland ( Supplementary Fig. 9c ). Five neural lineages in S. japonicum Unlike the scattered neuronal clusters revealed from the previously reported single-cell transcriptomic atlas of adult Schistosoma mansoni 21 , our datasets exhibit high connectivity among complex neuronal clusters ( Fig. 1b, and Supplementary Fig. 1 ), which is likely to enable cell lineage reconstruction of the whole schistosome nervous system from transcriptomic data. We first reclustered all the neural cells of males and females, including neural stem cells, neural precursor cells, nonciliated neurons, and ciliated neurons, identifying 19 subclusters and summarizing them into 5 lineages, which were named alpha lineage, beta lineage, gamma lineage, delta lineage, and epsilon lineage ( Supplementary Fig. 10a, and 11a ). Subsequently, we employed Slingshot to confirm the reconstructed cell differentiation lineage and infer pseudotime ( Supplementary Fig. 10b, and 11b ). Additionally, we performed RNA velocity analysis on the male and female datasets separately using scvelo algorithms 27 . We visualized the paths predicted by the mRNA velocity model on UMAP, predicting a potential differentiation trajectory from neural progenitor cells toward mature cell types ( Supplementary Fig. 10c, and 11c ). We then combined trajectory inference with RNA velocity to compute fate probabilities. In male S. japonicum , we identified 7 metastable states in the neural cells, with neural progenitor cells predicted as the initial state and delta-2 neuron, delta-3 neuron, delta-4 neuron, alpha -1 neuron, gamma-2 neuron, and beta-2 neuron inferred as six terminal states ( Supplementary Fig. 10d ). In female S. japonicum , we identified 7 metastable states in the neural cells, with neural progenitor cells predicted as the initial state and delta-1 neuron, delta-2 neuron, epsilon neuron, alpha -1 neuron, gamma-1 neuron, and beta-1 neuron inferred as six terminal states ( Supplementary Fig. 11d ). Using a partition-based graph abstraction (PAGA) approach, we further quantified the connectivity of cell populations and visualized the fate probability of each cell population as a pie chart (Supplementary Fig. 10e, and 11e ). In summary, we defined neural cell differentiation from an early neural progenitor state to several mature states and provided a mechanistic framework for characterizing worm neuronal cell fate decisions. Male-specific neuron clusters To explore the neuronal diversity in S. japonicum , we performed a subcluster analysis of neuronal cells in both male and female parasites ( Supplementary Fig. 12a) . From the integrated single-cell atlas of both sexes, we identified three neuron populations uniquely present in males: beta-2 , gamma-2 , and delta-3 ( Fig. 3a). The proportional distribution of neuronal subtypes in both sexes ( Supplementary Fig. 12b ) and the differential abundance analysis of neuronal cell types across genders ( Supplementary Fig. 12c ) further confirmed the existence of these male-specific neurons. We next investigated their spatial distribution within the parasite. From the enriched genes in each population, we first identified the potential markers for beta-2 , gamma-2 , and delta-3 ( Supplementary Fig. 13). We then conducted WISH and double-FISH using RNA probes targeting these markers, selecting specific markers to define each population: beta-2 ( Sjc-0003639 + Sjc-0000814 − ), gamma-2 ( Sjc-0001200 ), and delta-3 ( Sjc-0000814 ). As anticipated, all these cells expressed the pan-neuronal marker 7b2 21 ( Supplementary Fig. 14a-c ). Interestingly, all three of these neurons were primarily located in the gynecophoric canal, a specialized male structure that secures the female during pairing ( Fig. 3b, and 14a-c ). Specifically, unlike the delta-3 population, which was exclusively distributed throughout the male gynecophoric canal, the beta-2 and gamma-2 populations were also found in the head of male and female parasites ( Fig. 3b, and 14a-c ). Interestingly, the marker gene Sjc-0000814 of delta-3 is nrps ( nonribosomal peptide synthetase ) which encodes NRPS enzyme. In S. mansoni , NRPS enzyme has been reported to synthesize β-alanyl-tryptamine (BATT), a dipeptide pheromone that passed from male to induce female reproductive development 9 . Thus, our study clarified the delta-3 neurons are the key populations that regulate female sexual development by expressing the nrps to produce BATT. Furthermore, we identified the localization of additional mature neuronal populations using the RNA probes targeting specific markers, including beta-1 ( ano7 + ), gamma-1 ( Sjc-0006108 + ), delta-1 ( bmp, Sjc-0004182 + ), delta-4 ( fhl2, Sjc-0002320 + ) and epsilon ( Sjc-0000744 + ), in both sexes ( Supplementary Fig. 15 ). To summarize these findings, we generated schematic illustrations depicting the distribution patterns of these neurons ( Supplementary Fig. 16 ). To elucidate the potential functions of these three male-specific populations, we performed GO (Gene Ontology) enrichment analysis on the highly expressed genes within each neuronal subclass. These enriched genes indicated that beta-2 and gamma-2 populations exhibited typical neuronal functions, such as synaptic transmission and neurotransmitter transport. In contrast, gamma-2 population showed more specific enrichments, particularly in ion transport ( Fig. 3c, Supplementary Fig. 17, and Supplementary Data 9–10 ). Unlike them, the delta-3 population emerged as a specialized neuronal subtype, expressing genes closely related to cilium organization, cell projection assembly, and microtubule bundle formation ( Fig. 3c, and Supplementary Fig. 17 ). This finding aligns with previous observations in S. mansoni , where SEM studies identified Sm - nrps + cells with cilium-like protrusions, suggesting a conserved role of ciliated neurons in male-specific functions 9 . Collectively, these results indicate that these three types of neurons play different roles in male parasites. Considering the importance of the key enzyme coded by nrps in the delta-3 neuron that synthesizes the pheromone required for the female sexual development in schistosomes, we wondered whether there are essential genes maintaining the function of the delta-3 population. Male lsamp is essential for female sexual development To identify genes potentially driving the differentiation of delta-3 neuron, we analyzed the driver genes associated with the delta-3 neuron metastable state by correlating fate probabilities with gene expression using CellRank's compute_lineage_drivers function. Among these, genes such as nrps ( Sjc-0000814 ), ddc ( Sjc-0006754 , Aromatic L-amino acid decarboxylase ), and lsamp ( Sjc-0000373 , limbic system-associated membrane protein ) exhibited the highest correlations (Fig. 3d) . And these three genes are specifically expressed in delta-3 neurons as indicated in the UMAP (Fig. 3e) . The colocalization of nrps and ddc has been confirmed previously ( Supplementary Fig. 14c ). We performed double-FISH and found that lsamp is co-expressed with nrps and ddc in male delta-3 neurons ( Fig. 3f ). Additionally, lsamp and ddc also exhibited colocalization in the large cerebral ganglia ( Fig. 3f ). Nrps is a male-specific gene, and its expression is induced by pairing 9 . In S. japonicum , both ddc and lsamp displayed similar expression patterns to nrps , as reported in our previous study 10 ( Fig. 3g ). We therefore reasoned that lsamp or ddc may play an important role in the delta-3 neuron that may further affect the male-female interaction. By employing RNAi in vitro , we found the male worms treated with lsamp dsRNA instead of ddc dsRNA lost their ability to initiate female sexual development ( Fig. 4a-b, and Supplementary Fig. 18 ). These female worms paired with lsamp RNAi males displayed reduced Fast Blue BB staining in their vitellaria and possessed poorly developed ovaries ( Fig. 4b ). In addition, they barely laid eggs (Fig. 4c) . qRT-PCR result indicated a strong reduction of the lsamp expression in the male worms after the lsamp RNAi ( Fig. 4d ). The above results suggest that male lsamp is essential for female sexual development. To determine whether the female phenotype resulting from the male lsamp knockdown was due to a reduction in the number of delta-3 neurons, we performed FISH experiments to quantify the number of delta-3 neurons. We selected the marker gene ddc to detect delta-3 neurons number specifically. However, there was no significant difference in the lsamp silencing group compared to the control group (Fig. 4e) . Lsamp affects acetylated α-tubulin formation and vesicular transport within neurons Since lsamp colocalized with nrps in delta-3 neurons ( Fig. 3f ), and nrps has been recognized as the functional gene coding an enzyme to synthesize the male pheromone 9 , we therefore wondered whether the lsamp RNAi phenotype is caused by affecting the nrps expression. qRT-PCR results showed that the nrps expression was not affected in the lsamp knockdown males (Fig. 4f, and Supplementary Fig. 19) . According to Chen et al ., the NRPS enzyme used two simple precursors (β-alanine and tryptamine) to form the dipeptide pheromone BATT 9 . To explore why knocking down lsamp in male worms blocks female sexual development, we detected the levels of two precursors in worms but found no changes between the cntl and lsamp RNAi groups (Fig. 4g-h) . We then measured the amount of BATT, the product of the NRPS, that is made in males and can be released to the medium in vitro . Surprisingly, BATT contents were significantly reduced in the lsamp RNAi group, both in the male worms and the culture medium (Fig. 4i) , even though the nrps level was not changed ( Fig. 4f ). These findings suggest that lsamp impairs the ability of male delta-3 neurons to synthesize BATT, consequently failing to stimulate female sexual development, not through affecting nrps expression. To investigate the underlying mechanism of male lsamp in regulating BATT synthesis, we performed RNA-seq to overview the gene expression changes between paired lsamp RNAi and gfp RNAi males (D20). Silencing lsamp resulted in the downregulation of 50 genes and the upregulation of 19 genes ( Supplementary Fig. 20a-b, and Supplementary Data 11–12 ). Our RNA-seq data confirmed that the expression of nrps and ddc remained unaffected by lsamp knockdown (Supplementary Fig. 20c) , consistent with our previous results (Fig. 4f, and Supplementary Fig. 19) . Since the upregulation of nrps is triggered by physical contact between males and females 9 , these results suggest that the knockdown of lsamp does not impair male worms’ ability to sense females. Furthermore, GO term analysis of the downregulated genes in lsamp RNAi males revealed significant enrichment in biological processes, including the generation of precursor metabolites and energy, and peptide biosynthesis, as well as molecular functions such as structural constituents of the ribosome and ATPase-coupled ion transmembrane transporter activity (Supplementary Fig. 20d-e, and Supplementary Data 13–14) . KEGG pathway analysis of the down-regulated genes showed that lsamp mainly affects pathways involved in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease (Fig. 5a, and Supplementary Data 15) , both of which are closely related to the disorder of microtubule acetylation and axon vesicular transport 28 – 32 . In addition, GO enrichment on the highly expressed genes in the delta-3 population indicated it as a specialized neuron that expressed genes highly related to cilium organization, cell projection assembly, and microtubule bundle formation ( Fig. 3c, and Supplementary Fig. 17 ). Previous studies in mice and nematodes have shown that lsamp homologues play a role in synapse formation, axon guidance, and neuronal stability 33 – 37 . We therefore hypothesized that lsamp may affect the processes of microtubule formation and axon vesicular transport in male schistosomes. Acetylated α-tubulin is a protein that stabilizes microtubules and regulates cell morphology in nerve fibers. It plays a crucial role in various neuronal processes, including axonal formation, dendritic growth, and neuronal migration 28 . And acetylated α-tubulin plays a pivotal role in the transport of synaptic vesicles within neurons. It stabilizes microtubules, which serve as 'tracks' for motor proteins, facilitating the efficient movement of vesicles and ensuring the proper delivery of neurotransmitters to the synapse. In addition to classical neurotransmitters like dopamine, serotonin, and GABA, synaptic vesicles also transport other small molecules, such as the precursors for BATT (β-alanine and tryptamine) 38 , 39 . To further investigate the specific function of lsamp in schistosomes, we examined the acetylation status of microtubules and synapsin levels in male worms. As shown in Fig. 5b , acetylated α-tubulin antibody labeling revealed crisscrossed nerve fibers in the male gynecophoric canal. After knocking down the lsamp , the nerve fibers were markedly eliminated without affecting the nrps expression (Fig. 5b). These data suggest that LSAMP is essential for nerve fiber growth by stabilizing microtubules. Using a synapsin antibody, we detected disrupted synaptic protein distribution along nerve axons (Fig. 5b). Comparative co-staining of acetylated α-tubulin and synapsin revealed severe degeneration of neural network functionality in the lsamp knockdown group (Fig. 5b). We next examined the spatial relationship between these neural fibers and delta-3 neurons. By labeling delta-3 neurons with three markers— ddc , nrps , and lsamp —we observed that delta-3 neurons were closely associated with neural fibers marked by acetylated α-tubulin ( Fig. 5c ). Additionally, synaptic proteins localized along neural axons showed strong colocalization with delta-3 neurons, suggesting active vesicular transport between these axons and delta-3 cells ( Fig. 5d ). These findings indicate that the loss of lsamp in delta-3 neurons disrupts the integrity of the surrounding ventral neural network in male schistosomes. Lsamp guides the formation of ventral neural network in males LSAMP (Limbic system-associated membrane protein) is a glycosylated cell adhesion molecule of the IgLON family 40 . It is a GPI-anchored protein characterized by three immunoglobulin (Ig) domains 36 (Supplementary Fig. 21). Previous studies in mice and nematodes have demonstrated that lsamp plays essential roles in neuronal development and function, including promoting neuronal growth and guiding axon targeting 33 – 37 . To test this, we examined the development of the ventral neural fiber network in male worms from 14 to 26 dpi, covering key stages from male-female pairing to full sexual maturity. At 14 dpi, when males begin pairing with females, only a few elongated nerve fibers were detected within the male gynecophoric canal. As the males developed, a characteristic crisscrossed neural fiber network became evident by 22 dpi and was maintained thereafter (Fig. 5e ). Notably, the formation of this neural network coincided with a marked upregulation of lsamp expression (Fig. 3g, and Supplementary Fig. 22), suggesting that lsamp facilitates axon growth and attachment to delta-3 neurons, thereby contributing to the establishment of a functional ventral neural network ( Fig. 5f ). Overall, our study has profiled the dynamic single-cell atlas of male and female Schistosoma japonicum during pairing and sexual development. From a male-specific delta-3 neuron population, we identified the lsamp gene, which is crucial for female reproductive development and operates independently of a previously reported key regulator in males, nrps . We demonstrate that lsamp is essential for guiding the formation of ventral neural fibers in the male gynecophoric canal, where they connect to the delta-3 neurons (Fig. 5c-e, and Supplementary Fig. 22) . This nerve network functions as an interstate, facilitating the transport of BATT precursors to the delta-3 neurons, which produce the male pheromone to stimulate female reproductive development (Fig. 5f) . These findings not only deepen our understanding of the complex male-female interactions governing sexual development in schistosomes but also shed light on developing new strategies for combating schistosomiasis. Discussion Our study performed single-cell RNA sequencing across four key developmental stages of male and female S. japonicum , covering the critical period from pairing to egg production. For the first time, we present a dynamic single-cell transcriptome atlas of S. japonicum males and females, systematically describing the proportional changes in various cell types during their developmental process. Notably, differentiation trajectories were inferred for reproductive and neuronal cells, showing the most prominent changes. A set of marker genes was identified that can be used to distinguish the reproductive status of male and female worms, and we predicted gene sets associated with gametogenesis in both sexes. Additionally, we clarified that delta-3 neurons are nrps -producing neurons and that lsamp , a highly expressed gene in delta-3 neurons, is essential for male-induced female reproductive development by influencing axonal microtubule acetylation and vesicle transport, ultimately reducing BATT synthesis in males. The sexual development of schistosomes is a fascinating biological process in which female worms rely on pairing with male worms to initiate reproductive maturation. Pairing behavior begins around 14 dpi, during which females become encapsulated within the male’s gynecophoric canal. By 26 dpi, most females reach sexual maturity and begin laying eggs 10 . Between 14 and 26 dpi, both male and female worms undergo dramatic growth and development, not only in their reproductive organs but also in other tissues. Bulk RNA-seq is limited in its ability to resolve the molecular changes occurring at the cellular level. In the present study, we used single-cell RNA sequencing to systematically characterize the gene regulatory programs underlying dimorphic gametogenesis in S. japonicum . We identified both shared and sex-specific regulatory modules involved in germline development ( Supplementary Fig. 7 ). Among the shared modules, C2, C3, and C4 exhibit similar expression patterns in both sexes ( Supplementary Fig. 7b, and Supplementary Data 6 ). Notably, the C2 module includes nanos1 and nanos2 , which are critical regulators of germline stem cell differentiation 18 , 41 . The C3 module contains genes involved in cell cycle regulation ( e.g. , pcna ), energy metabolism ( e.g ., sdhb ), and RNA processing ( e.g ., srsf12 ), which are associated with cellular proliferation and maintenance of genomic stability 42 – 44 . The C4 module features genes such as smc3 and tubb4b , which are involved in chromosome segregation and cytoskeletal organization during meiosis 45 , 46 . These shared modules highlight the evolutionary conservation of core gametogenic processes in both male and female S. japonicum , including germline stem cell maintenance, epigenetic regulation, chromosome dynamics, and protein homeostasis. In contrast, some shared modules display sexually dimorphic expression patterns. For example, C1 genes are initially downregulated and then upregulated in females, whereas in males, their expression steadily declines ( Supplementary Fig. 7b, and Supplementary Data 6 ). Additionally, sex-specific modules reveal distinct features between male and female gametogenesis ( Supplementary Fig. 7c-d, and Supplementary Data 7–8 ). For instance, male-specific modules such as C5 include genes involved in flagellar assembly, which are directly associated with spermatogenesis and motility. Importantly, some unannotated genes within these clusters may represent Schistosoma -specific reproductive regulators that warrant further investigation. Schistosome neural development remains poorly understood, especially the differentiation trajectories of neuronal cells. While single-cell RNA sequencing has been conducted on adult S. mansoni , it primarily captured mature neuron populations 21 , limiting lineage tracing analysis. In this study, we profiled single-cell data from S. japonicum at 14, 18, 22, and 26 dpi, identifying various neural cell stages, including neural stem cells, precursors, and mature neurons ( Fig. 1a-c ). Pseudotime and RNA velocity analysis, reconstructed clear lineage connections, providing a detailed neural development map ( Supplementary Fig. 10–11 ). We further classified the nervous system of S. japonicum into five clear neuronal lineages, each exhibiting significant spatial heterogeneity ( Supplementary Fig. 10–11, and 13–16 ). Intriguingly, some neurons exhibit sex-specific distribution, such as delta-3 , gamma-2 , beta-2 , epsilon , and beta-1 neurons ( Fig. 3a-b, and Supplementary Fig. 13–16 ). Our data reveal that male-specific delta-3 neurons express nrps ( Fig. 3a-b, and d-e ), a gene rapidly upregulated upon pairing. Nrps encodes NRPS, an enzyme synthesizing the male pheromone BATT, which stimulates female sexual development 9 , 47 . Additionally, we identified other driver genes expressed in delta-3 neurons, such as ddc ( Sjc-0006754 ) and lsamp ( Sjc-0000373 ). These findings highlight the robust heterogeneity of schistosome neurons and suggest that neural differentiation occurs at early developmental stages, with no active neurogenesis at the adult stage. DDC is an enzyme involved in the synthesis of several monoamines, including serotonin (5-HT), tyramine, tryptamine, histamine, dopamine, and other catecholamines 48 , 49 . In planarians, inhibiting ddc expression results in the loss of ovaries and other female reproductive organs, including the yolk gland 50 . Similarly, in nematodes, egg-laying is regulated by serotonergic neurons 51 . However, in this study, ddc knockdown in male schistosomes did not cause developmental arrest in female reproduction ( Supplementary Fig. 18 ). Since biogenic amines, including 5-HT, are present in the culture medium, we hypothesized that female schistosomes might sustain reproductive development by utilizing exogenous 5-HT. To test this, we cultured mature S. japonicum worm pairs using 5-HT-supplemented and 5-HT-free mAB169 (1640) medium. After 12 days, females in the 5-HT-free group exhibited impaired vitellaria and produced significantly fewer eggs, compared to the 5-HT-supplemented group ( Supplementary Fig. 23 ). These findings indicate that 5-HT, a product of DDC activity, is essential for female reproductive development. Thus, while DDC and its product 5-HT are crucial for female sexual development, schistosome females can compensate for ddc knockdown by acquiring 5-HT from external sources under in vitro conditions. Previous studies in mice and nematodes have shown that LSAMP plays key roles in synapse formation, axon guidance, and neuronal stability 33 – 37 , but there have been no studies on this gene in schistosomes. Although S. japonicum LSAMP shares only ~ 27% overall sequence similarity with its homologs in Drosophila , Caenorhabditis elegans , and Mus musculus , its functional domains are highly conserved ( Supplementary Fig. 24a-b ), suggesting that its biological roles may be preserved. In this study, we found that lsamp is essential for establishing the specialized neural network within the male gynecophoral canal. As a glycosylated cell adhesion molecule of the IgLON family, LSAMP functions as a membrane protein ( Supplementary Fig. 24c, and 21 ). In C. elegans , SAX-3 (LSAMP homolog) has been shown to localize on the surface of PVD neurons, guiding axon outgrowth 36 , 37 . Here, we observed lsamp expression in the BATT-producing delta-3 neurons in schistosomes. In the absence of lsamp , the intricate neural network connecting delta-3 neurons failed to form ( Fig. 5b ). We propose that LSAMP acts as a selective adhesion molecule on the surface of delta-3 neurons, guiding axon growth and facilitating neuronal connectivity ( Supplementary Fig. 25 ). Beyond structural organization, vesicular transport of amino acids is crucial for intracellular movement and distribution, particularly in neurotransmission 52 , 53 . Specific transport proteins facilitate the transfer of neurotransmitters into synaptic vesicles for release into the synapse 53 – 55 . Although direct labeling of BATT precursors (β-alanine and tryptamine) failed, our LC-MS results provided definitive evidence for the inhibition of their transport. Together, this well-established neuronal network likely ensures the targeted transport of BATT precursors to their synthesis sites in delta-3 neurons. Interestingly, this specialized neural architecture is formed during male-female pairing ( Fig. 5e ), implying a direct role in reproductive coordination. Collectively, these findings provide novel insight into the male-specific neural circuitry involved in regulating female reproduction, thereby expanding our understanding of male-induced female sexual development in this parasitic flatworm. In conclusion, through the comprehensive single-cell transcriptomic analysis of Schistosoma japonicum , we decoded male-female interactions at single-cell resolution and delineated both conserved and sex-specific gene modules involved in gametogenesis. Furthermore, we uncovered a male-specific neural circuit that directly regulates female sexual development. Our study identified lsamp as a key regulator of axonal connectivity in pheromone-producing delta-3 neurons, which establish a specialized neural network essential for the transport of BATT precursors. These findings fundamentally reshape our understanding of parasite neurobiology by revealing how a primitive nervous system orchestrates reproductive processes, and they offer novel targets for disrupting the schistosome life cycle. Methods Ethics All experiments related to animals were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China (2006398) and were approved by the Animal Care and Use Committee of Fudan University (Fudan IACUC 201802158S) to ensure ethical and responsible treatment of the animals. S. japonicum parasites The cercariae of S. japonicum were provided by the Chinese Center for Disease Control and Prevention. Single-sex infections of S. japonicum were obtained by infecting mice with male or female cercariae obtained from Oncomelania hupensis snails (Anhui strain) infected with single miracidia. S. japonicum infected mice Six-week-old C57BL/6 mice were purchased from Shanghai Jeste Experimental Animal Co., Ltd. Fluorescence activated cell sorting Twenty female C57 mice were infected percutaneously through the abdomen with 80–200 cercariae. At each time point (14, 18, 22, and 26 dpi), five mice were sacrificed for the recovery of schistosomes 10 . Worms were collected from mice by perfusing the hepatic portal vein with 4℃ PBS (Sangon Biotech) and then rinsed in twice in DMEM (5% FBS) 10 , 47 , 56 , 57 . Freshly perfused male and female worms, except 14 dpi worms, which are difficult to distinguish gender under a light microscope, were separated by incubation in a 0.25% solution of tricaine for approximately 5 min. FACS sorting was performed as previously described with minor modifications. Worms were washed twice in 8 mL PBS, then triturated for approximately 15 min in 4 mL 0.125% Trypsin-EDTA (in PBS) until the solution became turbid and no large pieces of worms were left. The trypsin was inactivated by adding an 8 mL 10% FBS DMEM. The dissociated worms were then centrifuged at 500 g for 10 min at 4°C. Next, the worm cells were resuspended in 1 mL of PBS with 10 µL of RQ1 DNAse (Promega M6101) and incubated for 10 min at RT. The worm cells were centrifuged again at 500 g for 10 minutes at 4°C. Then the worm cells were resuspended in 1mL of PBS containing Fixable Viability Dye eFluor™ 506 (1µL/mL) (eBioscience) and incubated for 30 min at 4℃ in the dark. The worm cell suspension was centrifuged at 500 g for 10 minutes at 4°C, then washed twice in PBS, then filtered through a 40 µm cell strainer into a 12×75 mm FACS tube. Filtered cells were then sorted on a MoFlo XDP High-Speed Cell Sorter (BECKMAN COULTER) with 405/460/488/532/561/640nm lasers. Sorts were performed with a 100 µm nozzle, and cells were sorted into sorting media (0.04% BSA in PBS, pH = 7.40). Single-cell RNA sequencing FACS-sorted cells were centrifuged at 500 g for 10 minutes at 4°C and then resuspended in 0.04% BSA in PBS. Libraries were created using a Chromium Controller (10× Genomics) according to manufacturer guidelines and sequenced using a NovaSeq6000 (Illumina) to generate 150-bp paired-end reads. Raw data was processed and mapped to the Schistosoma japonicum genome (v3) using the Cell Ranger 3.0.0 (10× Genomics) pipeline. Single-cell RNA-seq data processing The Seurat package (v 3.1.5) was used to analyze the raw values of the matrix as follows 58 . Cells with greater than 30% mitochondrial reads or fewer than 500 genes were excluded from the analysis. Mitochondrial genes were identified with the prefix “^ND|^C|^ATP”. Each of the 7 individual datasets was scaled and transformed, and variable genes were identified using the SCTransform function with the parameter “variable.features.n = 3000”. All samples were combined using the functions “FingIntegrationAnchor” and “IntegrateData”. Then we performed principal component analysis using variable genes and used the first 100 principal components (PCs) to perform UMAP to embed the dataset into two dimensions. For clustering, we identified 76 clusters using the FindNeighbors and FindClusters function with the parameter “dims = 1:100, resolution = 2”. To determine cellular identity, we used the function FindAllMarkers with the parameter “test.use = "wilcox", only.pos = T, min.pct = 0.25, logfc.threshold = 0.25 ” to find DE genes for each cluster and compared to the known marker genes from the previous datasets 18 , 19 , 21 , 59 . GO (Gene Ontology) enrichment analysis was performed with the R package clusterProfiler 60 . The p-value was corrected for multiple hypothesis testing with the Benjamini–Hochberg false-discovery rate procedure (adjusted p-value ). GO terms with corrected p-value < 0.05 were considered significant. R packages ggplot2 ( https://ggplot2.tidyverse.org ) and tidyverse ( https://www.tidyverse.org ) were used to generate figures and intermediate data preprocessing. The density plots of cells from each sample over UMAP embedding were generated using a modified treecor_celldensityplot function from TreeCorTreat package ( https://github.com/byzhang23/TreeCorTreat ). Differential abundance analysis We tested for differences in cell-type abundances between female and male worms using the miloR package (v 1.4.0) 61 , which is a method for differential abundance analysis on KNN graphs from single-cell datasets. Briefly, we computed the k-nearest neighbor graph of cells in the integrated datasets based on latent embedding. Subsequently, cell neighborhoods were computed using miloR's makeNhoods function with parameter “ k = 25, prop = 0.2”. To leverage the variation in the number of cells between 18, 22, and 26 dpi samples, we counted the cells belonging to each sample in each neighborhood using the countCells function. We assigned to each neighborhood a cell-type label based on the majority voting of the cells belonging to that neighborhood. We then used the testNhoods function to test for enrichment of cells from the different sex datasets. Neighborhoods were enriched if the spatial FDR 1. Trajectory inference and RNA velocity To infer potential developmental trajectories within the neuronal lineages, we performed RNA Velocity analysis. In brief, we generated loom files of the spliced and unspliced RNA matrices using velocity (v 0.17.17) 62 with our CellRanger output. Then we applied scVelo (v 0.2.2) to estimate transient cell states and velocities using a generalized dynamical model following the recommended workflow. CellRank package (v 1.0.0) 63 was performed as described in ( https://cellrank.readthedocs.io/en/stable/index.html ) to characterize the cell fate decision process and determine initial and terminal states in data. The connectivity of cell populations was quantified using the partition-based graph abstraction (PAGA) method 64 . Then, we visualized the fate probability of each cell population as a pie chart using the “cellrank.pl.cluster_fates” functions with the parameter “mode = paga_pie”. To infer putative driver genes for any of these trajectories, we correlate expression values with fate probabilities using the “cellrank.tl.lineage_drivers” function with the parameter “method = Fischer”. In addition, we used the R package Slingshot (v 2.0.1) 65 as another independent method to analyze single-cell trajectories. We re-computed the UMAP embedding on subsetted neuronal and germline stem cell lineages identified in our integrated data set. Then, we used UMAP reduction to determine dimensionality and set Neuron stem cells or GSCs as start clusters to infer cell trajectory. Specifically, to identify temporally expressed genes changing in male and female gametogenesis, we used the GAM function implemented in the gam R package to investigate variable genes over pseudo time. To identify more significant genes along the pseudotime of germ cell lineage, we calculated the amplitude of change for each tested gene (with a cut-off of 0.5). We retained the top 25% of genes exhibiting the lowest q-values . Subsequently, we separately selected 750 and 693 variable genes from the male and female datasets, respectively, for visualization purposes. We then generated heatmaps of these genes using the R package ComplexHeatmap (v 2.12.0) 66 RNA interference (RNAi) DsRNA productions were essentially performed as previously described 47 . Oligo sequences used to generate dsRNA templates are listed in Supplementary Data 16. The dsRNA of the gfp ( green fluorescent protein ) fragment was used as a negative control for all RNAi experiments. Day 0 represents the first day of the experiment. Males were treated with 30 µg/mL dsRNA (on Day 0/2/4/6 with fresh medium) for one week in BM169 and then paired with virgin female parasites from Day 8 to Day 24 in ABC169 (fresh medium was replaced every other day). The eggs produced by the worms are retained when changing the medium. On Day 25, the egg number and the number of female parasites were counted to calculate egg/female/day. Bulk RNA-sequencing and data analysis We collected male worms from the gfp RNAi and lsamp RNAi groups on Day 20 for bulk RNA sequencing, with four biological replicates per group. Total RNA extraction, library construction, and RNA sequencing were performed by the Novogene Co., Ltd (Beijing, China). Briefly, RNA was extracted from worms using the AG RNAex Pro Reagent (Accurate Biotechnology (Hunan) Co., Ltd., China). The integrity and quality of total RNA were assessed using a Nanodrop ND-2000c spectrophotometer (Thermo Fisher Scientific, USA) and an Agilent 2100 Bioanalyzer (Agilent, USA). Pooled RNA was used to construct a library using Illumina TruSeq™ RNA sample prep Kit v2 (Illumina, USA) and the Ribo-Zero Plus rRNA Depletion Kit (Illumina, USA) for rRNA depletion, following the TruSeq RNA Sample Preparation Guide. The library was subsequently sequenced on the Illumina NovaSeq 6000 platforms to obtain 150 bp paired-end (PE) reads. QC (Quality control) of the raw sequencing data was performed using the FASTQC program ( http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Low-quality reads and adapter sequences were trimmed using the Fastp tool (v 0.20.1) (parameters: -q 15 -u 40 -n 5 -l 15) 67 . The clean reads were mapped to the chromosome-level S. japonicum reference genome ( Sj V3) using HISAT2 (v 2.1.0) with default parameters 68 . The gene-level assignment was then performed to estimate the transcript abundance using featureCounts 1.6.4 69 . PCA (Principal Component Analysis) was performed using the prcomp function in the R stats package (v 3.6.0). HCA (Hierarchical clustering analysis) was performed with pheatmap ( https://cran.r-project.org/web/packages/pheatmap/index.html ). Differential expression analysis was performed using DESeq2 70 , and the differentially expressed genes (DEGs) were defined with adjusted p-value 2. To identify the possible functions and pathways of DEGs, GO terms enrichment analysis and Gene Set Enrichment Analysis (GSEA) were performed using the clusterProfiler package 60 . Parasite labeling and imaging Colorimetric and fluorescence in situ hybridization were performed as previously described 22 , 57 . Riboprobes were synthesized from templates generated using primers listed in Supplementary Data 4 . Typically, all probes were applied at a concentration of 100 ng/mL in the hybridization buffer, with the concentrations of certain probes adjusted to optimize localization performance. The immunofluorescence assays for detecting Synapsin levels in the parasite were performed using anti-Synapsin (1:100, Clone 3C11, Developmental Studies Hybridoma Bank), and for detecting acetylated α-tubulin levels using anti-acetylated α-tubulin (1:500, Clone 6-11B-1, Santa Cruz) as previously described by 71 , 72 . Fast Blue BB labeling experiments were performed on females collected on Day 25, as previously reported 23 , 47 . All labeled parasites were counterstained with DAPI (1 µg/mL), cleared in 80% glycerol, and mounted on slides with 80% glycerol. Confocal imaging of fluorescently labeled samples was performed on Nikon A1 Laser Scanning Confocal Microscope or Olympus FV3000 Laser Scanning Confocal Microscope. Brightfield images were acquired on Olympus BX51 Microscope and Zeiss AxioZoom V16 Microscope. qRT-PCR (Quantitative Real-time PCR) All qPCR reactions were performed on a LightCycler® 96 Instrument (Roche, Switzerland) using 2× SYBR green qPCR master mix (Yeasen, China) according to the manufacturer's instructions. Each 20 µL qRT-PCR reaction mixture comprised 2 µL of cDNA(1:4), 10 µL 2× SYBR green master, 0.8 µL (5 µM) of each primer, and 6.4 µL ddH 2 O. The qRT-PCR cycle parameters were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s; melt curve analysis ranged from 60°C to 95°C to ensure that the specific product was amplified in each reaction. The 2 −ΔΔCt method was used to calculate the relative fold change of the differentially expressed transcripts. BATT synthesis and detection The synthesis of BATT was conducted by the Chinese Center for Disease Control and Prevention. Concentrated stock solutions (100 mM) of BATT were prepared in sterile DMSO and stored at -20℃. LC-MS/MS was employed for the detection of BATT in parasites (thirty male worms) and medium samples collected on Day 20, as previously described. BATT detection was performed on a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) employing negative ESI interface using QTRAP® 6500 + LC-MS/MS System, with the transitions set at m/z 232.4 → m/z 143.7, m/z 232.4 → m/z 185.1, m/z 232.4 → m/z 89.1, respectively. Quantification and statistical analysis GraphPad Prism software (USA) processed and presented the data as the mean with SD (Standard Deviation). Statistical significance was calculated by unpaired two-tailed parametric t -test. P values < 0.05 are considered significant ( ns , not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Error bars represent SD. Declarations Acknowledgements We would like to express our gratitude to the following individuals and institutions for their support and assistance: Bin Xu, Ying Wu, and Yang Hong from the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, for their support and assistance with experimental materials. The amino metabolome and BATT were quantified by Professor Huiru Tang's group as a paid service. Jingwei Zhang of Fudan University for his guidance and assistance in single-cell sample evaluation. This research was supported by National Natural Science Foundation of China (No. 31725025, and No. 31972699 to W.H.), the National Key Research and Development Program of China (No. 2021YFC2300800, and No. 2021YFC2300803 to J.P.W.), Science and Technology Leading Talent Team in Inner Mongolia Autonomous Region (No. 2022LJRC0009 to W.H.), and Shanghai Natural Science Foundation Young Scientists Program (No. 25ZR1402030 to Y.M.Y.). Author contributions W.H. and Y.M.Y. conceptualized this project. W.H. and J.P.W. supervised the overall research, and directed the bioinformatics analysis. W.H., J.P.W. and Y.M.Y. secured fundings. Y.M.Y. performed all experiments, and S.Y.C. analyzed the scRNA-seq and bulk RNA-seq data. Y.M.Y. and J.P.W. drafted the manuscript, with Y.M.Y., J.P.W., S.Y.C., and W.H. participating in manuscript revision. X.C., X.C., C.Y., and M.Q.C. provided experimental assistance in WISH and FISH. F.L., X.L.W., Y.X.X., W.B.Y., L.X., and M.J.G., assisted in sample processing for scRNA-seq. C.S.S. provided negative Oncomelania hupensis snails. L.L.H. contributed to BATT synthesis. G.W.C. and S.L. provided assistance in sample collection. All authors critically reviewed and approved the final manuscript. Competing interests The authors declare no competing interests. Data availability The single-cell RNA sequencing (scRNA-seq) data and the bulk RNA sequencing (RNA-seq) raw data for S. japonicum in this study can be accessed from NCBI Sequence Read Archive (SRA) using the accession number PRJNA1244463, The gene expression data for scRNA-seq have been deposited at the Gene Expression Omnibus (GEO) with the accession number GSE293642. References McManus, D.P. et al. Schistosomiasis. Nat Rev Dis Primers 4 , 13 (2018). Molehin, A.J. Schistosomiasis vaccine development: update on human clinical trials. J Biomed Sci 27 , 28 (2020). Bergquist, R., Utzinger, J. & Keiser, J. Controlling schistosomiasis with praziquantel: How much longer without a viable alternative? Infect Dis Poverty 6 , 74 (2017). Moore, D.V. & Sandground, J.H. The relative egg producing capacity of Schistosoma mansoni and Schistosoma japonicum. Am J Trop Med Hyg 5 , 831-840 (1956). Schwartz, C. & Fallon, P.G. Schistosoma "Eggs-Iting" the Host: Granuloma Formation and Egg Excretion. Front Immunol 9 , 2492 (2018). Popiel, I. & Basch, P.F. Reproductive development of female Schistosoma mansoni (Digenea: Schistosomatidae) following bisexual pairing of worms and worm segments. J Exp Zool 232 , 141-150 (1984). Armstrong, J.C. Mating Behavior and Development of Schistosomes in the Mouse. J Parasitol 51 , 605-616 (1965). AE, S. Sex studies on Schistosoma japonicum. Quarterly Journal of Microscopical Science s2 , 653 (1928). Chen, R. et al. A male-derived nonribosomal peptide pheromone controls female schistosome development. Cell 185 , 1506-1520.e1517 (2022). Wang, J. et al. Dynamic transcriptomes identify biogenic amines and insect-like hormonal regulation for mediating reproduction in Schistosoma japonicum. Nature Communications 8 , 14693 (2017). Waisberg, M. et al. Microarray analysis of gene expression induced by sexual contact in Schistosoma mansoni. BMC Genomics 8 , 181 (2007). Sun, J. et al. Transcriptome profilings of female Schistosoma japonicum reveal significant differential expression of genes after pairing. Parasitol Res 113 , 881-892 (2014). Gobert, G.N., Moertel, L., Brindley, P.J. & McManus, D.P. Developmental gene expression profiles of the human pathogen Schistosoma japonicum. BMC Genomics 10 , 128 (2009). Fitzpatrick, J.M., Johansen, M.V., Johnston, D.A., Dunne, D.W. & Hoffmann, K.F. Gender-associated gene expression in two related strains of Schistosoma japonicum. Mol Biochem Parasitol 136 , 191-209 (2004). Piao, X. et al. Global expression analysis revealed novel gender-specific gene expression features in the blood fluke parasite Schistosoma japonicum. PLoS One 6 , e18267 (2011). Hoffmann, K.F., Johnston, D.A. & Dunne, D.W. Identification of Schistosoma mansoni gender-associated gene transcripts by cDNA microarray profiling. Genome Biol 3 , RESEARCH0041 (2002). Anderson, L. et al. Schistosoma mansoni Egg, Adult Male and Female Comparative Gene Expression Analysis and Identification of Novel Genes by RNA-Seq. PLoS Negl Trop Dis 9 , e0004334 (2015). Wang, B. et al. Stem cell heterogeneity drives the parasitic life cycle of Schistosoma mansoni. eLife 7 , e35449 (2018). Diaz Soria, C.L. et al. Single-cell atlas of the first intra-mammalian developmental stage of the human parasite Schistosoma mansoni. Nat Commun 11 , 6411 (2020). Diaz Soria, C.L. et al. Single-cell transcriptomics of the human parasite Schistosoma mansoni first intra-molluscan stage reveals tentative tegumental and stem-cell regulators. Scientific Reports 14 (2024). Wendt, G. et al. A single-cell RNA-seq atlas of Schistosoma mansoni identifies a key regulator of blood feeding. Science 369 , 1644-1649 (2020). Collins, J.J., 3rd et al. Adult somatic stem cells in the human parasite Schistosoma mansoni. Nature 494 , 476-479 (2013). Wang, J., Chen, R. & Collins, J.J. Systematically improved in vitro culture conditions reveal new insights into the reproductive biology of the human parasite Schistosoma mansoni. PLOS Biology 17 , e3000254 (2019). Wang, J. & Collins, J.J., 3rd Identification of new markers for the Schistosoma mansoni vitelline lineage. Int J Parasitol 46 , 405-410 (2016). Hanumantha-Rao, K. Histochemistry of mehlis' gland and egg-shell formation in the liver flukeFasciola hepatica L. Experientia 15 , 464-465 (1959). Moczon, T., Swiderski, Z. & Huggel, H. Schistosoma mansoni: The chemical nature of the secretions produced by the Mehlis' gland and ootype as revealed by cytochemical studies. International Journal for Parasitology 22 , 65-73 (1992). Bergen, V., Lange, M., Peidli, S., Wolf, F.A. & Theis, F.J. Generalizing RNA velocity to transient cell states through dynamical modeling. Nature Biotechnology 38 , 1408-1414 (2020). Naren, P. et al. Microtubule acetylation dyshomeostasis in Parkinson’s disease. Translational Neurodegeneration 12 , 20 (2023). Deshpande, P. et al. N-Acetyltransferase 9 ameliorates Aβ42-mediated neurodegeneration in the Drosophila eye. Cell Death & Disease 14 (2023). Mondal, P. et al. Structure‐Based Discovery of A Small Molecule Inhibitor of Histone Deacetylase 6 (HDAC6) that Significantly Reduces Alzheimer's Disease Neuropathology. Advanced Science 11 (2024). Mazzetti, S. et al. Linking acetylated α-Tubulin redistribution to α-Synuclein pathology in brain of Parkinson’s disease patients. npj Parkinson's Disease 10 (2024). Esteves, A.R. et al. Acetylation as a major determinant to microtubule-dependent autophagy: Relevance to Alzheimer's and Parkinson disease pathology. Biochim Biophys Acta Mol Basis Dis 1865 , 2008-2023 (2019). Singh, K. et al. The combined impact of IgLON family proteins Lsamp and Neurotrimin on developing neurons and behavioral profiles in mouse. Brain Research Bulletin 140 , 5-18 (2018). Bregin, A. et al. Increased sensitivity to psychostimulants and GABAergic drugs in Lsamp-deficient mice. Pharmacology Biochemistry and Behavior 183 , 87-97 (2019). Bregin, A. et al. Expression and impact of Lsamp neural adhesion molecule in the serotonergic neurotransmission system. Pharmacology, Biochemistry, and Behavior 198 , 173017 (2020). Um, J.W. & Ko, J. Neural Glycosylphosphatidylinositol-Anchored Proteins in Synaptic Specification. Trends in Cell Biology 27 , 931-945 (2017). Chen, C.-H., Hsu, H.-W., Chang, Y.-H. & Pan, C.-L. Adhesive L1CAM-Robo Signaling Aligns Growth Cone F-Actin Dynamics to Promote Axon-Dendrite Fasciculation in C. elegans. Developmental Cell 48 , 215-228.e215 (2019). Schlager, M.A. & Hoogenraad, C.C. Basic mechanisms for recognition and transport of synaptic cargos. Molecular Brain 2 , 25 (2009). Peng, L. & Wang, T. Histamine synthesis and transport are coupled in axon terminals via a dual quality control system. The EMBO Journal 43 , 4472-4491 (2024). Pimenta, A.F. et al. The limbic system-associated membrane protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting. Neuron 15 , 287-297 (1995). Li, P. et al. Single-cell analysis of Schistosoma mansoni identifies a conserved genetic program controlling germline stem cell fate. Nature Communications 12 , 485 (2021). Naryzhny, S.N. Proliferating cell nuclear antigen: a proteomics view. Cellular and Molecular Life Sciences 65 , 3789-3808 (2008). Liu, C. et al. Research progress on the pathogenesis of the SDHB mutation and related diseases. Biomedicine & Pharmacotherapy 167 , 115500 (2023). Shkreta, L., Delannoy, A., Toutant, J. & Chabot, B. Regulatory interplay between SR proteins governs CLK1 kinase splice variants production. RNA 30 , 1596-1607 (2024). Sang, C. et al. Reversible acetylation of HDAC8 regulates cell cycle. EMBO Reports 25 , 3925-3943 (2024). Dodd, D.O. et al. Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules. Science 384 (2024). You, Y. et al. An improved medium for in vitro studies of female reproduction and oviposition in Schistosoma japonicum. Parasit Vectors 17 , 116 (2024). Li, X., Weth, O., Haeberlein, S. & Grevelding, C.G. Molecular characterization of Smtdc-1 and Smddc-1 discloses roles as male-competence factors for the sexual maturation of Schistosoma mansoni females. Frontiers in Cellular and Infection Microbiology 13 (2023). Nikishin, D.A., Alyoshina, N.M., Semenova, M.L. & Shmukler, Y.B. Analysis of Expression and Functional Activity of Aromatic L-Amino Acid Decarboxylase (DDC) and Serotonin Transporter (SERT) as Potential Sources of Serotonin in Mouse Ovary. International Journal of Molecular Sciences 20 , 3070 (2019). Khan, U.W. & Newmark, P.A. Somatic regulation of female germ cell regeneration and development in planarians. Cell Reports 38 , 110525 (2022). Alkema, M.J., Hunter-Ensor, M., Ringstad, N. & Horvitz, H.R. Tyramine Functions independently of octopamine in the Caenorhabditis elegans nervous system. Neuron 46 , 247-260 (2005). Martineau, M. et al. Storage and uptake of D-serine into astrocytic synaptic-like vesicles specify gliotransmission. J Neurosci 33 , 3413-3423 (2013). Li, D. et al. Astrocyte VAMP3 vesicles undergo Ca2+ -independent cycling and modulate glutamate transporter trafficking. The Journal of Physiology 593 , 2807-2832 (2015). Kumar, R. et al. Fibroblast Growth Factor 2‐Mediated Regulation of Neuronal Exosome Release Depends on VAMP3/Cellubrevin in Hippocampal Neurons. Advanced Science 7 , 1902372 (2020). Sneeggen, M. et al. WDFY2 restrains matrix metalloproteinase secretion and cell invasion by controlling VAMP3-dependent recycling. Nat Commun 10 , 2850 (2019). Luo, F. et al. A chromosome-level genome of the human blood fluke Schistosoma japonicum identifies the genomic basis of host-switching. Cell Reports 39 , 110638 (2022). Cheng, S. et al. Dynamic profiles of lncRNAs reveal a functional natural antisense RNA that regulates the development of Schistosoma japonicum. PLOS Pathogens 20 , e1011949 (2024). Satija, R., Farrell, J.A., Gennert, D., Schier, A.F. & Regev, A. Spatial reconstruction of single-cell gene expression data. Nature Biotechnology 33 , 495-502 (2015). Xia, J. et al. A single-cell resolution developmental atlas of hematopoietic stem and progenitor cell expansion in zebrafish. Proceedings of the National Academy of Sciences of the United States of America 118 , e2015748118 (2021). Yu, G., Wang, L.G., Han, Y. & He, Q.Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16 , 284-287 (2012). Dann, E., Henderson, N.C., Teichmann, S.A., Morgan, M.D. & Marioni, J.C. Differential abundance testing on single-cell data using k-nearest neighbor graphs. Nature Biotechnology 40 , 245-253 (2021). La Manno, G. et al. RNA velocity of single cells. Nature 560 , 494-498 (2018). Lange, M. et al. CellRank for directed single-cell fate mapping. Nat Methods 19 , 159-170 (2022). Wolf, F.A. et al. PAGA: graph abstraction reconciles clustering with trajectory inference through a topology preserving map of single cells. Genome Biol 20 , 59 (2019). Street, K. et al. Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics. BMC Genomics 19 , 477 (2018). Gu, Z., Eils, R. & Schlesner, M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32 , 2847-2849 (2016). Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34 , i884-i890 (2018). Kim, D., Paggi, J.M., Park, C., Bennett, C. & Salzberg, S.L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol 37 , 907-915 (2019). Liao, Y., Smyth, G.K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30 , 923-930 (2014). Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15 (2014). Collins, J.J., King, R.S., Cogswell, A., Williams, D.L. & Newmark, P.A. An Atlas for Schistosoma mansoni Organs and Life-Cycle Stages Using Cell Type-Specific Markers and Confocal Microscopy. PLoS Neglected Tropical Diseases 5 , e1009 (2011). Creppe, C. et al. Elongator Controls the Migration and Differentiation of Cortical Neurons through Acetylation of α-Tubulin. Cell 136 , 551-564 (2009). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryData1.xlsx Supplementary Data 1 SupplementaryData2.xlsx Supplementary Data 2 SupplementaryData3.xlsx Supplementary Data 3 SupplementaryData4.xlsx Supplementary Data 4 SupplementaryData5.xlsx Supplementary Data 5 SupplementaryData6.xlsx Supplementary Data 6 SupplementaryData7.xlsx Supplementary Data 7 SupplementaryData8.xlsx Supplementary Data 8 SupplementaryData9.xlsx Supplementary Data 9 SupplementaryData10.xlsx Supplementary Data 10 SupplementaryData11.xlsx Supplementary Data 11 SupplementaryData12.xlsx Supplementary Data 12 SupplementaryData13.xlsx Supplementary Data 13 SupplementaryData14.xlsx Supplementary Data 14 SupplementaryData15.xlsx Supplementary Data 15 SupplementaryData16.xlsx Supplementary Data 16 SupplementaryFig10.pdf SupplementaryFig11.pdf SupplementaryFig12.pdf SupplementaryFig1.pdf SupplementaryFig13.pdf SupplementaryFig14.pdf SupplementaryFig15.pdf SupplementaryFig16.pdf SupplementaryFig17.pdf SupplementaryFig18.pdf SupplementaryFig19.pdf SupplementaryFigsandDatalegends.docx SupplementaryFig2.pdf SupplementaryFig20.pdf SupplementaryFig21.pdf SupplementaryFig22.pdf SupplementaryFig23.pdf SupplementaryFig24.pdf SupplementaryFig25.pdf SupplementaryFig3.pdf SupplementaryFig4.pdf SupplementaryFig5.pdf SupplementaryFig6.pdf SupplementaryFig7.pdf SupplementaryFig8.pdf SupplementaryFig9.pdf Cite Share Download PDF Status: Published Journal Publication published 22 Jan, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6983189","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":482589473,"identity":"bebc60a6-e8da-4c63-b26b-01282a542e6d","order_by":0,"name":"Wei 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China","correspondingAuthor":false,"prefix":"","firstName":"Jipeng","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-06-26 11:50:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6983189/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6983189/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-68305-7","type":"published","date":"2026-01-22T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86391796,"identity":"be216e4b-68e5-4414-ab17-9972a32088cd","added_by":"auto","created_at":"2025-07-10 07:03:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":249233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDynamic single-cell transcriptomics of male and female\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e S. japonicum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e during the sexual development.\u003c/strong\u003e (a) Schematic workflow of the single-cell RNA sequencing (10× Genomics) for female and male\u003cem\u003e S. japonicum\u003c/em\u003e at 14 dpi (mixed sex), 18 dpi, 22 dpi, and 26 dpi\u003cem\u003e. \u003c/em\u003e(b) UMAP visualization of 104, 671 single-cell transcriptomes from seven samples, classified into 21 distinct cell types (including one unannotated “unknown” population). (c) Temporal changes in the proportions of male and female cell types across the four developmental stages. (d) Expression patterns of \u003cem\u003eeled \u003c/em\u003e(\u003cem\u003eSjc-0009220\u003c/em\u003e): (left) heatmap showing temporal expression dynamics; (right)\u003cem\u003e in situ\u003c/em\u003e hybridization localization in specific tissues (Ovary, Ov; Vitellocytes, Vi; Testis, Te). Scale bars = 100 μm.\u003c/p\u003e","description":"","filename":"Binder11.png","url":"https://assets-eu.researchsquare.com/files/rs-6983189/v1/687c6b93b4b629bac27f4ac3.png"},{"id":86391455,"identity":"d25a2d11-9270-462c-9c8a-a552064cb3e9","added_by":"auto","created_at":"2025-07-10 06:55:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":912857,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetailed lineage analysis of cells related to the reproductive development in both genders. \u003c/strong\u003e(a) (left) heatmaps showing tissue-specific markers and (right) \u003cem\u003ein situ\u003c/em\u003ehybridization of novel markers for characterizing the developmental status of reproductive organs: \u003cem\u003eSjc-0003040\u003c/em\u003e (Ovary, Ov), \u003cem\u003eSjc-0006798\u003c/em\u003e (\u003cem\u003eesg-2a\u003c/em\u003e) (Vitellocytes, Vi), \u003cem\u003eSjc-0000795\u003c/em\u003e (Mehlis’ gland, Mg), and \u003cem\u003eSjc-0000843\u003c/em\u003e(\u003cem\u003eribc2\u003c/em\u003e) (Testis, Te). Scale bars = 100 μm. (b) RNA velocity analysis of cells related to the reproductive development in both \u003cem\u003eS. japoncium\u003c/em\u003egenders. (c) Cell subclustering (left) and RNA velocity analysis (right) related to gametogenesis in\u003cem\u003e S. japonicum \u003c/em\u003efemales. (d) Cell subclustering (left) and RNA velocity analysis (right) related to gametogenesis in\u003cem\u003e S. japonicum \u003c/em\u003emales.\u003c/p\u003e","description":"","filename":"Binder12.png","url":"https://assets-eu.researchsquare.com/files/rs-6983189/v1/c0fa02eac2784fc62de5b217.png"},{"id":86391789,"identity":"266870b4-178e-48d8-8c8d-2081c917b616","added_by":"auto","created_at":"2025-07-10 07:03:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":553065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMale-specific neuron clusters in\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e S. japonicum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(a) Three neuron populations unique to male\u003cem\u003e S. japonicum \u003c/em\u003e(right) compared to females (left):\u003cem\u003e beta-2\u003c/em\u003e, \u003cem\u003egamma-2 \u003c/em\u003eand \u003cem\u003edelta-3\u003c/em\u003e. (b) FISH (Fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization) localization of neural marker genes (\u003cem\u003enrps\u003c/em\u003e, \u003cem\u003eSjc-0001200\u003c/em\u003e, \u003cem\u003eSjc-0003639\u003c/em\u003e) in adult male worms. Scale bars = 200 μm. (c) GO (Gene Ontology) enrichment analysis of highly expressed genes in \u003cem\u003ebeta-2\u003c/em\u003e, \u003cem\u003egamma-2 \u003c/em\u003eand \u003cem\u003edelta-3\u003c/em\u003e neurons. (d) Key driver genes (\u003cem\u003enrps\u003c/em\u003e, \u003cem\u003eSjc-0006754, \u003c/em\u003eand\u003cem\u003e Sjc-0000373\u003c/em\u003e) associated with the \u003cem\u003edelta-3\u003c/em\u003e neuron development. (e) Heatmaps showing three driver genes expressed in \u003cem\u003edelta-3\u003c/em\u003e neurons. (f) Localization of the \u003cem\u003elsamp\u003c/em\u003e gene in adult male worms: (left) \u003cem\u003ein situ\u003c/em\u003e hybridization; (right) double-FISH colocalization of \u003cem\u003elsamp\u003c/em\u003e with \u003cem\u003enrps\u003c/em\u003e or \u003cem\u003eddc \u003c/em\u003ein \u003cem\u003edelta-3\u003c/em\u003e neurons. Scale bars = 200 μm (left), Scale bars = 5 μm (right). (g) The expression profiles of\u003cem\u003e nrps\u003c/em\u003e, \u003cem\u003eddc\u003c/em\u003e, and \u003cem\u003elsamp\u003c/em\u003e in female (red line) and male (blue line)\u003cem\u003e S. japonicum\u003c/em\u003e based on previous RNA-seq data \u003csup\u003e10\u003c/sup\u003e. All of them exhibit male-specific and pairing-induced expression patterns.\u003c/p\u003e","description":"","filename":"Binder13.png","url":"https://assets-eu.researchsquare.com/files/rs-6983189/v1/da4b8f5b2e90ee31d5d29b3a.png"},{"id":86391485,"identity":"b31f1e63-4242-4b04-bd26-a7a966d3792b","added_by":"auto","created_at":"2025-07-10 06:55:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":592474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMale \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003elsamp\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e is essential for female sexual development.\u003c/strong\u003e (a) Flowchart of\u003cem\u003e in vitro \u003c/em\u003eRNAi interference and phenotypic observation. (b) Female worms paired with\u003cem\u003e lsamp\u003c/em\u003e RNAi males exhibited reduced FastBlue BB staining in vitellaria and underdeveloped ovaries. Scale bars = 200 μm. (c) Egg production was significantly reduced in female worms paired with \u003cem\u003elsamp\u003c/em\u003e RNAi-treated males. (d)\u003cem\u003e \u003c/em\u003eqRT-PCR validation of \u003cem\u003elsamp\u003c/em\u003e knockdown in male worms. (e) FISH localization of \u003cem\u003edelta-3\u003c/em\u003e neurons (\u003cem\u003eddc\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e cells) in the \u003cem\u003elsamp\u003c/em\u003e RNAi group and\u003cem\u003e gfp\u003c/em\u003e RNAi group (left), Scale bars = 50 μm; Quantification of \u003cem\u003edelta-3\u003c/em\u003e neurons (\u003cem\u003eddc\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e cells per 100 μm²) showed no significant change upon \u003cem\u003elsamp\u003c/em\u003e silencing (right). (f) qRT-PCR analysis confirmed that \u003cem\u003enrps\u003c/em\u003e expression was unaffected by \u003cem\u003elsamp\u003c/em\u003e downregulation. (g) LC-MS/MS results show levels of β-alanine remained unchanged in the \u003cem\u003elsamp\u003c/em\u003e RNAi group. (h) LC-MS/MS results show levels of tryptamine remained unchanged in the \u003cem\u003elsamp\u003c/em\u003e RNAi group. (i) LC-MS/MS results show BATT contents both in the worms (left) and the culture medium (right) were significantly reduced in the \u003cem\u003elsamp\u003c/em\u003e RNAi group.\u003cem\u003e ns\u003c/em\u003e, not significant; *,\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003et \u003c/em\u003etest; Error bars represent the SD based on three separate experiments.\u003c/p\u003e","description":"","filename":"Binder14.png","url":"https://assets-eu.researchsquare.com/files/rs-6983189/v1/0bfb48e6cfc77375aa97a35e.png"},{"id":86391800,"identity":"63659b35-cd22-4915-8926-c7a329486151","added_by":"auto","created_at":"2025-07-10 07:03:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1637030,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMale\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e lsamp \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eis essential for female sexual development by regulating acetylated α-tubulin levels and synaptic vesicle proteins. \u003c/strong\u003e(a) KEGG pathway analysis on the down-regulated genes set in \u003cem\u003elsamp\u003c/em\u003eRNAi worms, revealing significant involvement in neurodegenerative disease pathways (\u003cem\u003ee.g\u003c/em\u003e., Alzheimer's and Parkinson's diseases), which are associated with microtubule acetylation and axon vesicular transport. (b) Immunofluorescence co-staining of acetylated α-tubulin and synapsin revealed severe degeneration of neural network functionality in the\u003cem\u003e lsamp\u003c/em\u003eknockdown group. Scale bars = 20 μm. (c) The \u003cem\u003edelta-3\u003c/em\u003e neurons, labeled with \u003cem\u003enrps\u003c/em\u003e, \u003cem\u003eddc\u003c/em\u003e, and\u003cem\u003e lsamp\u003c/em\u003e probes individually, exhibited tight adherence to the neural axons labeled with acetylated α-tubulin. Scale bars = 10 μm. (d) The \u003cem\u003edelta-3\u003c/em\u003e neurons, labeled with \u003cem\u003enrps\u003c/em\u003e, \u003cem\u003eddc\u003c/em\u003e, and \u003cem\u003elsamp\u003c/em\u003e probes separately, showed tight adherence to the synapsin-labeled neural axons, demonstrating clear colocalization. Scale bars = 10 μm. (e) Development of ventral neural network in male schistosomes at 14, 18, 22, and 26 dpi, labeled with acetylated α-tubulin antibody, showing increasing robustness with maturation. 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07:03:09","extension":"pdf","order_by":41,"title":"","display":"","copyAsset":false,"role":"supplement","size":454696,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6983189/v1/4b151551700362609a90b85e.pdf"},{"id":86391808,"identity":"a1be17c6-994c-4f2a-a578-ecb4bfd508ff","added_by":"auto","created_at":"2025-07-10 07:03:08","extension":"pdf","order_by":42,"title":"","display":"","copyAsset":false,"role":"supplement","size":808389,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig9.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6983189/v1/95f31bee35d5930829a99e42.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Dynamic single-cell transcriptomics reveals lsamp-guided neural network formation in male S. japonicum driving female reproduction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSchistosomiasis is a prevalent and devastating neglected tropical disease caused by parasitic flatworms called \u003cem\u003eSchistosoma\u003c/em\u003e spp., which affects over 250\u0026nbsp;million people in disadvantaged areas \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Currently, no vaccine is available, and treatment relies on a single drug (praziquantel) \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, new therapeutic and preventive strategies are urgently needed. Eggs produced by mature female schistosomes drive the pathology and transmission of this disease. In a definitive mammalian host, one mature female parasite could produce hundreds to thousands of eggs daily that are deposited in the host tissues, \u003cem\u003ee.g\u003c/em\u003e., the liver, where they induce the local granuloma and further lead to tissue fibrosis \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Eggs excluded with feces or urine hatch in the water environment, and then the released miracidia invade the intermediate snail host before spreading. Therefore, developing strategies for obstructing the egg production of these parasites will conduce to the control of this disease.\u003c/p\u003e\u003cp\u003eSchistosomes have a unique reproductive feature. The pairing with the male worm initiates the sexual development of a female worm and maintains her mature state \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Unlike females, the male worm can reach sexual maturity independently \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Continued investigations have been carried out to uncover the underlying mechanism of this male-induced female reproduction since the first report of the phenomenon in \u003cem\u003eS. japonicum\u003c/em\u003e nearly a century ago \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Recently, a dipeptide pheromone (β-alanyl-tryptamine) synthesized and secreted by male worms has been identified in \u003cem\u003eS. mansoni\u003c/em\u003e that stimulates female sexual development \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The discovery of this neuron-derived key molecular that can be transmitted from males to females highlights the importance of understanding the complex network during male-female interplay.\u003c/p\u003e\u003cp\u003eThe direct physical connection between males and females occurs at their late juvenile stage when the male worm clasps the female through the gynecophoric canal to form a worm pair \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Thereafter, the coupled male and female continue to grow and turn to be sexually mature. Besides the extension of body length, a series of previous studies have observed dramatic gene expression changes in males and females upon pairing \u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. These changes are not only restricted to the sexual organs but also somatic tissues, such as neurons and muscles. Additionally, labor division is more apparent between the two genders at their adult stage, which is represented by the distinct sex-associated gene expression patterns \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Recently, researchers have revealed that the cell types of \u003cem\u003eS. mansoni\u003c/em\u003e expanded along with the growth and development of the parasite in the mammalian hosts \u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Notably, single-cell RNA sequencing showed distinguished cell clusters between the adult male and female and between the virgin female and mature female in \u003cem\u003eS. mansoni\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, which highly indicates a dynamic sex-related cellular differentiation during the male-female interaction. However, the whole picture of the molecular changes of males and females during their interaction at the single-cell level is still lacking.\u003c/p\u003e\u003cp\u003eIn this study, we profiled the dynamic single-cell atlas of male and female \u003cem\u003eS. japonicum\u003c/em\u003e across 4 time points throughout the reproductive development during their interplay. We sequenced 104, 671 single-cell transcriptomes and characterized 76 molecularly distinct cell populations. Along with the male-female interaction, the cell clusters of the reproductive and nervous systems undergo the most dramatic changes in both sexes. RNA velocity analysis revealed the detailed lineage of cells related to the reproductive development in both genders. We analyzed and predicted gene expression patterns and key regulatory factors involved in the maturation of male and female gametes. Furthermore, we noticed a complex heterogeneity and sex-biased characteristics of the nervous system during the maturation of the parasite. Notably, we identified a \u003cem\u003elsamp\u003c/em\u003e gene expressed within a male-specific neuron population, which triggers female reproductive development by fostering axon growth within the male gynecophoric canal in \u003cem\u003eS. japonicum\u003c/em\u003e. Taken together, our datasets provide the comprehensive single-cell atlas of male and female \u003cem\u003eS. japonicum\u003c/em\u003e across their maturation and reveal the key regulator of a male-specific neuron that is required for the stimulus of female sexual development, which will further prompt the innovation of strategies against this human parasite.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eDynamic single-cell transcriptomes of male and female\u003c/b\u003e \u003cb\u003eS. japonicum\u003c/b\u003e \u003cb\u003eduring their interplay\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eS. japonicum\u003c/em\u003e primarily undergoes pairing and sexual maturation between 14\u0026ndash;26 dpi (days post-infection) \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. To characterize the dynamic single-cell atlas of \u003cem\u003eS. japonicum\u003c/em\u003e throughout the female-male pairing and subsequential maturation process, we collected worms at four-time points (14, 18, 22, 26 dpi) from the mice model (\u003cb\u003eFig.\u0026nbsp;1a\u003c/b\u003e). Whole worms were separated by sex at each stage, except for those from 14 dpi (as gender could not be distinguished morphologically at this stage). Subsequently, we dissociated these samples and isolated live single cells using fluorescence-activated cell sorting (FACS). Utilizing a 10\u0026times; Genomics chromium controller, we generated 7 single-cell RNA-seq (scRNA-seq) libraries (\u003cb\u003eFig.\u0026nbsp;1a, and Supplementary Data 1\u003c/b\u003e). Through the analysis of these libraries using Cell Ranger, we obtained a total of 110,204 cells (\u003cb\u003eSupplementary Data 1\u003c/b\u003e). After excluding cells with more than 30% mitochondrial reads or fewer than 500 detected genes, the final count was 104, 671 cells, which were further clustered into 76 distinct cell clusters (\u003cb\u003eSupplementary Fig.\u0026nbsp;1, and Supplementary Data 1\u003c/b\u003e). Furthermore, we annotated the identities of these cell clusters by referring to cell markers defined in the single-cell sequencing of \u003cem\u003eS. mansoni\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eSupplementary Data 2\u003c/b\u003e). In total, the \u003cem\u003eS. japonicum\u003c/em\u003e single-cell atlas comprised 33 transcriptionally distinct neuron cell clusters, 14 neoblast clusters, 9 tegument cell clusters, 5 muscle cell clusters, 5 parenchyma clusters, 3 germ stem cell (GSC) clusters, 1 vitellocyte cluster, 1 male gamete cluster, 1 Mehlis' gland cell cluster, 1 gut cell cluster, 1 flame cell cluster, 1 esophageal gland cell cluster, and 1 unknown cluster (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eOur single-cell atlas provided detailed lineage information for the parasite\u0026rsquo;s specific tissues. Interestingly, our data unexpectedly revealed many schistosome neural stem cells and neural precursor cells for the first time. In comparison, the single-cell atlas of \u003cem\u003eS. mansoni\u003c/em\u003e depicts neural cells as scattered clusters lacking apparent lineage connections \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Based on their marked heterogeneity, we classified the neural cells into five categories: neural stem cells, neural precursor cells, \u003cem\u003ekk7\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e neurons, nonciliated neurons, and ciliated neurons (\u003cb\u003eFig.\u0026nbsp;1b)\u003c/b\u003e. The tegument cells are divided into tegument progenitor, tegument progeny 1, tegument progeny 2, syncytial 1, and syncytial 2 (\u003cb\u003eFig.\u0026nbsp;1b\u003c/b\u003e). In addition to the well-defined cell markers in previous studies on \u003cem\u003eS. mansoni\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, we further identified the marker genes as shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;2 and Supplementary Data 3\u0026ndash;4\u003c/b\u003e through WISH (whole-mount \u003cem\u003ein situ\u003c/em\u003e hybridization), including new markers such as \u003cem\u003eSjc-0003010\u003c/em\u003e and \u003cem\u003eSjc-0008393\u003c/em\u003e for GSCs (germline stem cells) (\u003cb\u003eSupplementary Fig.\u0026nbsp;3a\u003c/b\u003e), \u003cem\u003eSjc-0001566\u003c/em\u003e (\u003cem\u003eccdc\u003c/em\u003e) for male gametes (\u003cb\u003eSupplementary Fig.\u0026nbsp;3c\u003c/b\u003e), \u003cem\u003eSjc-0004922\u003c/em\u003e (\u003cem\u003eTubulin alpha-1 chain\u003c/em\u003e) for neoblasts (\u003cb\u003eSupplementary Fig.\u0026nbsp;3e\u003c/b\u003e), \u003cem\u003eSjc-0008047\u003c/em\u003e (\u003cem\u003eklf11\u003c/em\u003e) for neural stem cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;3f\u003c/b\u003e), \u003cem\u003eSjc-0005931\u003c/em\u003e (\u003cem\u003eacss2\u003c/em\u003e) and \u003cem\u003eSjc-0007590\u003c/em\u003e (\u003cem\u003epcdh7\u003c/em\u003e) for neural precursor cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;3g\u003c/b\u003e), \u003cem\u003eSjc-0002588\u003c/em\u003e (\u003cem\u003eslc6a5\u003c/em\u003e) for parenchyma cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;3i\u003c/b\u003e), \u003cem\u003eSjc-0001890\u003c/em\u003e (\u003cem\u003esgf1\u003c/em\u003e) for esophageal gland cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;3k\u003c/b\u003e), \u003cem\u003eSjc-0008416\u003c/em\u003e (\u003cem\u003ecpb\u003c/em\u003e) and \u003cem\u003eSjc-0000095\u003c/em\u003e (\u003cem\u003ecacnb1\u003c/em\u003e) for muscle cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;3m\u003c/b\u003e), \u003cem\u003eSjc-0008478\u003c/em\u003e and \u003cem\u003eSjc-0000277\u003c/em\u003e (\u003cem\u003eSlc7a14\u003c/em\u003e) for syncytial 2 of tegument cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;3n\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eWe first analyzed the distribution of cells in each sample, which allowed us to identify developmental differences at the cellular level between male and female worms at various stages (\u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e). The most significant differences observed between male and female worms were primarily related to cell types associated with the reproductive system. As the worms mature, the number of GSCs and vitellocyte cells in females gradually increases, while the number of male gametes rises in males (\u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e). To gain a general view of the changes in different cell clusters in male and female worms during their pairing, we analyzed the dynamics of the proportion for each cell type. In both sexes, neoblasts and neuronal cells individually accounted for ~\u0026thinsp;20% of the total cells, representing the two largest cell populations in \u003cem\u003eS. japonicum\u003c/em\u003e (\u003cb\u003eFig.\u0026nbsp;1c, and Supplementary Data 5\u003c/b\u003e). The proportion of neural stem cells and precursor cells decreased after 14 dpi, while the populations of differentiated neural cells (\u003cem\u003ee.g\u003c/em\u003e., nonciliated neurons and ciliated neurons) dramatically expanded (\u003cb\u003eFig.\u0026nbsp;1c, and Supplementary Data 5\u003c/b\u003e). This finding indicates that neural progenitor cells are active during the juvenile stage, but gradually degenerate in the adulthood. Interestingly, the proportion of mature neurons in male worms continues to rise, whereas in female worms, it initially increases before eventually declining (\u003cb\u003eFig.\u0026nbsp;1c, Supplementary Fig.\u0026nbsp;5, and Supplementary Data 5\u003c/b\u003e). In addition, the proportion of the reproduction-related cell types gradually increased throughout the sexual maturation process in each sex as expected, such as germline stem cells, female vitellocytes, and male gametes (\u003cb\u003eFig.\u0026nbsp;1c, Supplementary Fig.\u0026nbsp;5, and Supplementary Data 5)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eAdditionally, the proportions of parenchyma and flame cells were enlarged in females and males along with their development (\u003cb\u003eFig.\u0026nbsp;1c, Supplementary Fig.\u0026nbsp;5, and Supplementary Data 5\u003c/b\u003e). However, the percentage of muscle cells declined sharply in both genders after 14 dpi, especially in male worms (\u003cb\u003eFig.\u0026nbsp;1c, Supplementary Fig.\u0026nbsp;5, and Supplementary Data 5\u003c/b\u003e). For neoblasts, a clear decrease was observed in male parasites across all four-time points. In females, however, the decrease was evident from 14 to 18 dpi, followed by an expansion as female development progressed. We further examined the tegument, which includes well-defined cell types from the progenitors to the progenies. Unlike the neuronal populations, the proportion of tegument progenitors remained stable even as the proportion of their progenitor cells and differentiated syncytial cells increased for both branches \u003cb\u003e(Fig.\u0026nbsp;1c, Supplementary Fig.\u0026nbsp;5, and Supplementary Data 5)\u003c/b\u003e. This differing pattern of changes may be attributed to the tegument requiring renewal: as the interface between the host and parasite, the tegument needs rapid turnover to replace damaged or aging tissues, thereby maintaining the activity of progenitor cells. To validate the dynamic changes in cell populations along the male-female pairing indicated by our single-cell atlas, we performed WISH using the germline cell marker \u003cem\u003eeled\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. As shown in \u003cb\u003eFig.\u0026nbsp;1d\u003c/b\u003e, the expression region of \u003cem\u003eeled\u003c/em\u003e expanded significantly in both female and male sexual organs from 18 to 26 dpi, which was consistent with the single-cell analysis data.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDetailed lineage analysis of cells related to the reproductive development in both genders\u003c/h2\u003e\u003cp\u003eThe schistosome germ cells originate from a subset of stem cells at the asexual stage and eventually separate from somatic cell lineage at the juvenile stage \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Upon pairing, the committed GSCs in both sexes proliferate and differentiate into mature gametes. To demonstrate the cellular dynamics of the parasite's reproductive system and the relationship between germline and somatic lineages, we first subset the cells defined as stem cells (neoblasts) and those present in the reproductive organs, including GSCs, male gametes, female gametes, vitellocytes, and Mehlis' glands, from both female and male samples (18, 22, and 26 dpi), and performed sub-cluster analysis. Then, we identified 17 clusters with representative markers for each subtype based on well-defined markers from previous studies (\u003cb\u003eSupplementary Fig.\u0026nbsp;6a, and Supplementary Data 2)\u003c/b\u003e, such as the neoblast marker \u003cem\u003enanos-2\u003c/em\u003e (\u003cem\u003eSjc-0005962\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, the vitellocyte S1 cell maker \u003cem\u003evf1\u003c/em\u003e (\u003cem\u003eSjc-0000108\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, the germline stem cell marker \u003cem\u003enanos-1\u003c/em\u003e (\u003cem\u003eSjc-0006622\u003c/em\u003e) and \u003cem\u003eeled\u003c/em\u003e (\u003cem\u003eSjc-0009220\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;6b-c)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eWe also validated newly identified tissue-specific markers using WISH, including \u003cem\u003eSjc-0000843\u003c/em\u003e (\u003cem\u003eribc2\u003c/em\u003e, \u003cem\u003eRIB43A-like with coiled-coils protein 2\u003c/em\u003e) for male gametes, \u003cem\u003eSjc-0003040\u003c/em\u003e for female gametes, \u003cem\u003eesg-2a\u003c/em\u003e (\u003cem\u003eSjc-0006798\u003c/em\u003e) for vitelline cells, and \u003cem\u003eSjc-0000795\u003c/em\u003e for Mehlis' gland (\u003cb\u003eFig.\u0026nbsp;2a, and Supplementary Fig.\u0026nbsp;6b-c\u003c/b\u003e). These markers can effectively characterize the developmental status of each organ. Interestingly, female gametes were also observed in male worms, albeit in relatively low numbers (\u003cb\u003eSupplementary Fig.\u0026nbsp;6a, c-d\u003c/b\u003e). To confirm the presence of female gamete cells in males, we performed WISH using the female gamete marker gene \u003cem\u003eSjc-0004512\u003c/em\u003e (\u003cem\u003eclec, C-type lectin domain-containing protein\u003c/em\u003e) and observed a signal in the testes of male worms (\u003cb\u003eSupplementary Fig.\u0026nbsp;6d\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eBy performing RNA velocity analysis, we inferred the possible developmental trajectories from germline stem cells to mature gametes, separately for female and male worms (\u003cb\u003eFig.\u0026nbsp;2b\u003c/b\u003e). To further investigate the differences in the germline lineage, we further performed RNA velocity analysis on the GSCs, GSC progenies, and the gametes of males and females. It revealed the cell trajectory originating from the GSCs population into mature gametes, in both sexes (\u003cb\u003eFig.\u0026nbsp;2c-d)\u003c/b\u003e. Subsequently, we analyzed the expression patterns of genes involved in the differentiation of male or female gametes, including sex-specific highly expressed genes and highly expressed genes shared by both sexes (\u003cb\u003eSupplementary Fig.\u0026nbsp;7a, and Supplementary Data 6\u0026ndash;8\u003c/b\u003e). We found that male and female gametes shared 156 genes in their lineages that could be further clustered into five modules (\u003cb\u003eSupplementary Fig.\u0026nbsp;7b, and Supplementary Data 6\u003c/b\u003e). Most of these genes, represented in C2-C5, showed similar expression trends in both genders. Interestingly, genes in the C1 module exhibited opposite expression patterns during the maturation of male and female gametes. Highly expressed genes in female gametogenesis also could be clustered into five modules (\u003cb\u003eSupplementary Fig.\u0026nbsp;7c, and Supplementary Data 7\u003c/b\u003e): The C1 module shows a gradual downregulation in expression as gametes mature, such as \u003cem\u003eSjc-0003683\u003c/em\u003e and \u003cem\u003eSjc-0006860\u003c/em\u003e (\u003cem\u003ehist1h1a\u003c/em\u003e); The C2 module exhibits an initial slight downregulation followed by a rapid upregulation during gamete maturation, such as \u003cem\u003eSjc-0003078\u003c/em\u003e and \u003cem\u003eSjc-0009371\u003c/em\u003e (\u003cem\u003enars\u003c/em\u003e, \u003cem\u003eAsparagine-tRNA ligase, cytoplasmic\u003c/em\u003e); The C3 and C4 modules maintain a constant expression level in the early stages, followed by a rapid upregulation in the mid to late stages, such as \u003cem\u003eSjc-0003040, Sjc-0004512\u003c/em\u003e (\u003cem\u003eclec), Sjc-0004939\u003c/em\u003e (\u003cem\u003eerc2\u003c/em\u003e, \u003cem\u003eERC protein 2\u003c/em\u003e) and \u003cem\u003eSjc-0000072\u003c/em\u003e; The C5 module showed a positive correlation with gamete development, before stabilizing at a constant expression level during the later stages, as seen with \u003cem\u003eSjc-0008219\u003c/em\u003e (\u003cem\u003ehormad1\u003c/em\u003e, \u003cem\u003ehorma domain-containing protein 1\u003c/em\u003e) and \u003cem\u003eSjc-0001311.\u003c/em\u003e Highly expressed genes in male gametogenesis can also be clustered into five modules (\u003cb\u003eSupplementary Fig.\u0026nbsp;7d, and Supplementary Data 8\u003c/b\u003e): The gene expression trends in modules C1 to C4 are similar, showing a negative regulatory relationship with the gamete maturation, including \u003cem\u003eSjc-0003673\u003c/em\u003e (\u003cem\u003egpd1\u003c/em\u003e, \u003cem\u003eGlycerol-3-phosphate dehydrogenase\u003c/em\u003e) and \u003cem\u003eSjc-0004527\u003c/em\u003e (\u003cem\u003eeef1b\u003c/em\u003e, \u003cem\u003eElongation factor 1-beta\u003c/em\u003e), while the C5 module exhibits a positive regulation, as exemplified by \u003cem\u003eSjc-0002056\u003c/em\u003e (\u003cem\u003ezinc finger CCCH domain-containing protein 31\u003c/em\u003e) and \u003cem\u003eSjc-0000843\u003c/em\u003e (\u003cem\u003eribc2\u003c/em\u003e). The above results demonstrate that the development of male and female gametes is a complex process regulated by multiple gene modules. Further functional validation is required to explore the mechanisms of common and specific genes related to female or male gametogenesis.\u003c/p\u003e\u003cp\u003eVitellaria is the largest reproductive organ in adult female schistosomes that occupies two-thirds of the body's length. We found that both vitellaria S1 cells and GSCs express a certain level of \u003cem\u003eeled\u003c/em\u003e (\u003cem\u003eSjc-0009220\u003c/em\u003e) (\u003cb\u003eSupplementary Fig.\u0026nbsp;6b-c\u003c/b\u003e), suggesting a potential shared origin from a population of \u003cem\u003eeled\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e stem cells, as previously reported \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. We analyzed the developmental relationship between the vitellaria and the female germline cells through RNA velocity. However, we did not find any connection between S1 vitellaria cells and GSCs in \u003cb\u003eFig.\u0026nbsp;2b\u003c/b\u003e, as the two cell clusters were spatially distant.\u003c/p\u003e\u003cp\u003eVitellaria maturation is crucial for egg production in schistosomes. Previous studies have shown that vitellaria development can be divided into stages S1-S4, all of which express \u003cem\u003evf1\u003c/em\u003e (\u003cem\u003eSjc-0000108\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In the single-cell transcriptomic analysis of \u003cem\u003eS. mansoni\u003c/em\u003e, the vitellaria development process was further categorized into five stages: S1, S1 progeny, early vitellocytes, late vitellocytes, and mature vitellocytes \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, the detailed differentiation trajectory of vitellaria cells has not been fully elucidated at the single-cell level. In our study, we classified the vitellaria development process into four stages based on clustering results: S1 vitellocytes, S1 vitellocyte progeny, early vitellocytes (early vi), and late vitellocytes (late vi). Using RNA velocity, we inferred the developmental trajectory of vitellaria, revealing that S1 vitellocyte cells (\u003cem\u003evf1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e \u003cem\u003eesg-2a\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e) gradually differentiate into S1 vitellocyte progenies (\u003cem\u003evf1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e \u003cem\u003eesg-2a\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e), which subsequently give rise to early vitellocytes and late vitellocytes \u003cb\u003e(Fig.\u0026nbsp;2b, and Supplementary Fig.\u0026nbsp;8)\u003c/b\u003e. This trajectory aligns with previous findings and provides new insights into the molecular mechanisms underlying vitellaria maturation. Further functional studies on key markers such as \u003cem\u003evf1\u003c/em\u003e and \u003cem\u003eesg-2a\u003c/em\u003e will enhance our understanding of their roles in vitellaria development and their potential as therapeutic targets.\u003c/p\u003e\u003cp\u003eMehlis' gland, a specialized organ unique to trematodes and cestodes, has been long speculated to play an essential role in eggshell formation and the release of eggshell granules \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. However, research on its function in schistosomes remains limited. In our study, we observed that Mehlis' gland cells emerge earlier than vitelline cells, appearing in large numbers by 18 dpi, while vitelline cells do not proliferate in large numbers until 22 dpi (\u003cb\u003eSupplementary Fig.\u0026nbsp;9a-b\u003c/b\u003e). These findings were further validated through WISH experiments using the marker gene \u003cem\u003eSjc-0000795\u003c/em\u003e for Mehlis' gland, the marker gene \u003cem\u003eesg-2a\u003c/em\u003e for vitellaria and the \u003cem\u003emarker gene Sjc-0000278\u003c/em\u003e (\u003cem\u003erreb1\u003c/em\u003e) for both vitellaria and Mehlis' gland (\u003cb\u003eSupplementary Fig.\u0026nbsp;9c\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFive neural lineages in\u003c/b\u003e \u003cb\u003eS. japonicum\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUnlike the scattered neuronal clusters revealed from the previously reported single-cell transcriptomic atlas of adult \u003cem\u003eSchistosoma mansoni\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, our datasets exhibit high connectivity among complex neuronal clusters (\u003cb\u003eFig.\u0026nbsp;1b, and Supplementary Fig.\u0026nbsp;1\u003c/b\u003e), which is likely to enable cell lineage reconstruction of the whole schistosome nervous system from transcriptomic data. We first reclustered all the neural cells of males and females, including neural stem cells, neural precursor cells, nonciliated neurons, and ciliated neurons, identifying 19 subclusters and summarizing them into 5 lineages, which were named \u003cem\u003ealpha\u003c/em\u003e lineage, \u003cem\u003ebeta\u003c/em\u003e lineage, \u003cem\u003egamma\u003c/em\u003e lineage, \u003cem\u003edelta\u003c/em\u003e lineage, and \u003cem\u003eepsilon\u003c/em\u003e lineage (\u003cb\u003eSupplementary Fig.\u0026nbsp;10a, and 11a\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eSubsequently, we employed Slingshot to confirm the reconstructed cell differentiation lineage and infer pseudotime (\u003cb\u003eSupplementary Fig.\u0026nbsp;10b, and 11b\u003c/b\u003e). Additionally, we performed RNA velocity analysis on the male and female datasets separately using scvelo algorithms \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. We visualized the paths predicted by the mRNA velocity model on UMAP, predicting a potential differentiation trajectory from neural progenitor cells toward mature cell types (\u003cb\u003eSupplementary Fig.\u0026nbsp;10c, and 11c\u003c/b\u003e). We then combined trajectory inference with RNA velocity to compute fate probabilities. In male \u003cem\u003eS. japonicum\u003c/em\u003e, we identified 7 metastable states in the neural cells, with neural progenitor cells predicted as the initial state and \u003cem\u003edelta-2\u003c/em\u003e neuron, \u003cem\u003edelta-3\u003c/em\u003e neuron, \u003cem\u003edelta-4\u003c/em\u003e neuron, \u003cem\u003ealpha\u003c/em\u003e-1 neuron, \u003cem\u003egamma-2\u003c/em\u003e neuron, and \u003cem\u003ebeta-2\u003c/em\u003e neuron inferred as six terminal states (\u003cb\u003eSupplementary Fig.\u0026nbsp;10d\u003c/b\u003e). In female \u003cem\u003eS. japonicum\u003c/em\u003e, we identified 7 metastable states in the neural cells, with neural progenitor cells predicted as the initial state and \u003cem\u003edelta-1\u003c/em\u003e neuron, \u003cem\u003edelta-2\u003c/em\u003e neuron, \u003cem\u003eepsilon\u003c/em\u003e neuron, \u003cem\u003ealpha\u003c/em\u003e-1 neuron, \u003cem\u003egamma-1\u003c/em\u003e neuron, and \u003cem\u003ebeta-1\u003c/em\u003e neuron inferred as six terminal states (\u003cb\u003eSupplementary Fig.\u0026nbsp;11d\u003c/b\u003e). Using a partition-based graph abstraction (PAGA) approach, we further quantified the connectivity of cell populations and visualized the fate probability of each cell population as a pie chart \u003cb\u003e(Supplementary Fig.\u0026nbsp;10e, and 11e\u003c/b\u003e). In summary, we defined neural cell differentiation from an early neural progenitor state to several mature states and provided a mechanistic framework for characterizing worm neuronal cell fate decisions.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMale-specific neuron clusters\u003c/h3\u003e\n\u003cp\u003eTo explore the neuronal diversity in \u003cem\u003eS. japonicum\u003c/em\u003e, we performed a subcluster analysis of neuronal cells in both male and female parasites (\u003cb\u003eSupplementary Fig.\u0026nbsp;12a)\u003c/b\u003e. From the integrated single-cell atlas of both sexes, we identified three neuron populations uniquely present in males: \u003cem\u003ebeta-2\u003c/em\u003e, \u003cem\u003egamma-2\u003c/em\u003e, and \u003cem\u003edelta-3\u003c/em\u003e (\u003cb\u003eFig.\u0026nbsp;3a).\u003c/b\u003e The proportional distribution of neuronal subtypes in both sexes (\u003cb\u003eSupplementary Fig.\u0026nbsp;12b\u003c/b\u003e) and the differential abundance analysis of neuronal cell types across genders (\u003cb\u003eSupplementary Fig.\u0026nbsp;12c\u003c/b\u003e) further confirmed the existence of these male-specific neurons. We next investigated their spatial distribution within the parasite. From the enriched genes in each population, we first identified the potential markers for \u003cem\u003ebeta-2\u003c/em\u003e, \u003cem\u003egamma-2\u003c/em\u003e, and \u003cem\u003edelta-3\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;13).\u003c/b\u003e We then conducted WISH and double-FISH using RNA probes targeting these markers, selecting specific markers to define each population: \u003cem\u003ebeta-2\u003c/em\u003e (\u003cem\u003eSjc-0003639\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eSjc-0000814\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e), \u003cem\u003egamma-2\u003c/em\u003e (\u003cem\u003eSjc-0001200\u003c/em\u003e), and \u003cem\u003edelta-3\u003c/em\u003e (\u003cem\u003eSjc-0000814\u003c/em\u003e). As anticipated, all these cells expressed the pan-neuronal marker \u003cem\u003e7b2\u003c/em\u003e \u003csup\u003e21\u003c/sup\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;14a-c\u003c/b\u003e). Interestingly, all three of these neurons were primarily located in the gynecophoric canal, a specialized male structure that secures the female during pairing (\u003cb\u003eFig.\u0026nbsp;3b, and 14a-c\u003c/b\u003e). Specifically, unlike the \u003cem\u003edelta-3\u003c/em\u003e population, which was exclusively distributed throughout the male gynecophoric canal, the \u003cem\u003ebeta-2\u003c/em\u003e and \u003cem\u003egamma-2\u003c/em\u003e populations were also found in the head of male and female parasites (\u003cb\u003eFig.\u0026nbsp;3b, and 14a-c\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eInterestingly, the marker gene \u003cem\u003eSjc-0000814\u003c/em\u003e of \u003cem\u003edelta-3\u003c/em\u003e is \u003cem\u003enrps\u003c/em\u003e (\u003cem\u003enonribosomal peptide synthetase\u003c/em\u003e) which encodes NRPS enzyme. In \u003cem\u003eS. mansoni\u003c/em\u003e, NRPS enzyme has been reported to synthesize β-alanyl-tryptamine (BATT), a dipeptide pheromone that passed from male to induce female reproductive development \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Thus, our study clarified the \u003cem\u003edelta-3\u003c/em\u003e neurons are the key populations that regulate female sexual development by expressing the \u003cem\u003enrps\u003c/em\u003e to produce BATT.\u003c/p\u003e\u003cp\u003eFurthermore, we identified the localization of additional mature neuronal populations using the RNA probes targeting specific markers, including \u003cem\u003ebeta-1\u003c/em\u003e (\u003cem\u003eano7\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e), \u003cem\u003egamma-1\u003c/em\u003e (\u003cem\u003eSjc-0006108\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e), \u003cem\u003edelta-1\u003c/em\u003e (\u003cem\u003ebmp, Sjc-0004182\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e), \u003cem\u003edelta-4\u003c/em\u003e (\u003cem\u003efhl2, Sjc-0002320\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e) and \u003cem\u003eepsilon\u003c/em\u003e (\u003cem\u003eSjc-0000744\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e), in both sexes (\u003cb\u003eSupplementary Fig.\u0026nbsp;15\u003c/b\u003e). To summarize these findings, we generated schematic illustrations depicting the distribution patterns of these neurons (\u003cb\u003eSupplementary Fig.\u0026nbsp;16\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eTo elucidate the potential functions of these three male-specific populations, we performed GO (Gene Ontology) enrichment analysis on the highly expressed genes within each neuronal subclass. These enriched genes indicated that \u003cem\u003ebeta-2\u003c/em\u003e and \u003cem\u003egamma-2\u003c/em\u003e populations exhibited typical neuronal functions, such as synaptic transmission and neurotransmitter transport. In contrast, \u003cem\u003egamma-2\u003c/em\u003e population showed more specific enrichments, particularly in ion transport (\u003cb\u003eFig.\u0026nbsp;3c, Supplementary Fig.\u0026nbsp;17, and Supplementary Data 9\u0026ndash;10\u003c/b\u003e). Unlike them, the \u003cem\u003edelta-3\u003c/em\u003e population emerged as a specialized neuronal subtype, expressing genes closely related to cilium organization, cell projection assembly, and microtubule bundle formation (\u003cb\u003eFig.\u0026nbsp;3c, and Supplementary Fig.\u0026nbsp;17\u003c/b\u003e). This finding aligns with previous observations in \u003cem\u003eS. mansoni\u003c/em\u003e, where SEM studies identified \u003cem\u003eSm\u003c/em\u003e-\u003cem\u003enrps\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e cells with cilium-like protrusions, suggesting a conserved role of ciliated neurons in male-specific functions \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Collectively, these results indicate that these three types of neurons play different roles in male parasites. Considering the importance of the key enzyme coded by \u003cem\u003enrps\u003c/em\u003e in the \u003cem\u003edelta-3\u003c/em\u003e neuron that synthesizes the pheromone required for the female sexual development in schistosomes, we wondered whether there are essential genes maintaining the function of the \u003cem\u003edelta-3\u003c/em\u003e population.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMale\u003c/b\u003e \u003cb\u003elsamp\u003c/b\u003e \u003cb\u003eis essential for female sexual development\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify genes potentially driving the differentiation of \u003cem\u003edelta-3\u003c/em\u003e neuron, we analyzed the driver genes associated with the \u003cem\u003edelta-3\u003c/em\u003e neuron metastable state by correlating fate probabilities with gene expression using CellRank's compute_lineage_drivers function. Among these, genes such as \u003cem\u003enrps\u003c/em\u003e (\u003cem\u003eSjc-0000814\u003c/em\u003e), \u003cem\u003eddc\u003c/em\u003e (\u003cem\u003eSjc-0006754\u003c/em\u003e, \u003cem\u003eAromatic L-amino acid decarboxylase\u003c/em\u003e), and \u003cem\u003elsamp\u003c/em\u003e (\u003cem\u003eSjc-0000373\u003c/em\u003e, \u003cem\u003elimbic system-associated membrane protein\u003c/em\u003e) exhibited the highest correlations \u003cb\u003e(Fig.\u0026nbsp;3d)\u003c/b\u003e. And these three genes are specifically expressed in \u003cem\u003edelta-3\u003c/em\u003e neurons as indicated in the UMAP \u003cb\u003e(Fig.\u0026nbsp;3e)\u003c/b\u003e. The colocalization of \u003cem\u003enrps\u003c/em\u003e and \u003cem\u003eddc\u003c/em\u003e has been confirmed previously (\u003cb\u003eSupplementary Fig.\u0026nbsp;14c\u003c/b\u003e). We performed double-FISH and found that \u003cem\u003elsamp\u003c/em\u003e is co-expressed with \u003cem\u003enrps\u003c/em\u003e and \u003cem\u003eddc\u003c/em\u003e in male \u003cem\u003edelta-3\u003c/em\u003e neurons (\u003cb\u003eFig.\u0026nbsp;3f\u003c/b\u003e). Additionally, \u003cem\u003elsamp\u003c/em\u003e and \u003cem\u003eddc\u003c/em\u003e also exhibited colocalization in the large cerebral ganglia (\u003cb\u003eFig.\u0026nbsp;3f\u003c/b\u003e). \u003cem\u003eNrps\u003c/em\u003e is a male-specific gene, and its expression is induced by pairing \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eS. japonicum\u003c/em\u003e, both \u003cem\u003eddc\u003c/em\u003e and \u003cem\u003elsamp\u003c/em\u003e displayed similar expression patterns to \u003cem\u003enrps\u003c/em\u003e, as reported in our previous study \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eFig.\u0026nbsp;3g\u003c/b\u003e). We therefore reasoned that \u003cem\u003elsamp\u003c/em\u003e or \u003cem\u003eddc\u003c/em\u003e may play an important role in the \u003cem\u003edelta-3\u003c/em\u003e neuron that may further affect the male-female interaction.\u003c/p\u003e\u003cp\u003eBy employing RNAi \u003cem\u003ein vitro\u003c/em\u003e, we found the male worms treated with \u003cem\u003elsamp\u003c/em\u003e dsRNA instead of \u003cem\u003eddc\u003c/em\u003e dsRNA lost their ability to initiate female sexual development (\u003cb\u003eFig.\u0026nbsp;4a-b, and Supplementary Fig.\u0026nbsp;18\u003c/b\u003e). These female worms paired with \u003cem\u003elsamp\u003c/em\u003e RNAi males displayed reduced Fast Blue BB staining in their vitellaria and possessed poorly developed ovaries (\u003cb\u003eFig.\u0026nbsp;4b\u003c/b\u003e). In addition, they barely laid eggs \u003cb\u003e(Fig.\u0026nbsp;4c)\u003c/b\u003e. qRT-PCR result indicated a strong reduction of the \u003cem\u003elsamp\u003c/em\u003e expression in the male worms after the \u003cem\u003elsamp\u003c/em\u003e RNAi (\u003cb\u003eFig.\u0026nbsp;4d\u003c/b\u003e). The above results suggest that male \u003cem\u003elsamp\u003c/em\u003e is essential for female sexual development.\u003c/p\u003e\u003cp\u003eTo determine whether the female phenotype resulting from the male \u003cem\u003elsamp\u003c/em\u003e knockdown was due to a reduction in the number of \u003cem\u003edelta-3\u003c/em\u003e neurons, we performed FISH experiments to quantify the number of \u003cem\u003edelta-3\u003c/em\u003e neurons. We selected the marker gene \u003cem\u003eddc\u003c/em\u003e to detect \u003cem\u003edelta-3\u003c/em\u003e neurons number specifically. However, there was no significant difference in the \u003cem\u003elsamp\u003c/em\u003e silencing group compared to the control group \u003cb\u003e(Fig.\u0026nbsp;4e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLsamp\u003c/b\u003e \u003cb\u003eaffects acetylated α-tubulin formation and vesicular transport within neurons\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSince \u003cem\u003elsamp\u003c/em\u003e colocalized with \u003cem\u003enrps\u003c/em\u003e in \u003cem\u003edelta-3\u003c/em\u003e neurons (\u003cb\u003eFig.\u0026nbsp;3f\u003c/b\u003e), and \u003cem\u003enrps\u003c/em\u003e has been recognized as the functional gene coding an enzyme to synthesize the male pheromone \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, we therefore wondered whether the \u003cem\u003elsamp\u003c/em\u003e RNAi phenotype is caused by affecting the \u003cem\u003enrps\u003c/em\u003e expression. qRT-PCR results showed that the \u003cem\u003enrps\u003c/em\u003e expression was not affected in the \u003cem\u003elsamp\u003c/em\u003e knockdown males \u003cb\u003e(Fig.\u0026nbsp;4f, and Supplementary Fig.\u0026nbsp;19)\u003c/b\u003e. According to Chen \u003cem\u003eet al\u003c/em\u003e., the NRPS enzyme used two simple precursors (β-alanine and tryptamine) to form the dipeptide pheromone BATT \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. To explore why knocking down \u003cem\u003elsamp\u003c/em\u003e in male worms blocks female sexual development, we detected the levels of two precursors in worms but found no changes between the \u003cem\u003ecntl\u003c/em\u003e and \u003cem\u003elsamp\u003c/em\u003e RNAi groups \u003cb\u003e(Fig.\u0026nbsp;4g-h)\u003c/b\u003e. We then measured the amount of BATT, the product of the NRPS, that is made in males and can be released to the medium \u003cem\u003ein vitro\u003c/em\u003e. Surprisingly, BATT contents were significantly reduced in the \u003cem\u003elsamp\u003c/em\u003e RNAi group, both in the male worms and the culture medium \u003cb\u003e(Fig.\u0026nbsp;4i)\u003c/b\u003e, even though the \u003cem\u003enrps\u003c/em\u003e level was not changed (\u003cb\u003eFig.\u0026nbsp;4f\u003c/b\u003e). These findings suggest that \u003cem\u003elsamp\u003c/em\u003e impairs the ability of male \u003cem\u003edelta-3\u003c/em\u003e neurons to synthesize BATT, consequently failing to stimulate female sexual development, not through affecting \u003cem\u003enrps\u003c/em\u003e expression.\u003c/p\u003e\u003cp\u003eTo investigate the underlying mechanism of male \u003cem\u003elsamp\u003c/em\u003e in regulating BATT synthesis, we performed RNA-seq to overview the gene expression changes between paired \u003cem\u003elsamp\u003c/em\u003e RNAi and \u003cem\u003egfp\u003c/em\u003e RNAi males (D20). Silencing \u003cem\u003elsamp\u003c/em\u003e resulted in the downregulation of 50 genes and the upregulation of 19 genes (\u003cb\u003eSupplementary Fig.\u0026nbsp;20a-b, and Supplementary Data 11\u0026ndash;12\u003c/b\u003e). Our RNA-seq data confirmed that the expression of \u003cem\u003enrps\u003c/em\u003e and \u003cem\u003eddc\u003c/em\u003e remained unaffected by \u003cem\u003elsamp\u003c/em\u003e knockdown \u003cb\u003e(Supplementary Fig.\u0026nbsp;20c)\u003c/b\u003e, consistent with our previous results \u003cb\u003e(Fig.\u0026nbsp;4f, and Supplementary Fig.\u0026nbsp;19)\u003c/b\u003e. Since the upregulation of \u003cem\u003enrps\u003c/em\u003e is triggered by physical contact between males and females \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, these results suggest that the knockdown of \u003cem\u003elsamp\u003c/em\u003e does not impair male worms\u0026rsquo; ability to sense females. Furthermore, GO term analysis of the downregulated genes in \u003cem\u003elsamp\u003c/em\u003e RNAi males revealed significant enrichment in biological processes, including the generation of precursor metabolites and energy, and peptide biosynthesis, as well as molecular functions such as structural constituents of the ribosome and ATPase-coupled ion transmembrane transporter activity \u003cb\u003e(Supplementary Fig.\u0026nbsp;20d-e, and Supplementary Data 13\u0026ndash;14)\u003c/b\u003e. KEGG pathway analysis of the down-regulated genes showed that \u003cem\u003elsamp\u003c/em\u003e mainly affects pathways involved in neurodegenerative diseases such as Alzheimer\u0026rsquo;s disease and Parkinson\u0026rsquo;s disease \u003cb\u003e(Fig.\u0026nbsp;5a, and Supplementary Data 15)\u003c/b\u003e, both of which are closely related to the disorder of microtubule acetylation and axon vesicular transport \u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In addition, GO enrichment on the highly expressed genes in the \u003cem\u003edelta-3\u003c/em\u003e population indicated it as a specialized neuron that expressed genes highly related to cilium organization, cell projection assembly, and microtubule bundle formation (\u003cb\u003eFig.\u0026nbsp;3c, and Supplementary Fig.\u0026nbsp;17\u003c/b\u003e). Previous studies in mice and nematodes have shown that \u003cem\u003elsamp\u003c/em\u003e homologues play a role in synapse formation, axon guidance, and neuronal stability \u003csup\u003e\u003cspan additionalcitationids=\"CR34 CR35 CR36\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. We therefore hypothesized that \u003cem\u003elsamp\u003c/em\u003e may affect the processes of microtubule formation and axon vesicular transport in male schistosomes.\u003c/p\u003e\u003cp\u003eAcetylated α-tubulin is a protein that stabilizes microtubules and regulates cell morphology in nerve fibers. It plays a crucial role in various neuronal processes, including axonal formation, dendritic growth, and neuronal migration \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. And acetylated α-tubulin plays a pivotal role in the transport of synaptic vesicles within neurons. It stabilizes microtubules, which serve as 'tracks' for motor proteins, facilitating the efficient movement of vesicles and ensuring the proper delivery of neurotransmitters to the synapse. In addition to classical neurotransmitters like dopamine, serotonin, and GABA, synaptic vesicles also transport other small molecules, such as the precursors for BATT (β-alanine and tryptamine) \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. To further investigate the specific function of \u003cem\u003elsamp\u003c/em\u003e in schistosomes, we examined the acetylation status of microtubules and synapsin levels in male worms. As shown in \u003cb\u003eFig.\u0026nbsp;5b\u003c/b\u003e, acetylated α-tubulin antibody labeling revealed crisscrossed nerve fibers in the male gynecophoric canal. After knocking down the \u003cem\u003elsamp\u003c/em\u003e, the nerve fibers were markedly eliminated without affecting the \u003cem\u003enrps\u003c/em\u003e expression \u003cb\u003e(Fig.\u0026nbsp;5b).\u003c/b\u003e These data suggest that LSAMP is essential for nerve fiber growth by stabilizing microtubules. Using a synapsin antibody, we detected disrupted synaptic protein distribution along nerve axons \u003cb\u003e(Fig.\u0026nbsp;5b). Comparative co-staining of acetylated α-tubulin and synapsin revealed severe degeneration of neural network functionality in the\u003c/b\u003e \u003cb\u003elsamp\u003c/b\u003e \u003cb\u003eknockdown group (Fig.\u0026nbsp;5b).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next examined the spatial relationship between these neural fibers and \u003cem\u003edelta-3\u003c/em\u003e neurons. By labeling \u003cem\u003edelta-3\u003c/em\u003e neurons with three markers\u0026mdash;\u003cem\u003eddc\u003c/em\u003e, \u003cem\u003enrps\u003c/em\u003e, and \u003cem\u003elsamp\u003c/em\u003e\u0026mdash;we observed that \u003cem\u003edelta-3\u003c/em\u003e neurons were closely associated with neural fibers marked by acetylated α-tubulin (\u003cb\u003eFig.\u0026nbsp;5c\u003c/b\u003e). Additionally, synaptic proteins localized along neural axons showed strong colocalization with \u003cem\u003edelta-3\u003c/em\u003e neurons, suggesting active vesicular transport between these axons and \u003cem\u003edelta-3\u003c/em\u003e cells (\u003cb\u003eFig.\u0026nbsp;5d\u003c/b\u003e). These findings indicate that the loss of \u003cem\u003elsamp\u003c/em\u003e in \u003cem\u003edelta-3\u003c/em\u003e neurons disrupts the integrity of the surrounding ventral neural network in male schistosomes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLsamp\u003c/b\u003e \u003cb\u003eguides the formation of ventral neural network in males\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLSAMP (Limbic system-associated membrane protein) is a glycosylated cell adhesion molecule of the IgLON family\u003c/b\u003e \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. \u003cb\u003eIt is a GPI-anchored protein characterized by three immunoglobulin (Ig) domains\u003c/b\u003e \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e \u003cb\u003e(Supplementary Fig.\u0026nbsp;21). Previous studies in mice and nematodes have demonstrated that\u003c/b\u003e \u003cb\u003elsamp\u003c/b\u003e \u003cb\u003eplays essential roles in neuronal development and function, including promoting neuronal growth and guiding axon targeting\u003c/b\u003e \u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR34 CR35 CR36\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. \u003cb\u003eTo test this, we examined the development of the ventral neural fiber network in male worms from 14 to 26 dpi, covering key stages from male-female pairing to full sexual maturity. At 14 dpi, when males begin pairing with females, only a few elongated nerve fibers were detected within the male gynecophoric canal. As the males developed, a characteristic crisscrossed neural fiber network became evident by 22 dpi and was maintained thereafter (Fig.\u0026nbsp;5e\u003c/b\u003e). \u003cb\u003eNotably, the formation of this neural network coincided with a marked upregulation of\u003c/b\u003e \u003cb\u003elsamp\u003c/b\u003e \u003cb\u003eexpression (Fig.\u0026nbsp;3g, and Supplementary Fig.\u0026nbsp;22), suggesting that\u003c/b\u003e \u003cb\u003elsamp\u003c/b\u003e \u003cb\u003efacilitates axon growth and attachment to\u003c/b\u003e \u003cb\u003edelta-3\u003c/b\u003e \u003cb\u003eneurons, thereby contributing to the establishment of a functional ventral neural network\u003c/b\u003e (\u003cb\u003eFig.\u0026nbsp;5f\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eOverall, our study has profiled the dynamic single-cell atlas of male and female \u003cem\u003eSchistosoma japonicum\u003c/em\u003e during pairing and sexual development. From a male-specific \u003cem\u003edelta-3\u003c/em\u003e neuron population, we identified the \u003cem\u003elsamp\u003c/em\u003e gene, which is crucial for female reproductive development and operates independently of a previously reported key regulator in males, \u003cem\u003enrps\u003c/em\u003e. We demonstrate that \u003cem\u003elsamp\u003c/em\u003e is essential for guiding the formation of ventral neural fibers in the male gynecophoric canal, where they connect to the \u003cem\u003edelta-3\u003c/em\u003e neurons \u003cb\u003e(Fig.\u0026nbsp;5c-e, and Supplementary Fig.\u0026nbsp;22)\u003c/b\u003e. This nerve network functions as an interstate, facilitating the transport of BATT precursors to the \u003cem\u003edelta-3\u003c/em\u003e neurons, which produce the male pheromone to stimulate female reproductive development \u003cb\u003e(Fig.\u0026nbsp;5f)\u003c/b\u003e. These findings not only deepen our understanding of the complex male-female interactions governing sexual development in schistosomes but also shed light on developing new strategies for combating schistosomiasis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study performed single-cell RNA sequencing across four key developmental stages of male and female \u003cem\u003eS. japonicum\u003c/em\u003e, covering the critical period from pairing to egg production. For the first time, we present a dynamic single-cell transcriptome atlas of \u003cem\u003eS. japonicum\u003c/em\u003e males and females, systematically describing the proportional changes in various cell types during their developmental process. Notably, differentiation trajectories were inferred for reproductive and neuronal cells, showing the most prominent changes. A set of marker genes was identified that can be used to distinguish the reproductive status of male and female worms, and we predicted gene sets associated with gametogenesis in both sexes. Additionally, we clarified that \u003cem\u003edelta-3\u003c/em\u003e neurons are \u003cem\u003enrps\u003c/em\u003e-producing neurons and that \u003cem\u003elsamp\u003c/em\u003e, a highly expressed gene in \u003cem\u003edelta-3\u003c/em\u003e neurons, is essential for male-induced female reproductive development by influencing axonal microtubule acetylation and vesicle transport, ultimately reducing BATT synthesis in males.\u003c/p\u003e\u003cp\u003eThe sexual development of schistosomes is a fascinating biological process in which female worms rely on pairing with male worms to initiate reproductive maturation. Pairing behavior begins around 14 dpi, during which females become encapsulated within the male\u0026rsquo;s gynecophoric canal. By 26 dpi, most females reach sexual maturity and begin laying eggs \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Between 14 and 26 dpi, both male and female worms undergo dramatic growth and development, not only in their reproductive organs but also in other tissues. Bulk RNA-seq is limited in its ability to resolve the molecular changes occurring at the cellular level. In the present study, we used single-cell RNA sequencing to systematically characterize the gene regulatory programs underlying dimorphic gametogenesis in \u003cem\u003eS. japonicum\u003c/em\u003e. We identified both shared and sex-specific regulatory modules involved in germline development (\u003cb\u003eSupplementary Fig.\u0026nbsp;7\u003c/b\u003e). Among the shared modules, C2, C3, and C4 exhibit similar expression patterns in both sexes (\u003cb\u003eSupplementary Fig.\u0026nbsp;7b, and Supplementary Data 6\u003c/b\u003e). Notably, the C2 module includes \u003cem\u003enanos1\u003c/em\u003e and \u003cem\u003enanos2\u003c/em\u003e, which are critical regulators of germline stem cell differentiation \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The C3 module contains genes involved in cell cycle regulation (\u003cem\u003ee.g.\u003c/em\u003e, \u003cem\u003epcna\u003c/em\u003e), energy metabolism (\u003cem\u003ee.g\u003c/em\u003e., \u003cem\u003esdhb\u003c/em\u003e), and RNA processing (\u003cem\u003ee.g\u003c/em\u003e., \u003cem\u003esrsf12\u003c/em\u003e), which are associated with cellular proliferation and maintenance of genomic stability \u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The C4 module features genes such as \u003cem\u003esmc3\u003c/em\u003e and \u003cem\u003etubb4b\u003c/em\u003e, which are involved in chromosome segregation and cytoskeletal organization during meiosis \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. These shared modules highlight the evolutionary conservation of core gametogenic processes in both male and female \u003cem\u003eS. japonicum\u003c/em\u003e, including germline stem cell maintenance, epigenetic regulation, chromosome dynamics, and protein homeostasis. In contrast, some shared modules display sexually dimorphic expression patterns. For example, C1 genes are initially downregulated and then upregulated in females, whereas in males, their expression steadily declines (\u003cb\u003eSupplementary Fig.\u0026nbsp;7b, and Supplementary Data 6\u003c/b\u003e). Additionally, sex-specific modules reveal distinct features between male and female gametogenesis (\u003cb\u003eSupplementary Fig.\u0026nbsp;7c-d, and Supplementary Data 7\u0026ndash;8\u003c/b\u003e). For instance, male-specific modules such as C5 include genes involved in flagellar assembly, which are directly associated with spermatogenesis and motility. Importantly, some unannotated genes within these clusters may represent \u003cem\u003eSchistosoma\u003c/em\u003e-specific reproductive regulators that warrant further investigation.\u003c/p\u003e\u003cp\u003eSchistosome neural development remains poorly understood, especially the differentiation trajectories of neuronal cells. While single-cell RNA sequencing has been conducted on adult \u003cem\u003eS. mansoni\u003c/em\u003e, it primarily captured mature neuron populations \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, limiting lineage tracing analysis. In this study, we profiled single-cell data from \u003cem\u003eS. japonicum\u003c/em\u003e at 14, 18, 22, and 26 dpi, identifying various neural cell stages, including neural stem cells, precursors, and mature neurons (\u003cb\u003eFig.\u0026nbsp;1a-c\u003c/b\u003e). Pseudotime and RNA velocity analysis, reconstructed clear lineage connections, providing a detailed neural development map (\u003cb\u003eSupplementary Fig.\u0026nbsp;10\u0026ndash;11\u003c/b\u003e). We further classified the nervous system of \u003cem\u003eS. japonicum\u003c/em\u003e into five clear neuronal lineages, each exhibiting significant spatial heterogeneity (\u003cb\u003eSupplementary Fig.\u0026nbsp;10\u0026ndash;11, and 13\u0026ndash;16\u003c/b\u003e). Intriguingly, some neurons exhibit sex-specific distribution, such as \u003cem\u003edelta-3\u003c/em\u003e, \u003cem\u003egamma-2\u003c/em\u003e, \u003cem\u003ebeta-2\u003c/em\u003e, \u003cem\u003eepsilon\u003c/em\u003e, and \u003cem\u003ebeta-1\u003c/em\u003e neurons (\u003cb\u003eFig.\u0026nbsp;3a-b, and Supplementary Fig.\u0026nbsp;13\u0026ndash;16\u003c/b\u003e). Our data reveal that male-specific \u003cem\u003edelta-3\u003c/em\u003e neurons express \u003cem\u003enrps\u003c/em\u003e (\u003cb\u003eFig.\u0026nbsp;3a-b, and d-e\u003c/b\u003e), a gene rapidly upregulated upon pairing. \u003cem\u003eNrps\u003c/em\u003e encodes NRPS, an enzyme synthesizing the male pheromone BATT, which stimulates female sexual development \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Additionally, we identified other driver genes expressed in \u003cem\u003edelta-3\u003c/em\u003e neurons, such as \u003cem\u003eddc\u003c/em\u003e (\u003cem\u003eSjc-0006754\u003c/em\u003e) and \u003cem\u003elsamp\u003c/em\u003e (\u003cem\u003eSjc-0000373\u003c/em\u003e). These findings highlight the robust heterogeneity of schistosome neurons and suggest that neural differentiation occurs at early developmental stages, with no active neurogenesis at the adult stage.\u003c/p\u003e\u003cp\u003eDDC is an enzyme involved in the synthesis of several monoamines, including serotonin (5-HT), tyramine, tryptamine, histamine, dopamine, and other catecholamines \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In planarians, inhibiting \u003cem\u003eddc\u003c/em\u003e expression results in the loss of ovaries and other female reproductive organs, including the yolk gland \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Similarly, in nematodes, egg-laying is regulated by serotonergic neurons \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. However, in this study, \u003cem\u003eddc\u003c/em\u003e knockdown in male schistosomes did not cause developmental arrest in female reproduction (\u003cb\u003eSupplementary Fig.\u0026nbsp;18\u003c/b\u003e). Since biogenic amines, including 5-HT, are present in the culture medium, we hypothesized that female schistosomes might sustain reproductive development by utilizing exogenous 5-HT. To test this, we cultured mature \u003cem\u003eS. japonicum\u003c/em\u003e worm pairs using 5-HT-supplemented and 5-HT-free mAB169 (1640) medium. After 12 days, females in the 5-HT-free group exhibited impaired vitellaria and produced significantly fewer eggs, compared to the 5-HT-supplemented group (\u003cb\u003eSupplementary Fig.\u0026nbsp;23\u003c/b\u003e). These findings indicate that 5-HT, a product of DDC activity, is essential for female reproductive development. Thus, while DDC and its product 5-HT are crucial for female sexual development, schistosome females can compensate for \u003cem\u003eddc\u003c/em\u003e knockdown by acquiring 5-HT from external sources under \u003cem\u003ein vitro\u003c/em\u003e conditions.\u003c/p\u003e\u003cp\u003ePrevious studies in mice and nematodes have shown that LSAMP plays key roles in synapse formation, axon guidance, and neuronal stability \u003csup\u003e\u003cspan additionalcitationids=\"CR34 CR35 CR36\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, but there have been no studies on this gene in schistosomes. Although \u003cem\u003eS. japonicum\u003c/em\u003e LSAMP shares only\u0026thinsp;~\u0026thinsp;27% overall sequence similarity with its homologs in \u003cem\u003eDrosophila\u003c/em\u003e, \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e, and \u003cem\u003eMus musculus\u003c/em\u003e, its functional domains are highly conserved (\u003cb\u003eSupplementary Fig.\u0026nbsp;24a-b\u003c/b\u003e), suggesting that its biological roles may be preserved. In this study, we found that \u003cem\u003elsamp\u003c/em\u003e is essential for establishing the specialized neural network within the male gynecophoral canal. As a glycosylated cell adhesion molecule of the IgLON family, LSAMP functions as a membrane protein (\u003cb\u003eSupplementary Fig.\u0026nbsp;24c, and 21\u003c/b\u003e). In \u003cem\u003eC. elegans\u003c/em\u003e, SAX-3 (LSAMP homolog) has been shown to localize on the surface of PVD neurons, guiding axon outgrowth \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Here, we observed \u003cem\u003elsamp\u003c/em\u003e expression in the BATT-producing \u003cem\u003edelta-3\u003c/em\u003e neurons in schistosomes. In the absence of \u003cem\u003elsamp\u003c/em\u003e, the intricate neural network connecting \u003cem\u003edelta-3\u003c/em\u003e neurons failed to form (\u003cb\u003eFig.\u0026nbsp;5b\u003c/b\u003e). We propose that LSAMP acts as a selective adhesion molecule on the surface of \u003cem\u003edelta-3\u003c/em\u003e neurons, guiding axon growth and facilitating neuronal connectivity (\u003cb\u003eSupplementary Fig.\u0026nbsp;25\u003c/b\u003e). Beyond structural organization, vesicular transport of amino acids is crucial for intracellular movement and distribution, particularly in neurotransmission \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Specific transport proteins facilitate the transfer of neurotransmitters into synaptic vesicles for release into the synapse \u003csup\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Although direct labeling of BATT precursors (β-alanine and tryptamine) failed, our LC-MS results provided definitive evidence for the inhibition of their transport. Together, this well-established neuronal network likely ensures the targeted transport of BATT precursors to their synthesis sites in \u003cem\u003edelta-3\u003c/em\u003e neurons. Interestingly, this specialized neural architecture is formed during male-female pairing (\u003cb\u003eFig.\u0026nbsp;5e\u003c/b\u003e), implying a direct role in reproductive coordination. Collectively, these findings provide novel insight into the male-specific neural circuitry involved in regulating female reproduction, thereby expanding our understanding of male-induced female sexual development in this parasitic flatworm.\u003c/p\u003e\u003cp\u003eIn conclusion, through the comprehensive single-cell transcriptomic analysis of \u003cem\u003eSchistosoma japonicum\u003c/em\u003e, we decoded male-female interactions at single-cell resolution and delineated both conserved and sex-specific gene modules involved in gametogenesis. Furthermore, we uncovered a male-specific neural circuit that directly regulates female sexual development. Our study identified \u003cem\u003elsamp\u003c/em\u003e as a key regulator of axonal connectivity in pheromone-producing \u003cem\u003edelta-3\u003c/em\u003e neurons, which establish a specialized neural network essential for the transport of BATT precursors. These findings fundamentally reshape our understanding of parasite neurobiology by revealing how a primitive nervous system orchestrates reproductive processes, and they offer novel targets for disrupting the schistosome life cycle.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eEthics\u003c/h2\u003e\n \u003cp\u003eAll experiments related to animals were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People\u0026rsquo;s Republic of China (2006398) and were approved by the Animal Care and Use Committee of Fudan University (Fudan IACUC 201802158S) to ensure ethical and responsible treatment of the animals.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eS. japonicum\u003c/strong\u003e \u003cstrong\u003eparasites\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe cercariae of \u003cem\u003eS. japonicum\u003c/em\u003e were provided by the Chinese Center for Disease Control and Prevention. Single-sex infections of \u003cem\u003eS. japonicum\u003c/em\u003e were obtained by infecting mice with male or female cercariae obtained from \u003cem\u003eOncomelania hupensis\u003c/em\u003e snails (Anhui strain) infected with single miracidia.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eS. japonicum\u003c/strong\u003e \u003cstrong\u003einfected mice\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eSix-week-old C57BL/6 mice were purchased from Shanghai Jeste Experimental Animal Co., Ltd.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eFluorescence activated cell sorting\u003c/h2\u003e\n \u003cp\u003eTwenty female C57 mice were infected percutaneously through the abdomen with 80\u0026ndash;200 cercariae. At each time point (14, 18, 22, and 26 dpi), five mice were sacrificed for the recovery of schistosomes \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eWorms were collected from mice by perfusing the hepatic portal vein with 4℃ PBS (Sangon Biotech) and then rinsed in twice in DMEM (5% FBS) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Freshly perfused male and female worms, except 14 dpi worms, which are difficult to distinguish gender under a light microscope, were separated by incubation in a 0.25% solution of tricaine for approximately 5 min. FACS sorting was performed as previously described with minor modifications. Worms were washed twice in 8 mL PBS, then triturated for approximately 15 min in 4 mL 0.125% Trypsin-EDTA (in PBS) until the solution became turbid and no large pieces of worms were left. The trypsin was inactivated by adding an 8 mL 10% FBS DMEM. The dissociated worms were then centrifuged at 500 \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C. Next, the worm cells were resuspended in 1 mL of PBS with 10 \u0026micro;L of RQ1 DNAse (Promega M6101) and incubated for 10 min at RT. The worm cells were centrifuged again at 500 \u003cem\u003eg\u003c/em\u003e for 10 minutes at 4\u0026deg;C. Then the worm cells were resuspended in 1mL of PBS containing Fixable Viability Dye eFluor\u0026trade; 506 (1\u0026micro;L/mL) (eBioscience) and incubated for 30 min at 4℃ in the dark. The worm cell suspension was centrifuged at 500 \u003cem\u003eg\u003c/em\u003e for 10 minutes at 4\u0026deg;C, then washed twice in PBS, then filtered through a 40 \u0026micro;m cell strainer into a 12\u0026times;75 mm FACS tube. Filtered cells were then sorted on a MoFlo XDP High-Speed Cell Sorter (BECKMAN COULTER) with 405/460/488/532/561/640nm lasers. Sorts were performed with a 100 \u0026micro;m nozzle, and cells were sorted into sorting media (0.04% BSA in PBS, pH\u0026thinsp;=\u0026thinsp;7.40).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSingle-cell RNA sequencing\u003c/h3\u003e\n\u003cp\u003eFACS-sorted cells were centrifuged at 500 \u003cem\u003eg\u003c/em\u003e for 10 minutes at 4\u0026deg;C and then resuspended in 0.04% BSA in PBS. Libraries were created using a Chromium Controller (10\u0026times; Genomics) according to manufacturer guidelines and sequenced using a NovaSeq6000 (Illumina) to generate 150-bp paired-end reads. Raw data was processed and mapped to the \u003cem\u003eSchistosoma japonicum\u003c/em\u003e genome (v3) using the Cell Ranger 3.0.0 (10\u0026times; Genomics) pipeline.\u003c/p\u003e\n\u003ch3\u003eSingle-cell RNA-seq data processing\u003c/h3\u003e\n\u003cp\u003eThe Seurat package (v 3.1.5) was used to analyze the raw values of the matrix as follows \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Cells with greater than 30% mitochondrial reads or fewer than 500 genes were excluded from the analysis. Mitochondrial genes were identified with the prefix \u0026ldquo;^ND|^C|^ATP\u0026rdquo;. Each of the 7 individual datasets was scaled and transformed, and variable genes were identified using the SCTransform function with the parameter \u0026ldquo;variable.features.n\u0026thinsp;=\u0026thinsp;3000\u0026rdquo;. All samples were combined using the functions \u0026ldquo;FingIntegrationAnchor\u0026rdquo; and \u0026ldquo;IntegrateData\u0026rdquo;. Then we performed principal component analysis using variable genes and used the first 100 principal components (PCs) to perform UMAP to embed the dataset into two dimensions. For clustering, we identified 76 clusters using the FindNeighbors and FindClusters function with the parameter \u0026ldquo;dims\u0026thinsp;=\u0026thinsp;1:100, resolution\u0026thinsp;=\u0026thinsp;2\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003eTo determine cellular identity, we used the function FindAllMarkers with the parameter \u0026ldquo;test.use = \u0026quot;wilcox\u0026quot;, only.pos\u0026thinsp;=\u0026thinsp;T, min.pct\u0026thinsp;=\u0026thinsp;0.25, logfc.threshold\u0026thinsp;=\u0026thinsp;0.25 \u0026rdquo; to find DE genes for each cluster and compared to the known marker genes from the previous datasets \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. GO (Gene Ontology) enrichment analysis was performed with the R package clusterProfiler \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003ep-value\u003c/em\u003e was corrected for multiple hypothesis testing with the Benjamini\u0026ndash;Hochberg false-discovery rate procedure (adjusted \u003cem\u003ep-value\u003c/em\u003e). GO terms with corrected \u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant. R packages ggplot2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ggplot2.tidyverse.org\u003c/span\u003e\u003c/span\u003e) and tidyverse (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.tidyverse.org\u003c/span\u003e\u003c/span\u003e) were used to generate figures and intermediate data preprocessing.\u003c/p\u003e\n\u003cp\u003eThe density plots of cells from each sample over UMAP embedding were generated using a modified treecor_celldensityplot function from TreeCorTreat package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/byzhang23/TreeCorTreat\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eDifferential abundance analysis\u003c/h2\u003e\n \u003cp\u003eWe tested for differences in cell-type abundances between female and male worms using the miloR package (v 1.4.0) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, which is a method for differential abundance analysis on KNN graphs from single-cell datasets. Briefly, we computed the k-nearest neighbor graph of cells in the integrated datasets based on latent embedding. Subsequently, cell neighborhoods were computed using miloR\u0026apos;s makeNhoods function with parameter \u0026ldquo;\u003cem\u003ek\u003c/em\u003e\u0026thinsp;=\u0026thinsp;25, prop\u0026thinsp;=\u0026thinsp;0.2\u0026rdquo;. To leverage the variation in the number of cells between 18, 22, and 26 dpi samples, we counted the cells belonging to each sample in each neighborhood using the countCells function. We assigned to each neighborhood a cell-type label based on the majority voting of the cells belonging to that neighborhood. We then used the testNhoods function to test for enrichment of cells from the different sex datasets. Neighborhoods were enriched if the spatial FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.2 and \u003cstrong\u003e|\u003c/strong\u003elog\u003csub\u003e2\u003c/sub\u003eFC\u003cstrong\u003e|\u003c/strong\u003e\u0026gt;1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eTrajectory inference and RNA velocity\u003c/h2\u003e\n \u003cp\u003eTo infer potential developmental trajectories within the neuronal lineages, we performed RNA Velocity analysis. In brief, we generated loom files of the spliced and unspliced RNA matrices using velocity (v 0.17.17) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e with our CellRanger output. Then we applied scVelo (v 0.2.2) to estimate transient cell states and velocities using a generalized dynamical model following the recommended workflow. CellRank package (v 1.0.0) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e was performed as described in (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cellrank.readthedocs.io/en/stable/index.html\u003c/span\u003e\u003c/span\u003e) to characterize the cell fate decision process and determine initial and terminal states in data. The connectivity of cell populations was quantified using the partition-based graph abstraction (PAGA) method \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Then, we visualized the fate probability of each cell population as a pie chart using the \u0026ldquo;cellrank.pl.cluster_fates\u0026rdquo; functions with the parameter \u0026ldquo;mode\u0026thinsp;=\u0026thinsp;paga_pie\u0026rdquo;. To infer putative driver genes for any of these trajectories, we correlate expression values with fate probabilities using the \u0026ldquo;cellrank.tl.lineage_drivers\u0026rdquo; function with the parameter \u0026ldquo;method\u0026thinsp;=\u0026thinsp;Fischer\u0026rdquo;.\u003c/p\u003e\n \u003cp\u003eIn addition, we used the R package Slingshot (v 2.0.1) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e as another independent method to analyze single-cell trajectories. We re-computed the UMAP embedding on subsetted neuronal and germline stem cell lineages identified in our integrated data set. Then, we used UMAP reduction to determine dimensionality and set Neuron stem cells or GSCs as start clusters to infer cell trajectory. Specifically, to identify temporally expressed genes changing in male and female gametogenesis, we used the GAM function implemented in the gam R package to investigate variable genes over pseudo time. To identify more significant genes along the pseudotime of germ cell lineage, we calculated the amplitude of change for each tested gene (with a cut-off of 0.5). We retained the top 25% of genes exhibiting the lowest \u003cem\u003eq-values\u003c/em\u003e. Subsequently, we separately selected 750 and 693 variable genes from the male and female datasets, respectively, for visualization purposes. We then generated heatmaps of these genes using the R package ComplexHeatmap (v 2.12.0) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA interference (RNAi)\u003c/h2\u003e\n \u003cp\u003eDsRNA productions were essentially performed as previously described \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Oligo sequences used to generate dsRNA templates are listed in \u003cstrong\u003eSupplementary Data 16.\u003c/strong\u003e The dsRNA of the \u003cem\u003egfp\u003c/em\u003e (\u003cem\u003egreen fluorescent protein\u003c/em\u003e) fragment was used as a negative control for all RNAi experiments. Day 0 represents the first day of the experiment. Males were treated with 30 \u0026micro;g/mL dsRNA (on Day 0/2/4/6 with fresh medium) for one week in BM169 and then paired with virgin female parasites from Day 8 to Day 24 in ABC169 (fresh medium was replaced every other day). The eggs produced by the worms are retained when changing the medium. On Day 25, the egg number and the number of female parasites were counted to calculate egg/female/day.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eBulk RNA-sequencing and data analysis\u003c/h2\u003e\n \u003cp\u003eWe collected male worms from the \u003cem\u003egfp\u003c/em\u003e RNAi and \u003cem\u003elsamp\u003c/em\u003e RNAi groups on Day 20 for bulk RNA sequencing, with four biological replicates per group. Total RNA extraction, library construction, and RNA sequencing were performed by the Novogene Co., Ltd (Beijing, China). Briefly, RNA was extracted from worms using the AG RNAex Pro Reagent (Accurate Biotechnology (Hunan) Co., Ltd., China). The integrity and quality of total RNA were assessed using a Nanodrop ND-2000c spectrophotometer (Thermo Fisher Scientific, USA) and an Agilent 2100 Bioanalyzer (Agilent, USA). Pooled RNA was used to construct a library using Illumina TruSeq\u0026trade; RNA sample prep Kit v2 (Illumina, USA) and the Ribo-Zero Plus rRNA Depletion Kit (Illumina, USA) for rRNA depletion, following the TruSeq RNA Sample Preparation Guide. The library was subsequently sequenced on the Illumina NovaSeq 6000 platforms to obtain 150 bp paired-end (PE) reads.\u003c/p\u003e\n \u003cp\u003eQC (Quality control) of the raw sequencing data was performed using the FASTQC program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003c/span\u003e). Low-quality reads and adapter sequences were trimmed using the Fastp tool (v 0.20.1) (parameters: -q 15 -u 40 -n 5 -l 15) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. The clean reads were mapped to the chromosome-level \u003cem\u003eS. japonicum\u003c/em\u003e reference genome (\u003cem\u003eSj\u003c/em\u003eV3) using HISAT2 (v 2.1.0) with default parameters \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. The gene-level assignment was then performed to estimate the transcript abundance using featureCounts 1.6.4 \u003csup\u003e69\u003c/sup\u003e. PCA (Principal Component Analysis) was performed using the prcomp function in the R stats package (v 3.6.0). HCA (Hierarchical clustering analysis) was performed with pheatmap (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cran.r-project.org/web/packages/pheatmap/index.html\u003c/span\u003e\u003c/span\u003e). Differential expression analysis was performed using DESeq2 \u003csup\u003e70\u003c/sup\u003e, and the differentially expressed genes (DEGs) were defined with adjusted \u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2FC|\u0026gt;2. To identify the possible functions and pathways of DEGs, GO terms enrichment analysis and Gene Set Enrichment Analysis (GSEA) were performed using the clusterProfiler package \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eParasite labeling and imaging\u003c/h2\u003e\n \u003cp\u003eColorimetric and fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization were performed as previously described \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Riboprobes were synthesized from templates generated using primers listed in \u003cstrong\u003eSupplementary Data 4\u003c/strong\u003e. Typically, all probes were applied at a concentration of 100 ng/mL in the hybridization buffer, with the concentrations of certain probes adjusted to optimize localization performance. The immunofluorescence assays for detecting Synapsin levels in the parasite were performed using anti-Synapsin (1:100, Clone 3C11, Developmental Studies Hybridoma Bank), and for detecting acetylated \u0026alpha;-tubulin levels using anti-acetylated \u0026alpha;-tubulin (1:500, Clone 6-11B-1, Santa Cruz) as previously described by \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Fast Blue BB labeling experiments were performed on females collected on Day 25, as previously reported \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. All labeled parasites were counterstained with DAPI (1 \u0026micro;g/mL), cleared in 80% glycerol, and mounted on slides with 80% glycerol. Confocal imaging of fluorescently labeled samples was performed on Nikon A1 Laser Scanning Confocal Microscope or Olympus FV3000 Laser Scanning Confocal Microscope. Brightfield images were acquired on Olympus BX51 Microscope and Zeiss AxioZoom V16 Microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eqRT-PCR (Quantitative Real-time PCR)\u003c/h2\u003e\n \u003cp\u003eAll qPCR reactions were performed on a LightCycler\u0026reg; 96 Instrument (Roche, Switzerland) using 2\u0026times; SYBR green qPCR master mix (Yeasen, China) according to the manufacturer\u0026apos;s instructions. Each 20 \u0026micro;L qRT-PCR reaction mixture comprised 2 \u0026micro;L of cDNA(1:4), 10 \u0026micro;L 2\u0026times; SYBR green master, 0.8 \u0026micro;L (5 \u0026micro;M) of each primer, and 6.4 \u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO. The qRT-PCR cycle parameters were as follows: 95\u0026deg;C for 3 min, followed by 40 cycles of 95\u0026deg;C for 15 s, 60\u0026deg;C for 30 s; melt curve analysis ranged from 60\u0026deg;C to 95\u0026deg;C to ensure that the specific product was amplified in each reaction. The 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method was used to calculate the relative fold change of the differentially expressed transcripts.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eBATT synthesis and detection\u003c/h2\u003e\n \u003cp\u003eThe synthesis of BATT was conducted by the Chinese Center for Disease Control and Prevention. Concentrated stock solutions (100 mM) of BATT were prepared in sterile DMSO and stored at -20℃. LC-MS/MS was employed for the detection of BATT in parasites (thirty male worms) and medium samples collected on Day 20, as previously described. BATT detection was performed on a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) employing negative ESI interface using QTRAP\u0026reg; 6500\u003csup\u003e+\u003c/sup\u003e LC-MS/MS System, with the transitions set at m/z 232.4 \u0026rarr; m/z 143.7, m/z 232.4 \u0026rarr; m/z 185.1, m/z 232.4 \u0026rarr; m/z 89.1, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e\n \u003cp\u003eGraphPad Prism software (USA) processed and presented the data as the mean with SD (Standard Deviation). Statistical significance was calculated by unpaired two-tailed parametric \u003cem\u003et\u003c/em\u003e-test. \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 are considered significant (\u003cem\u003ens\u003c/em\u003e, not significant; *, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Error bars represent SD.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to the following individuals and institutions for their support and assistance: Bin Xu, Ying Wu, and Yang Hong from the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, for their support and assistance with experimental materials.\u0026nbsp;The amino metabolome and BATT were quantified by \u0026nbsp; \u0026nbsp; Professor Huiru Tang's group as a paid service. Jingwei Zhang of Fudan University for his guidance and assistance in single-cell sample evaluation. This research was supported by National Natural Science Foundation of China (No. 31725025, and No. 31972699 to W.H.), the National Key Research and Development Program of China (No. 2021YFC2300800, and No.\u0026nbsp;2021YFC2300803 to J.P.W.), Science and Technology Leading Talent Team in Inner Mongolia Autonomous Region (No. 2022LJRC0009 to W.H.), and Shanghai Natural Science Foundation Young Scientists Program (No. 25ZR1402030 to Y.M.Y.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.H. and Y.M.Y. conceptualized this project. W.H. and J.P.W. supervised the overall research, and directed the bioinformatics analysis. W.H., J.P.W. and Y.M.Y. secured fundings. Y.M.Y. performed all experiments, and S.Y.C. analyzed the scRNA-seq and bulk RNA-seq data. Y.M.Y. and J.P.W. drafted the manuscript, with Y.M.Y., J.P.W., S.Y.C., and W.H. participating in manuscript revision. X.C., X.C., C.Y., and M.Q.C. provided experimental assistance in WISH and FISH. F.L., X.L.W., Y.X.X., W.B.Y., L.X., and M.J.G., assisted in sample processing for scRNA-seq. C.S.S. provided negative \u003cem\u003eOncomelania\u003c/em\u003e \u003cem\u003ehupensis\u003c/em\u003e snails. L.L.H. contributed to BATT synthesis. G.W.C. and S.L. provided assistance in sample collection. All authors critically reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe single-cell RNA sequencing (scRNA-seq) data and the bulk RNA sequencing (RNA-seq) raw data for\u003cem\u003e\u0026nbsp;S. japonicum\u003c/em\u003e in this study can be accessed from NCBI Sequence Read Archive (SRA) using the accession number PRJNA1244463, The gene expression data for scRNA-seq have been deposited at the Gene Expression Omnibus (GEO) with the accession number GSE293642.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcManus, D.P.\u003cem\u003e et al.\u003c/em\u003e Schistosomiasis. \u003cem\u003eNat Rev Dis Primers\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 13 (2018).\u003c/li\u003e\n\u003cli\u003eMolehin, A.J. Schistosomiasis vaccine development: update on human clinical trials. \u003cem\u003eJ Biomed Sci\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 28 (2020).\u003c/li\u003e\n\u003cli\u003eBergquist, R., Utzinger, J. \u0026amp; Keiser, J. Controlling schistosomiasis with praziquantel: How much longer without a viable alternative? \u003cem\u003eInfect Dis Poverty\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 74 (2017).\u003c/li\u003e\n\u003cli\u003eMoore, D.V. \u0026amp; Sandground, J.H. The relative egg producing capacity of Schistosoma mansoni and Schistosoma japonicum. \u003cem\u003eAm J Trop Med Hyg\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 831-840 (1956).\u003c/li\u003e\n\u003cli\u003eSchwartz, C. \u0026amp; Fallon, P.G. Schistosoma \u0026quot;Eggs-Iting\u0026quot; the Host: Granuloma Formation and Egg Excretion. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 2492 (2018).\u003c/li\u003e\n\u003cli\u003ePopiel, I. \u0026amp; Basch, P.F. Reproductive development of female Schistosoma mansoni (Digenea: Schistosomatidae) following bisexual pairing of worms and worm segments. \u003cem\u003eJ Exp Zool\u003c/em\u003e \u003cstrong\u003e232\u003c/strong\u003e, 141-150 (1984).\u003c/li\u003e\n\u003cli\u003eArmstrong, J.C. Mating Behavior and Development of Schistosomes in the Mouse. \u003cem\u003eJ Parasitol\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, 605-616 (1965).\u003c/li\u003e\n\u003cli\u003eAE, S. Sex studies on Schistosoma japonicum. \u003cem\u003eQuarterly Journal of Microscopical Science \u003c/em\u003e\u003cstrong\u003es2\u003c/strong\u003e, 653 (1928).\u003c/li\u003e\n\u003cli\u003eChen, R.\u003cem\u003e et al.\u003c/em\u003e A male-derived nonribosomal peptide pheromone controls female schistosome development. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e185\u003c/strong\u003e, 1506-1520.e1517 (2022).\u003c/li\u003e\n\u003cli\u003eWang, J.\u003cem\u003e et al.\u003c/em\u003e Dynamic transcriptomes identify biogenic amines and insect-like hormonal regulation for mediating reproduction in Schistosoma japonicum. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 14693 (2017).\u003c/li\u003e\n\u003cli\u003eWaisberg, M.\u003cem\u003e et al.\u003c/em\u003e Microarray analysis of gene expression induced by sexual contact in Schistosoma mansoni. \u003cem\u003eBMC Genomics\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 181 (2007).\u003c/li\u003e\n\u003cli\u003eSun, J.\u003cem\u003e et al.\u003c/em\u003e Transcriptome profilings of female Schistosoma japonicum reveal significant differential expression of genes after pairing. \u003cem\u003eParasitol Res\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e, 881-892 (2014).\u003c/li\u003e\n\u003cli\u003eGobert, G.N., Moertel, L., Brindley, P.J. \u0026amp; McManus, D.P. Developmental gene expression profiles of the human pathogen Schistosoma japonicum. \u003cem\u003eBMC Genomics\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 128 (2009).\u003c/li\u003e\n\u003cli\u003eFitzpatrick, J.M., Johansen, M.V., Johnston, D.A., Dunne, D.W. \u0026amp; Hoffmann, K.F. Gender-associated gene expression in two related strains of Schistosoma japonicum. \u003cem\u003eMol Biochem Parasitol\u003c/em\u003e \u003cstrong\u003e136\u003c/strong\u003e, 191-209 (2004).\u003c/li\u003e\n\u003cli\u003ePiao, X.\u003cem\u003e et al.\u003c/em\u003e Global expression analysis revealed novel gender-specific gene expression features in the blood fluke parasite Schistosoma japonicum. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, e18267 (2011).\u003c/li\u003e\n\u003cli\u003eHoffmann, K.F., Johnston, D.A. \u0026amp; Dunne, D.W. Identification of Schistosoma mansoni gender-associated gene transcripts by cDNA microarray profiling. \u003cem\u003eGenome Biol\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, RESEARCH0041 (2002).\u003c/li\u003e\n\u003cli\u003eAnderson, L.\u003cem\u003e et al.\u003c/em\u003e Schistosoma mansoni Egg, Adult Male and Female Comparative Gene Expression Analysis and Identification of Novel Genes by RNA-Seq. \u003cem\u003ePLoS Negl Trop Dis\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, e0004334 (2015).\u003c/li\u003e\n\u003cli\u003eWang, B.\u003cem\u003e et al.\u003c/em\u003e Stem cell heterogeneity drives the parasitic life cycle of Schistosoma mansoni. \u003cem\u003eeLife\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, e35449 (2018).\u003c/li\u003e\n\u003cli\u003eDiaz Soria, C.L.\u003cem\u003e et al.\u003c/em\u003e Single-cell atlas of the first intra-mammalian developmental stage of the human parasite Schistosoma mansoni. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 6411 (2020).\u003c/li\u003e\n\u003cli\u003eDiaz Soria, C.L.\u003cem\u003e et al.\u003c/em\u003e Single-cell transcriptomics of the human parasite Schistosoma mansoni first intra-molluscan stage reveals tentative tegumental and stem-cell regulators. \u003cem\u003eScientific Reports\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e (2024).\u003c/li\u003e\n\u003cli\u003eWendt, G.\u003cem\u003e et al.\u003c/em\u003e A single-cell RNA-seq atlas of Schistosoma mansoni identifies a key regulator of blood feeding. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e369\u003c/strong\u003e, 1644-1649 (2020).\u003c/li\u003e\n\u003cli\u003eCollins, J.J., 3rd\u003cem\u003e et al.\u003c/em\u003e Adult somatic stem cells in the human parasite Schistosoma mansoni. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e494\u003c/strong\u003e, 476-479 (2013).\u003c/li\u003e\n\u003cli\u003eWang, J., Chen, R. \u0026amp; Collins, J.J. Systematically improved in vitro culture conditions reveal new insights into the reproductive biology of the human parasite Schistosoma mansoni. \u003cem\u003ePLOS Biology\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, e3000254 (2019).\u003c/li\u003e\n\u003cli\u003eWang, J. \u0026amp; Collins, J.J., 3rd Identification of new markers for the Schistosoma mansoni vitelline lineage. \u003cem\u003eInt J Parasitol\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 405-410 (2016).\u003c/li\u003e\n\u003cli\u003eHanumantha-Rao, K. Histochemistry of mehlis\u0026apos; gland and egg-shell formation in the liver flukeFasciola hepatica L. \u003cem\u003eExperientia\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 464-465 (1959).\u003c/li\u003e\n\u003cli\u003eMoczon, T., Swiderski, Z. \u0026amp; Huggel, H. Schistosoma mansoni: The chemical nature of the secretions produced by the Mehlis\u0026apos; gland and ootype as revealed by cytochemical studies. \u003cem\u003eInternational Journal for Parasitology\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 65-73 (1992).\u003c/li\u003e\n\u003cli\u003eBergen, V., Lange, M., Peidli, S., Wolf, F.A. \u0026amp; Theis, F.J. Generalizing RNA velocity to transient cell states through dynamical modeling. \u003cem\u003eNature Biotechnology\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 1408-1414 (2020).\u003c/li\u003e\n\u003cli\u003eNaren, P.\u003cem\u003e et al.\u003c/em\u003e Microtubule acetylation dyshomeostasis in Parkinson\u0026rsquo;s disease. \u003cem\u003eTranslational Neurodegeneration\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 20 (2023).\u003c/li\u003e\n\u003cli\u003eDeshpande, P.\u003cem\u003e et al.\u003c/em\u003e N-Acetyltransferase 9 ameliorates A\u0026beta;42-mediated neurodegeneration in the Drosophila eye. \u003cem\u003eCell Death \u0026amp;amp; Disease\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e (2023).\u003c/li\u003e\n\u003cli\u003eMondal, P.\u003cem\u003e et al.\u003c/em\u003e Structure‐Based Discovery of A Small Molecule Inhibitor of Histone Deacetylase 6 (HDAC6) that Significantly Reduces Alzheimer\u0026apos;s Disease Neuropathology. \u003cem\u003eAdvanced Science\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e (2024).\u003c/li\u003e\n\u003cli\u003eMazzetti, S.\u003cem\u003e et al.\u003c/em\u003e Linking acetylated \u0026alpha;-Tubulin redistribution to \u0026alpha;-Synuclein pathology in brain of Parkinson\u0026rsquo;s disease patients. \u003cem\u003enpj Parkinson\u0026apos;s Disease\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e (2024).\u003c/li\u003e\n\u003cli\u003eEsteves, A.R.\u003cem\u003e et al.\u003c/em\u003e Acetylation as a major determinant to microtubule-dependent autophagy: Relevance to Alzheimer\u0026apos;s and Parkinson disease pathology. \u003cem\u003eBiochim Biophys Acta Mol Basis Dis\u003c/em\u003e \u003cstrong\u003e1865\u003c/strong\u003e, 2008-2023 (2019).\u003c/li\u003e\n\u003cli\u003eSingh, K.\u003cem\u003e et al.\u003c/em\u003e The combined impact of IgLON family proteins Lsamp and Neurotrimin on developing neurons and behavioral profiles in mouse. \u003cem\u003eBrain Research Bulletin\u003c/em\u003e \u003cstrong\u003e140\u003c/strong\u003e, 5-18 (2018).\u003c/li\u003e\n\u003cli\u003eBregin, A.\u003cem\u003e et al.\u003c/em\u003e Increased sensitivity to psychostimulants and GABAergic drugs in Lsamp-deficient mice. \u003cem\u003ePharmacology Biochemistry and Behavior\u003c/em\u003e \u003cstrong\u003e183\u003c/strong\u003e, 87-97 (2019).\u003c/li\u003e\n\u003cli\u003eBregin, A.\u003cem\u003e et al.\u003c/em\u003e Expression and impact of Lsamp neural adhesion molecule in the serotonergic neurotransmission system. \u003cem\u003ePharmacology, Biochemistry, and Behavior\u003c/em\u003e \u003cstrong\u003e198\u003c/strong\u003e, 173017 (2020).\u003c/li\u003e\n\u003cli\u003eUm, J.W. \u0026amp; Ko, J. Neural Glycosylphosphatidylinositol-Anchored Proteins in Synaptic Specification. \u003cem\u003eTrends in Cell Biology\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 931-945 (2017).\u003c/li\u003e\n\u003cli\u003eChen, C.-H., Hsu, H.-W., Chang, Y.-H. \u0026amp; Pan, C.-L. Adhesive L1CAM-Robo Signaling Aligns Growth Cone F-Actin Dynamics to Promote Axon-Dendrite Fasciculation in C. elegans. \u003cem\u003eDevelopmental Cell\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 215-228.e215 (2019).\u003c/li\u003e\n\u003cli\u003eSchlager, M.A. \u0026amp; Hoogenraad, C.C. Basic mechanisms for recognition and transport of synaptic cargos. \u003cem\u003eMolecular Brain\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 25 (2009).\u003c/li\u003e\n\u003cli\u003ePeng, L. \u0026amp; Wang, T. Histamine synthesis and transport are coupled in axon terminals via a dual quality control system. \u003cem\u003eThe EMBO Journal\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 4472-4491 (2024).\u003c/li\u003e\n\u003cli\u003ePimenta, A.F.\u003cem\u003e et al.\u003c/em\u003e The limbic system-associated membrane protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 287-297 (1995).\u003c/li\u003e\n\u003cli\u003eLi, P.\u003cem\u003e et al.\u003c/em\u003e Single-cell analysis of Schistosoma mansoni identifies a conserved genetic program controlling germline stem cell fate. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 485 (2021).\u003c/li\u003e\n\u003cli\u003eNaryzhny, S.N. Proliferating cell nuclear antigen: a proteomics view. \u003cem\u003eCellular and Molecular Life Sciences\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 3789-3808 (2008).\u003c/li\u003e\n\u003cli\u003eLiu, C.\u003cem\u003e et al.\u003c/em\u003e Research progress on the pathogenesis of the SDHB mutation and related diseases. \u003cem\u003eBiomedicine \u0026amp;amp; Pharmacotherapy\u003c/em\u003e \u003cstrong\u003e167\u003c/strong\u003e, 115500 (2023).\u003c/li\u003e\n\u003cli\u003eShkreta, L., Delannoy, A., Toutant, J. \u0026amp; Chabot, B. Regulatory interplay between SR proteins governs \u003cem\u003eCLK1 \u003c/em\u003ekinase splice variants production. \u003cem\u003eRNA\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1596-1607 (2024).\u003c/li\u003e\n\u003cli\u003eSang, C.\u003cem\u003e et al.\u003c/em\u003e Reversible acetylation of HDAC8 regulates cell cycle. \u003cem\u003eEMBO Reports\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 3925-3943 (2024).\u003c/li\u003e\n\u003cli\u003eDodd, D.O.\u003cem\u003e et al.\u003c/em\u003e Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e384\u003c/strong\u003e (2024).\u003c/li\u003e\n\u003cli\u003eYou, Y.\u003cem\u003e et al.\u003c/em\u003e An improved medium for in vitro studies of female reproduction and oviposition in Schistosoma japonicum. \u003cem\u003eParasit Vectors\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 116 (2024).\u003c/li\u003e\n\u003cli\u003eLi, X., Weth, O., Haeberlein, S. \u0026amp; Grevelding, C.G. Molecular characterization of Smtdc-1 and Smddc-1 discloses roles as male-competence factors for the sexual maturation of Schistosoma mansoni females. \u003cem\u003eFrontiers in Cellular and Infection Microbiology\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e (2023).\u003c/li\u003e\n\u003cli\u003eNikishin, D.A., Alyoshina, N.M., Semenova, M.L. \u0026amp; Shmukler, Y.B. Analysis of Expression and Functional Activity of Aromatic L-Amino Acid Decarboxylase (DDC) and Serotonin Transporter (SERT) as Potential Sources of Serotonin in Mouse Ovary. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 3070 (2019).\u003c/li\u003e\n\u003cli\u003eKhan, U.W. \u0026amp; Newmark, P.A. Somatic regulation of female germ cell regeneration and development in planarians. \u003cem\u003eCell Reports\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 110525 (2022).\u003c/li\u003e\n\u003cli\u003eAlkema, M.J., Hunter-Ensor, M., Ringstad, N. \u0026amp; Horvitz, H.R. Tyramine Functions independently of octopamine in the Caenorhabditis elegans nervous system. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 247-260 (2005).\u003c/li\u003e\n\u003cli\u003eMartineau, M.\u003cem\u003e et al.\u003c/em\u003e Storage and uptake of D-serine into astrocytic synaptic-like vesicles specify gliotransmission. \u003cem\u003eJ Neurosci\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 3413-3423 (2013).\u003c/li\u003e\n\u003cli\u003eLi, D.\u003cem\u003e et al.\u003c/em\u003e Astrocyte VAMP3 vesicles undergo Ca2+ -independent cycling and modulate glutamate transporter trafficking. \u003cem\u003eThe Journal of Physiology\u003c/em\u003e \u003cstrong\u003e593\u003c/strong\u003e, 2807-2832 (2015).\u003c/li\u003e\n\u003cli\u003eKumar, R.\u003cem\u003e et al.\u003c/em\u003e Fibroblast Growth Factor 2‐Mediated Regulation of Neuronal Exosome Release Depends on VAMP3/Cellubrevin in Hippocampal Neurons. \u003cem\u003eAdvanced Science\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 1902372 (2020).\u003c/li\u003e\n\u003cli\u003eSneeggen, M.\u003cem\u003e et al.\u003c/em\u003e WDFY2 restrains matrix metalloproteinase secretion and cell invasion by controlling VAMP3-dependent recycling. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 2850 (2019).\u003c/li\u003e\n\u003cli\u003eLuo, F.\u003cem\u003e et al.\u003c/em\u003e A chromosome-level genome of the human blood fluke Schistosoma japonicum identifies the genomic basis of host-switching. \u003cem\u003eCell Reports\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 110638 (2022).\u003c/li\u003e\n\u003cli\u003eCheng, S.\u003cem\u003e et al.\u003c/em\u003e Dynamic profiles of lncRNAs reveal a functional natural antisense RNA that regulates the development of Schistosoma japonicum. \u003cem\u003ePLOS Pathogens\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, e1011949 (2024).\u003c/li\u003e\n\u003cli\u003eSatija, R., Farrell, J.A., Gennert, D., Schier, A.F. \u0026amp; Regev, A. Spatial reconstruction of single-cell gene expression data. \u003cem\u003eNature Biotechnology\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 495-502 (2015).\u003c/li\u003e\n\u003cli\u003eXia, J.\u003cem\u003e et al.\u003c/em\u003e A single-cell resolution developmental atlas of hematopoietic stem and progenitor cell expansion in zebrafish. \u003cem\u003eProceedings of the National Academy of Sciences of the United States of America\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, e2015748118 (2021).\u003c/li\u003e\n\u003cli\u003eYu, G., Wang, L.G., Han, Y. \u0026amp; He, Q.Y. clusterProfiler: an R package for comparing biological themes among gene clusters. \u003cem\u003eOMICS\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 284-287 (2012).\u003c/li\u003e\n\u003cli\u003eDann, E., Henderson, N.C., Teichmann, S.A., Morgan, M.D. \u0026amp; Marioni, J.C. Differential abundance testing on single-cell data using k-nearest neighbor graphs. \u003cem\u003eNature Biotechnology\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 245-253 (2021).\u003c/li\u003e\n\u003cli\u003eLa Manno, G.\u003cem\u003e et al.\u003c/em\u003e RNA velocity of single cells. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e560\u003c/strong\u003e, 494-498 (2018).\u003c/li\u003e\n\u003cli\u003eLange, M.\u003cem\u003e et al.\u003c/em\u003e CellRank for directed single-cell fate mapping. \u003cem\u003eNat Methods\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 159-170 (2022).\u003c/li\u003e\n\u003cli\u003eWolf, F.A.\u003cem\u003e et al.\u003c/em\u003e PAGA: graph abstraction reconciles clustering with trajectory inference through a topology preserving map of single cells. \u003cem\u003eGenome Biol\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 59 (2019).\u003c/li\u003e\n\u003cli\u003eStreet, K.\u003cem\u003e et al.\u003c/em\u003e Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics. \u003cem\u003eBMC Genomics\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 477 (2018).\u003c/li\u003e\n\u003cli\u003eGu, Z., Eils, R. \u0026amp; Schlesner, M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 2847-2849 (2016).\u003c/li\u003e\n\u003cli\u003eChen, S., Zhou, Y., Chen, Y. \u0026amp; Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, i884-i890 (2018).\u003c/li\u003e\n\u003cli\u003eKim, D., Paggi, J.M., Park, C., Bennett, C. \u0026amp; Salzberg, S.L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. \u003cem\u003eNat Biotechnol\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 907-915 (2019).\u003c/li\u003e\n\u003cli\u003eLiao, Y., Smyth, G.K. \u0026amp; Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 923-930 (2014).\u003c/li\u003e\n\u003cli\u003eLove, M.I., Huber, W. \u0026amp; Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. \u003cem\u003eGenome Biology\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e (2014).\u003c/li\u003e\n\u003cli\u003eCollins, J.J., King, R.S., Cogswell, A., Williams, D.L. \u0026amp; Newmark, P.A. An Atlas for Schistosoma mansoni Organs and Life-Cycle Stages Using Cell Type-Specific Markers and Confocal Microscopy. \u003cem\u003ePLoS Neglected Tropical Diseases\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, e1009 (2011).\u003c/li\u003e\n\u003cli\u003eCreppe, C.\u003cem\u003e et al.\u003c/em\u003e Elongator Controls the Migration and Differentiation of Cortical Neurons through Acetylation of \u0026alpha;-Tubulin. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e136\u003c/strong\u003e, 551-564 (2009).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Schistosomes, single-cell RNA sequencing, male-female interaction, reproductive development, ventral neural fibers","lastPublishedDoi":"10.21203/rs.3.rs-6983189/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6983189/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSexual maturation and egg production in schistosomes require sustained male-female pairing, yet the cellular dynamics underlying this process remain poorly understood. Here, we generated a high-resolution single-cell transcriptomic atlas of 104,671 cells from male and female \u003cem\u003eSchistosoma japonicum\u003c/em\u003e across four developmental stages, identifying 76 distinct cell populations and mapping lineage trajectories in reproductive and neural systems. Notably, we discovered three male-specific neuron subtypes, including \u003cem\u003edelta-3\u003c/em\u003e neurons located in the gynecophoric canal, and identified \u003cem\u003elsamp \u003c/em\u003eas a critical gene co-expressed with \u003cem\u003enrps\u003c/em\u003e in \u003cem\u003edelta-3\u003c/em\u003e neurons. Loss of \u003cem\u003elsamp \u003c/em\u003eimpaired axonal growth and disrupted the male ventral nerve network, reducing the targeted delivery of pheromone BATT precursors to \u003cem\u003edelta-3\u003c/em\u003e neurons, thereby hindering female reproductive maturation. This work maps schistosome male-female interactions at single-cell resolution, revealing a male ventral nerve network that facilitates intersex communication and promotes female reproductive development, offering the new strategies to disrupt parasite reproduction and combat schistosomiasis.\u003c/p\u003e","manuscriptTitle":"Dynamic single-cell transcriptomics reveals lsamp-guided neural network formation in male S. japonicum driving female reproduction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 06:54:59","doi":"10.21203/rs.3.rs-6983189/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8242195a-3f1b-44de-85ca-f696e41fb7f7","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51240453,"name":"Biological sciences/Microbiology/Parasitology/Parasite biology"},{"id":51240454,"name":"Biological sciences/Molecular biology/Transcriptomics"},{"id":51240455,"name":"Biological sciences/Microbiology/Pathogens"}],"tags":[],"updatedAt":"2026-02-14T08:11:22+00:00","versionOfRecord":{"articleIdentity":"rs-6983189","link":"https://doi.org/10.1038/s41467-026-68305-7","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-01-22 05:00:00","publishedOnDateReadable":"January 22nd, 2026"},"versionCreatedAt":"2025-07-10 06:54:59","video":"","vorDoi":"10.1038/s41467-026-68305-7","vorDoiUrl":"https://doi.org/10.1038/s41467-026-68305-7","workflowStages":[]},"version":"v1","identity":"rs-6983189","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6983189","identity":"rs-6983189","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}