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In living cells, the genetic information stored in the DNA sequence is always associated with chromosomal and extra-chromosomal epigenetic information. Chromatin is formed by the DNA and associated proteins, in particular histones. Covalent histone modifications are important bearers of epigenetic information and as such have been increasingly studied since about the year 2000. One of the principal techniques to gather information about the association between DNA and modified histones is chromatin immunoprecipitation (ChIP), also combined with massive sequencing (ChIP-Seq). Automated ChIPmentation procedure is a convenient alternative to native chromatin immunoprecipitation (N-ChIP). It is now routinely used for ChIP-Seq in many model species, using in general roughly 106 cells per experiment. Such high cell numbers are sometimes difficult to produce. Using the human parasite Schistosoma mansoni, whose production requires sacrificing animals and should therefore be kept to a minimum, we show here that automated ChIPmentation is suitable for limited biological material. We define the operational limit as ≥20,000 Schistosoma cells. We also present a streamlined protocol for the preparation of ChIP input libraries.

The rhesus macaque provides a unique model of acquired immunity against schistosomes, which afflict >200 million people worldwide. By monitoring bloodstream levels of parasite-gut-derived antigen, we show that from week 10 onwards an established infection with Schistosoma mansoni is cleared in an exponential manner, eliciting resistance to reinfection. Secondary challenge at week 42 demonstrates that protection is strong in all animals and complete in some. Antibody profiles suggest that antigens mediating protection are the released products of developing schistosomula. In culture they are killed by addition of rhesus plasma, collected from week 8 post-infection onwards, and even more efficiently with post-challenge plasma. Furthermore, cultured schistosomula lose chromatin activating marks at the transcription start site of genes related to worm development and show decreased expression of genes related to lysosomes and lytic vacuoles involved with autophagy. Overall, our results indicate that enhanced antibody responses against the challenge migrating larvae mediate the naturally acquired protective immunity and will inform the route to an effective vaccine., To date there is only one single drug with modest efficacy and no vaccine available to protect from schistosomiasis. Here, Amaral et al. characterize the self-cure process of rhesus macaques following primary infection and secondary challenge with Schistosoma mansoni to inform future vaccine development studies.

Differentiated sex chromosomes are accompanied by a difference in gene dose between X/Z-specific and autosomal genes. At the transcriptomic level, these sex-linked genes can lead to expression imbalance, or gene dosage can be compensated by epigenetic mechanisms and results into expression level equalization. Schistosoma mansoni has been previously described as a ZW species (i.e., female heterogamety, in opposition to XY male heterogametic species) with a partial dosage compensation, but underlying mechanisms are still unexplored. Here, we combine transcriptomic (RNA-Seq) and epigenetic data (ChIP-Seq against H3K4me3, H3K27me3, and H4K20me1 histone marks) in free larval cercariae and intravertebrate parasitic stages. For the first time, we describe differences in dosage compensation status in ZW females, depending on the parasitic status: free cercariae display global dosage compensation, whereas intravertebrate stages show a partial dosage compensation. We also highlight regional differences of gene expression along the Z chromosome in cercariae, but not in the intravertebrate stages. Finally, we feature a consistent permissive chromatin landscape of the Z chromosome in both sexes and stages. We argue that dosage compensation in schistosomes is characterized by chromatin remodeling mechanisms in the Z-specific region.

The objective of this work is to evaluate the action of original Roundup® on the snail Biomphalaria glabrata infected and not infected by Schistosoma mansoni by observing its mortality and reproductive biology. Snails were submitted for 30 days to aqueous solutions of original Roundup at concentrations of 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.018, 0.016, 0.014, 0.012 and 0.01 (%) to determine LC50. To analyze the reproductive activity of B. glabrata, against the pesticide, four B. glabrata groups were formed: infected and exposed to the pesticide; infected and not exposed (control); not infected and exposed; and not infected and not exposed(control). LC50 (0.012% aqueous solution of original Roundup) caused mortality of at most up to 95% for 24, 48 and 72 h during the experimental period (30 days), and lost effect totally after 27 days. The reproductive activity of the non-infected and exposed snails was 30.3 ± 20.5 eggs in 2.7 ± 1.2 egg masses, and there was a reduction of 51% in the average number of hatched snails (11.5 ± 7.5) in comparison to the control group (23.9 ± 18.0) for four weeks (p = 0.003). The results indicate that the lethal concentration (LC50) of the 0.012% aqueous solution of original Roundup has a significant action on Biomphalaria glabrata for up to 27 days. Roundup significantly diminished the fertility of infected and exposed snails in comparison with the control group. O presente estudo teve como objetivo avaliar a ação do Roundup® original sobre o molusco Biomphalaria glabrata, infectado e não infectado por Schistosoma mansoni, observando a mortalidade e a biologia reprodutiva. Os caramujos foram submetidos às concentrações aquosas: 0,1; 0,09; 0,08; 0,07; 0,06; 0,05; 0,04; 0,03; 0,02, 0,018; 0,016; 0,014; 0,012 e 0,01 (%) do Roundup Original® para a determinação da CL50, sendo essa concentração testada durante 30 dias sobre os moluscos. Para a observação da atividade reprodutiva frente à exposição ao agrotóxico, quatro grupos de B. glabrata foram formados: infectados e expostos ao agrotóxicos, infectados e não expostos (controle), não infectados e expostos e não infectados e não expostos (controle). A CL50 (0,012% da solução aquosa do Roundup original) apresentou mortalidade que não ultrapassou 95%, 24, 48 e 72 h de exposição durante os 30 dias e perdendo totalmente o efeito após o 27º dia de teste. A atividade reprodutiva de caramujos não infectados e expostos foi de 30,3 ± 20,5 ovos em 2,7 ± 1,2 massas ovígeras, sendo observada redução de 51% na média de moluscos eclodidos (11,5 ± 7,5) em comparação ao grupo controle (23,9 ± 18,0) ao longo de quatro semanas (p = 0,003). Os resultados indicam que a concentração letal (CL50) da solução aquosa 0,012% do Roundup Original tem ação significativa sobre B. glabrata durante 27 dias. O Roundup diminui significativamente a fertilidade dos moluscos infectados e expostos em comparação com o grupo controle.

version 2; peer review: 2 approved; International audience; We describe here a protocol for the generation of sequence-ready libraries for population epigenomics studies, and the analysis of alignment results. We show that the protocol can be used to monitor chromatin structure changes in populations when exposed to environmental cues. The protocol is a streamlined version of the Assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) that provides a positive display of accessible, presumably euchromatic regions. The protocol is straightforward and can be used with small individuals such as daphnia and schistosome worms, and probably many other biological samples of comparable size (~10,000 cells), and it requires little molecular biology handling expertise.

Phenotypic plasticity is an important feature of biological systems that is likely to play a major role in the future adaptation of organisms to the ongoing global changes. It may allow an organism to produce alternative phenotypes in responses to environmental cues. Modifications in the phenotype can be reversible but are sometimes enduring and can even span over generations. The notion of phenotypic plasticity was conceptualized in the early 20thcentury by Richard Woltereck. He introduced the idea that the combined relations of a phenotypic character and all environmental gradients that influence on it can be defined as “norm of reaction”. Norms of reaction are specific to species and to lineages within species, and they are heritable. He postulated that reaction norms can progressively be shifted over generations depending on the environmental conditions. One of his biological models was the water-fleedaphnia. Woltereck proposed that enduring phenotypic modifications and gene mutations could have similar adaptive effects, and he postulated that their molecular bases would be different. Mutations occurred in genes, while enduring modifications were based on something he called theMatrix. He suggested that this matrix (i) was associated with the chromosomes, (ii) that it was heritable, (iii) it changed during development of the organisms, and (iv) that changes of the matrix could be simple chemical substitutions of an unknown, but probably polymeric molecule. We reasoned that the chromatin has all postulated features of this matrix and revisited Woltereck’s classical experiments withdaphnia. We developed a robust and rapid ATAC-seq technique that allows for analyzing chromatin of individual daphnia and show here (i) that this technique can be used with minimal expertise in molecular biology, and (ii) we used it to identify open chromatin structure in daphnia exposed to different environmental cues. Our result indicates that chromatin structure changes consistently in daphnia upon this exposure confirming Woltereck’s classical postulate.

Correction to: Chapter 9 in: David J. Timson (ed.), Schistosoma mansoni: Methods and Protocols, Methods in Molecular Biology, vol. 2151.

El objetivo de este trabajo fue estudiar la composición elemental del látex liofilizado obtenido de Euphorbia milii var. hislopii (N.E.Br.) Ursch & Leandri) y el tegumento del macho adulto de Schistosoma mansoni Sambon, 1907 originado a partir cercarias previamente expuesta al látex. La composición y las concentraciones de 15 elementos se caracterizaron mediante microscopía electrónica de barrido (SEM) y la espectroscopía de dispersión de energía (EDS). El magnesio, potasio y sodio se encontraron en un alto porcentaje en peso en el látex liofilizado. Altos niveles de los elementos sodio, magnesio y nitrógeno se detectaron, respectivamente, en el control de los machos adultos de S. mansoni. La exposición de cercarias de S. mansoni al látex de E. milii cambió la composición elemental del parásito adulto, que mostró un mayor nivel de nitrógeno en lugar de sodio como se observó en los parásitos normales. Además, se compararon los elementos del grupo de control y de los gusanos adultos de las cercarias expuestas al látex y mostraron una disminución significativa de nitrógeno y un aumento de sodio, potasio y magnesio. El presente trabajo representa el primer análisis de la composición elemental del látex liofilizado de E. milii var. hislopii y su impacto en el macho de S. mansoni por la Sonda de microanálisis de Electrones de Rayos X.

International audience; We tested a new approach to describe how a sublethal dose of the Euphorbia milii Des Moul. latex can affect cercariae, inducing changes in the adult Schistosoma mansoni Sambon 1907. We analyzed the effects on biology and used proteomic tools to identify the expression of different proteins. The treatment reduced the development of adults (males, 74.1%; females, 76.3%) and decreased the number of eggs released in 89.9%. The protein profile between adult males of S. mansoni from the control and exposed groups detected 1,020 spots, 26 were differently expressed and 10 were identified and validated. The proteins downregulated after exposition included actin, 14-3-3 epsilon and aldehyde dehydrogenase. The hsp70 was the only protein that was upregulated. The changes in the proteomic profile of adult males of S. mansoni may be related to a muscular fragility of the body. This work provides new insight for schistosomiasis control using a natural, water-soluble product at sublethal concentrations for mollusks. This approach is inexpensive, ecological and efficient as an indirect therapy for schistosomiasis.; Hemos probado un nuevo enfoque para describir cómo una dosis subletal del látex de Euphorbia milii Des Moul. puede afectar a las cercarias, induciendo cambios en el Schistosoma mansoni Sambon 1907 adulto. Se analizaron los efectos sobre la biología y se utilizaron herramientas proteómicas para identificar la expresión de diferentes proteínas. El tratamiento redujo el desarrollo de adultos (machos 74,1%, hembras 76,3%) y disminuyó el número de huevos liberados en 89,9%. El perfil proteico entre machos adultos de S. mansoni de los grupos control y expuestos detectó 1.020 spots, 26 se expresaron de forma diferente y 10 fueron identificados y validados. Las proteínas menos abundante después de la exposición incluyeron actina, 14-3-3 epsilon y aldehído deshidrogenasa. La hsp70 fue la única proteína que se más abundante. Los cambios en el perfil proteómico de los machos adultos de S. mansoni pueden estar relacionados con una fragilidad muscular del cuerpo. Este trabajo proporciona una nueva visión para el control de la esquistosomiasis utilizando un producto natural, soluble en agua a concentraciones subletales para moluscos. Este enfoque es barato, ecológico y eficiente como terapia indirecta para la esquistosomiasis

DNA-binding proteins play critical roles in many major processes such as development and sexual biology of Schistosoma mansoni and are important for the pathogenesis of schistosomiasis. Chromatin immunoprecipitation (ChIP) experiments followed by sequencing (ChIP-seq) are useful to characterize the association of genomic regions with posttranslational chemical modifications of histone proteins. Challenges in the standard ChIP protocol have motivated recent enhancements in this approach, such as reducing the number of cells required and increasing the resolution. In this chapter, we describe the latest advances made by our group in the ChIP methods to improve the standard ChIP protocol to reduce the number of input cells required and to increase the resolution and robustness of ChIP in S. mansoni.

Correction to: Chapter 9 in: David J. Timson (ed.), Schistosoma mansoni: Methods and Protocols, Methods in Molecular Biology, vol. 2151.

International audience; DNA-binding proteins play critical roles in many major processes such as development and sexual biology of Schistosoma mansoni, and are important for the pathogenesis of schistosomiasis. Chromatin immunoprecipitation (ChIP) experiments followed by sequencing (ChIP-seq) are useful to characterize the association of genomic regions with posttranslational chemical modifications of histone proteins. Challenges in the standard ChIP protocol have motivated recent enhancements in this approach, such as reducing the number of cells required and increasing the resolution. In this chapter, we describe the latest advances made by our group in the ChIP methods to improve the standard ChIP protocol to reduce the number of input cells required and to increase the resolution and robustness of ChIP in S. mansoni.

We describe here a step-by-step protocol for the generation of sequence-ready libraries for the Assay for Transposase Accessible Chromatin (ATAC-seq) that provides a positive display of accessible, presumably euchromatic regions. The protocol is straightforward and can be used with small individuals such as daphnia and schistosome worms, and probably many other biological samples of comparable size. This makes the method suitable for population epigenomics studies. This protocol is based on the protocol from Corces et al. (2016), Buenrostro et al. (2015) and Nextera DNA Library Prep Kit (2017) with some modifications. Controls must be done without organisms as input.

International audience; As one of the most successful invasive land snail species, Achatina (Lissachatina) fulica Bowdich, 1822 has achieved wide global distribution, particularly in (sub)tropical regions, with further dispersal likely due to climate change. This species of giant African snails (up to 17 cm shell length) is a pest that has extensive negative impact on agriculture and can serve as vector for several parasites, including Angiostrongylus cantonensis, a nematode parasite that causes (human) eosinophilic meningitis, an emergent disease. Investigation showed that A. cantonensis infection negatively impacts the metabolism of A. fulica by depleting polysaccharide stores of the intermediate host, compromising the energy balance of the snail. A review of the literature indicates that A. fulica possesses potent innate type immune defenses to counter infection, including phagocytic hemocytes capable of deploying reactive oxygen species and lectins for non-self recognition, a serine protease-dependent coagulation response (not observed in other taxa of gastropods), as well as antimicrobial proteins including achacin, an antimicrobial protein. A recent chromosome level genome assembly will facilitate progressively detailed characterization of these immune features of A. fulica. We strongly encourage further immunological studies of A. fulica, ranging from organismal level to molecular biology to gain better understanding of the A. fulica internal defense response to nematode pathogens like A. cantonensis and the contribution of immune function to the invasiveness of (snail) species. Characterization of immunity of A. fulica, representing the understudied Stylommatophora (panpulmonate landsnails) will also broaden the comparative immunology of Gastropoda.

The trematode Schistosoma mansoni belongs to the group of digenetic parasites which need obligatory multiple hosts to develop. They transit between hosts as free-swimming stages in fresh water ecosystems. They generate phenotypically different developmental stages throughout their lifecycle and receive hugly heterogenous environmental cues. Each developmental stage is characterized by specific posttranslational histone modifications, in particular methylations. The combination of the different marks result in stage specific chromatin structure that is essential for development, sexual biology and pathogenesis. Histone methylation also responds to environmental changes and seems to be involved in an adaptive reponse or adjustment to the environment. Histone methylation thus represent promising source of therapeutic targets. In this chapter we will present the state-of-the-art of how the dynamics of histone methylation are involved in multiple factors of the schistosome’s development, as well as what is still lacking for better understanding it.

Evidence of how environmental cues affect the phenotypes of, and compatibility between Schistosoma mansoni and their hosts come from studies in environmental parasitology and research on host diet and chemotherapeutic treatment. Schistosomes deal with a multitude of signals from the water environment as well as cues that come from their hosts, particularly in response to molecules that serve to recognize and destroy them, i.e., those molecules that arise from their hosts’ immune systems. These interactions shape, not only the parasite’s morphology, metabolism and behavior in the short-term, but also their infection success and development into different stage-specific phenotypes later in their life cycle, through the modification of the parasite’s inheritance system. Developmental phenotypic plasticity of S. mansoni is based on epigenetic mechanisms which are also sensitive to environmental cues, but are poorly understood. Here, we argue that specific cues from the environment could lead to changes in parasite development and infectivity, and consequently, environmental signals that come from environmental control measures could be used to influence S. mansoni dynamics and transmission. This approach poses a challenge since epigenetic modification can lead to unexpected and undesired outcomes. However, we suggest that a better understanding of how environmental cues are interpreted by epigenome during schistosome development and host interactions could potentially be applied to control parasite’s virulence. We review evidence about the role of environmental cues on the phenotype of S. mansoni and the compatibility between this parasite and its intermediate and definitive hosts.

Schistosomiasis is an important parasitic disease, touching roughly 200 million people worldwide. The causative agents are different Schistosoma species. Schistosomes have a complex life cycle, with a freshwater snail as intermediate host. After infection, sporocysts develop inside the snail host and give rise to human dwelling larvae. We present here a detailed step-by-step video instruction in English, French, Spanish and Portuguese that shows how these sporocysts can be manipulated and transferred from one snail to another. This procedure provides a technical basis for different types of ex vivo modifications, such as those used in functional genomics studies.

Background: Two aspects need to be considered for schistosomiasis control: morbidity and transmission. In this context, many soluble substances have been tested and Euphorbia milii latex is one of the most promising Brazilian molluscicides. Phytochemical studies involving simulation of the applicability of this latex on all aquatic forms of the S. mansoni life cycle are rare in the literature. The aim of this work was to evaluate the effect of E. milii latex on S. mansoni in the egg, miracidium and different developmental stages in Biomphalaria glabrata. Methods: The laboratory study was designed to simulate the different forms of exposure of the life cycle stages of S. mansoni to the LC50 of E. milii latex; we tested the exposition from four situations of S. mansoni contact with the latex and observed the exposure on different snails’ infection stage too. All snails were analyzed weekly for cercarial shedding and reproductive biology. Results: The results showed that contact of S. mansoni eggs and miracidia with the LC50 of E. milii negatively influenced the development of the parasite life cycle in the intermediate host, with consequent reduction of cercarial shedding. The exposure of infected snails affected the reproductive biology and cercarial shedding in all intra-mollusk development stages of S. mansoni, but the reduction was greater in the first, fourth, fifth, sixth, seventh and eighth weeks of infection. The LC50 of E. milii latex had toxic action on eggs and miracidia, and the number of cercariae shed by snails during the study period declined by about 80%. Conclusions: We can conclude that the use of natural biodegradable compounds containing low concentrations of substances already characterized as having eco-toxico- logical potential can be an important tool to reduce the transmission of Schistosomiasis.

Background Schistosomiasis has been reported in 78 endemic countries and affects 240 million people worldwide. The digenetic parasite Schistosoma mansoni needs fresh water to compete its life cycle. There, it is susceptible to soluble compounds that can affect directly and/or indirectly the parasite’s biology. The cercariae stage is one of the key points in which the parasite is vulnerable to different soluble compounds that can significantly alter the parasite’s life cycle. Molluscicides are recommended by the World Health Organization for the control of schistosomiasis transmission and Euphorbia milii latex is effective against snails intermediate hosts. Methodology/Principal findings We used parasitological tools and electron microscopy to verify the effects of cercariae exposure to natural molluscicide (Euphorbia milii latex) on morphology, physiology and fitness of adult parasite worms. In order to generate insights into key metabolic pathways that lead to the observed phenotypes we used comparative transcriptomics and proteomics. Conclusions/Significance We describe here that the effect of latex on the adult is not due to direct toxicity but it triggers an early change in developmental trajectory and perturbs cell memory, mobility, energy metabolism and other key pathways. We conclude that latex has not only an effect on the vector but applies also long lasting schistosomastatic action. We believe that these results are of interest not only to parasitologists since it shows that natural compounds, presumably without side effects, can have an impact that occurred unexpectedly on developmental processes. Such collateral damage is in this case positive, since it impacts the true target of the treatment campaign. This type of treatment could also provide a rational for the control of other pests. Our results will contribute to enforce the use of E. milii latex in Brazil and other endemic countries as cheap alternative or complement to mass drug treatment with Praziquantel, the only available drug to cure the patients (without preventing re-infection)., Author summary Intestinal schistosomiasis is among the most important parasitic disease caused by helminthes, affecting 67 million people worldwide. Vector and intermediate host of the parasitic worm are fresh water snails. WHO recommends use of molluscicides for control of local transmission. Among those, natural plant extracts such as Euphorbia milii latex have attracted particular attention since they are sustainable and cheap. We had anecdotic evidence that E. milii latex also impacts infection outcome if treated snails were infected with S. mansoni. We show here that transient exposure of the human dwelling larvae (cercariae) to the latex at doses that do not affect its infectivity has effects 60 days later on the morphology, physiology and fitness of the adult parasite worms. In order to generate insights into key metabolic pathways that lead to the observed phenotypes we used comparative transcriptomics and proteomics. We show that the effect of latex on the adult is not due to direct toxicity but it triggers an early change in developmental trajectory and perturbs cell memory, mobility, energy metabolism and other key pathways. We conclude that latex has not only an effect on the vector but applies also long lasting schistosomastatic action. The present work might also provide insights on targets with implications for developing new interventions for schistosomiasis control.

This article presents an improvement to the Kato-Katz (KK) method, making it faster and more efficient for the visualisation of fertile eggs in stool samples. This modified KK method uses sodium acetate formalin as a fixative and reveals the intensity of infection in less than 1 h, reducing the diagnostic time without increasing the cost. This modified method may contribute to future epidemiological studies in both hospitals and the field due to its rapid and precise diagnostic, which allow for immediate treatment.

The aim of this study was to evaluate the influence of population density and food intake on the survival and reproduction of uninfected Biomphalaria glabrata and the effect of calcium carbonate availability on cercarial emergence from experimentally infected snails, to define conditions to maximize snails’ breeding and cercarial production for future studies about this model. The results observed in this study indicate that increased population density has a negative effect on the survival and reproductive activity of B. glabrata. In addition, the quantity of lettuce offered to the snails altered the number of eggs laid per snail. There was a significant relation between the amount of lettuce eaten per day and the number of eggs produced per snail, as well as the number of egg masses per snail, and eggs per egg mass. Furthermore, the snail’s survival was directly associated with the amount of calcium carbonate and the cercarial emergence was inversely related to the calcium carbonate amount. This study might help to understand the influence of population density and food intake on the reproductive biology of captive snail populations. In relation to cercarial emergence, calcium supplies must not be provided to Schistosoma mansoni-infected snails while maintaining S. mansoni under laboratory conditions because it decreases cercarial emergence.

Calcium plays a fundamental role in the life of snails, by regulating different process such as motility, growth, and cell division. Calcium directly influences the shell growth, fecundity, oviposition, mortality, internal metabolism and homeostasis. The aim of this work was to describe the pearl formation in Biomphalaria glabrata exposed to different amounts of calcium carbonate in laboratory conditions. The pearls were observed in the digestive gland and gut in the exposed group given 20 and 60 mg/L after 45 days. The results show that the snails produce pearls as a reservoir of calcium carbonate before reaching sexual maturity.