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A multistrain approach to studying the mechanisms underlying compatibility in the interaction between Biomphalaria glabrata and Schistosoma mansoni ArchiMer
Galinier, Richard; Roger, Emmanuel; Mone, Yves; Duval, David; Portet, Anais; Pinaud, Silvain; Chaparro, Cristian; Grunau, Christoph; Genthon, Clemence; Dubois, Emeric; Rognon, Anne; Arancibia, Nathalie; Dejean, Bernard; Theron, Andre; Gourbal, Benjamin; Mitta, Guillaume.
In recent decades, numerous studies have sought to better understand the mechanisms underlying the compatibility between Biomphalaria glabrata and Schistosoma mansoni. The developments of comparative transcriptomics, comparative genomics, interactomics and more targeted approaches have enabled researchers to identify a series of candidate genes. However, no molecular comparative work has yet been performed on multiple populations displaying different levels of compatibility. Here, we seek to fill this gap in the literature. We focused on B. glabrata FREPs and S. mansoni SmPoMucs, which were previously demonstrated to be involved in snail/schistosome compatibility. We studied the expression and polymorphisms of these factors in combinations of snail and...
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Ano: 2017 URL: https://archimer.ifremer.fr/doc/00388/49951/50515.pdf
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A Novel Bacterial Pathogen of Biomphalaria glabrata: A Potential Weapon for Schistosomiasis Control? ArchiMer
Duval, David; Galinier, Richard; Mouahid, Gabriel; Toulza, Eve; Allienne, Jean Francois; Portela, Julien; Calvayrac, Christophe; Rognon, Anne; Arancibia, Nathalie; Mitta, Guillaume; Theron, Andre; Gourbal, Benjamin.
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...
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Ano: 2015 URL: https://archimer.ifremer.fr/doc/00615/72716/71712.pdf
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A Shift from Cellular to Humoral Responses Contributes to Innate Immune Memory in the Vector Snail Biomphalaria glabrata ArchiMer
Pinaud, Silvain; Portela, Julien; Duval, David; Nowacki, Fanny C.; Olive, Marie-aude; Allienne, Jean-francois; Galinier, Richard; Dheilly, Nolwenn M.; Kieffer-jaquinod, Sylvie; Mitta, Guillaume; Theron, Andre; Gourbal, Benjamin.
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...
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Ano: 2016 URL: https://archimer.ifremer.fr/doc/00315/42664/71872.pdf
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A Sustained Immune Response Supports Long-Term Antiviral Immune Priming in the Pacific Oyster, Crassostrea gigas ArchiMer
Lafont, Maxime; Vergnes, Agnes; Vidal-dupiol, Jeremie; De Lorgeril, Julien; Gueguen, Yannick; Haffner, Philippe; Petton, Bruno; Chaparro, Cristian; Barrachina, Celia; Destoumieux-garzon, Delphine; Mitta, Guillaume; Gourbal, Benjamin; Montagnani, Caroline.
Over the last decade, innate immune priming has been evidenced in many invertebrate phyla. If mechanistic models have been proposed, molecular studies aiming to substantiate these models have remained scarce. We reveal here the transcriptional signature associated with immune priming in the oyster Crassostrea gigas. Oysters were fully protected against Ostreid herpesvirus 1 (OsHV-1), a major oyster pathogen, after priming with poly(I·C), which mimics viral double-stranded RNA. Global analysis through RNA sequencing of oyster and viral genes after immune priming and viral infection revealed that poly(I·C) induces a strong antiviral response that impairs OsHV-1 replication. Protection is based on a sustained upregulation of immune genes, notably genes...
Tipo: Text Palavras-chave: Innate immunity; Priming; OsHV-1; Antiviral response; Immune memory; Oyster; POMS; Poly(I·C); Interferon; Transcriptomic.
Ano: 2020 URL: https://archimer.ifremer.fr/doc/00614/72580/71541.pdf
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BgTEP: An Antiprotease Involved in Innate Immune Sensing in Biomphalaria glabrata ArchiMer
Portet, Anais; Galinier, Richard; Pinaud, Silvain; Portela, Julien; Nowacki, Fanny; Gourbal, Benjamin; Duval, David.
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...
Tipo: Text Palavras-chave: Thioester-containing protein; Complement-like protein; Biomphalaria glabrata; Interaction host/pathogens; Innate immunity.
Ano: 2018 URL: https://archimer.ifremer.fr/doc/00444/55548/71780.tif
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Deciphering the molecular mechanisms of mother-to-egg immune protection in the mealworm beetle Tenebrio molitor ArchiMer
Tetreau, Guillaume; Dhinaut, Julien; Galinier, Richard; Audant-lacour, Pascaline; Voisin, Sébastien N; Arafah, Karim; Chogne, Manon; Hilliou, Frédérique; Bordes, Anaïs; Sabarly, Camille; Chan, Philippe; Walet-balieu, Marie-laure; Vaudry, David; Duval, David; Bulet, Philippe; Coustau, Christine; Moret, Yannick; Gourbal, Benjamin.
In a number of species, individuals exposed to pathogens can mount an immune response and transmit this immunological experience to their offspring, thereby protecting them against persistent threats. Such vertical transfer of immunity, named trans-generational immune priming (TGIP), has been described in both vertebrates and invertebrates. Although increasingly studied during the last decade, the mechanisms underlying TGIP in invertebrates are still elusive, especially those protecting the earliest offspring life stage, i.e. the embryo developing in the egg. In the present study, we combined different proteomic and transcriptomic approaches to determine whether mothers transfer a “signal” (such as fragments of infecting bacteria), mRNA and/or...
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Ano: 2020 URL: https://archimer.ifremer.fr/doc/00655/76684/77815.pdf
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Fasciola hepatica-Pseudosuccinea columella interaction: effect of increasing parasite doses, successive exposures and geographical origin on the infection outcome of susceptible and naturally-resistant snails from Cuba ArchiMer
Alba, Annia; Vazquez, Antonio A.; Sanchez, Jorge; Duval, David; Hernandez, Hilda M.; Sabourin, Emeline; Vittecoq, Marion; Hurtrez-bousses, Sylvie; Gourbal, Benjamin.
Background: Pseudosuccinea columella is one of the most widespread vectors of Fasciola hepatica, a globally distributed trematode that affects humans, livestock and wildlife. The exclusive occurrence in Cuba of susceptible and naturally-resistant populations to F. hepatica within this snail species, offers a fascinating model for evolutionary biology, health sciences and vector control strategies. In particular, resistance in P. columella is characterized by the encapsulation of the parasite by host's immune cells and has been experimentally tested using different Cuban F. hepatica isolates with no records of successful infection. Here, we aimed to explore for the first time, the effect of different parasite doses, successive exposures and different...
Tipo: Text Palavras-chave: Snail-trematode interaction; Lymnaeidae; Liver fluke; Experimental infection; Immune priming; Compatibility; Allopatric parasites.
Ano: 2018 URL: https://archimer.ifremer.fr/doc/00467/57894/71760.pdf
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Hemocyte siRNA uptake is increased by 5′ cholesterol-TEG addition in Biomphalaria glabrata , snail vector of schistosome ArchiMer
Portet, Anaïs; Galinier, Richard; Lassalle, Damien; Faille, Alexandre; Gourbal, Benjamin; Duval, David.
Biomphalaria glabrata is one of the snail intermediate hosts of Schistosoma mansoni, the causative agent of intestinal schistosomiasis disease. Numerous molecular studies using comparative approaches between susceptible and resistant snails to S. mansoni infection have helped identify numerous snail key candidates supporting such susceptible/resistant status. The functional approach using RNA interference (RNAi) remains crucial to validate the function of such candidates. CRISPR-Cas systems are still under development in many laboratories, and RNA interference remains the best tool to study B. glabrata snail genetics. Herein, we describe the use of modified small interfering RNA (siRNA) molecules to enhance cell delivery, especially into hemocytes, the...
Tipo: Text Palavras-chave: Biomphalaria glabrata; Cholesteryl TEG; RNAi interference; BgTEP1; Hemocyte.
Ano: 2021 URL: https://archimer.ifremer.fr/doc/00682/79376/81902.pdf
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Long-lasting antiviral innate immune priming in the Lophotrochozoan Pacific oyster, Crassostrea gigas ArchiMer
Lafont, Maxime; Petton, Bruno; Vergnes, Agnes; Pauletto, Marianna; Segarra, Amelie; Gourbal, Benjamin; Montagnani, Caroline.
In the last decade, a paradigm shift has emerged in comparative immunology. Invertebrates can no longer be considered to be devoid of specific recognition and immune memory. However, we still lack a comprehensive view of these phenomena and their molecular mechanisms across phyla, especially in terms of duration, specificity, and efficiency in a natural context. In this study, we focused on a Lophotrochozoan/virus interaction, as antiviral priming is mostly overlooked in molluscs. Juvenile Crassostrea gigas oysters experience reoccurring mass mortalities events from Ostreid herpes virus 1 with no existing therapeutic treatment. Our results showed that various nucleic acid injections can prime oysters to trigger an antiviral state ultimately protecting them...
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Ano: 2017 URL: https://archimer.ifremer.fr/doc/00407/51811/52413.pdf
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Molluscicidal and parasiticidal activities of Eryngium triquetrum essential oil on Schistosoma mansoni and its intermediate snail host Biomphalaria glabrata, a double impact ArchiMer
De Carvalho Augusto, Ronaldo; Merad, Nadjiya; Rognon, Anne; Gourbal, Benjamin; Bertrand, Cédric; Djabou, Nassim; Duval, David.
Background Freshwater snails are the intermediate hosts of a large variety of trematode flukes such as Schistosoma mansoni responsible for one of the most important parasitic diseases caused by helminths, affecting 67 million people worldwide. Recently, the WHO Global Vector Control Response 2017–2030 (GVCR) programme reinforced its message for safer molluscicides as part of required strategies to strengthen vector control worldwide. Here, we present the essential oil from Eryngium triquetrum as a powerful product with molluscicide and parasiticide effect against S. mansoni and the snail intermediate host Biomphalaria glabrata. Methods In the present study, we describe using several experimental approaches, the chemical composition of E. triquetrum...
Tipo: Text Palavras-chave: Biomphalaria glabrata; Eryngium triquetrum; Molluscicide; Oil; Parasiticide; Schistosoma mansoni.
Ano: 2020 URL: https://archimer.ifremer.fr/doc/00652/76382/77391.pdf
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Natural resistance to Fasciola hepatica (Trematoda) in Pseudosuccinea columella snails: A review from literature and insights from comparative “omic” analyses ArchiMer
Alba, Annia; Tetreau, Guillaume; Chaparro, Cristian; Sánchez, Jorge; Vázquez, Antonio A.; Gourbal, Benjamin.
The snail Pseudosuccinea columella is one of the main vectors of the medically-important trematode Fasciola hepatica. In Cuba, the existence of natural P. columella populations that are either susceptible or resistant to F. hepatica infection offers a unique snail-parasite for study of parasite-host compatibility and immune function in gastropods. Here, we review all previous literature on this system and present new “omic” data that provide a molecular baseline of both P. columella phenotypes from naïve snails. Comparison of whole snail transcriptomes (RNAseq) and the proteomes of the albumen gland (2D-electrophoresis, MS) revealed that resistant and susceptible strains differed mainly in an enrichment of particular biological processes/functions and a...
Tipo: Text Palavras-chave: Transcriptome; Albumen gland; Allocation of resources; Response to stress; Immune defense; Cost of resistance.
Ano: 2019 URL: https://archimer.ifremer.fr/doc/00509/62039/66180.pdf
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New Insights Into Biomphalysin Gene Family Diversification in the Vector Snail Biomphalaria glabrata ArchiMer
Pinaud, Silvain; Tetreau, Guillaume; Poteaux, Pierre; Galinier, Richard; Chaparro, Cristian; Lassalle, Damien; Portet, Anaïs; Simphor, Elodie; Gourbal, Benjamin; Duval, David.
Aerolysins initially characterized as virulence factors in bacteria are increasingly found in massive genome and transcriptome sequencing data from metazoans. Horizontal gene transfer has been demonstrated as the main way of aerolysin-related toxins acquisition in metazoans. However, only few studies have focused on their potential biological functions in such organisms. Herein, we present an extensive characterization of a multigene family encoding aerolysins - named biomphalysin - in Biomphalaria glabrata snail, the intermediate host of the trematode Schistosoma mansoni. Our results highlight that duplication and domestication of an acquired bacterial toxin gene in the snail genome result in the acquisition of a novel and diversified toxin family....
Tipo: Text Palavras-chave: Biomphalaria; Biomphalysin; Aerolysin; Invertebrate immunity; Pore-forming toxin (PFT); Structure.
Ano: 2021 URL: https://archimer.ifremer.fr/doc/00687/79909/82814.pdf
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Outer membrane vesicles are vehicles for the delivery of Vibrio tasmaniensis virulence factors to oyster immune cells ArchiMer
Vanhove, Audrey; Duperthuy, Marylise; Charriere, Guillaume; Le Roux, Frederique; Goudenege, David; Gourbal, Benjamin; Kieffer-jaquinod, Sylvie; Coute, Yohann; Wai, Sun Nyunt; Destoumieux-garzon, Delphine.
Vibrio tasmaniensis LGP32, a facultative intracellular pathogen of oyster haemocytes, was shown here to release outer membrane vesicles (OMVs) both in the extracellular milieu and inside haemocytes. Intracellular release of OMVs occurred inside phagosomes of intact haemocytes having phagocytosed few vibrios as well as in damaged haemocytes containing large vacuoles heavily loaded with LGP32. The OMV proteome of LGP32 was shown to be rich in hydrolases (25%) including potential virulence factors such as proteases, lipases, phospholipases, haemolysins and nucleases. One major caseinase/gelatinase named Vsp for vesicular serine protease was found to be specifically secreted through OMVs in which it is enclosed. Vsp was shown to participate in the virulence...
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Ano: 2015 URL: http://archimer.ifremer.fr/doc/00199/31009/29459.pdf
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Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba ArchiMer
Alba, Annia; Vázquez, Antonio A.; Sánchez, Jorge; Lounnas, Manon; Pointier, Jean-pierre; Hurtrez-boussès, Sylvie; Gourbal, Benjamin.
Pseudosuccinea columella snails transmit the trematode Fasciola hepatica, but in Cuba, six naturally occurring populations successfully resist parasite infection. Here, we present an updated distribution of P. columella in Cuba; 68 positive sites with the earliest records more abundant in west-central Cuba and with east-central populations generally corresponding to the newest samples. No records were found farther east. The IPA site reported 10.5% prevalence of F. hepatica-infected snails. Population genetics, studied through microsatellites, showed low allelic and multilocus genotypic richness (MLGT), mainly in susceptible populations, strong deviations from panmixia and high self-fertilization rates. Susceptible individuals were grouped in one major...
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Ano: 2019 URL: https://archimer.ifremer.fr/doc/00584/69660/67514.pdf
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Schistosomiasis Vector Snails and Their Microbiota Display a Phylosymbiosis Pattern ArchiMer
Huot, Camille; Clerissi, Camille; Gourbal, Benjamin; Galinier, Richard; Duval, David; Toulza, Eve.
Planorbidae snails are the intermediate host for the trematode parasite of the Schistosoma genus, which is responsible for schistosomiasis, a disease that affects both humans and cattle. The microbiota for Schistosoma has already been described as having an effect on host/parasite interactions, specifically through immunological interactions. Here, we sought to characterize the microbiota composition of seven Planorbidae species and strains. Individual snail microbiota was determined using 16S ribosomal DNA amplicon sequencing. The bacterial composition was highly specific to the host strain with limited interindividual variation. In addition, it displayed complete congruence with host phylogeny, revealing a phylosymbiosis pattern. These results were...
Tipo: Text Palavras-chave: Microbiota; Phylosymbiosis; Metabarcoding; Planorbid snails; Tripartite interactions; Schistosomiasis.
Ano: 2020 URL: https://archimer.ifremer.fr/doc/00606/71807/70295.pdf
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Specific Pathogen Recognition by Multiple Innate Immune Sensors in an Invertebrate ArchiMer
Tetreau, Guillaume; Pinaud, Silvain; Portet, Anais; Galinier, Richard; Gourbal, Benjamin; Duval, David.
Detection of pathogens by all living organisms is the primary step needed to implement a coherent and efficient immune response. This implies a mediation by different soluble and/or membrane-anchored proteins related to innate immune receptors called PRRs (pattern-recognition receptors) to trigger immune signaling pathways. In most invertebrates, their roles have been inferred by analogy to those already characterized in vertebrate homologs. Despite the induction of their gene expression upon challenge and the presence of structural domains associated with the detection of pathogen-associated molecular patterns in their sequence, their exact role in the induction of immune response and their binding capacity still remain to be demonstrated. To this...
Tipo: Text Palavras-chave: Invertebrate innate immunity; Interactome; Pathogen sensing; Biomphalaria glabrata; Pattern-recognition receptor; Proteomic profiling; Immune specificity; Hemocyte-free hemolymph.
Ano: 2017 URL: https://archimer.ifremer.fr/doc/00405/51633/71796.pdf
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Sympatric versus allopatric evolutionary contexts shape differential immune response in Biomphalaria / Schistosoma interaction ArchiMer
Portet, Anais; Pinaud, Silvain; Chaparro, Cristian; Galinier, Richard; Dheilly, Nolwenn M.; Portela, Julien; Charriere, Guillaume; Allienne, Jean-francois; Duval, David; Gourbal, Benjamin.
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...
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Ano: 2019 URL: https://archimer.ifremer.fr/doc/00488/59982/63214.pdf
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Trans-generational Immune Priming in Invertebrates: Current Knowledge and Future Prospects ArchiMer
Tetreau, Guillaume; Dhinaut, Julien; Gourbal, Benjamin; Moret, Yannick.
Trans-generational immune priming (TGIP) refers to the transfer of the parental immunological experience to its progeny. This may result in offspring protection from repeated encounters with pathogens that persist across generations. Although extensively studied in vertebrates for over a century, this phenomenon has only been identified 20 years ago in invertebrates. Since then, invertebrate TGIP has been the focus of an increasing interest, with half of studies published during the last few years. TGIP has now been tested in several invertebrate systems using various experimental approaches and measures to study it at both functional and evolutionary levels. However, drawing an overall picture of TGIP from available studies still appears to be a difficult...
Tipo: Text Palavras-chave: Trans-generational immune priming; Invertebrate immunity; Host-pathogens interaction; Ecology and evolution; Molecular mechanisms; Scenarios.
Ano: 2019 URL: https://archimer.ifremer.fr/doc/00512/62369/66642.pdf
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Whole genome analysis of a schistosomiasis-transmitting freshwater snail ArchiMer
Adema, Coen M.; Hillier, Ladeana W.; Jones, Catherine S.; Loker, Eric S.; Knight, Matty; Minx, Patrick; Oliveira, Guilherme; Raghavan, Nithya; Shedlock, Andrew; Do Amaral, Laurence Rodrigues; Arican-goktas, Halime D.; Assis, Juliana G.; Baba, Elio Hideo; Baron, Olga L.; Bayne, Christopher J.; Bickham-wright, Utibe; Biggar, Kyle K.; Blouin, Michael; Bonning, Bryony C.; Botka, Chris; Bridger, Joanna M.; Buckley, Katherine M.; Buddenborg, Sarah K.; Caldeira, Roberta Lima; Carleton, Julia; Carvalho, Omar S.; Castillo, Maria G.; Chalmers, Iain W.; Christensens, Mikkel; Clifton, Sandra; Cosseau, Celine; Coustau, Christine; Cripps, Richard M.; Cuesta-astroz, Yesid; Cummins, Scott F.; Di Stephano, Leon; Dinguirard, Nathalie; Duval, David; Emrich, Scott; Feschotte, Cedric; Feyereisen, Rene; Fitzgerald, Peter; Fronick, Catrina; Fulton, Lucinda; Galinier, Richard; Gava, Sandra G.; Geusz, Michael; Geyer, Kathrin K.; Giraldo-calderon, Gloria I.; Gomes, Matheus De Souza; Gordy, Michelle A.; Gourbal, Benjamin; Grunau, Christoph; Hanington, Patrick C.; Hoffmann, Karl F.; Hughes, Daniel; Humphries, Judith; Jackson, Daniel J.; Jannotti-passos, Liana K.; Jeremias, Wander De Jesus; Jobling, Susan; Kamel, Bishoy; Kapusta, Aurelie; Kaur, Satwant; Koene, Joris M.; Kohn, Andrea B.; Lawson, Dan; Lawton, Scott P.; Liang, Di; Limpanont, Yanin; Liu, Sijun; Lockyer, Anne E.; Lovato, Tyanna L.; Ludolf, Fernanda; Magrini, Vince; Mcmanus, Donald P.; Medina, Monica; Misra, Milind; Mitta, Guillaume; Mkoji, Gerald M.; Montague, Michael J.; Montelongo, Cesar; Moroz, Leonid L.; Munoz-torres, Monica C.; Niazi, Umar; Noble, Leslie R.; Oliveira, Francislon S.; Pais, Fabiano S.; Papenfuss, Anthony T.; Peace, Rob; Pena, Janeth J.; Pila, Emmanuel A.; Quelais, Titouan; Raney, Brian J.; Rast, Jonathan P.; Rollinson, David; Rosse, Izinara C.; Rotgans, Bronwyn; Routledge, Edwin J.; Ryan, Kathryn M.; Scholte, Larissa L. S.; Storey, Kenneth B.; Swain, Martin; Tennessen, Jacob A.; Tomlinson, Chad; Trujillo, Damian L.; Volpi, Emanuela V.; Walker, Anthony J.; Wang, Tianfang; Wannaporn, Ittiprasert; Warren, Wesley C.; Wu, Xiao-jun; Yoshino, Timothy P.; Yusuf, Mohammed; Zhang, Si-ming; Zhao, Min; Wilson, Richard K..
Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of...
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Ano: 2017 URL: https://archimer.ifremer.fr/doc/00387/49840/71816.pdf
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