Development of environmental loop-mediated isothermal amplification (eLAMP) diagnostic tool for Bulinus truncatus field detection.

BACKGROUND: Global changes are reshaping the distribution of vector-borne diseases by spreading vectors to previously non-endemic areas. Since 2013, urogenital schistosomiasis has emerged in Corsica and threatens European countries. Gastropod vectors release schistosome larvae that can infect humans who come into contact with freshwater bodies. Monitoring schistosomiasis host vectors is a prerequisite to understand and subsequently to control this pathogen transmission. Because malacological surveys are time consuming and require special expertise, the use of a simple molecular method is desirable. METHODS: The aim of this study is to develop a ready-to-use protocol using the LAMP (loop-mediated isothermal amplification) method to detect environmental DNA of Bulinus truncatus, vector of Schistosoma haematobium. Interestingly, LAMP method possesses all the characteristics required for adaptability to field conditions particularly in low-income countries: speed, simplicity, lyophilized reagents, low cost and robustness against DNA amplification inhibitors. We have tested this new method on Corsican water samples previously analysed by qPCR and ddPCR. RESULTS: We demonstrate that our diagnostic tool B. truncatus eLAMP (Bt-eLAMP) can detect the eDNA of Bulinus truncatus as effectively as the two other methods. Bt-eLAMP can even detect 1/4 of positive samples not detectable by qPCR. Moreover, the complete Bt-eLAMP protocol (sampling, sample pre-process, amplification and revelation) does not require sophisticated equipment and can be done in 1 ½ h. CONCLUSIONS: LAMP detection of environmental DNA provides large-scale sensitive surveillance of urogenital schistosomiasis possible by identifying potentially threatened areas. More generally, eLAMP method has great potential in vector-borne diseases and ecology.

Alkoxyamines Designed as Potential Drugs against Plasmodium and Schistosoma Parasites.

Malaria and schistosomiasis are major infectious causes of morbidity and mortality in the tropical and sub-tropical areas. Due to the widespread drug resistance of the parasites, the availability of new efficient and affordable drugs for these endemic pathologies is now a critical public health issue. In this study, we report the design, the synthesis and the preliminary biological evaluation of a series of alkoxyamine derivatives as potential drugs against Plasmodium and Schistosoma parasites. The compounds (RS/SR)-2F, (RR/SS)-2F, and 8F, having IC50 values in nanomolar range against drug-resistant P. falciparum strains, but also five other alkoxyamines, inducing the death of all adult worms of S. mansoni in only 1 h, can be considered as interesting chemical starting points of the series for improvement of the activity, and further structure activity, relationship studies. Moreover, investigation of the mode of action and the rate constants kd for C-ON bond homolysis of new alkoxyamines is reported, showing a possible alkyl radical mediated biological activity. A theoretical chemistry study allowed us to design new structures of alkoxyamines in order to improve the selectivity index of these drugs.

Sympatric versus allopatric evolutionary contexts shape differential immune response in Biomphalaria / Schistosoma interaction.

Selective pressures between hosts and their parasites can result in reciprocal evolution or adaptation of specific life history traits. Local adaptation of resident hosts and parasites should lead to increase parasite infectivity/virulence (higher compatibility) when infecting hosts from the same location (in sympatry) than from a foreign location (in allopatry). Analysis of geographic variations in compatibility phenotypes is the most common proxy used to infer local adaptation. However, in some cases, allopatric host-parasite systems demonstrate similar or greater compatibility than in sympatry. In such cases, the potential for local adaptation remains unclear. Here, we study the interaction between Schistosoma and its vector snail Biomphalaria in which such discrepancy in local versus foreign compatibility phenotype has been reported. Herein, we aim at bridging this gap of knowledge by comparing life history traits (immune cellular response, host mortality, and parasite growth) and molecular responses in highly compatible sympatric and allopatric Schistosoma/Biomphalaria interactions originating from different geographic localities (Brazil, Venezuela and Burundi). We found that despite displaying similar prevalence phenotypes, sympatric schistosomes triggered a rapid immune suppression (dual-RNAseq analyses) in the snails within 24h post infection, whereas infection by allopatric schistosomes (regardless of the species) was associated with immune cell proliferation and triggered a non-specific generalized immune response after 96h. We observed that, sympatric schistosomes grow more rapidly. Finally, we identify miRNAs differentially expressed by Schistosoma mansoni that target host immune genes and could be responsible for hijacking the host immune response during the sympatric interaction. We show that despite having similar prevalence phenotypes, sympatric and allopatric snail-Schistosoma interactions displayed strong differences in their immunobiological molecular dialogue. Understanding the mechanisms allowing parasites to adapt rapidly and efficiently to new hosts is critical to control disease emergence and risks of Schistosomiasis outbreaks.

BgTEP: An Antiprotease Involved in Innate Immune Sensing in Biomphalaria glabrata.

Insect thioester-containing protein (iTEP) is the most recently defined group among the thioester-containing protein (TEP) superfamily. TEPs are key components of the immune system, and iTEPs from flies and mosquitoes were shown to be major immune weapons. Initially characterized from insects, TEP genes homologous to iTEP were further described from several other invertebrates including arthropods, cniderians, and mollusks albeit with few functional characterizations. In the freshwater snail Biomphalaria glabrata, a vector of the schistosomiasis disease, the presence of a TEP protein (BgTEP) was previously described in a well-defined immune complex involving snail lectins (fibrinogen-related proteins) and schistosome parasite mucins (SmPoMuc). To investigate the potential role of BgTEP in the immune response of the snail, we first characterized its genomic organization and its predicted protein structure. A phylogenetic analysis clustered BgTEP in a well-conserved subgroup of mollusk TEP. We then investigated the BgTEP expression profile in different snail tissues and followed immune challenges using different kinds of intruders during infection kinetics. Results revealed that BgTEP is particularly expressed in hemocytes, the immune-specialized cells in invertebrates, and is secreted into the hemolymph. Transcriptomic results further evidenced an intruder-dependent differential expression pattern of BgTEP, while interactome experiments showed that BgTEP is capable of binding to the surface of different microbes and parasite either in its full length form or in processed forms. An immunolocalization approach during snail infection by the Schistosoma mansoni parasite revealed that BgTEP is solely expressed by a subtype of hemocytes, the blast-like cells. This hemocyte subtype is present in the hemocytic capsule surrounding the parasite, suggesting a potential role in the parasite clearance by encapsulation. Through this work, we report the first characterization of a snail TEP. Our study also reveals that BgTEP may display an unexpected functional dual role. In addition to its previously characterized anti-protease activity, we demonstrate that BgTEP can bind to the intruder surface membrane, which supports a likely opsonin role.

A Shift from Cellular to Humoral Responses Contributes to Innate Immune Memory in the Vector Snail Biomphalaria glabrata.

Discoveries made over the past ten years have provided evidence that invertebrate antiparasitic responses may be primed in a sustainable manner, leading to the failure of a secondary encounter with the same pathogen. This phenomenon called "immune priming" or "innate immune memory" was mainly phenomenological. The demonstration of this process remains to be obtained and the underlying mechanisms remain to be discovered and exhaustively tested with rigorous functional and molecular methods, to eliminate all alternative explanations. In order to achieve this ambitious aim, the present study focuses on the Lophotrochozoan snail, Biomphalaria glabrata, in which innate immune memory was recently reported. We provide herein the first evidence that a shift from a cellular immune response (encapsulation) to a humoral immune response (biomphalysin) occurs during the development of innate memory. The molecular characterisation of this process in Biomphalaria/Schistosoma system was undertaken to reconcile mechanisms with phenomena, opening the way to a better comprehension of innate immune memory in invertebrates. This prompted us to revisit the artificial dichotomy between innate and memory immunity in invertebrate systems.

A novel bacterial pathogen of Biomphalaria glabrata: a potential weapon for schistosomiasis control?

BACKGROUND: Schistosomiasis is the second-most widespread tropical parasitic disease after malaria. Various research strategies and treatment programs for achieving the objective of eradicating schistosomiasis within a decade have been recommended and supported by the World Health Organization. One of these approaches is based on the control of snail vectors in endemic areas. Previous field studies have shown that competitor or predator introduction can reduce snail numbers, but no systematic investigation has ever been conducted to identify snail microbial pathogens and evaluate their molluscicidal effects. METHODOLOGY/PRINCIPAL FINDINGS: In populations of Biomphalaria glabrata snails experiencing high mortalities, white nodules were visible on snail bodies. Infectious agents were isolated from such nodules. Only one type of bacteria, identified as a new species of Paenibacillus named Candidatus Paenibacillus glabratella, was found, and was shown to be closely related to P. alvei through 16S and Rpob DNA analysis. Histopathological examination showed extensive bacterial infiltration leading to overall tissue disorganization. Exposure of healthy snails to Paenibacillus-infected snails caused massive mortality. Moreover, eggs laid by infected snails were also infected, decreasing hatching but without apparent effects on spawning. Embryonic lethality was correlated with the presence of pathogenic bacteria in eggs. CONCLUSIONS/SIGNIFICANCE: This is the first account of a novel Paenibacillus strain, Ca. Paenibacillus glabratella, as a snail microbial pathogen. Since this strain affects both adult and embryonic stages and causes significant mortality, it may hold promise as a biocontrol agent to limit schistosomiasis transmission in the field.

Biomphalysin, a new ß pore-forming toxin involved in Biomphalaria glabrata immune defense against Schistosoma mansoni.

Aerolysins are virulence factors belonging to the ß pore-forming toxin (ß-PFT) superfamily that are abundantly distributed in bacteria. More rarely, ß-PFTs have been described in eukaryotic organisms. Recently, we identified a putative cytolytic protein in the snail, Biomphalaria glabrata, whose primary structural features suggest that it could belong to this ß-PFT superfamily. In the present paper, we report the molecular cloning and functional characterization of this protein, which we call Biomphalysin, and demonstrate that it is indeed a new eukaryotic ß-PFT. We show that, despite weak sequence similarities with aerolysins, Biomphalysin shares a common architecture with proteins belonging to this superfamily. A phylogenetic approach revealed that the gene encoding Biomphalysin could have resulted from horizontal transfer. Its expression is restricted to immune-competent cells and is not induced by parasite challenge. Recombinant Biomphalysin showed hemolytic activity that was greatly enhanced by the plasma compartment of B. glabrata. We further demonstrated that Biomphalysin with plasma is highly toxic toward Schistosoma mansoni sporocysts. Using in vitro binding assays in conjunction with Western blot and immunocytochemistry analyses, we also showed that Biomphalysin binds to parasite membranes. Finally, we showed that, in contrast to what has been reported for most other members of the family, lytic activity of Biomphalysin is not dependent on proteolytic processing. These results provide the first functional description of a mollusk immune effector protein involved in killing S. mansoni.

Evidence for specific genotype-dependent immune priming in the lophotrochozoan Biomphalaria glabrata snail.

Historically, the prevailing view in the field of invertebrate immunity was that invertebrates that do not possess acquired adaptive immunity rely on innate mechanisms with low specificity and no memory. Several recent studies have shaken this paradigm and suggested that the immune defenses of invertebrates are more complex and specific than previously thought. Mounting evidence has shown that at least some invertebrates (mainly Ecdysozoa) show high levels of specificity in their immune responses to different pathogens, and that subsequent reexposure may result in enhanced protection (recently called 'immune priming'). Here, we investigated immune priming in the Lophotrochozoan snail species Biomphalaria glabrata, following infection by the trematode pathogen Schistosoma mansoni. We confirmed that snails were protected against a secondary homologous infection whatever the host strain. We then investigated how immune priming occurs and the level of specificity of B. glabrata immune priming. In this report we confirmed that immune priming exists and we identified a genotype-dependent immune priming in the fresh-water snail B. glabrata.

Chromatin structural changes around satellite repeats on the female sex chromosome in Schistosoma mansoni and their possible role in sex chromosome emergence.

BACKGROUND: In the leuphotrochozoan parasitic platyhelminth Schistosoma mansoni, male individuals are homogametic (ZZ) whereas females are heterogametic (ZW). To elucidate the mechanisms that led to the emergence of sex chromosomes, we compared the genomic sequence and the chromatin structure of male and female individuals. As for many eukaryotes, the lower estimate for the repeat content is 40%, with an unknown proportion of domesticated repeats. We used massive sequencing to de novo assemble all repeats, and identify unambiguously Z-specific, W-specific and pseudoautosomal regions of the S. mansoni sex chromosomes. RESULTS: We show that 70 to 90% of S. mansoni W and Z are pseudoautosomal. No female-specific gene could be identified. Instead, the W-specific region is composed almost entirely of 36 satellite repeat families, of which 33 were previously unknown. Transcription and chromatin status of female-specific repeats are stage-specific: for those repeats that are transcribed, transcription is restricted to the larval stages lacking sexual dimorphism. In contrast, in the sexually dimorphic adult stage of the life cycle, no transcription occurs. In addition, the euchromatic character of histone modifications around the W-specific repeats decreases during the life cycle. Recombination repression occurs in this region even if homologous sequences are present on both the Z and W chromosomes. CONCLUSION: Our study provides for the first time evidence for the hypothesis that, at least in organisms with a ZW type of sex chromosomes, repeat-induced chromatin structure changes could indeed be the initial event in sex chromosome emergence.

Antischistosomal activity of trioxaquines: in vivo efficacy and mechanism of action on Schistosoma mansoni.

Schistosomiasis is among the most neglected tropical diseases, since its mode of spreading tends to limit the contamination to people who are in contact with contaminated waters in endemic countries. Here we report the in vitro and in vivo anti-schistosomal activities of trioxaquines. These hybrid molecules are highly active on the larval forms of the worms and exhibit different modes of action, not only the alkylation of heme. The synergy observed with praziquantel on infected mice is in favor of the development of these trioxaquines as potential anti-schistosomal agents.

Trioxaquine PA1259 alkylates heme in the blood-feeding parasite Schistosoma mansoni.

Trioxaquine PA1259 is an efficient drug on larval- and adult-stage schistosomes, able to alkylate heme inside worms treated with it, leading to the formation of covalent heme-drug adducts. Such a mechanism, similar to one reported for other trioxaquines in Plasmodium, indicates that heme may be a common target of these trioxane-based drugs in different blood-feeding parasites.

Mating system drives negative associations between morphological features in Schistosomatidae.

BACKGROUND: Sexual morphological features are known to be associated with the mating systems of several animal groups. However, it has been suggested that morphological features other than sexual characteristics could also be constrained by the mating system as a consequence of negative associations. Schistosomatidae are parasitic organisms that vary in mating system and can thus be used to explore links between the mating system and negative associations with morphological features. RESULTS: A comparative analysis of Schistosomatidae morphological features revealed an association between the mating system (monogamous versus polygynandrous) and morphological characteristics of reproduction, nutrition, and locomotion. CONCLUSIONS: The mating system drives negative associations between somatic and sexual morphological features. In monogamous species, males display a lower investment in sexual tissues and a higher commitment of resources to tissues involved in female transport, protection, and feeding assistance. In contrast, males of polygynandrous species invest to a greater extent in sexual tissues at the cost of reduced commitment to female care.

Follow-up of the genetic diversity and snail infectivity of a Schistosoma mansoni strain from field to laboratory.

Schistosoma mansoni is an endoparasite causing a serious human disease called schistosomiasis. The quantification of parasite genetic diversity is an essential component to understand schistosomiasis epidemiology and disease transmission patterns but some studies on parasite genetic diversity are performed using parasite laboratory strains. However, a potential discrepancy in level of genetic variation between field populations and laboratory strains may have various implications in our deductions. In this study, 246 adult worms were analysed on 15 microsatellite markers to investigate variation of genetic diversity between a founder field isolate and the nine successive laboratory generations during three years of laboratory maintenance. In parallel, we measured a parasite life trait (snail infectivity) at each generation in order to test a potential link between inbreeding and snail infectivity. Our genetic analyses demonstrate a significant genetic differentiation between all parasite generations and a significant isolation by time associated with a decrease in neutral genetic diversity that is likely to be the result of successive bottleneck events. However, while snail infectivity decreases sharply between field isolate and the first laboratory generation, this parasite life trait does not evolve between other laboratory generations and appeared disconnected from this continuous neutral genetic diversity loss. We hypothesize that a sufficient level of compatibility polymorphism at a genomic level is maintained independently of an increase of inbreeding, ensuring the stability in the parasite life trait.

Whole-genome in-silico subtractive hybridization (WISH)--using massive sequencing for the identification of unique and repetitive sex-specific sequences: the example of Schistosoma mansoni.

BACKGROUND: Emerging methods of massive sequencing that allow for rapid re-sequencing of entire genomes at comparably low cost are changing the way biological questions are addressed in many domains. Here we propose a novel method to compare two genomes (genome-to-genome comparison). We used this method to identify sex-specific sequences of the human blood fluke Schistosoma mansoni. RESULTS: Genomic DNA was extracted from male and female (heterogametic) S. mansoni adults and sequenced with a Genome Analyzer (Illumina). Sequences are available at the NCBI sequence read archive http://www.ncbi.nlm.nih.gov/Traces/sra/ under study accession number SRA012151.6. Sequencing reads were aligned to the genome, and a pseudogenome composed of known repeats. Straightforward comparative bioinformatics analysis was performed to compare male and female schistosome genomes and identify female-specific sequences. We found that the S. mansoni female W chromosome contains only few specific unique sequences (950 Kb i.e. about 0.2% of the genome). The majority of W-specific sequences are repeats (10.5 Mb i.e. about 2.5% of the genome). Arbitrarily selected W-specific sequences were confirmed by PCR. Primers designed for unique and repetitive sequences allowed to reliably identify the sex of both larval and adult stages of the parasite. CONCLUSION: Our genome-to-genome comparison method that we call "whole-genome in-silico subtractive hybridization" (WISH) allows for rapid identification of sequences that are specific for a certain genotype (e.g. the heterogametic sex). It can in principle be used for the detection of any sequence differences between isolates (e.g. strains, pathovars) or even closely related species.