Identification and characterisation of the tegument-expressed aldehyde dehydrogenase SmALDH_312 of Schistosoma mansoni, a target of disulfiram.

Schistosomiasis is an infectious disease caused by blood flukes of the genus Schistosoma and affects approximately 200 million people worldwide. Since Praziquantel (PZQ) is the only drug for schistosomiasis, alternatives are needed. By a biochemical approach, we identified a tegumentally expressed aldehyde dehydrogenase (ALDH) of S. mansoni, SmALDH_312. Molecular analyses of adult parasites showed Smaldh_312 transcripts in both genders and different tissues. Physiological and cell-biological experiments exhibited detrimental effects of the drug disulfiram (DSF), a known ALDH inhibitor, on larval and adult schistosomes in vitro. DSF also reduced stem-cell proliferation and caused severe tegument damage in treated worms. In silico-modelling of SmALDH_312 and docking analyses predicted DSF binding, which we finally confirmed by enzyme assays with recombinant SmALDH_312. Furthermore, we identified compounds of the Medicine for Malaria Venture (MMV) pathogen box inhibiting SmALDH_312 activity. Our findings represent a promising starting point for further development towards new drugs for schistosomiasis.

The impact of species-wide gene expression variation on Caenorhabditis elegans complex traits.

Phenotypic variation in organism-level traits has been studied in Caenorhabditis elegans wild strains, but the impacts of differences in gene expression and the underlying regulatory mechanisms are largely unknown. Here, we use natural variation in gene expression to connect genetic variants to differences in organismal-level traits, including drug and toxicant responses. We perform transcriptomic analyses on 207 genetically distinct C. elegans wild strains to study natural regulatory variation of gene expression. Using this massive dataset, we perform genome-wide association mappings to investigate the genetic basis underlying gene expression variation and reveal complex genetic architectures. We find a large collection of hotspots enriched for expression quantitative trait loci across the genome. We further use mediation analysis to understand how gene expression variation could underlie organism-level phenotypic variation for a variety of complex traits. These results reveal the natural diversity in gene expression and possible regulatory mechanisms in this keystone model organism, highlighting the promise of using gene expression variation to understand how phenotypic diversity is generated.

Evaluation of the in vitro susceptibility of various filarial nematodes to emodepside.

Filariae are vector-borne nematodes responsible for an enormous burden of disease. Human lymphatic filariasis, caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori, and onchocerciasis (caused by Onchocerca volvulus) are neglected parasitic diseases of major public health significance in tropical regions. To date, therapeutic efforts to eliminate human filariasis have been hampered by the lack of a drug with sufficient macrofilaricidal and/or long-term sterilizing effects that is suitable for use in mass drug administration (MDA) programs, particularly in areas co-endemic with Loa loa, the causative agent of loiasis. Emodepside, a semi-synthetic cyclooctadepsipeptide, has been shown to have broad-spectrum efficacy against gastrointestinal nematodes in a variety of mammalian hosts, and has been approved as an active ingredient in dewormers for cats and dogs. This paper evaluates, compares (where appropriate) and summarizes the in vitro effects of emodepside against a range of filarial nematodes at various developmental stages. Emodepside inhibited the motility of all tested stages of filariae frequently used as surrogate species for preclinical investigations (Acanthocheilonema viteae, Brugia pahangi, Litomosoides sigmodontis, Onchocerca gutturosa, and Onchocerca lienalis), human-pathogenic filariae (B. malayi) and filariae of veterinary importance (Dirofilaria immitis) in a concentration-dependent manner. While motility of all filariae was inhibited, both stage- and species-specific differences were observed. However, whether these differences were detected because of stage- and/or species-specific factors or as a consequence of variations in protocol parameters among the participating laboratories (such as purification of the parasites, read-out units, composition of media, incubation conditions, duration of incubation etc.) remains unclear. This study, however, clearly shows that emodepside demonstrates broad-spectrum in vitro activity against filarial nematode species across different genera and can therefore be validated as a promising candidate for the treatment of human filariases, including onchocerciasis and lymphatic filariasis.

Development of emodepside as a possible adulticidal treatment for human onchocerciasis-The fruit of a successful industrial-academic collaboration.

Current mass drug administration (MDA) programs for the treatment of human river blindness (onchocerciasis) caused by the filarial worm Onchocerca volvulus rely on ivermectin, an anthelmintic originally developed for animal health. These treatments are primarily directed against migrating microfilariae and also suppress fecundity for several months, but fail to eliminate adult O. volvulus. Therefore, elimination programs need time frames of decades, well exceeding the life span of adult worms. The situation is worsened by decreased ivermectin efficacy after long-term therapy. To improve treatment options against onchocerciasis, a drug development candidate should ideally kill or irreversibly sterilize adult worms. Emodepside is a broad-spectrum anthelmintic used for the treatment of parasitic nematodes in cats and dogs (Profender and Procox). Our current knowledge of the pharmacology of emodepside is the result of more than 2 decades of intensive collaborative research between academia and the pharmaceutical industry. Emodepside has a novel mode of action with a broad spectrum of activity, including against extraintestinal nematode stages such as migrating larvae or macrofilariae. Therefore, emodepside is considered to be among the most promising candidates for evaluation as an adulticide treatment against onchocerciasis. Consequently, in 2014, Bayer and the Drugs for Neglected Diseases initiative (DNDi) started a collaboration to develop emodepside for the treatment of patients suffering from the disease. Macrofilaricidal activity has been demonstrated in various models, including Onchocerca ochengi in cattle, the parasite most closely related to O. volvulus. Emodepside has now successfully passed Phase I clinical trials, and a Phase II study is planned. This Bayer-DNDi partnership is an outstanding example of "One World Health," in which experience gained in veterinary science and drug development is translated to human health and leads to improved tools to combat neglected tropical diseases (NTDs) and shorten development pathways and timelines in an otherwise neglected area.

Comparison of electrophysiological and motility assays to study anthelmintic effects in Caenorhabditis elegans.

Currently, only a few chemical drug classes are available to control the global burden of nematode infections in humans and animals. Most of these drugs exert their anthelmintic activity by interacting with proteins such as ion channels, and the nematode neuromuscular system remains a promising target for novel intervention strategies. Many commonly-used phenotypic readouts such as motility provide only indirect insight into neuromuscular function and the site(s) of action of chemical compounds. Electrophysiological recordings provide more specific information but are typically technically challenging and lack high throughput for drug discovery. Because drug discovery relies strongly on the evaluation and ranking of drug candidates, including closely related chemical derivatives, precise assays and assay combinations are needed for capturing and distinguishing subtle drug effects. Past studies show that nematode motility and pharyngeal pumping (feeding) are inhibited by most anthelmintic drugs. Here we compare two microfluidic devices ("chips") that record electrophysiological signals from the nematode pharynx (electropharyngeograms; EPGs) ─ the ScreenChip™ and the 8-channel EPG platform ─ to evaluate their respective utility for anthelmintic research. We additionally compared EPG data with whole-worm motility measurements obtained with the wMicroTracker instrument. As references, we used three macrocyclic lactones (ivermectin, moxidectin, and milbemycin oxime), and levamisole, which act on different ion channels. Drug potencies (IC50 and IC95 values) from concentration-response curves, and the time-course of drug effects, were compared across platforms and across drugs. Drug effects on pump timing and EPG waveforms were also investigated. These experiments confirmed drug-class specific effects of the tested anthelmintics and illustrated the relative strengths and limitations of the different assays for anthelmintic research.

Emodepside targets SLO-1 channels of Onchocerca ochengi and induces broad anthelmintic effects in a bovine model of onchocerciasis.

Onchocerciasis (river blindness), caused by the filarial worm Onchocerca volvulus, is a neglected tropical disease mostly affecting sub-Saharan Africa and is responsible for >1.3 million years lived with disability. Current control relies almost entirely on ivermectin, which suppresses symptoms caused by the first-stage larvae (microfilariae) but does not kill the long-lived adults. Here, we evaluated emodepside, a semi-synthetic cyclooctadepsipeptide registered for deworming applications in companion animals, for activity against adult filariae (i.e., as a macrofilaricide). We demonstrate the equivalence of emodepside activity on SLO-1 potassium channels in Onchocerca volvulus and Onchocerca ochengi, its sister species from cattle. Evaluation of emodepside in cattle as single or 7-day treatments at two doses (0.15 and 0.75 mg/kg) revealed rapid activity against microfilariae, prolonged suppression of female worm fecundity, and macrofilaricidal effects by 18 months post treatment. The drug was well tolerated, causing only transiently increased blood glucose. Female adult worms were mostly paralyzed; however, some retained metabolic activity even in the multiple high-dose group. These data support ongoing clinical development of emodepside to treat river blindness.

Two novel loci underlie natural differences in Caenorhabditis elegans abamectin responses.

Parasitic nematodes cause a massive worldwide burden on human health along with a loss of livestock and agriculture productivity. Anthelmintics have been widely successful in treating parasitic nematodes. However, resistance is increasing, and little is known about the molecular and genetic causes of resistance for most of these drugs. The free-living roundworm Caenorhabditis elegans provides a tractable model to identify genes that underlie resistance. Unlike parasitic nematodes, C. elegans is easy to maintain in the laboratory, has a complete and well annotated genome, and has many genetic tools. Using a combination of wild isolates and a panel of recombinant inbred lines constructed from crosses of two genetically and phenotypically divergent strains, we identified three genomic regions on chromosome V that underlie natural differences in response to the macrocyclic lactone (ML) abamectin. One locus was identified previously and encodes an alpha subunit of a glutamate-gated chloride channel (glc-1). Here, we validate and narrow two novel loci using near-isogenic lines. Additionally, we generate a list of prioritized candidate genes identified in C. elegans and in the parasite Haemonchus contortus by comparison of ML resistance loci. These genes could represent previously unidentified resistance genes shared across nematode species and should be evaluated in the future. Our work highlights the advantages of using C. elegans as a model to better understand ML resistance in parasitic nematodes.

Caenorhabditis elegans in anthelmintic research - Old model, new perspectives.

For more than four decades, the free-living nematode Caenorhabditis elegans has been extensively used in anthelmintic research. Classic genetic screens and heterologous expression in the C. elegans model enormously contributed to the identification and characterization of molecular targets of all major anthelmintic drug classes. Although these findings provided substantial insights into common anthelmintic mechanisms, a breakthrough in the treatment and control of parasitic nematodes is still not in sight. Instead, we are facing increasing evidence that the enormous diversity within the phylum Nematoda cannot be recapitulated by any single free-living or parasitic species and the development of novel broad-spectrum anthelmintics is not be a simple goal. In the present review, we summarize certain milestones and challenges of the C. elegans model with focus on drug target identification, anthelmintic drug discovery and identification of resistance mechanisms. Furthermore, we present new perspectives and strategies on how current progress in C. elegans research will support future anthelmintic research.

Quantitative benzimidazole resistance and fitness effects of parasitic nematode beta-tubulin alleles.

Infections by parasitic nematodes inflict a huge burden on the health of humans and livestock throughout the world. Anthelmintic drugs are the first line of defense against these infections. Unfortunately, resistance to these drugs is rampant and continues to spread. To improve treatment strategies, we must understand the genetics and molecular mechanisms that underlie resistance. Studies of the fungus Aspergillus nidulans and the free-living nematode Caenorhabditis elegans discovered that a beta-tubulin gene is mutated in benzimidazole (BZ) resistant strains. In parasitic nematode populations, three beta-tubulin alleles, F167Y, E198A, and F200Y, have long been correlated with resistance. Additionally, improvements in sequencing technologies have identified new alleles - E198V, E198L, E198K, E198I, and E198Stop - also correlated with BZ resistance. However, none of these alleles have been proven to cause resistance. To empirically demonstrate this point, we independently introduced the F167Y, E198A, and F200Y alleles as well as two of the newly identified alleles, E198V and E198L, into the BZ susceptible C. elegans N2 genetic background using the CRISPR-Cas9 system. These genome-edited strains were exposed to both albendazole and fenbendazole to quantitatively measure animal responses to BZs. We used a range of concentrations for each BZ compound to define response curves and found that all five of the alleles conferred resistance to BZ compounds equal to a loss of the entire beta-tubulin gene. These results prove that the parasite beta-tubulin alleles cause resistance. The E198V allele is found at low frequencies along with the E198L allele in natural parasite populations, suggesting that it could affect fitness. We performed competitive fitness assays and demonstrated that the E198V allele reduces animal health, supporting the hypothesis that this allele might be less fit in field populations. Overall, we present a powerful platform to quantitatively assess anthelmintic resistance and effects of specific resistance alleles on organismal fitness in the presence or absence of the drug.

Deep sampling of Hawaiian Caenorhabditis elegans reveals high genetic diversity and admixture with global populations.

Hawaiian isolates of the nematode species Caenorhabditis elegans have long been known to harbor genetic diversity greater than the rest of the worldwide population, but this observation was supported by only a small number of wild strains. To better characterize the niche and genetic diversity of Hawaiian C. elegans and other Caenorhabditis species, we sampled different substrates and niches across the Hawaiian islands. We identified hundreds of new Caenorhabditis strains from known species and a new species, Caenorhabditis oiwi. Hawaiian C. elegans are found in cooler climates at high elevations but are not associated with any specific substrate, as compared to other Caenorhabditis species. Surprisingly, admixture analysis revealed evidence of shared ancestry between some Hawaiian and non-Hawaiian C. elegans strains. We suggest that the deep diversity we observed in Hawaii might represent patterns of ancestral genetic diversity in the C. elegans species before human influence.

Identification of a new panel of reference genes to study pairing-dependent gene expression in Schistosoma mansoni.

Facilitated by the Schistosoma mansoni genome project, multiple transcriptomic studies were performed over the last decade to elucidate gene expression patterns among different developmental stages of the complex schistosome life cycle. While these analyses enable the identification of candidate genes with key functions in schistosome biology, a diverse molecular tool set is needed that allows comprehensive functional characterization at the single gene level. This includes the availability of reliable reference genes to confirm changes in the transcription of genes of interest over different biological samples and experimental conditions. In particular, the investigation of one key aspect of schistosome biology, the pairing-dependent gene expression in females and males, requires knowledge on reference genes that are expressed independently of both pairing and of in vitro culture effects. Therefore, the present study focused on the identification of quantitative reverse transcription (qRT)-PCR reference genes suitable for the investigation of pairing-dependent gene expression in the S. mansoni male. The "pipeline" we present here is based on qRT-PCR analyses of high biological replication combined with three different statistical analysis tools, BestKeeper, geNorm, and NormFinder. Our approach resulted in a statistically robust ranking of 15 selected reference genes with respect to their transcription stability between pairing-unexperienced and -experienced males. We further tested the top seven candidate genes for their transcription stability during invitro culture of adult S. mansoni. Of these, the two most suitable reference genes were used to investigate the influence of the pairing contact on the transcription of genes of interest, comprising a tyrosine decarboxylase gene Smtdc1, an ebony ortholog Smebony, and the follistatin ortholog Smfst in S. mansoni males. Performing pairing, separation and re-pairing experiments with adult S. mansoni in vitro, our results indicate for the first time that pairing can act as a molecular on/off-switch of specific genes to strictly control their expression in schistosome males.

Insects in anthelminthics research: Lady beetle-derived harmonine affects survival, reproduction and stem cell proliferation of Schistosoma mansoni.

Natural products have moved into the spotlight as possible sources for new drugs in the treatment of helminth infections including schistosomiasis. Surprisingly, insect-derived compounds have largely been neglected so far in the search for novel anthelminthics, despite the generally recognized high potential of insect biotechnology for drug discovery. This motivated us to assess the antischistosomal capacity of harmonine, an antimicrobial alkaloid from the harlequin ladybird Harmonia axyridis that raised high interest in insect biotechnology in recent years. We observed remarkably pleiotropic effects of harmonine on physiological, cellular, and molecular processes in adult male and female Schistosoma mansoni at concentrations as low as 5 µM in vitro. This included tegumental damage, gut dilatation, dysplasia of gonads, a complete stop of egg production at 10 µM, and increased production of abnormally shaped eggs at 5 µM. Motility was reduced with an EC50 of 8.8 µM and lethal effects occurred at 10-20 µM within 3 days of culture. Enzyme inhibition assays revealed acetylcholinesterase (AChE) as one potential target of harmonine. To assess possible effects on stem cells, which represent attractive anthelminthic targets, we developed a novel in silico 3D reconstruction of gonads based on confocal laser scanning microscopy of worms after EdU incorporation to allow for quantification of proliferating stem cells per organ. Harmonine significantly reduced the number of proliferating stem cells in testes, ovaries, and also the number of proliferating parenchymal neoblasts. This was further supported by a downregulated expression of the stem cell markers nanos-1 and nanos-2 in harmonine-treated worms revealed by quantitative real-time PCR. Our data demonstrate a multifaceted antischistosomal activity of the lady beetle-derived compound harmonine, and suggest AChE and stem cell genes as possible targets. Harmonine is the first animal-derived alkaloid detected to have antischistosomal capacity. This study highlights the potential of exploiting insects as a source for the discovery of anthelminthics.

Extreme allelic heterogeneity at a Caenorhabditis elegans beta-tubulin locus explains natural resistance to benzimidazoles.

Benzimidazoles (BZ) are essential components of the limited chemotherapeutic arsenal available to control the global burden of parasitic nematodes. The emerging threat of BZ resistance among multiple nematode species necessitates the development of novel strategies to identify genetic and molecular mechanisms underlying this resistance. All detection of parasitic helminth resistance to BZ is focused on the genotyping of three variant sites in the orthologs of the ß-tubulin gene found to confer resistance in the free-living nematode Caenorhabditis elegans. Because of the limitations of laboratory and field experiments in parasitic nematodes, it is difficult to look beyond these three sites to identify additional mechanisms that might contribute to BZ resistance in the field. Here, we took an unbiased genome-wide mapping approach in the free-living nematode species C. elegans to identify the genetic underpinnings of natural resistance to the commonly used BZ, albendazole (ABZ). We found a wide range of natural variation in ABZ resistance in natural C. elegans populations. In agreement with known mechanisms of BZ resistance in parasites, we found that a majority of the variation in ABZ resistance among wild C. elegans strains is caused by variation in the ß-tubulin gene ben-1. This result shows empirically that resistance to ABZ naturally exists and segregates within the C. elegans population, suggesting that selection in natural niches could enrich for resistant alleles. We identified 25 distinct ben-1 alleles that are segregating at low frequencies within the C. elegans population, including many novel molecular variants. Population genetic analyses indicate that ben-1 variation arose multiple times during the evolutionary history of C. elegans and provide evidence that these alleles likely occurred recently because of local selective pressures. Additionally, we find purifying selection at all five ß-tubulin genes, despite predicted loss-of-function variants in ben-1, indicating that BZ resistance in natural niches is a stronger selective pressure than loss of one ß-tubulin gene. Furthermore, we used genome-editing to show that the most common parasitic nematode ß-tubulin allele that confers BZ resistance, F200Y, confers resistance in C. elegans. Importantly, we identified a novel genomic region that is correlated with ABZ resistance in the C. elegans population but independent of ben-1 and the other ß-tubulin loci, suggesting that there are multiple mechanisms underlying BZ resistance. Taken together, our results establish a population-level resource of nematode natural diversity as an important model for the study of mechanisms that give rise to BZ resistance.

Tissue-specific transcriptome analyses provide new insights into GPCR signalling in adult Schistosoma mansoni.

Schistosomes are blood-dwelling trematodes with global impact on human and animal health. Because medical treatment is currently based on a single drug, praziquantel, there is urgent need for the development of alternative control strategies. The Schistosoma mansoni genome project provides a platform to study and connect the genetic repertoire of schistosomes to specific biological functions essential for successful parasitism. G protein-coupled receptors (GPCRs) form the largest superfamily of transmembrane receptors throughout the Eumetazoan phyla, including platyhelminths. Due to their involvement in diverse biological processes, their pharmacological importance, and proven druggability, GPCRs are promising targets for new anthelmintics. However, to identify candidate receptors, a more detailed understanding of the roles of GPCR signalling in schistosome biology is essential. An updated phylogenetic analysis of the S. mansoni GPCR genome (GPCRome) is presented, facilitated by updated genome data that allowed a more precise annotation of GPCRs. Additionally, we review the current knowledge on GPCR signalling in this parasite and provide new insights into the potential roles of GPCRs in schistosome reproduction based on the findings of a recent tissue-specific transcriptomic study in paired and unpaired S. mansoni. According to the current analysis, GPCRs contribute to gonad-specific functions but also to nongonad, pairing-dependent processes. The latter may regulate gonad-unrelated functions during the multifaceted male-female interaction. Finally, we compare the schistosome GPCRome to that of another parasitic trematode, Fasciola, and discuss the importance of GPCRs to basic and applied research. Phylogenetic analyses display GPCR diversity in free-living and parasitic platyhelminths and suggest diverse functions in schistosomes. Although their roles need to be substantiated by functional studies in the future, the data support the selection of GPCR candidates for basic and applied studies, invigorating the exploitation of this important receptor class for drug discovery against schistosomes but also other trematodes.

A gene expression atlas of adult Schistosoma mansoni and their gonads.

RNA-Seq has proven excellence in providing information about the regulation and transcript levels of genes. We used this method for profiling genes in the flatworm Schistosoma mansoni. This parasite causes schistosomiasis, an infectious disease of global importance for human and animals. The pathology of schistosomiasis is associated with the eggs, which are synthesized as a final consequence of male and female adults pairing. The male induces processes in the female that lead to the full development of its gonads as a prerequisite for egg production. Unpaired females remain sexually immature. Based on an organ-isolation method we obtained gonad tissue for RNA extraction from paired and unpaired schistosomes, with whole adults included as controls. From a total of 23 samples, we used high-throughput cDNA sequencing (RNA-Seq) on the Illumina platform to profile gene expression between genders and tissues, with and without pairing influence. The data obtained provide a wealth of information on the reproduction biology of schistosomes and a rich resource for exploitation through basic and applied research activities.

First characterization of SmOPG1, a novel protein involved in gonad-associated processes in Schistosoma mansoni.

In the context of investigating the exceptional reproductive biology of schistosomes, several studies have focused on the identification and characterization of involved molecules. Among these are cellular tyrosine kinases (CTKs) which control differentiation processes in the female gonads. On the way to unravel CTK-mediated signaling processes in more detail, several upstream- and downstream partners of these CTKs were identified. In this context we present here first data characterizing the novel orphan gene Sm opg1. Annotated as hypothetical protein, SmOPG1 was identified as an interaction partner of the CTK SmTK6 by yeast two-hybrid library screening. Y2/3H interaction studies showed that SmTK6 binds with its SH2 domain to a specific binding motif within the C-terminus of SmOPG1. Additionally, in situ-hybridization and organ-specific RT-PCR analyses demonstrated the co-localization of SmOPG1 and SmTK6 transcripts in the gonads of adult S. mansoni. Finally, SmOPG1 knock-down provided first hints for a function in the ovary.

Evidence for Integrin - Venus Kinase Receptor 1 Alliance in the Ovary of Schistosoma mansoni Females Controlling Cell Survival.

In metazoan integrin signaling is an important process of mediating extracellular and intracellular communication processes. This can be achieved by cooperation of integrins with growth factor receptors (GFRs). Schistosoma mansoni is a helminth parasite inducing schistosomiasis, an infectious disease of worldwide significance for humans and animals. First studies on schistosome integrins revealed their role in reproductive processes, being involved in spermatogenesis and oogenesis. With respect to the roles of eggs for maintaining the parasite´s life cycle and for inducing the pathology of schistosomiasis, elucidating reproductive processes is of high importance. Here we studied the interaction of the integrin receptor Smß-Int1 with the venus kinase receptor SmVKR1 in S. mansoni. To this end we cloned and characterized SmILK, SmPINCH, and SmNck2, three putative bridging molecules for their role in mediating Smß-Int1/SmVKR1 cooperation. Phylogenetic analyses showed that these molecules form clusters that are specific for parasitic platyhelminths as it was shown for integrins before. Transcripts of all genes colocalized in the ovary. In Xenopus oocytes germinal vesicle breakdown (GVBD) was only induced if all members were simultaneously expressed. Coimmunoprecipitation results suggest that a Smß-Int1-SmILK-SmPINCH-SmNck2-SmVKR1 complex can be formed leading to the phosphorylation and activation of SmVKR1. These results indicate that SmVKR1 can be activated in a ligand-independent manner by receptor-complex interaction. RNAi and inhibitor studies to knock-down SmILK as a representative complex member concurrently revealed effects on the extracellular matrix surrounding the ovary and oocyte localization within the ovary, oocyte survival, and egg production. By TUNEL assays, confocal laser scanning microscopy (CLSM), Caspase-3 assay, and transcript profiling of the pro-apoptotic BCL-2 family members BAK/BAX we obtained first evidence for roles of this signaling complex in mediating cell death in immature and primary oocytes. These results suggest that the Smß-Int1/SmVKR1 signaling complex is important for differentiation and survival in oocytes of paired schistosomes.

SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways.

Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis.

Schistosome sex matters: a deep view into gonad-specific and pairing-dependent transcriptomes reveals a complex gender interplay.

As a key event for maintaining life cycles, reproduction is a central part of platyhelminth biology. In case of parasitic platyhelminths, reproductive processes can also contribute to pathology. One representative example is the trematode Schistosoma, which causes schistosomiasis, an infectious disease, whose pathology is associated with egg production. Among the outstanding features of schistosomes is their dioecious lifestyle and the pairing-dependent differentiation of the female gonads which finally leads to egg synthesis. To analyze the reproductive biology of Schistosoma mansoni in-depth we isolated complete ovaries and testes from paired and unpaired schistosomes for comparative RNA-seq analyses. Of >7,000 transcripts found in the gonads, 243 (testes) and 3,600 (ovaries) occurred pairing-dependently. Besides the detection of genes transcribed preferentially or specifically in the gonads of both genders, we uncovered pairing-induced processes within the gonads including stem cell-associated and neural functions. Comparisons to work on neuropeptidergic signaling in planarian showed interesting parallels but also remarkable differences and highlights the importance of the nervous system for flatworm gonad differentiation. Finally, we postulated first functional hints for 235 hypothetical genes. Together, these results elucidate key aspects of flatworm reproductive biology and will be relevant for basic as well as applied, exploitable research aspects.

Correction: Venus Kinase Receptors Control Reproduction in the Platyhelminth Parasite Schistosoma mansoni.

[This corrects the article DOI: 10.1371/journal.ppat.1004138.].