ARGONAUTE2 Localizes to Sites of Sporocysts in the Schistosome-Infected Snail, Biomphalaria glabrata.

MicroRNAs (miRNAs) are a class of small regulatory RNA that are generated via core protein machinery. The miRNAs direct gene-silencing mechanisms to mediate an essential role in gene expression regulation. In mollusks, miRNAs have been demonstrated to be required to regulate gene expression in various biological processes, including normal development, immune responses, reproduction, and stress adaptation. In this study, we aimed to establishment the requirement of the miRNA pathway as part of the molecular response of exposure of Biomphalaria glabrata (snail host) to Schistosoma mansoni (trematode parasite). Initially, the core pieces of miRNA pathway protein machinery, i.e., Drosha, DGCR8, Exportin-5, Ran, and Dicer, together with the central RNA-induced silencing complex (RISC) effector protein Argonaute2 (Ago2) were elucidated from the B. glabrata genome. Following exposure of B. glabrata to S. mansoni miracidia, we identified significant expression up-regulation of all identified pieces of miRNA pathway protein machinery, except for Exportin-5, at 16 h post exposure. For Ago2, we went on to show that the Bgl-Ago2 protein was localized to regions surrounding the sporocysts in the digestive gland of infected snails 20 days post parasite exposure. In addition to documenting elevated miRNA pathway protein machinery expression at the early post-exposure time point, a total of 13 known B. glabrata miRNAs were significantly differentially expressed. Of these thirteen B. glabrata miRNAs responsive to S. mansoni miracidia exposure, five were significantly reduced in their abundance, and correspondingly, these five miRNAs were determined to putatively target six genes with significantly elevated expression and that have been previously associated with immune responses in other animal species, including humans. In conclusion, this study demonstrates the central importance of a functional miRNA pathway in snails, which potentially forms a critical component of the immune response of snails to parasite exposure. Further, the data reported in this study provide additional evidence of the complexity of the molecular response of B. glabrata to S. mansoni infection: a molecular response that could be targeted in the future to overcome parasite infection and, in turn, human schistosomiasis.

Metabolic disruptions in Biomphalaria glabrata induced by Heterorhabditis bacteriophora HP88: Implications for entomopathogenic nematodes in biological control.

Research on the use of entomopathogenic nematodes (EPNs) as a potential tool for the biological control of invertebrates has been growing in recent years, including studies involving snails with One Health importance. In this study, the effect of exposure time (24 or 48 h) of Heterorhabditis bacteriophora HP88 on the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as the concentration of total proteins, uric acid, and urea in the hemolymph of Biomphalaria glabrata, were investigated. The concentrations of these metabolic markers were measured weekly until the end of the third week after exposure. Along with a significant reduction in total protein levels, a significant increase (p < 0.01) in uric acid and urea contents in the hemolymph of B. glabrata exposed to H. bacteriophora was observed. The accumulation of urea in these mollusks could lead to deleterious effects due to its high toxicity, inducing significant cell damage. Variations in transaminase activities were also observed, with snails exposed to EPNs showing significantly higher values (p < 0.01) than individuals in the control group, both for ALT and AST. These results indicate that experimental exposure to infective juveniles of H. bacteriophora causes significant alterations in the metabolic pattern of B. glabrata, compromising the maintenance of its homeostasis. Finally, exposure for 48 h caused more damage to the planorbid in question compared to snails exposed for 24 h, suggesting that the exposure time may influence the intensity of the host's response.

Different metazoan parasites, different transcriptomic responses, with new insights on parasitic castration by digenetic trematodes in the schistosome vector snail Biomphalaria glabrata.

BACKGROUND: Gastropods of the genus Biomphalaria (Family Planorbidae) are exploited as vectors by Schistosoma mansoni, the most common causative agent of human intestinal schistosomiasis. Using improved genomic resources, overviews of how Biomphalaria responds to S. mansoni and other metazoan parasites can provide unique insights into the reproductive, immune, and other systems of invertebrate hosts, and their responses to parasite challenges. RESULTS: Using Illumina-based RNA-Seq, we compared the responses of iM line B. glabrata at 2, 8, and 40 days post-infection (dpi) to single infections with S. mansoni, Echinostoma paraensei (both digenetic trematodes) or Daubaylia potomaca (a nematode parasite of planorbid snails). Responses were compared to unexposed time-matched control snails. We observed: (1) each parasite provoked a distinctive response with a predominance of down-regulated snail genes at all time points following exposure to either trematode, and of up-regulated genes at 8 and especially 40dpi following nematode exposure; (2) At 2 and 8dpi with either trematode, several snail genes associated with gametogenesis (particularly spermatogenesis) were down-regulated. Regarding the phenomenon of trematode-mediated parasitic castration in molluscs, we define for the first time a complement of host genes that are targeted, as early as 2dpi when trematode larvae are still small; (3) Differential gene expression of snails with trematode infection at 40dpi, when snails were shedding cercariae, was unexpectedly modest and revealed down-regulation of genes involved in the production of egg mass proteins and peptide processing; and (4) surprisingly, D. potomaca provoked up-regulation at 40dpi of many of the reproduction-related snail genes noted to be down-regulated at 2 and 8dpi following trematode infection. Happening at a time when B. glabrata began to succumb to D. potomaca, we hypothesize this response represents an unexpected form of fecundity compensation. We also document expression patterns for other Biomphalaria gene families, including fibrinogen domain-containing proteins (FReDs), C-type lectins, G-protein coupled receptors, biomphalysins, and protease and protease inhibitors. CONCLUSIONS: Our study is relevant in identifying several genes involved in reproduction that are targeted by parasites in the vector snail B. glabrata and that might be amenable to manipulation to minimize their ability to serve as vectors of schistosomes.

Combining a transcriptomic approach and a targeted metabolomics approach for deciphering the molecular bases of compatibility phenotype in the snail Biomphalaria glabrata toward Schistosoma mansoni.

Biomphalaria glabrata is a freshwater snail and the obligatory intermediate host of Schistosoma mansoni parasite, the etiologic agent of intestinal Schistosomiasis, in South America and Caribbean. Interestingly in such host-parasite interactions, compatibility varies between populations, strains or individuals. This observed compatibility polymorphism is based on a complex molecular-matching-phenotype, the molecular bases of which have been investigated in numerous studies, notably by comparing between different strains or geographical isolates or clonal selected snail lines. Herein we propose to decipher the constitutive molecular support of this interaction in selected non-clonal resistant and susceptible snail strain originating from the same natural population from Brazil and thus having the same genetic background. Thanks to a global RNAseq transcriptomic approach on whole snail, we identified a total of 328 differentially expressed genes between resistant and susceptible phenotypes among which 129 were up-regulated and 199 down-regulated. Metabolomic studies were used to corroborate the RNAseq results. The activation of immune genes and specific metabolic pathways in resistant snails might provide them with the capacity to better respond to parasite infection.

How do microplastics alter molluscicidal activity? Effects of weathered microplastics and niclosamide in developing freshwater snails.

Despite the wide distribution and persistence of microplastics (MPs), their interactive effects with molluscicides are unknown. Schistosomiasis, a neglected tropical disease, affects 236.6 million people worldwide. Niclosamide (NCL) is the only molluscicide recommended by the World Health Organization (WHO) and it is used to control the population of Schistosoma spp.'s intermediate host. Thus, this study aimed to evaluate of the interaction between polyethylene (PE) MPs and NCL, and their associated toxicity in the freshwater snail Biomphalaria glabrata (Say 1818). Weathered PE MPs were characterized and theoretical analysis of NCL-MP adsorption nature was made using quantum mechanical calculations. The toxicity of NCL isolated (0.0265 to 0.0809 mg L-1) and under interaction with PE MPs (3400 µg L-1) in B. glabrata embryos and newly hatched snails was analyzed. In silico analysis confirmed the adsorption mechanisms of NCL into PE MPs. PE MPs decreased the NCL toxicity to both B. glabrata developmental stages, increasing their survival and NCL lethal concentrations, indicating concerns regarding NCL use as molluscicide in aquatic environments polluted by MPs. In conclusion, MPs may change the efficiency of chemicals used in snail control programs.

Potential of Ocotea indecora (Schott) Mez essential oil nanoemulsion in schistosomiasis control: Molluscicidal effects.

Schistosomiasis is a neglected disease transmitted through contaminated water in populations with low basic sanitation. The World Health Organization recommends controlling the intermediate host snails of the Biomphalaria genus with the molluscicide niclosamide. This work aims to evaluate the biocidal potential of the nanoemulsion prepared with the essential oil of Ocotea indecora leaves for the control of the mollusk Biomphalaria glabrata, intermediate host of the Schistosoma mansoni, the etiologic agent of schistosomiasis.

Exploring the immune interactions between Oncomelania hupensis and Schistosoma japonicum, with a cross-comparison of immunological research progress in other intermediate host snails.

Schistosomiasis, the second largest parasitic disease in the world after malaria, poses a significant threat to human health and causes public health issues. The disease primarily affects populations in economically underdeveloped tropical regions, earning it the title of "neglected tropical disease". Schistosomiasis is difficult to eradicate globally if medication alone is used. One of the essential elements of thorough schistosomiasis prevention and control is the management and disruption of the life cycle of intermediate host snails. The key approach to controlling the transmission of schistosomiasis is to control the intermediate hosts of the schistosome to disrupt its life cycle. We believe that approaching it from the perspective of the intermediate host's immunity could be an environmentally friendly and potentially effective method. Currently, globally significant intermediate host snails for schistosomes include Oncomelania hupensis, Biomphalaria glabrata, and Bulinus truncatus. The immune interaction research between B. glabrata and Schistosoma mansoni has a history of several decades, and the complete genome sequencing of both B. glabrata and B. truncatus has been accomplished. We have summarized the immune-related factors and research progress primarily studied in B. glabrata and B. truncatus and compared them with several humoral immune factors that O. hupensis research focuses on: macrophage migration inhibitory factor (MIF), Toll-like receptors (TLRs), and thioredoxin (Trx). We believe that continued exploration of the immune interactions between O. hupensis and Schistosoma japonicum is valuable. This comparative analysis can provide some direction and clues for further in-depth research. Comparative immunological studies between them not only expand our understanding of the immune defense responses of snails that act as intermediaries for schistosomes but also facilitate the development of more comprehensive and integrated strategies for schistosomiasis prevention and control. Furthermore, it offers an excellent opportunity to study the immune system of gastropods and their co-evolution with pathogenic organisms.

Inhibition of carbonic anhydrase using aspirin is a novel method to block schistosomiasis infection of the parasitic trematode, Schistosoma mansoni, in the intermediate snail host, Biomphalaria glabrata.

Schistosomiasis is a major public health concern worldwide. Although praziquantel is currently available as the only treatment option for schistosomiasis, the absence of reliable diagnostic and prognostic tools highlights the need for the identification and characterization of new drug targets. Recently, we identified the B. glabrata homolog (accession number XP_013075832.1) of human CAXIV, showing 37% amino acid sequence identity, from a BLAST search in NCBI (National Center for Biotechnology Information). Carbonic Anhydrases (CAs) are metalloenzymes that catalyze the reversible hydration/dehydration of CO2/HCO3. These enzymes are associated with many physiological processes, and their role in tumorigenesis has been widely implicated. CAs create an acidic extracellular environment that facilitates the survival, metastasis, and growth of cancer cells. In this study, we investigated the role of CA inhibition in B. glabrata snails exposed to S. mansoni miracidia. We analyzed the expression of the B. glabrata CA encoding transcript in juvenile susceptible and resistant snails, with and without exposure to S. mansoni. Our results showed that the expression of the CA mRNA encoding transcript was upregulated during early and prolonged infection in susceptible snails (BBO2), but not in the resistant BS-90 stock. Notably, sodium salicylate, a form of aspirin, inhibited the expression of CA, post-exposure, to the parasite. Increasing research between parasites and cancer has shown that schistosomes and cancer cells share similarities in their capacity to proliferate, survive, and evade host immune mechanisms. Here, we show that this model system is a potential new avenue for understanding the role of CA in the metastasis and proliferation of cancer cells. Further studies are needed to explore the potential of CA as a biomarker for infection in other schistosomiasis-causing parasites, including S. japonicum and S. haematobium.

Effects of Commercial Sunscreens on Survival, Reproduction and Embryonic Development of the Aquatic Snail Biomphalaria glabrata (SAY, 1818).

Over the past few years, there has been a significant increase in the use of sunscreens. Consequently, the occurrence in aquatic environments of ultraviolet filters has also increased. The present study aims to evaluate the toxicity of two commercial sunscreens to the aquatic snail Biomphalaria glabrata. Acute assays were performed with adult snails exposed to solutions of the two products in synthetic soft water. Reproduction and development assays were carried out, involving individual adult and egg masses exposure to assess fertility and embryonic development. Sunscreen A showed a LC50-96 h of 6.8 g/L and reduction in number of eggs and egg masses per individual in the concentration of 0.3 g/L. Sunscreen B presented higher malformation rates in 0.4 g/L with 63% of malformed embryos. Results indicate that the formulation used in sunscreens is an important factor in aquatic toxicity and needs to be evaluated before the final product is commercialized.

Inhibition of alternative oxidase disrupts the development and oviposition of Biomphalaria glabrata snails.

BACKGROUND: Biomphalaria glabrata is one of the main intermediate hosts of Schistosoma mansoni, the most widespread species of Schistosoma. Our previous studies proved that alternative oxidase (AOX), the terminal oxidase in the mitochondrial respiratory chain, widely exists in several species of intermediate host snails of Schistosoma. Meanwhile, inhibition of AOX activity in Oncomelania hupensis snails could dramatically enhance the molluscicidal effect of niclosamide. As a hermaphroditic aquatic mollusc, the high fecundity and population density of B. glabrata increase the difficulty of snail control, which is one of the critical strategies for schistosomiasis elimination. The present study aimed to investigate the possible role of AOX in the development and fecundity of B. glabrata snail, which could be manipulated more manageable than other species of intermediate host snails of Schistosoma. METHODS: The dynamic expression of the AOX gene was investigated in different developmental stages and tissues of B. glabrata, with morphological change and oviposition behaviour observed from juvenile to adult snails. Furtherly, dsRNA-mediated knockdown of BgAOX mRNA and the AOX protein activity inhibiting was performed to investigate the effect of AOX on the development and oviposition of snails. RESULTS: The BgAOX gene expression profile is highly related to the development from late juveniles to adults, especially to the reproductive system of snails, with a positive correlation of 0.975 between egg production and BgAOX relative expression in ovotestis of snails. The inhibition of BgAOX at the transcriptional level and AOX activity could efficiently inhibit snail growth. However, the interference at the BgAOX protein activity level led to more severe tissue damage and more significant inhibition of oviposition than at the transcriptional level. This inhibition of growth and oviposition decreased gradually with the increase in the snail size. CONCLUSIONS: The inhibition of AOX could efficiently disrupt the development and oviposition of B. glabrata snails, and the intervention targeting AOX at the juvenile stage is more effective for snails. This investigation explored the role of AOX in the growth and development of snails. It would benefit snail control in the future by providing a potential target while using molluscicides more efficiently.

Expression and Characterisation of the First Snail-Derived UDP-Gal: Glycoprotein-N-acetylgalactosamine ß-1,3-Galactosyltransferase (T-Synthase) from Biomphalaria glabrata.

UDP-Gal: glycoprotein-N-acetylgalactosamine ß-1,3-galactosyltransferase (T-synthase, EC 2.4.1.122) catalyses the transfer of the monosaccharide galactose from UDP-Gal to GalNAc-Ser/Thr, synthesizing the core 1 mucin type O-glycan. Such glycans play important biological roles in a number of recognition processes. The crucial role of these glycans is acknowledged for mammals, but a lot remains unknown regarding invertebrate and especially mollusc O-glycosylation. Although core O-glycans have been found in snails, no core 1 ß-1,3-galactosyltransferase has been described so far. Here, the sequence of the enzyme was identified by a BlastP search of the NCBI Biomphalaria glabrata database using the human T-synthase sequence (NP_064541.1) as a template. The obtained gene codes for a 388 amino acids long transmembrane protein with two putative N-glycosylation sites. The coding sequence was synthesised and expressed in Sf9 cells. The expression product of the putative enzyme displayed core 1 ß-1,3-galactosyltransferase activity using pNP-α-GalNAc as the substrate. The enzyme showed some sequence homology (49.40% with Homo sapiens, 53.69% with Drosophila melanogaster and 49.14% with Caenorhabditis elegans) and similar biochemical parameters with previously characterized T-synthases from other phyla. In this study we present the identification, expression and characterisation of the UDP-Gal: glycoprotein-N-acetylgalactosamine ß-1,3-galactosyltransferase from the fresh-water snail Biomphalaria glabrata, which is the first cloned T-synthase from mollusc origin.

Two novel mollusk short-form ApeC-containing proteins act as pattern recognition proteins for peptidoglycan.

The Apextrin C-terminal (ApeC) domain is a new protein domain largely specific to aquatic invertebrates. In amphioxus, a short-form ApeC-containing protein (ACP) family is capable of binding peptidoglycan (PGN) and agglutinating bacteria via its ApeC domain. However, the functions of ApeC in other phyla remain unknown. Here we examined 130 ACPs from gastropods and bivalves, the first and second biggest mollusk classes. They were classified into nine groups based on their phylogenetics and architectures, including three groups of short-form ACPs, one group of apextrins and two groups of ACPs of complex architectures. No groups have orthologs in other phyla and only four groups have members in both gastropods and bivalves, suggesting that mollusk ACPs are highly diversified. We selected one bivalve ACP (CgACP1; from the oyster Crossostrea gigas) and one gastropod ACP (BgACP1; from the snail Biomphalaria glabrata) for functional experiments. Both are highly-expressed, secreted short-form ACPs and hence comparable to the amphioxus ACPs previously reported. We found that recombinant CgACP1 and BgACP1 bound with yeasts and several bacteria with different affinities. They also agglutinated these microbes, but showed no inhibiting or killing effects. Further analyses show that both ACPs had high affinities to the Lys-type PGN from S. aureus but weak or no affinities to the DAP-type PGN from Bacillus subtilis. Both recombinant ACPs displayed weak or no affinities to other microbial cell wall components, including lipopolysaccharide (LPS), lipoteichoic acid (LTA), zymosan A, chitin, chitosan and cellulose, as well as to several PGN moieties, including muramyl dipeptide (MDP), N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Besides, CgACP1 had the highest expression in the gill and could be greatly up-regulated quickly after bacterial challenge. This is reminiscent of the amphioxus ACP1/2 which serve as essential mucus lectins in the gill. Taken together, the current findings from mollusk and amphioxus ACPs suggest several basic common traits for the ApeC domains, including the high affinity to Lys-type PGN, the bacterial binding and agglutinating capacity, and the role as mucus proteins to protect the mucosal surface.

Identification of Schistosoma mansoni miracidia attractant candidates in infected Biomphalaria glabrata using behaviour-guided comparative proteomics.

Schistosomiasis, caused by infection with Schistosoma digenetic trematodes, is one of the deadliest neglected tropical diseases in the world. The Schistosoma lifecycle involves the miracidial infection of an intermediate freshwater snail host, such as Biomphalaria glabrata. Dispersing snail host-derived Schistosoma miracidia attractants has been considered a method of minimising intermediate host infections and, by extension, human schistosomiasis. The attractiveness of B. glabrata to miracidia is known to be reduced following infection; however, the relationship between duration of infection and attractiveness is unclear. Excretory-secretory proteins (ESPs) most abundant in attractive snail conditioned water (SCW) are key candidates to function as miracidia attractants. This study analysed SCW from B. glabrata that were naïve (uninfected) and at different time-points post-miracidia exposure (PME; 16h, 1-week, 2-weeks and 3-weeks PME) to identify candidate ESPs mediating Schistosoma mansoni miracidia behaviour change, including aggregation and chemoklinokinesis behaviour (random motion, including slowdown and increased turning rate and magnitude). Miracidia behaviour change was only observed post-addition of naïve and 3W-PME SCW, with other treatments inducing significantly weaker behaviour changes. Therefore, ESPs were considered attractant candidates if they were shared between naïve and 3W-PME SCW (or exclusive to the former), contained a predicted N-terminal signal peptide and displayed low identity (<50%) to known proteins outside of the Biomphalaria genus. Using these criteria, a total of 6 ESP attractant candidates were identified, including acetylcholine binding protein-like proteins and uncharacterised proteins. Tissue-specific RNA-seq analysis of the genes encoding these 6 ESPs indicated relatively high gene expression within various B. glabrata tissues, including the foot, mantle and kidney. Acetylcholine binding protein-like proteins were highly promising due to their high abundance in naïve and 3W-PME SCW, high specificity to B. glabrata and high expression in the ovotestis, from which attractants have been previously identified. In summary, this study used proteomics, guided by behavioural assays, to identify miracidia attractant candidates that should be further investigated as potential biocontrols to disrupt miracidia infection and minimise schistosomiasis.

Snail juvenile growth rate as a rapid predictor of the transmission potential of parasitizing human schistosomes.

Host and parasite traits that are sensitive to environmental perturbations merit special attention in the mitigation of diseases. While life table experiments allow a practical evaluation of variability of these traits with environmental change, they are cost and resource intensive. Here, we use a model snail host-trematode parasite system to test the efficacy of an expeditious alternative. Rapidly changing host traits (such as juvenile growth rate) can be used as effective predictors of parasite transmission potential across a range of environmental factors. This approach can be applied to anticipate epidemiological changes under diverse environmental scenarios.

Molecular characterization of thioester-containing proteins in Biomphalaria glabrata and their differential gene expression upon Schistosoma mansoni exposure.

Schistosomiasis is a disease caused by trematode parasites of the genus Schistosoma that affects approximately 200 million people worldwide. Schistosomiasis has been a persistent problem in endemic areas as there is no vaccine available, currently used anti-helmintic medications do not prevent reinfection, and most concerning, drug resistance has been documented in laboratory and field isolates. Thus, alternative approaches to curtail this human disease are warranted. Understanding the immunobiology of the obligate intermediate host of these parasites, which include the freshwater snail Biomphalaria glabrata, may facilitate the development of novel methods to stop or reduce transmission to humans. Molecules from the thioester-containing protein (TEP) superfamily have been shown to be involved in immunological functions in many animals including corals and humans. In this study we identified, characterized, and compared TEP transcripts and their expression upon S. mansoni exposure in resistant and susceptible strains of B. glabrata snails. Results showed the expression of 11 unique TEPs in B. glabrata snails. These transcripts present high sequence identity at the nucleotide and putative amino acid levels between susceptible and resistant strains. Further analysis revealed differences in several TEPs' constitutive expression levels between resistant and susceptible snail strains, with C3-1, C3-3, and CD109 having higher constitutive expression levels in the resistant (BS90) strain, whereas C3-2 and TEP-1 showed higher constitutive expression levels in the susceptible (NMRI) strain. Furthermore, TEP-specific response to S. mansoni miracidia exposure reiterated their differential expression, with resistant snails upregulating the expression of both TEP-4 and TEP-3 at 2 h and 48 h post-exposure, respectively. Further understanding the diverse TEP genes and their functions in invertebrate animal vectors will not only expand our knowledge in regard to this ancient family of immune proteins, but also offer the opportunity to identify novel molecular targets that could aid in the efforts to develop control methods to reduce schistosomiasis transmission.

Mating Interactions between Schistosoma bovis and S. mansoni and Compatibility of Their F1 Progeny with Biomphalaria glabrata and Bulinus truncatus.

Contrary to the majority of other Trematoda, Schistosoma species are gonochoric. Consequently, in endemic areas where several schistosome species overlap and can co-infect the same definitive host, there may be frequent opportunities for interspecific pairing. Our experimental study provides novel insight on the pairing behavior between Schistosoma bovis and S. mansoni in mixed infections in mice. We used six mate choice experiments to assess mating interactions between the two schistosome species. We show that mating between the two Schistosoma species is not random and that S. mansoni exhibits greater mate recognition compared to S. bovis. We also performed reciprocal crosses (male S. mansoni × female S. bovis) and (female S. mansoni × male S. bovis) that produce active swimming miracidia. These miracidia were genotyped by ITS2 sequencing and proposed for mollusc infection. Molecular analyses show that all the miracidia are parthenogenetically produced (i.e., their harbor the mother ITS2 genotype) and as a consequence can only infect the mollusc of the maternal species. Offspring produced by male S. mansoni × female S. bovis pairing can only infect Bulinus truncatus whereas offspring produced by female S. mansoni × male S. bovis can only infect Biomphalaria glabrata snails. Evolutionary and epidemiological consequences are discussed.

A new accumulation assay of Schistosoma mansoni miracidia using square capillary glass tubes.

Current control measures for schistosomiasis have only been partially successful in endemic areas due to socioeconomic constraints. One possibility for controlling the disease is to aim at the miracidial stage of the trematode to avoid infecting intermediate snail hosts by introducing more attractive substances for miracidia in the environment. Here, we introduce an accumulation assay of Schistosoma mansoni miracidia using a square glass tube for analysis of the positive responses of miracidia toward several substances, including snail-conditioned water of Biomphalaria glabrata, Bulinus globosus and insusceptible snails collected in the Nagasaki area in Japan. The substances are not proteins because miracidia accumulated in boiled snail-conditioned water and the secretion or emission level of substances depended on the feeding conditions of Biomphalaria glabrata. The present study also showed that substances emitted from Biomphalaria glabrata with a molecular weight around 10 kDa accumulated Schistosoma mansoni miracidia. Further, we showed that Schistosoma mansoni miracidia did not accumulate in response to mono- or disaccharides tested in the study.

Recombinant Snail Sialic Acid Aldolase is Promiscuous towards Aliphatic Aldehydes.

Aldolases are enzymes that reversibly catalyze the cleavage of carbon-carbon bonds. Here we describe a recombinant sialic acid aldolase originating from the freshwater snail Biomphalaria glabrata (sNPL), and compare its substrate spectrum with a sialic acid aldolase originating from chicken (chNPL). In contrast to vertebrate animals which can synthesize, degrade, and incorporate sialic acids on glycoconjugate ubiquitously, snails (as all mollusks) cannot synthesize sialic acids endogenously, and therefore the biological function and substrate scope of sNPL ought to differ significantly from vertebrate sialic aldolases such as chNPL. sNPL was active towards a series of sialic acid derivatives but was in contrast to chNPL unable to catalyze the cleavage of N-acetylneuraminic acid into N-acetylmannosamine and pyruvate. Interestingly, chNPL and sNPL showed contrasting C4(R)/(S) diastereoselectivity towards the substrates d-mannose and d-galactose in the presence of pyruvate. In addition, sNPL was able to synthesize a series of 4-hydroxy-2-oxoates using the corresponding aliphatic aldehyde substrates in the presence of pyruvate, which could be not achieved by chNPL.

Metagenome-Assembled Genomes Reveal Mechanisms of Carbohydrate and Nitrogen Metabolism of Schistosomiasis-Transmitting Vector Biomphalaria Glabrata.

Biomphalaria glabrata transmits schistosomiasis mansoni which poses considerable risks to hundreds of thousands of people worldwide, and is widely used as a model organism for studies on the snail-schistosome relationship. Gut microbiota plays important roles in multiple aspects of host including development, metabolism, immunity, and even behavior; however, detailed information on the complete diversity and functional profiles of B. glabrata gut microbiota is still limited. This study is the first to reveal the gut microbiome of B. glabrata based on metagenome-assembled genome (MAG). A total of 28 gut samples spanning diet and age were sequenced and 84 individual microbial genomes with ≥ 70% completeness and ≤ 5% contamination were constructed. Bacteroidota and Proteobacteria were the dominant bacteria in the freshwater snail, unlike terrestrial organisms harboring many species of Firmicutes and Bacteroidota. The microbial consortia in B. glabrata helped in the digestion of complex polysaccharide such as starch, hemicellulose, and chitin for energy supply, and protected the snail from food poisoning and nitrate toxicity. Both microbial community and metabolism of B. glabrata were significantly altered by diet. The polysaccharide-degrading bacterium Chryseobacterium was enriched in the gut of snails fed with high-digestibility protein and high polysaccharide diet (HPHP). Notably, B. glabrata as a mobile repository can escalate biosafety issues regarding transmission of various pathogens such as Acinetobacter nosocomialis and Vibrio parahaemolyticus as well as multiple antibiotic resistance genes in the environment and to other organisms. IMPORTANCE The spread of aquatic gastropod Biomphalaria glabrata, an intermediate host of Schistosoma mansoni, exacerbates the burden of schistosomiasis disease worldwide. This study provides insights into the importance of microbiome for basic biological activities of freshwater snails, and offers a valuable microbial genome resource to fill the gap in the analysis of the snail-microbiota-parasite relationship. The results of this study clarified the reasons for the high adaptability of B. glabrata to diverse environments, and further illustrated the role of B. glabrata in accumulation of antibiotic resistance in the environment and spread of various pathogens. These findings have important implications for further exploration of the control of snail dissemination and schistosomiasis from a microbial perspective.

Molluscicidal and cercaricidal activities of the essential oil of Dysphania ambrosioides (L.) Mosyakin & Clemants: Implications for the control of schistosomiasis.

Schistosomiasis is one of the most important tropical diseases. A fundamental strategy to control its spread is the use of natural products against its vectors, which are snails of the genus Biomphalaria. The present study evaluated the chemical composition, the molluscicidal and cercaricidal effects, and the ecotoxicity of the essential oil from the aerial parts of Dysphania ambrosioides (L.) Mosyakin & Clemants (DAEO). The essential oil was obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). Molluscicidal and cercaricidal activities were determined by the immersion method. Environmental toxicity was assessed from bioassays using Artemia salina larvae and Danio rerio fish. DAEO presented a 0.8% yield. The GC-MS analysis revealed the predominance of hydrocarbon monoterpenes in the oil. A total of 32 constituents was identified, with α-terpinene (50.69%) being the major compound, followed by p-cymene (13.27%) and ascaridole (10.26%). DAEO was active against adult Biomphalaria glabrata snails and demonstrated lethal effect against Schistosoma mansoni cercariae, with LC50 values of 25.2 (22.7-27.8) and 62.4 (61.8-62.9) µg/mL, respectively. Regarding toxicity to non-target aquatic organisms, the oil showed LC50 values of 86.9 (84.7-87.6) and 18.6 µg/mL (15.5-22.8) for A. salina and D. rerio, respectively. DAEO proved to be a promising natural product for the control of schistosomiasis, acting on both the vectors and the etiological agent of the disease. However, the use of the oil is safer in transmission sites where there are no non-target organisms, as it has showed toxicity to D. rerio fish.