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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.

Schistosomiasis still affects a lot of people in many developing countries. Reducing the disease dissemination has been the target of various studies. As methyl gallate has antioxidant properties, it is assumed that it can be a good candidate for stimulating the immune response of snails. So, the aim of this work is to investigate the potential of using methyl gallate as an immunostimulant to Biomphalaria alexandrina snails in order to prevent the development of invading miracidia into infective cercariae. The infected snails were exposed to three concentrations of methyl gallate for two periods: 24 and 72 h. The results indicated that the most effective concentration was the lowest one: 125 mg/L of methyl gallate for 72 h, as it reduced both infection rate and mean number of shed cercariae. Also, it increased the total number of snails' hemocytes in hemolymph, which were observed in head-foot region and digestive gland of treated snails surrounding degenerated sporocysts and cercariae. In addition, hydrogen peroxide showed its highest content in tissues of snails exposed to 125 mg/L of methyl gallate for 72 h. In conclusion, methyl gallate can be considered as one of the most promising immunostimulants of B. alexandrina snails against infection with Schistosoma mansoni.

Schistosoma mansoni, which causes human intestinal schistosomiasis, continues to be a major public health concern in the Lake Victoria basin in western Kenya, with Biomphalaria sudanica (a shoreline inhabiting snail) and Biomphalaria choanomphala (a deep-water snail) playing roles in transmission. A recent study showed that B. sudanica was abundantly present near all study villages on the lakeshore, but B. choanomphala was significantly more abundant near villages known to be persistent transmission hotspots. The present study investigated the relative compatibility of B. sudanica and B. choanomphala with S. mansoni. A reciprocal cross-infection experiment used young adult F1 generation B. sudanica and B. choanomphala that were exposed to either 1, 5, or 10 sympatric or allopatric human-derived S. mansoni miracidia. Three weeks post-exposure (PE) and weekly thereafter, the snails were counted and screened for schistosome cercariae, and at 7 wk PE, total cercariae shed during a 2 hr period by each infected snail was determined. Pre-patent periods for S. mansoni in both B. sudanica and B. choanomphala were similar, and most snails in all exposure combinations started shedding cercariae 5 wk PE. Prevalences were significantly higher in B. choanomphala (12.2-80.9%) than in B. sudanica (5.2-18.6%) at each dose, regardless of whether miracidia were of an allopatric or a sympatric source (P < 0.0001). Overall, the odds of a snail becoming infected with 5 or 10 miracidia were significantly higher than the odds of being infected with 1 miracidium, (P < 0.0001), and fewer cercariae were produced by snails exposed to single as compared to 5 or 10 miracidia. On average, B. choanomphala produced more cercariae ( = 458, SD = 414) than B. sudanica ( = 238, SD = 208) (P < 0.0001). These results suggest that B. choanomphala is more compatible with S. mansoni than B. sudanica. Though B. choanomphala can be found in shallow shoreline waters, it is, for the most part, a deeper-water taxon. Because dredging is a relatively inefficient means of sampling, B. choanomphala is likely underestimated with respect to its population size, the number of S. mansoni-positive snails, and its role in maintaining transmission., (© American Society of Parasitologists 2021.)

Achieving a deeper understanding of the factors controlling the defense responses of invertebrate vectors to the human-infecting pathogens they transmit will provide needed new leads to pursue for control. Consequently, we provide new genomic and transcriptomic insights regarding FReDs (containing a fibrinogen domain) and FREPs (fibrinogen domain and one or two IgSF domains) from the planorbid snail Biomphalaria glabrata, a Neotropical vector of Schistosoma mansoni, causative agent of human intestinal schistosomiasis. Using new bioinformatics approaches to improve annotation applied to both genome and RNA-Seq data, we identify 73 FReD genes, 39 of which are FREPs. We provide details of domain structure and consider relationships and homologies of B. glabrata FBG and IgSF domains. We note that schistosome-resistant (BS-90) snails mount complex FREP responses following exposure to S. mansoni infection whereas schistosome-susceptible (M line) snails do not. We also identify several coding differences between BS-90 and M line snails in three FREPs (2, 3.1 and 3.2) repeatedly implicated in other studies of anti-schistosome responses. In combination with other results, our study provides a strong impetus to pursue particular FREPs (2, 3.1, 3.2 and 4) as candidate resistance factors to be considered more broadly with respect to schistosome control efforts, including involving other Biomphalaria species vectoring S. mansoni in endemic areas in Africa., Competing Interests: The authors have declared that no competing interests exist.

Planorbid freshwater snails are important intermediate hosts for parasitic diseases caused by parasitic worms, most notably schistosomiasis. There are numerous reports of snails, specifically Biomphalaria glabrata, having compromised defences against schistosomes after being exposed to thermal stress. Environmental modifications to the defenses of schistosome transmitting snails could have negative ramifications for human disease risk in the context of climate change. Here the effects of heat shock on the production of hydrogen peroxide, a primary anti-microbial effector in many molluscs, were examined. The present findings show that heat shock increases NADPH oxidase 2 mRNA levels and hydrogen peroxide produced by snail hemocytes, and that both of these phenotypes could be reversed by an HSP-90 inhibitor. These findings indicate that snail defense systems are altered by heat shock at a molecular level in B. glabrata, and that snail immunity to many pathogens may be altered by the rapid variations in temperature that are associated with global climate change., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Schistosomiasis is a debilitating parasitic disease infecting hundreds of millions of people. Schistosomes use aquatic snails as intermediate hosts. A promising avenue for disease control involves leveraging innate host mechanisms to reduce snail vectorial capacity. In a genome-wide association study of Biomphalaria glabrata snails, we identify genomic region PTC2 which exhibits the largest known correlation with susceptibility to parasite infection (>15 fold effect). Using new genome assemblies with substantially higher contiguity than the Biomphalaria reference genome, we show that PTC2 haplotypes are exceptionally divergent in structure and sequence. This variation includes multi-kilobase indels containing entire genes, and orthologs for which most amino acid residues are polymorphic. RNA-Seq annotation reveals that most of these genes encode single-pass transmembrane proteins, as seen in another resistance region in the same species. Such groups of hyperdiverse snail proteins may mediate host-parasite interaction at the cell surface, offering promising targets for blocking the transmission of schistosomiasis., Competing Interests: JT, SB, EP, BK, CS, BH, MB No competing interests declared, (© 2020, Tennessen et al.)

The freshwater snail, Biomphalaria glabrata, is an important intermediate host in the life cycle for the human parasite Schistosoma mansoni, the causative agent of schistosomiasis. Current treatment and prevention strategies have not led to a significant decrease in disease transmission. However, the genome of B. glabrata was recently sequenced to provide additional resources to further our understanding of snail biology. This review presents an overview of recently published, post-genome studies related to the topic of snail immunity. Many of these reports expand on findings originated from the genome characterization. These novel studies include a complementary gene linkage map, analysis of the genome of the B. glabrata embryonic (Bge) cell line, as well as transcriptomic and proteomic studies looking at snail-parasite interactions and innate immune memory responses towards schistosomes. Also included are biochemical investigations on snail pheromones, neuropeptides, and attractants, as well as studies investigating the frontiers of molluscan epigenetics and cell signaling were also included. Findings support the current hypotheses on snail-parasite strain compatibility, and that snail host resistance to schistosome infection is dependent not only on genetics and expression, but on the ability to form multimeric molecular complexes in a timely and tissue-specific manner. The relevance of cell immunity is reinforced, while the importance of humoral factors, especially for secondary infections, is supported. Overall, these studies reflect an improved understanding on the diversity, specificity, and complexity of molluscan immune systems., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Immune factors in snails of the genus Biomphalaria are critical for combating Schistosoma mansoni , the predominant cause of human intestinal schistosomiasis. Independently, many of these factors play an important role in, but do not fully define, the compatibility between the model snail B. glabrata , and S. mansoni . Here, we demonstrate association between four previously characterized humoral immune molecules; Bg FREP3, Bg TEP1, Bg FREP2 and Biomphalysin. We also identify unique immune determinants in the plasma of S. mansoni -resistant B. glabrata that associate with the incompatible phenotype. These factors coordinate to initiate haemocyte-mediated destruction of S. mansoni sporocysts via production of reactive oxygen species. The inclusion of Bg FREP2 in a Bg FREP3-initiated complex that also includes Bg TEP1 almost completely explains resistance to S. mansoni in this model. Our study unifies many independent lines of investigation to provide a more comprehensive understanding of the snail immune system in the context of infection by this important human parasite., Competing Interests: HL, JH, EP, AG, JF, XW, PH No competing interests declared, (© 2020, Li et al.)

Biomphalaria glabrata is a freshwater Planorbidae snail. In its environment, this mollusk faces numerous microorganisms or pathogens, and has developed sophisticated innate immune mechanisms to survive. The mechanisms of recognition are quite well understood in Biomphalaria glabrata , but immune effectors have been seldom described. In this study, we analyzed a new family of potential immune effectors and characterized five new genes that were named Glabralysins. The five Glabralysin genes showed different genomic structures and the high degree of amino acid identity between the Glabralysins, and the presence of the conserved ETX/MTX2 domain, support the hypothesis that they are pore-forming toxins. In addition, tertiary structure prediction confirms that they are structurally related to a subset of Cry toxins from Bacillus thuringiensis , including Cry23, Cry45, and Cry51. Finally, we investigated their gene expression profiles in snail tissues and demonstrated a mosaic transcription. We highlight the specificity in Glabralysin expression following immune stimulation with bacteria, yeast or trematode parasites. Interestingly, one Glabralysin was found to be expressed in immune-specialized hemocytes, and two others were induced following parasite exposure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

For many years, the immune response of invertebrates was considered to lack any mechanism of memory. However, the study of their response has shown a kind of acquired immunity, which is not so well understood given the lack of knowledge of the invertebrate defense system. This event can be called "innate immune memory." Recent studies using Biomphalaria glabrata snails have reported this phenomenon, relating it to an increase in humoral products, but no focus was given to hemocyte response or to other species of snails. In this study, we focus on hemocyte dynamics and some humoral factors in the species B. glabrata and B. straminea, the most widespread species in Brazil, sensitized and non-sensitized to the Schistosoma mansoni worm. We report a change in the prevalent hemocyte type after sensitization, through an increase in the proportion of granulocytes, as well as a change in the total number of hemocytes caused by a second exposure to the parasite. We also showed that melanization is not a key factor in Biomphalaria snail defense and varies little after the second exposure event. The data reported in this article confirm the effect of immune priming on these snails and suggest that the increase of humoral products shown in the literature is accompanied by variation in hemocytes after sensitization.

Mansonic schistosomiasis is a neglected disease transmitted by Biomphalaria spp. snails. Understanding what happens inside the intermediate host is important to develop more efficient ways of reducing schistosomiasis prevalence. Our purpose was to characterize metabolic and immunological changes in Biomphalaria glabrata 24 h after exposure to Schistosoma mansoni. For this purpose, proteins were extracted from snails' whole tissue with Tris-Urea buffer and digested with tripsin. Mass spectrometry was performed and analyzed with MaxQuant and Perseus software. Also, the hemolymph of five snails 24 h post exposure was collected, and the numbers of hemocytes, levels of urea, uric acid, nitric oxide, calcium, glycogen and alanine and aspartate aminotransferases activities were assessed. Snails were also dissected for measurement of glycogen content in the cephalopodal region and gonoda-digestive gland complex. Globin domain proteins were found to be up-regulated; also the number of circulating hemocytes was significantly higher after 24 h of exposure to the parasite. NO levels were higher 24 h post exposure. Several proteins associated with energy metabolism were found to be up-regulated. Glycogen analysis showed a significant decrease in the gonad-digestive gland complex glycogen content. We found several proteins which seem to be associated with the host immune response, most of which were up-regulated, however some were down-regulated, which may represent an important clue in understanding B. glabrata - S. mansoni compatibility., (Copyright © 2019 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Albino strains of Biomphalaria glabrata that are compatible with Schistosoma mansoni are commonly used to investigate snail-schistosome interactions, but whether they are all equally compatible is not known. In this study, compatibility with the Naval Medical Research Institute (NMRI) strain of S. mansoni was compared among 3 widely used albino strains: NMRI (the normal laboratory host for NMRI S. mansoni ), M line, and University of Massachusetts Lowell (UML). Compatibility was assessed on the basis of infection prevalence following exposure to miracidia, the histological fate of sporocysts, and mitotic response in the snail amebocyte-producing organ (APO), a component of the internal defense system (IDS). Infection prevalence in UML was nearly identical to that in NMRI but was significantly lower in M line. Although the APO of UML showed no response to infection over the course of 9 days, mitotic activity was elevated in the APO of NMRI and M line, with that in M line being higher and more prolonged than in the APO of resistant BS-90 snails. Finally, hemocyte responses against some small primary sporocysts occurred at 1 and 3 days post-exposure (DPE) in all 3 strains, but in 2 of 5 M line a response also occurred against large primary sporocysts at 6 DPE. Thus, based on infection prevalence and tissue responses, compatibility with NMRI S. mansoni is lowest in M line, whereas UML and NMRI show the same degree of compatibility, despite decades of maintenance of this parasite strain in NMRI snails. The elevated mitotic response in the APO of M line and NMRI snails suggests that a response of the IDS can occur even in a compatible host-parasite relationship.

Freshwater snails are obligate intermediate hosts for numerous parasitic trematodes, most notably schistosomes. Schistosomiasis is a devastating human and veterinary illness, which is primarily controlled by limiting the transmission of these parasites from their intermediate snail hosts. Understanding how this transmission occurs, as well as the basic immunobiology of these snails may be important for controlling this disease in the future. Allelic variation in the Guadeloupe resistance complex (GRC) of Biomphalaria glabrata partially determines their susceptibility to parasitic infection, and can influence the microbiome diversity and microbial defenses in the hemolymph of these snails. In the present study, we examine the most abundant proteins present in the hemolymph of snails that are resistant or susceptible to schistosomes, as determined by their GRC genotype. Using proteomic analysis, we found that snails with different GRC genotypes have differentially abundant hemolymph proteins that are not explained by differences in transcription. There are 13 revealed hemolymph proteins that differ significantly between resistant and susceptible genotypes, nearly 40% of which are involved in immune responses. These findings build on the mounting evidence that genes in the GRC region have multiple physiological roles, and likely contribute more extensively to the general immune response than previously believed. These data also raise the intriguing possibility that the GRC region controls resistance to schistosomes, not directly, but indirectly via its effects on the snail's proteome and potentially its microbiome., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

Invertebrate immune response may be primed by a current infection in a sustained manner, leading to the failure of a secondary infection with the same pathogen. The present study focuses on the Schistosomiasis vector snail Biomphalaria glabrata, in which a specific genotype-dependent immunological memory was demonstrated as a shift from a cellular to a humoral immune response. Herein, we investigate the complex molecular bases associated with this genotype-dependant immunological memory response. We demonstrate that Biomphalaria regulates a polymorphic set of immune recognition molecules and immune effector repertoires to respond to different strains of Schistosoma parasites. These results suggest a combinatorial usage of pathogen recognition receptors (PRRs) that distinguish different strains of parasites during the acquisition of immunological memory. Immunizations also show that snails become resistant after exposure to parasite extracts. Hemolymph transfer and a label-free proteomic analysis proved that circulating hemolymph compounds can be produced and released to more efficiently kill the newly encountered parasite of the same genetic lineage., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The immune system of snails is highly sensitive to pollutants, which can suppress its immune response. We investigated the effects of exposure to the glyphosate-based herbicide Roundup® Original on the snail Biomphalaria glabrata infected by the platyhelminth Echinostoma paraensei by evaluating changes in the snail's internal defense system. Four cohorts were studied: control group, infected snails, snails treated with Roundup®, and snails infected and treated with Roundup®. The hemocyte viability was assessed, morphological differentiation of cells was observed and flow cytometry was performed to determine the morphology, viability and the lectin expression profiles. The frequencies of dead hemocytes were lower in the infected group and higher in both pesticide treated groups. Three cell types were identified: blast-like cells, hyalinocytes and granulocytes. The highest number of all types of hemocytes, as well as the highest number of dead cells, were observed in the infected, pesticide-treated group. The association between infection and herbicide exposure greatly increased the frequency of dead hemocytes, suggesting that this condition impairs the internal defense system of B. glabrata making the snails more vulnerable to parasitic infections., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Circulating hemocytes of the snail Biomphalaria glabrata , a major intermediate host for the blood fluke Schistosoma mansoni , represent the primary immune effector cells comprising the host's internal defense system. Within hours of miracidial entry into resistant B. glabrata strains, hemocytes infiltrate around developing sporocysts forming multi-layered cellular capsules that results in larval death, typically within 24-48 h post-infection. Using an in vitro model of hemocyte-sporocyst encapsulation that recapitulates in vivo events, we conducted a comparative proteomic analysis on the responses of hemocytes from inbred B. glabrata strains during the encapsulation of S. mansoni primary sporocysts. This was accomplished by a combination of Laser-capture microdissection (LCM) to isolate sections of hemocyte capsules both in the presence and absence of sporocysts, in conjunction with mass spectrometric analyses to establish protein expression profiles. Comparison of susceptible NMRI snail hemocytes in the presence and absence of sporocysts revealed a dramatic downregulation of proteins in during larval encapsulation, especially those involved in protein/CHO metabolism, immune-related, redox and signaling pathways. One of 4 upregulated proteins was arginase, competitor of nitric oxide synthetase and inhibitor of larval-killing NO production. By contrast, when compared to control capsules, sporocyst-encapsulating hemocytes of resistant BS-90 B. glabrata exhibited a more balanced profile with enhanced expression of shared proteins involved in protein synthesis/processing, immunity, and redox, and unique expression of anti-microbial/anti-parasite proteins. A final comparison of NMRI and BS-90 host hemocyte responses to co-cultured sporocysts demonstrated a decrease or downregulation of 77% of shared proteins by NMRI cells during encapsulation compared to those of the BS-90 strain, including lipopolysaccharide-binding protein, thioredoxin reductase 1 and hemoglobins 1 and 2. Overall, using this in vitro model, results of our proteomic analyses demonstrate striking differences in proteins expressed by susceptible NMRI and resistant BS-90 snail hemocytes to S. mansoni sporocysts during active encapsulation, with NMRI hemocytes exhibiting extensive downregulation of protein expression and a lower level of constitutively expressed immune-relevant proteins (e.g., FREP2) compared to BS-90. Our data suggest that snail strain differences in hemocyte protein expression during the encapsulation process account for observed differences in their cytotoxic capacity to interact with and kill sporocysts.

Several genes have recently been shown to affect the innate resistance of laboratory strains of Biomphalaria glabrata to infection with Schistosoma mansoni, including Hsp90, the expression of which following a brief exposure to elevated temperature can interfere with resistance in juvenile BS-90 snails. Because a prior study failed to see a similar effect in adult snails, juvenile BS-90 snails were exposed to 10 or 50 miracidia following a 4.5 to 6 hr incubation at 33 or 37 C. Snails were then monitored for production of secondary sporocysts or release of cercariae. In addition, snails exposed to 33 C were examined histologically between 1 and 15 days post exposure (DPE) to 30 miracidia to assess the fate of primary sporocysts. Other than elevated numbers of viable primary sporocysts in the tissues of heat shocked snails at 3 DPE, no statistically significant effect of elevated temperature was observed. This discrepancy with regard to prior studies is hypothesized to result from genetic divergence in different laboratory colonies of the same strain of snail.

Freshwater snails are the intermediate hosts for numerous parasitic worms that are detrimental to human and agricultural health. Understanding the immune responses of these snails could be vital for finding ways to block transmission of those parasites. Allelic variation in a recently discovered genomic region in the snail, Biomphalaria glabrata, influences their susceptibility to schistosomes. Here we tested whether genes in that region, termed the Guadeloupe Resistance Complex (GRC), are involved in recognition of common pathogen-associated molecules that have been shown to be stimulants of the hydrogen peroxide defense pathway. We show that hemocytes extracted from individuals with one of the three GRC genotypes released less hydrogen peroxide than the other two genotypes, after stimulation with galactose. This difference was not observed after stimulation with several other microbial-associated carbohydrates, despite those ligands sharing the same putative pathway for hydrogen peroxide release. Therefore, we conclude that allelic variation in the GRC region may influence the recognition of galactose, rather than the conserved downstream steps in the hydrogen peroxide pathway. These results thus are consistent with the hypothesis that proteins produced by this region are involved in pathogen recognition., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The snail's immune response is an important determinant of schistosome infection success, acting in concert with host, parasite, and environmental factors. Coordinated by haemocytes and humoral factors, it possesses immunological hallmarks such as pattern recognition receptors, and predicted gastropod-unique factors like the immunoglobulin superfamily domain-containing fibrinogen-related proteins. Investigations into mechanisms that underpin snail-schistosome compatibility have advanced quickly, contributing functional insight to many observational studies. While the snail's immune response is important to continue studying from the perspective of evolutionary immunology, as the foundational determinants of snail-schistosome compatibility continue to be discovered, the possibility of exploiting the snail for schistosomiasis control moves closer into reach. Here, we review the current understanding of immune mechanisms that influence compatibility between Schistosoma mansoni and Biomphalaria glabrata., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The fresh water snail Biomphalaria glabrata is one of the vectors of the trematode pathogen Schistosoma mansoni, which is one of the agents responsible of human schistosomiasis. In this host-parasite interaction, co-evolutionary dynamic results into an infectivity mosaic known as compatibility polymorphism. Integrative approaches including large scale molecular approaches have been conducted in recent years to improve our understanding of the mechanisms underlying compatibility. This review presents the combination of integrated Multi-Omic approaches leading to the discovery of two repertoires of polymorphic and/or diversified interacting molecules: the parasite antigens S. mansoni polymorphic mucins (SmPoMucs) and the B. glabrata immune receptors fibrinogen-related proteins (FREPs). We argue that their interactions may be major components for defining the compatible/incompatible status of a specific snail/schistosome combination., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 schistosomiasis.

Interactions between early developing Schistosoma mansoni larval stages and the hemolymph of its snail intermediate host represent the first molecular encounter with the snail's immune system. To gain a more comprehensive understanding of this early parasite-host interaction, biotinylated sporocyst tegumental membrane (Mem) proteins and larval transformation proteins (LTP) were affixed to streptavidin-agarose beads and used as affinity matrices to enrich for larval-reactive plasma proteins from susceptible (NMRI) and resistant (BS-90) strains of the snail Biomphalaria glabrata. Nano-LC/MS-MS proteomic analyses of isolated plasma proteins revealed a diverse array of 94 immune-and nonimmune-related plasma proteins. Included among the immune-related subset were pattern recognition receptors (lectins, LPS-binding protein, thioester-containing proteins-TEPs), stress proteins (HSP60 and 70), adhesion proteins (dermatopontins), metalloproteases (A Disintegrin And Metalloproteinase (ADAM), ADAM-related Zn proteinases), cytotoxins (biomphalysin) and a Ca2+-binding protein (neo-calmodulin). Variable immunoglobulin and lectin domain (VIgL) gene family members, including fibrinogen-related proteins (FREPs), galectin-related proteins (GREPs) and C-type lectin-related proteins (CREPs), were the most prevalent of larval-reactive immune lectins present in plasma. FREPs were highly represented, although only a subset of FREP subfamilies (FREP 2, 3 and 12) were identified, suggesting potential selectivity in the repertoire of plasma lectins recognizing larval glycoconjugates. Other larval-binding FREP-like and CREP-like proteins possessing a C-terminal fibrinogen-related domain (FReD) or C-type lectin binding domain, respectively, and an Ig-fold domain also were identified as predicted proteins from the B. glabrata genome, although incomplete sequence data precluded their placement into specific FREP/CREP subfamilies. Similarly, a group of FReD-containing proteins (angiopoeitin-4, ficolin-2) that lacked N-terminal Ig-fold(s) were identified as a distinct group of FREP-like proteins, separate from the VIgL lectin family. Finally, differential appearance of GREPs in BS-90 plasma eluates, and others proteins exclusively found in eluates of the NMRI strain, suggested snail strain differences in the expression of select larval-reactive immune proteins. This hypothesis was supported by the finding that differential gene expression of the GREP in BS-90 and ADAM in NMRI snail strains generally correlated with their patterns of protein expression. In summary, this study is the first to provide a global comparative proteomic analysis of constitutively expressed plasma proteins from susceptible and resistant B. glabrata strains capable of binding early-expressed larval S. mansoni proteins. Identified proteins, especially those exhibiting differential expression, may play a role in determining immune compatibility in this snail host-parasite system. A complete listing of raw peptide data are available via ProteomeXchange using identifier PXD004942.

In this study, both digestive and hermaphrodite glands in Biomphalaria alexandrina snails were examined by light microscope (LM) and scanning electron microscopy (SEM) in a trial to clarify its immunological role as defense mechanism against the parasite infection. B. alexandrina snails used were exposed individually to Schistosoma mansoni miracidia; according to their responsq they were classified into infected snails (shed cercariae) and non-infected snails (failed to shed cercariae). Snails not exposed to S. mansoni miracidia used as control. LM showing great histological alteration in tissues, of the both glands in infected snails, these changes represented by degenerated both oocyte and spermatocyte in hermaphrodite gland in addition to degenerated digestive gland while non-infected snails showing mild degenerated ova with normal spermatocytes also degenerated miracidia. inside granuloma like structure with concentric layers of fibroblast and haemocyte could be observed. By SEM we could detect extensively damaged and fibrosed non-motile cilia with exfoliation of tegumental surface in in- fected B.alexandrina while non-infected ones showing attached numerous cilia with some ballooning of the folds.

Schistosomiasis is one of the most important neglected tropical diseases. Despite effective chemotherapeutic treatments, this disease continues to afflict hundreds of millions of people. Understanding the natural intermediate snail hosts of schistosome parasites is vital to the suppression of this disease. A recently identified genomic region in Caribbean Biomphalaria glabrata snails strongly influences their resistance to infection by Schistosoma mansoni. This region contains novel genes having structural similarity to known pathogen recognition proteins. Here we elaborate on the probable structure and role of one of these genes, grctm6. We characterised the expression of Grctm6 in a population of Caribbean snails, and performed a siRNA knockdown of Grctm6. We show that this protein is not only expressed in B. glabrata hemolymph, but that it also has a role in modulating the number of S. mansoni cercariae released by infected snails, making it a possible target for the biological control of schistosomiasis.

Schistosomiasis is a public health problem, in many developing: countries including Egypt, Determination of the antigenic relationship between S mansoni and its intermediate snail host (IMH) Biomphalaria alexandrina can open a new field for diagnosis and control of the dis- ease. In the present study infected and non-infected B. alexandrina foot and visceral hump tissue as well as S. mansion crude Ag (SWAg) were fractionated using SDS-PAGE. It's specific and cross reacted protein fractions were determine using EITB versus experimentally prepared mice hyper immune sera (HIS) versus each antigen. After treatment of fractionated S.mansoni crude worm antigens (SWAg) versus HIS produced after vaccination of mice by the same Ag, 8 kda protein fractions ranged from 35-140 kda were reacted specifically. Treatment of fractionated B.alexandrina infected and non-infected foot and visceral hump Ag versus previous HIS revealed presence of common polypeptides bands between SWAg and non-infected snail antigens. The fraction at 135 kda, 68 kda, were detected in all cases, while that at 40-42 kda and that at 35 kda was diagnosed in SWAg and that of infected snails only. The fraction at 68 kda was reacted specifically between SWAg and all tested fractionated snail antigens either that of foot or visceral hump when they treated separately by HIS of mice vaccinated by each snail A eparately. The fraction at 135 kda was common between SWAg and snail (infected and non-infected) visceral hump antigen. The fraction at the level of 110 kda was diagnosed inSWAg, in non-infected foot antigen and visceral hump Ag. The fraction at the level of 46-48 (da are common between SWAg and snail foot and visceral hump Ag after treated by HIS of mice vaccinated by foot Ag, Presence of common antigenic fractions between snail tissues and Schistosoma species can prefer an easily source of antigen valuable for diaguosis or vaccination as well as can be considered as new tool for determination to the snail IMH of new discovered trematode Darasites.

Climate change may affect the internal defense system (IDS) of freshwater snails, and as a result their capacity to transmit disease. We examined effects of short-term exposure to supra- and sub-optimal temperatures or starvation on 3 parameters of the IDS of the schistosome-resistant Salvador strain of Biomphalaria glabrata - hemocyte concentrations, cell division in the amebocyte-producing organ (APO), and resistance to infection with Schistosoma mansoni. Adult snails were exposed to 1 of 3 temperatures, 20°C, 27°C (controls), or 33°C, for 1 or 2weeks, with food. A fourth group was maintained at 27°C, but without food. Compared to the controls, starved snails had significantly higher hemocyte counts at both 1 and 2weeks, although mitotic activity in the APO was significantly lower at both time periods. Exposure to 20°C or 33°C for 1 or 2weeks did not affect hemocyte numbers. However, APO mitotic activity in snails exposed to 20°C was significantly higher at both 1 and 2weeks, whereas mitotic activity in snails exposed to 33°C was significantly lower at 1week but normal at 2weeks. None of the treatments altered the resistance phenotype of Salvador snails. In a follow-up experiment, exposure to 33°C for 4-5h, a treatment previously reported to both induce expression of heat shock proteins (Hsps) and abrogate resistance to infection, caused immediate upregulation of Hsp 70 and Hsp 90 expression, but did not alter resistance, and Hsp expression levels returned to baseline after 2weeks at 33°C. Results of this study indicate that abnormal environmental conditions can have both stimulatory and inhibitory effects on the IDS in adult B. glabrata, and that some degree of acclimation to abnormal temperatures may occur., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Blood flukes are the causative agent of schistosomiasis - a major neglected tropical disease that remains endemic in numerous countries of the tropics and sub-tropics. During the past decade, a concerted effort has been made to control the spread of schistosomiasis, using a drug intervention program aimed at curtailing transmission. These efforts notwithstanding, schistosomiasis has re-emerged in southern Europe, raising concerns that global warming could contribute to the spread of this disease to higher latitude countries where transmission presently does not take place. Vaccines against schistosomiasis are not currently available and reducing transmission by drug intervention programs alone does not prevent reinfection in treated populations. These challenges have spurred awareness that new interventions to control schistosomiasis are needed, especially since the World Health Organization hopes to eradicate the disease by 2025. For one of the major species of human schistosomes, Schistosoma mansoni, the causative agent of hepatointestinal schistosomiasis in Africa and the Western Hemisphere, freshwater snails of the genus Biomphalaria serve as the obligate intermediate host of this parasite. To determine mechanisms that underlie parasitism by S. mansoni of Biomphalaria glabrata, which might be manipulated to block the development of intramolluscan larval stages of the parasite, we focused effort on the impact of schistosome infection on the epigenome of the snail. Results to date reveal a complex relationship, manifested by the ability of the schistosome to manipulate the snail genome, including the expression of specific genes. Notably, the parasite subverts the stress response of the host to ensure productive parasitism. Indeed, in isolates of B. glabrata native to central and South America, susceptible to infection with S. mansoni, the heat shock protein 70 (Bg-HSP70) gene of this snail is rapidly relocated in the nucleus and transcribed to express HSP70. Concurrently, hypomethylation of the CpG sites, within the Bg-HSP70 intergenic DNA region, proceeds by conveying epigenetic and spatio-epigenetic mechanisms in temporal concordance. It is notable that this is only the second example reported where a pathogen has been shown to control host cell spatio-epigenetics for its own advantage. Nonetheless, the remarkable mechanisms through which genes become activated i.e. DNA and chromatin remodeling and relocation to a nuclear compartment conducive to gene expression may represent novel intervention targets., (Copyright © 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The anterior pericardial wall of the snail Biomphalaria glabrata has been identified as a site of hemocyte production, hence has been named the amebocyte-producing organ (APO). A number of studies have shown that exogenous abiotic and biotic substances, including pathogen associated molecular patterns (PAMPs), are able to stimulate APO mitotic activity and/or enlarge its size, implying a role for the APO in innate immunity. The molecular mechanisms underlying such responses have not yet been explored, in part due to the difficulty in obtaining sufficient APO tissue for gene expression studies. By using a modified RNA extraction technique and microarray technology, we investigated transcriptomic responses of APOs dissected from snails at 24 h post-injection with two bacterial PAMPs, lipopolysaccharide (LPS) and peptidoglycan (PGN), or with fucoidan (FCN), which may mimic fucosyl-rich glycan PAMPs on sporocysts of Schistosoma mansoni. Based upon the number of genes differentially expressed, LPS exhibited the strongest activity, relative to saline-injected controls. A concurrent activation of genes involved in cell proliferation, immune response and detoxification metabolism was observed. A gene encoding checkpoint 1 kinase, a key regulator of mitosis, was highly expressed after stimulation by LPS. Also, seven different aminoacyl-tRNA synthetases that play an essential role in protein synthesis were found to be highly expressed. In addition to stimulating genes involved in cell proliferation, the injected substances, especially LPS, also induced expression of a number of immune-related genes including arginase, peptidoglycan recognition protein short form, tumor necrosis factor receptor, ficolin, calmodulin, bacterial permeability increasing proteins and E3 ubiquitin-protein ligase. Importantly, significant up-regulation was observed in four GiMAP (GTPase of immunity-associated protein) genes, a result which provides the first evidence suggesting an immune role of GiMAP in protostome animals. Moreover, altered expression of genes encoding cytochrome P450, glutathione-S-transferase, multiple drug resistance protein as well as a large number of genes encoding enzymes associated with degradation and detoxification metabolism was elicited in response to the injected substances., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Despite extensive control efforts, schistosomiasis continues to be a major public health problem in developing nations in the tropics and sub-tropics. The miracidium, along with the cercaria, both of which are water-borne and free-living, are the only two stages in the life-cycle of Schistosoma mansoni which are involved in host invasion. Miracidia penetrate intermediate host snails and develop into sporocysts, which lead to cercariae that can infect humans. Infection of the snail host by the miracidium represents an ideal point at which to interrupt the parasite's life-cycle. This research focuses on an analysis of the miracidium proteome, including those proteins that are secreted. We have identified a repertoire of proteins in the S. mansoni miracidium at 2 hours post-hatch, including proteases, venom allergen-like proteins, receptors and HSP70, which might play roles in snail-parasite interplay. Proteins involved in energy production and conservation were prevalent, as were proteins predicted to be associated with defence. This study also provides a strong foundation for further understanding the roles that neurohormones play in host-seeking by schistosomes, with the potential for development of novel anthelmintics that interfere with its various life-cycle stages.

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.

Biomphalaria glabrata acts as the intermediate host to the parasite, Schistosoma mansoni, and for this reason, the immune system of B. glabrata has been researched extensively. Several studies have demonstrated that the transcriptome profile of B. glabrata changes following exposure to a variety of pathogens, yet very little is known regarding the regulation of gene expression in this species. Nuclear factor kappaB (NF-κB) homologues have recently been identified in B. glabrata but few functional studies have been carried out on this family of transcription factors. The aims of this study therefore were to identify NF-κB binding sites (κB motifs) in B. glabrata and examine them via functional assays. Two different κB motifs were predicted. Furthermore, the Rel homology domain (RHD) of a B. glabrata NF-κB was able to bind these κB motifs in EMSAs, as well as a vertebrate κB motif., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: New strategies to combat the global scourge of schistosomiasis may be revealed by increased understanding of the mechanisms by which the obligate snail host can resist the schistosome parasite. However, few molecular markers linked to resistance have been identified and characterized in snails., Methodology/principal Findings: Here we test six independent genetic loci for their influence on resistance to Schistosoma mansoni strain PR1 in the 13-16-R1 strain of the snail Biomphalaria glabrata. We first identify a genomic region, RADres, showing the highest differentiation between susceptible and resistant inbred lines among 1611 informative restriction-site associated DNA (RAD) markers, and show that it significantly influences resistance in an independent set of 439 outbred snails. The additive effect of each RADres resistance allele is 2-fold, similar to that of the previously identified resistance gene sod1. The data fit a model in which both loci contribute independently and additively to resistance, such that the odds of infection in homozygotes for the resistance alleles at both loci (13% infected) is 16-fold lower than the odds of infection in snails without any resistance alleles (70% infected). Genome-wide linkage disequilibrium is high, with both sod1 and RADres residing on haplotype blocks >2 Mb, and with other markers in each block also showing significant effects on resistance; thus the causal genes within these blocks remain to be demonstrated. Other candidate loci had no effect on resistance, including the Guadeloupe Resistance Complex and three genes (aif, infPhox, and prx1) with immunological roles and expression patterns tied to resistance, which must therefore be trans-regulated., Conclusions/significance: The loci RADres and sod1 both have strong effects on resistance to S. mansoni. Future approaches to control schistosomiasis may benefit from further efforts to characterize and harness this natural genetic variation.

This review summarizes the research progress made over the past decade in the field of gastropod immunity resulting from investigations of the interaction between the snail Biomphalaria glabrata and its trematode parasites. A combination of integrated approaches, including cellular, genetic and comparative molecular and proteomic approaches have revealed novel molecular components involved in mediating Biomphalaria immune responses that provide insights into the nature of host-parasite compatibility and the mechanisms involved in parasite recognition and killing. The current overview emphasizes that the interaction between B. glabrata and its trematode parasites involves a complex molecular crosstalk between numerous antigens, immune receptors, effectors and anti-effector systems that are highly diverse structurally and extremely variable in expression between and within host and parasite populations. Ultimately, integration of these molecular signals will determine the outcome of a specific interaction between a B. glabrata individual and its interacting trematodes. Understanding these complex molecular interactions and identifying key factors that may be targeted to impairment of schistosome development in the snail host is crucial to generating new alternative schistosomiasis control strategies., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Fibrinogen-related proteins or FREPs constitute a large family of molecules, defined by the presence of a fibrinogen-related domain (FReD). These molecules are found in all animals and are diverse in both form and function. Here, we review the current understanding of gastropod FREPs, which are characterized by the presence of a fibrinogen domain connected to one or two immunoglobulin superfamily domains by way of a short interceding region. We present a historical perspective on the discovery of FREPs in gastropods followed by a summary of advances made in the nearly two decades of research focused on the characterization of FREPs in Biomphalaria glabrata (BgFREPs). Topics covered include BgFREP genomic architecture, predicted structure and known functions, structural comparisons between BgFREPs, and evidence of somatic diversification. Also examined are the expression patterns of BgFREPs during snail development and immunological challenges. Recent functional characterization of the role BgFREPs play in the defence response against digenean trematodes is also presented, as well as new data investigating the nucleotide-level genomic conservation of FREPs among Pulmonate gastropods. Finally, we identify areas in need of further research. These include confirming and identifying the specific binding targets of BgFREPs and elucidating how they later engage snail haemocytes to elicit an immunological response, precise mechanisms and importance of BgFREP diversification, characterizing the tissue expression patterns of BgFREPs, as well as addressing whether gastropod FREPs retain immunological importance in alternative snail-trematode associations or more broadly in snail-pathogen interactions., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Histamine appears to be an important transmitter throughout the Animal Kingdom. Gastropods, in particular, have been used in numerous studies establishing potential roles for this biogenic amine in the nervous system and showing its involvement in the generation of diverse behaviours. And yet, the distribution of histamine has only previously been described in a small number of molluscan species. The present study examined the localization of histamine-like immunoreactivity in the central and peripheral nervous systems of pulmonate snails of the genus Biomphalaria. This investigation demonstrates immunoreactive cells throughout the buccal, cerebral, pedal, left parietal and visceral ganglia, indicative of diverse regulatory functions in Biomphalaria. Immunoreactivity was also present in statocyst hair cells, supporting a role for histamine in graviception. In the periphery, dense innervation by immunoreactive fibers was observed in the anterior foot, perioral zone, and other regions of the body wall. This study thus shows that histamine is an abundant transmitter in these snails and its distribution suggest involvement in numerous neural circuits. In addition to providing novel subjects for comparative studies of histaminegic neurons in gastropods, Biomphalaria is also the major intermediate host for the digenetic trematode parasite, which causes human schistosomiasis. The study therefore provides a foundation for understanding potential roles for histamine in interactions between the snail hosts and their trematode parasites.

Gastropod immunology is informed importantly by the study of the frequent encounters snails endure with digeneans (digenetic trematodes). One of the hallmarks of gastropod-digenean associations is their specificity: any particular digenean parasite species is transmitted by a limited subset of snail taxa. We discuss the nature of this specificity, including its immunological basis. We then review studies of the model gastropod Biomphalaria glabrata indicating that the baseline responses of snails to digeneans can be elevated in a specific manner. Studies incorporating molecular and functional approaches are then highlighted, and are further suggestive of the capacity for specific gastropod immune responses. These studies have led to the compatibility polymorphism hypothesis: the interactions between diversified fibrinogen-related proteins (FREPs) and diverse carbohydrate-decorated polymorphic parasite antigens determine recognition and trigger specific immunity. Complex glycan structures are also likely to play a role in the host specificity typifying snail-digenean interactions. We conclude by noting the dynamic and consequential interactions between snails and digeneans can be considered as drivers of diversification of digenean parasites and in the development and maintenance of specific immunity in gastropods., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Technical limitations have hindered comprehensive studies of highly variable immune response molecules that are thought to have evolved due to pathogen-mediated selection such as fibrinogen-related proteins (FREPs) from Biomphalaria glabrata. FREPs combine upstream immunoglobulin superfamily (IgSF) domains with a C-terminal fibrinogen-related domain (FreD) and participate in reactions against trematode parasites. From RNAseq data we assembled a de novo reference transcriptome of B. glabrata to investigate the diversity of FREP transcripts. This study increased over two fold the number of bonafide FREP subfamilies and revealed important sequence diversity within FREP12 subfamily. We also report the discovery of related molecules that feature one or two IgSF domains associated with different C-terminal lectin domains, named C-type lectin-related proteins (CREPs) and Galectin-related protein (GREP). Together, the highly similar FREPs, CREPs and GREP were designated VIgL (Variable Immunoglobulin and Lectin domain containing molecules)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The arms race between vertebrate hosts and parasites has led to diversification systems able to generate huge repertoires of immune recognition receptors and antigenic variants. Until recently, the invertebrate immunity was considered to be poorly specific, and consequently, antigenic variability was not expected to be high for their respective parasites. In the present chapter, we show how the study of the interaction between the snail Biomphalaria glabrata and its parasite Schistosome mansoni has shaken this paradigm. We show that the fate of the interaction between the snail and its parasite is at least partly the result of the concordance of highly variable repertoires of immune recognition receptors in the snail and corresponding antigenic variants in the parasite. We call these antigenic variants of the schistosome Schistosoma mansoni polymorphic mucins (SmPoMucs). We show that their high level of diversification is the result of a complex cascade of mechanisms, thus presenting evidence for antigenic variation in a parasite infecting an invertebrate species.

Anti-parasite responses of the snail Biomphalaria glabrata involve antigen-reactive plasma lectins termed fibrinogen-related proteins (FREPs) comprising a C-terminal fibrinogen (FBG) domain and one or two upstream immunoglobulin domains. FREPs are highly polymorphic; they derive from several gene families with multiple loci and alleles that are diversified by exon loss, alternative splicing, and random somatic mutation (gene conversion and point mutations). Individual B. glabrata snails have dynamically distinct FREP sequence repertoires. The immune relevance of B. glabrata FREPs is indicated by FREP binding to polymorphic antigens of (snail-specific) digenean parasites and altered resistance of B. glabrata to digeneans following RNAi knockdown of FREPs. The compatibility polymorphism hypothesis proposes that FREP mutation increases the range of germline-encoded immune recognition in B. glabrata to counter antigenically-varied parasites. Somatic mutation may result from sequence exchange among tandemly arranged FREP genes in the genome, and analysis of sequence variants also suggests involvement of cytidine deaminase-like activity or epigenetic regulation. Without current indications of selection or retention of effective sequence variants toward immunological memory, FREP diversification is thought to afford B. glabrata immunity that is anticipatory but not adaptive. More remains to be learned about this system; other mollusks elaborate diversified lectins consisting of single FBG domains, and bona fide FREPs were reported from additional gastropod species, but these may not be diversified. Future comparative immunological studies and gene discovery driven by next-generation sequencing will further clarify taxonomic distribution of FREP diversification and the underlying mutator mechanisms as a component of immune function in mollusks.

Biomphalaria species that act as an intermediate host for Schistosoma mansoni have different degrees of susceptibility and different internal defense system responses against parasites. Of these species, Biomphalaria alexandrina represents the only intermediate host in Egypt. Given the limited data on the efficacy of the B. alexandrina internal defense system in comparison to that of other species, we sought to better understand its defense against S. mansoni. We performed in vitro hemocyte adherence assay using whole hemolymph and in vitro reaction using the hemocyte-free hemolymph of susceptible and resistant snails against transformed mother sporocysts. The results demonstrated that the interacting factors between the parasite and the hemolymph of the resistant and susceptible snails do not act in a similar manner. Destruction of the parasite was a restricted function of the hemocytes among resistant snails only. This study demonstrates the key role played by snail hemocytes as a first line of defense against the parasite. The incubation of the hemocyte-free hemolymph of both susceptible and resistant snails with the sporocysts did not lead to any changes in the sporocysts shape or integrity. This immunological variance demonstrated between susceptible and resistant snails could be useful to differentiate between susceptible and resistant snails in future field studies. In addition, the results may help further studies to explain the process of attraction, encapsulation and subsequent killing of S. mansoni in its intermediate host., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Continuing transmission of human intestinal schistosomiasis depends on the parasite's access to susceptible snail intermediate hosts (often Biomphalaria glabrata). Transmission fails when parasite larvae enter resistant individuals in wild snail populations. The genetic basis for differences in snail susceptibility/resistance is being intensively investigated as a means to devise novel control strategies based on resistance genes. Reactive oxygen species produced by the snail's defence cells (haemocytes) are effectors of resistance. We hypothesised that genes relevant to production and consumption of reactive oxygen species would be expressed differentially in the haemocytes of snail hosts with different susceptibility/resistance phenotypes. By restricting the genetic diversity of snails, we sought to facilitate identification of resistance genes. By inbreeding, we procured from a 13-16-R1 snail population with both susceptible and resistant individuals 52 lines of B. glabrata (expected homozygosity ~87.5%), and determined the phenotype of each in regard to susceptibility/resistance to Schistosoma mansoni. The inbred lines were found to have line-specific differences in numbers of spreading haemocytes; these were enumerated in both juvenile and adult snails. Lines with high cell numbers were invariably resistant to S. mansoni, whereas lines with lower cell numbers could be resistant or susceptible. Transcript levels in haemocytes were quantified for 18 potentially defence-related genes. Among snails with low cell numbers, the different susceptibility/resistance phenotypes correlated with differences in transcript levels for two redox-relevant genes: an inferred phagocyte oxidase component and a peroxiredoxin. Allograft inflammatory factor (potentially a regulator of leucocyte activation) was expressed at higher levels in resistant snails regardless of spread cell number. Having abundant spreading haemocytes is inferred to enable a snail to kill parasite sporocysts. In contrast, snails with fewer spreading haemocytes seem to achieve resistance only if specific genes are expressed constitutively at levels that are high for the species., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

During its life cycle, the helminth parasite Schistosoma mansoni uses the freshwater snail Biomphalaria glabrata as an intermediate host to reproduce asexually generating cercariae for infection of the human definitive host. Following invasion of the snail, the parasite develops from a miracidium to a mother sporocyst and releases excretory-secretory products (ESPs) that likely influence the outcome of host infection. To better understand molecular interactions between these ESPs and the host snail defence system, we determined gene expression profiles of haemocytes from S. mansoni-resistant or -susceptible strains of B. glabrata exposed in vitro to S. mansoni ESPs (20 μg/ml) for 1 h, using a 5K B. glabrata cDNA microarray. Ninety-eight genes were found differentially expressed between haemocytes from the two snail strains, 57 resistant specific and 41 susceptible specific, 60 of which had no known homologue in GenBank. Known differentially expressed resistant-snail genes included the nuclear factor kappa B subunit Relish, elongation factor 1α, 40S ribosomal protein S9, and matrilin; known susceptible-snail specific genes included cathepsins D and L, and theromacin. Comparative analysis with other gene expression studies revealed 38 of the 98 identified genes to be uniquely differentially expressed in haemocytes in the presence of ESPs, thus identifying for the first time schistosome ESPs as important molecules that influence global snail host-defence cell gene expression profiles. Such immunomodulation may benefit the schistosome, enabling its survival and successful development in the snail host.

Injection of crude lipopolysaccharide (LPS) from Escherichia coli into the hemocoel of Biomphalaria glabrata stimulates cell proliferation in the amebocyte-producing organ (APO). However, it is not known if mitogenic activity resides in the lipid A or O-polysaccharide component of LPS. Moreover, the possible role of substances that commonly contaminate crude LPS and that are known to stimulate innate immune responses in mammals, e.g., peptidoglycan (PGN), protein, or bacterial DNA, is unclear. Therefore, we tested the effects of the following injected substances on the snail APO: crude LPS, ultrapurified LPS (lacking lipoprotein contamination), two forms of lipid A, (diphosphoryl lipid A and Kdo2-lipid A), O-polysaccharide, Gram negative PGN, both crude and ultrapurified (with and without endotoxin activity, respectively), Gram positive PGN, PGN components Tri-DAP and muramyl dipeptide, and bacterial DNA. Whereas crude LPS, ultrapurified LPS, and crude PGN were mitogenic, ultrapurified PGN was not. Moreover, LPS components, PGN components, and bacterial DNA were inactive. These results suggest that it is the intact LPS molecule which stimulates cell division in the APO., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Biomphalaria glabrata susceptibility to Schistosoma mansoni has a strong genetic component, offering the possibility for investigating host-parasite interactions at the molecular level, perhaps leading to novel control approaches. The identification, mapping and molecular characterisation of genes that influence the outcome of parasitic infection in the intermediate snail host is, therefore, seen as fundamental to the control of schistosomiasis. To better understand the evolutionary processes driving disease resistance/susceptibility phenotypes, we previously identified polymorphic random amplification of polymorphic DNA and genomic simple sequence repeats from B. glabrata. In the present study we identified and characterised polymorphic expressed simple sequence repeats markers (Bg-eSSR) from existing B. glabrata expressed sequence tags. Using these markers, and with previously identified genomic simple sequence repeats, genetic linkage mapping for parasite refractory and susceptibility phenotypes, the first known for B. glabrata, was initiated. Data mining of 54,309 expressed sequence tag, produced 660 expressed simple sequence repeats of which dinucleotide motifs (TA)n were the most common (37.88%), followed by trinucleotide (29.55%), mononucleotide (18.64%) and tetranucleotide (10.15%). Penta- and hexanucleotide motifs represented <3% of the Bg-eSSRs identified. While the majority (71%) of Bg-eSSRs were monomorphic between resistant and susceptible snails, several were, however, useful for the construction of a genetic linkage map based on their inheritance in segregating F2 progeny snails derived from crossing juvenile BS-90 and NMRI snails. Polymorphic Bg-eSSRs assorted into six linkage groups at a logarithm of odds score of 3. Interestingly, the heritability of four markers (Prim1&#95;910, Prim1&#95;771, Prim6&#95;1024 and Prim7&#95;823) with juvenile snail resistance were, by t-test, significant (P<0.05) while an allelic marker, Prim24&#95;524, showed linkage with the juvenile snail susceptibility phenotype. On the basis of our results it is possible that the gene(s) controlling juvenile resistance and susceptibility to S. mansoni infection in B. glabrata are not only on the same linkage group but lie within a short distance (42cM) of each other., (Copyright © 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Aerolysins are virulence factors belonging to the β pore-forming toxin (β-PFT) superfamily that are abundantly distributed in bacteria. More rarely, β-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 β-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 β-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.

Vertebrate females transfer antibodies via the placenta, colostrum and milk or via the egg yolk to protect their immunologically immature offspring against pathogens. This evolutionarily important transfer of immunity is poorly documented in invertebrates and basic questions remain regarding the nature and extent of parental protection of offspring. In this study, we show that a lipopolysaccharide binding protein/bactericidal permeability increasing protein family member from the invertebrate Biomphalaria glabrata (BgLBP/BPI1) is massively loaded into the eggs of this freshwater snail. Native and recombinant proteins displayed conserved LPS-binding, antibacterial and membrane permeabilizing activities. A broad screening of various pathogens revealed a previously unknown biocidal activity of the protein against pathogenic water molds (oomycetes), which is conserved in human BPI. RNAi-dependent silencing of LBP/BPI in the parent snails resulted in a significant reduction of reproductive success and extensive death of eggs through oomycete infections. This work provides the first functional evidence that a LBP/BPI is involved in the parental immune protection of invertebrate offspring and reveals a novel and conserved biocidal activity for LBP/BPI family members.

Populations of Biomphalaria straminea, Biomphalaria peregrina , Biomphalaria tenagophila, Biomphalaria orbignyi, and Biomphalaria oligoza from different Argentine localities were exposed to miracidia of Schistosoma mansoni EC strain, and Biomphalaria tenagophila, in addition to the SJ2 strain. Biomphalaria straminea and B. tenagophila displayed different susceptibility and compatibility (Frandsen's total cercariae production index class 0-II), whereas B. orbigny and B. oligoza were incompatible. Although B. peregrina and B. tenagophila were found naturally infected with the amphistome Zygocotyle lunata, all 5 species could be experimentally infected with Z. lunata. Exposure to Z. lunata infections with S. mansoni were obtained in natural populations of B. straminea and B. tenagophila with the EC strain (13.5-17.1% and 1.2%), respectively, and in B. tenagophila with the SJ2 strain (2.6%), 60 days postexposure [PE]), and in B. orbignyi and B. oligoza (31.1% and 26.7% 60 days PE, respectively, including single infections with S. mansoni and double infections with Z. lunata). The high susceptibility of B. orbignyi and B. oligoza is noteworthy, as these 2 species are considered resistant to S. mansoni .

Plagiorchis elegans has been shown to decrease the fecundity and survivorship of the incompatible snail host Biomphalaria glabrata. Furthermore, a prior infection with P. elegans was shown to render the snails resistant to the compatible parasite Schistosoma mansoni. Here, we test the hypothesis that infection with P. elegans stimulates the immune system of B. glabrata. Our findings indicate that infection by P. elegans significantly increased the number of free hemocytes in the hemolymph of B. glabrata by an average of ~60%. Furthermore, this immuno-stimulated state lasted from the first day post-infection (PI) to some time between 7 and 21 days PI. This is one of the few reported examples of a parasite stimulating the immune response of an incompatible host.

Scores of millions of people around the world are infected by Schistosoma mansoni causing considerable morbidity, mortality and loss of productivity. Safe chemotherapeutic agents have been used though there are challenges of re-infection due to resistance. Both epidemiological and experimental data suggest that acquired cell mediated immunity play significant roles in regulating the intensity of S. mansoni infection as well as its patho-physiologic sequelae. Improved control of this trematode parasite may be obtained with immunization to enhance the resistance of individuals to risk of infection. This study investigated the cellular responses of mice immunized with soluble proteins from foot and digestive gland of the vector snail and challenged with S. mansoni. The proteins were used to immunize the experimental groups then challenged with the S. mansoni. The experimental groups were FT (immunized with foot protein) and DG (immunized with digestive gland). The parameters, which were analyzed to demonstrate protection, included; the worm counts and cellular (IFN-γ, IL-5 cytokines) responses. It was observed that, the experimental groups showed significant protection in terms of worm reduction and immune responses. The group vaccinated with foot protein showed higher protection (87.5%) as compared to the group vaccinated with the digestive gland (50%) in terms of worm reduction. Cytokines (IFN-γ and IL-5) production was present in different levels during the assay time points which showed an aspect of protection. The Foot protein of the vector showed more immunizing power than the digestive gland. Research towards utilizing the two proteins as feasible vaccine candidates is encouraged.