Advances in gastropod immunity from the study of the interaction between the snail Biomphalaria glabrata and its parasites: A review of research progress over the last decade.

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.

Transcriptomics of mussel transmissible cancer MtrBTN2 suggests accumulation of multiple cancer traits and oncogenic pathways shared among bilaterians.

Transmissible cancer cell lines are rare biological entities giving rise to diseases at the crossroads of cancer and parasitic diseases. These malignant cells have acquired the amazing capacity to spread from host to host. They have been described only in dogs, Tasmanian devils and marine bivalves. The Mytilus trossulus bivalve transmissible neoplasia 2 (MtrBTN2) lineage has even acquired the capacity to spread inter-specifically between marine mussels of the Mytilus edulis complex worldwide. To identify the oncogenic processes underpinning the biology of these atypical cancers we performed transcriptomics of MtrBTN2 cells. Differential expression, enrichment, protein-protein interaction network, and targeted analyses were used. Overall, our results suggest the accumulation of multiple cancerous traits that may be linked to the long-term evolution of MtrBTN2. We also highlight that vertebrate and lophotrochozoan cancers could share a large panel of common drivers, which supports the hypothesis of an ancient origin of oncogenic processes in bilaterians.

Vertebrate host protective immunity drives genetic diversity and antigenic polymorphism in Schistosoma mansoni.

Schistosomes are gonochoric blood parasites with a complex life cycle responsible for a disease of considerable medical and veterinary importance in tropical and subtropical regions. Understanding the evolution of schistosome genetic diversity is clearly of fundamental importance to interpreting schistosomiasis epidemiology and disease transmission patterns of this parasite. In this article, we investigated the putative role of the host immune system in the selection of male genetic diversity. We demonstrated the link between genetic dissimilarity and the protective effect among male worms. We then compared the proteomes of three male clones with different genotypes and differing by their capacity to protect against reinfection. The identified differences correspond mainly to antigens known or supposed to be involved in the induction of protective immunity. These results underline the role played by host immune system in the selection of schistosome genetic diversity that is linked to antigenic diversity. We discuss the evolutionary consequences in the context of schistosome infection.

Oyster hemolymph is a complex and dynamic ecosystem hosting bacteria, protists and viruses.

BACKGROUND: The impact of the microbiota on host fitness has so far mainly been demonstrated for the bacterial microbiome. We know much less about host-associated protist and viral communities, largely due to technical issues. However, all microorganisms within a microbiome potentially interact with each other as well as with the host and the environment, therefore likely affecting the host health. RESULTS: We set out to explore how environmental and host factors shape the composition and diversity of bacterial, protist and viral microbial communities in the Pacific oyster hemolymph, both in health and disease. To do so, five oyster families differing in susceptibility to the Pacific oyster mortality syndrome were reared in hatchery and transplanted into a natural environment either before or during a disease outbreak. Using metabarcoding and shotgun metagenomics, we demonstrate that hemolymph can be considered as an ecological niche hosting bacterial, protist and viral communities, each of them shaped by different factors and distinct from the corresponding communities in the surrounding seawater. Overall, we found that hemolymph microbiota is more strongly shaped by the environment than by host genetic background. Co-occurrence network analyses suggest a disruption of the microbial network after transplantation into natural environment during both non-infectious and infectious periods. Whereas we could not identify a common microbial community signature for healthy animals, OsHV-1 µVar virus dominated the hemolymph virome during the disease outbreak, without significant modifications of other microbiota components. CONCLUSION: Our study shows that oyster hemolymph is a complex ecosystem containing diverse bacteria, protists and viruses, whose composition and dynamics are primarily determined by the environment. However, all of these are also shaped by oyster genetic backgrounds, indicating they indeed interact with the oyster host and are therefore not only of transient character. Although it seems that the three microbiome components respond independently to environmental conditions, better characterization of hemolymph-associated viruses could change this picture.

The compatibiuty polymorphism in invertebrate host/trematodes interactions: research of molecular determinants.

The co-evolutionary dynamics that exist in many host-parasite interactions sometimes leads to compatibility polymorphism. This phenomenon is well documented in mollusc/trematodes interactions but its molecular base is unknown. In order to identify key molecules involved in this phenomenon, we developed several molecular approaches comparing compatible or incompatible strains of mollusc or parasite. These comparisons led to the identification of numerous candidate genes listed and discussed (some of them) in the present review.

The Compatibility Between Biomphalaria glabrata Snails and Schistosoma mansoni: An Increasingly Complex Puzzle.

This review reexamines the results obtained in recent decades regarding the compatibility polymorphism between the snail, Biomphalaria glabrata, and the pathogen, Schistosoma mansoni, which is one of the agents responsible for human schistosomiasis. Some results point to the snail's resistance as explaining the incompatibility, while others support a "matching hypothesis" between the snail's immune receptors and the schistosome's antigens. We propose here that the two hypotheses are not exclusive, and that the compatible/incompatible status of a particular host/parasite couple probably reflects the balance of multiple molecular determinants that support one hypothesis or the other. Because these genes are involved in a coevolutionary arms race, we also propose that the underlying mechanisms can vary. Finally, some recent results show that environmental factors could influence compatibility. Together, these results make the compatibility between B. glabrata and S. mansoni an increasingly complex puzzle. We need to develop more integrative approaches in order to find targets that could potentially be manipulated to control the transmission of schistosomiasis.

Evidence for a genetic sex determination in Cnidaria, the Mediterranean red coral (Corallium rubrum).

Sexual reproduction is widespread among eukaryotes, and the sex-determining processes vary greatly among species. While genetic sex determination (GSD) has been intensively described in bilaterian species, no example has yet been recorded among non-bilaterians. However, the quasi-ubiquitous repartition of GSD among multicellular species suggests that similar evolutionary forces can promote this system, and that these forces could occur also in non-bilaterians. Studying sex determination across the range of Metazoan diversity is indeed important to understand better the evolution of this mechanism and its lability. We tested the existence of sex-linked genes in the gonochoric red coral (Corallium rubrum, Cnidaria) using restriction site-associated DNA sequencing. We analysed 27 461 single nucleotide polymorphisms (SNPs) in 354 individuals from 12 populations including 53 that were morphologically sexed. We found a strong association between the allele frequencies of 472 SNPs and the sex of individuals, suggesting an XX/XY sex-determination system. This result was confirmed by the identification of 435 male-specific loci. An independent test confirmed that the amplification of these loci enabled us to identify males with absolute certainty. This is the first demonstration of a GSD system among non-bilaterian species and a new example of its convergence in multicellular eukaryotes.

Compatibility in the Biomphalaria glabrata/Echinostoma caproni model: potential involvement of adhesion genes.

Because susceptibility or resistance of Biomphalaria glabrata to the trematode Echinostoma caproni correlates with differential hemocytic adhesive properties, we compared the expression of genes involved in adhesion processes between hemocytes from susceptible and resistant snails. Quantitative reverse transcriptase-PCR analysis revealed four genes whose transcripts were differentially represented between hemocytes from resistant and susceptible snails. These genes encode two dermatopontin-like, one matrilin-like and one cadherin-like proteins. Expression analyses performed following parasite exposure suggested that dermatopontins may be involved in the compatibility differences between these strains. We also investigated expression levels on whole snails of different genes potentially involved in extracellular matrix structure or coagulation. Our results support the hypothesis that susceptible snails possess a hemolymph coagulation-like system that is more potent than that of resistant snails. This system may prevent hemocyte migration towards the parasite larvae and therefore facilitate parasite settlement in susceptible snails.

Compatibility in the Biomphalaria glabrata/Echinostoma caproni model: Potential involvement of proteins from hemocytes revealed by a proteomic approach.

As an approach to investigate the suspected involvement of cellular factors in Biomphalaria glabrata resistance/susceptibility to Echinostoma caproni, we compared protein patterns from hemocytes collected from susceptible and resistant snails. This proteomic approach revealed that twelve hemocytic proteins exhibited significant differences in their apparent abundance. The genes corresponding to five of them were characterized by a combination of mass spectrometry and molecular cloning. They encode an aldolase, an intermediate filament protein, a cytidine deaminase, the ribosomal protein P1 and the histone H4. Furthermore, we investigated their expression in parasite-exposed or -unexposed snails. These last experiments revealed changes in transcript levels corresponding to intermediate filament and histone H4 proteins post-infection.

Transcriptional changes in Crassostrea gigas oyster spat following a parental exposure to the herbicide diuron.

The Pacific oyster Crassostrea gigas is the main oyster species produced in the world, and a key coastal economic resource in France. High mortalities affect Pacific oysters since 2008 in France and Europe. Their origins have been attributed to a combination of biotic and abiotic factors, underlining the importance of environment quality. The impact of water pollution has been pointed out and one of the pollutants, the genotoxic herbicide diuron, occurs at high concentrations all along the French coasts. Previous work has revealed that a parental exposure to diuron had a strong impact on hatching rates and offspring development even if spats were not exposed to diuron themselves. In this study, we explored for the first time the transcriptional changes occurring in oyster spats (non exposed) originating from genitors exposed to an environmentally relevant concentration of diuron during gametogenesis using the RNAseq methodology. We identified a transcriptomic remodeling revealing an effect of the herbicide. Different molecular pathways involved in energy production, translation and cell proliferation are particularly disturbed. This analysis revealed modulated candidate genes putatively involved in response to oxidative stress and mitochondrial damage in offspring of genitors exposed to diuron. Complementary measures of the activity of enzymes involved in these latter processes corroborate the results obtained at the transcriptomic level. In addition, our results suggested an increase in energy production and mitotic activity in 5-month-spats from diuron-exposed genitors. These results could correspond to a "catch-up growth" phenomenon allowing the spats from diuron-exposed genitors, which displayed a growth delay at 3 months, to gain a normal size when they reach the age of 6 months. These results indicate that exposure to a concentration of diuron that is frequently encountered in the field during the oyster's gametogenesis stage can impact the next generation and may result in fitness disturbance.

Gene discovery and expression analysis of immune-relevant genes from Biomphalaria glabrata hemocytes.

The immune effector cells (hemocytes) of the snail host Biomphalaria glabrata are known to play a key role in recognition and elimination of larval helminths such as the human blood fluke Schistosoma mansoni. To identify novel immune-relevant genes, we undertook an expressed sequence tag program. A hemocyte cDNA library was constructed using snails that were not exposed to a particular pathogen or parasite but maintained in non-axenic conditions. Putative function could be assigned to 53% of the 1613 high quality cDNAs analysed. Based on sequence similarities, we identified 31 immune-relevant genes corresponding either to cellular defence effectors, proteases and protease inhibitors, pattern recognition receptors, cell adhesion molecules or immune regulators. In order to further investigate the potential involvement of these genes in snail-trematode immunobiological interactions, we analysed their expression in unchallenged and parasite-challenged snails, using the immunosuppressive trematode Echinostoma caproni and snail strains selected for resistance or susceptibility to this parasite. Real-time PCR analysis of expression ratios at 7 time-points post-exposure revealed both (i) genes displaying constitutive expression differences between the two strains; and (ii) genes differentially modulated after parasite exposure.

Characterisation of proteins differentially present in the plasma of Biomphalaria glabrata susceptible or resistant to Echinostoma caproni.

Snail immune responses towards a trematode infection are known to rely on both plasmatic and cellular host factors. As an approach to further investigate the suspected involvement of plasmatic factors in Biomphalaria glabrata resistance/susceptibility to Echinostoma caproni, we compared protein patterns of plasma collected from susceptible and resistant snails. This proteomic approach revealed that 13 plasmatic proteins exhibited significant differences in their apparent representativity. The genes corresponding to five of them were characterised by a combination of mass spectrometry and molecular cloning. They encode two isoforms of a glycolytic enzyme, two isoforms of a calcium binding protein and an inhibitor of cysteine protease. Furthermore, we investigated gene expression in parasite-exposed or -unexposed snails as well as in various tissues by quantitative PCR. This study showed that: (i) differential representation of plasma proteins between the snail strains was correlated with a differential level of transcripts; (ii) expression of these genes after parasite exposure was differentially regulated in the two strains; and (iii) these genes were expressed predominantly in the albumen gland.

Original involvement of antimicrobial peptides in mussel innate immunity.

Recently, the existence and extended diversity of antimicrobial peptides has been revealed in two mussel species. These molecules are classified into four groups according to common features of their primary structure: defensins, mytilins, myticins and mytimycin. In Mytilus galloprovincialis, gene structure reveals synthesis as precursors in circulating hemocytes. Synthesised even in absence of challenge, the precursors mature and the peptides are stored in granules as active forms. The different peptides are engaged in the destruction of bacteria inside phagocytes, before being released into hemolymph to participate in systemic responses. Such involvement in anti-infectious responses is unique, and apparently more related to those of mammalian phagocytes than to those of insects.

Selective mRNA degradation by antisense oligonucleotide-2,5A chimeras: involvement of RNase H and RNase L.

Antisense oligonucleotides (ON) allow the specific control of gene expression and phosphorothioate derivatives are currently being evaluated for possible clinical applications. Numerous second generation ON analogues with improved pharmacological properties have been described. Most of them, however, do not recruit RNase H, which is known to increase ON potency by eliciting the specific degradation of the target RNA. Silverman, Torrence and colleagues have conjugated 2,5A to natural antisense ON and demonstrated the preferential cleavage of a target RNA in cell-free and intact cell experiments. We have established for the first time that RNase H-incompetent ON, viz. alpha-anomeric ON analogues, can be converted into sequence-specific nucleases upon conjugation to 2,5A. The use of alpha-ON- and beta-ON-2,5A chimeras has allowed us to delineate the part played by RNase H and RNase L in target RNA degradation and translation arrest. Finally, the present studies have revealed limitations which are encountered in the choice of a suitable target for such ON-2,5A chimeras.

Mytilin B and MGD2, two antimicrobial peptides of marine mussels: gene structure and expression analysis.

Previous research has shown that mytilins and MGDs are two types of 4-kDa, cysteine-rich, cationic antimicrobial peptides, which are abundant in hemocytes of the mussels, Mytilus galloprovincialis and M. edulis. The expression of the genes encoding these peptides has been analyzed in the hemocytes of animals subjected to various stress factors, as well as during larval development. Variations in gene expression in adult mussels have been tested under conditions of physical stress, bacterial challenge and heat shock. The results suggest that in adult mussels, the MGD2 gene may be over-expressed with physical and temperature stress, but that reduced expression occurs with bacterial challenge. Gene expression during development has been analyzed using different larval and post-larval stages, ranging from 4-day-old veliger larvae to 32-day-old post-larvae. The results show that the expression of both mytilin B and MGD2 is developmentally regulated, but neither gene is expressed in mussels until after larval settlement and metamorphosis. Finally, the genes encoding two isoforms of these peptides have been cloned and sequenced, revealing that both genes contain four exons and three introns.

Use of individual polymorphism to validate potential functional markers: case of a candidate lectin (BgSel) differentially expressed in susceptible and resistant strains of Biomphalaria glabrata.

BgSel has been identified in Biomphalaria glabrata as a candidate adhesion molecule exhibiting both an Ig-like domain and a carbohydrate recognition domain showing similarities with the l domain of C-type lectins. As susceptibility or resistance of B. glabrata to the trematode Echinostoma caproni correlates with a differential hemocytic adhesive behavior, we investigated the expression of BgSel in snails selected for their susceptibility or resistance. Semi-quantitative RT-PCR analysis of BgSel expression revealed that (i) BgSel expression level was high in susceptible snails and almost undetectable in resistant snails, and that (ii) exposure to the parasite did not affect the expression level of BgSel in either strain. In order to validate this apparent association between low levels of BgSel expression and resistance, we used Real-Time PCR to characterize the relative expression of BgSel in individual snails segregating for susceptibility/resistance. Results established that differential expression of BgSel represents a functional strain marker, but is not a marker of resistance/susceptibility. It is suggested that this correlative approach may be a rapid and efficient alternative to complete functional analyses, and may facilitate the validation of candidate transcripts potentially identified through the numerous differential analyses of animal transcriptomes.

Multi-parasite host susceptibility and multi-host parasite infectivity: a new approach of the Biomphalaria glabrata/Schistosoma mansoni compatibility polymorphism.

In this study, we analyze the degree of susceptibility/un-susceptibility of five strains of Biomphalaria glabrata from different geographical origins successively challenged with a panel of 4 Schistosoma mansoni strains. A total of 20 homopatric and heteropatric host-parasite combinations were tested with exposure doses of 1, 10, 20, 30 and 50 miracidia per individual host. By doing this, we characterized each B. glabrata strain by its "multi-parasite susceptibility phenotype" that reflects better the efficiency of their defense mechanism against not only one, but a diversity of schistosome stocks. In the same time, all the S. mansoni strains used were characterized, by their "multi-host infectivity phenotype" that reflects the level of infectivity they display when confronted to diverse snail populations. Based on these results it is possible to select different homogenous stocks of snails with different spectrum of susceptibility/un-susceptibility for several parasite strains. This will be a useful tool for future functional studies conducted to understand the genetics and molecular basis of the compatibility polymorphism in this host/parasite model.

Compatibility polymorphism in snail/schistosome interactions: From field to theory to molecular mechanisms.

Coevolutionary dynamics in host-parasite interactions potentially lead to an arms race that results in compatibility polymorphism. The mechanisms underlying compatibility have remained largely unknown in the interactions between the snail Biomphalaria glabrata and Schistosoma mansoni, one of the agents of human schistosomiasis. This review presents a combination of data obtained from field and laboratory studies arguing in favor of a matching phenotype model to explain compatibility polymorphism. Investigations focused on the molecular determinants of compatibility have revealed two repertoires of polymorphic and/or diversified molecules that have been shown to interact: the parasite antigens S. mansoni polymorphic mucins and the B. glabrata fibrinogen-related proteins immune receptors. We hypothesize their interactions define the compatible/incompatible status of a specific snail/schistosome combination. This line of thought suggests concrete approaches amenable to testing in field-oriented studies attempting to control schistosomiasis by disrupting schistosome-snail compatibility.

Compatibility in the Biomphalaria glabrata/Echinostoma caproni model: new candidate genes evidenced by a suppressive subtractive hybridization approach.

In order to elucidate mechanisms underlying snail/echinostome compatibility, numerous molecular studies comparing transcripts and proteins of Biomphalaria glabrata susceptible or resistant to Echinostoma caproni were undertaken. These studies focused on plasma and haemocytes of the two strains and revealed that some transcripts and/or proteins were differentially expressed between strains. The aim of the present study was to develop a complementary transcriptomic approach by constructing subtractive libraries. This work revealed some candidate transcripts already identified in previous studies (calcium-binding proteins and glycolytic enzymes) as well as novel candidate transcripts that were differentially represented between strains of B. glabrata. Among these newly identified genes, we revealed several genes potentially involved in immune processes encoding proteases, protease inhibitors, a lectin, an aplysianin-like molecule, and cell adhesion molecules.

The strong induction of metallothionein gene following cadmium exposure transiently affects the expression of many genes in Eisenia fetida: a trade-off mechanism?

Metal pollution causes disturbances at various levels of biological organization in most species. Important physiological functions could be affected in the exposed individuals and among the main physiological functions, immunity may provide one (or more) effector(s) whose expression can be directly affected by a metal exposure in various macroinvertebrates. Protein expressions were studied in order to test them as molecular biomarkers of metal exposure in Eisenia fetida. Selected effectors were calmodulin, heat shock proteins, superoxide dismutase, catalase, metallothionein, beta-adrenergic receptor kinase, pyruvate carboxylase, transcriptionally controlled tumor protein, protein kinase C, ubiquitin and cyclophilin-A. The level of expression of each gene was analysed in whole organism following exposures to cadmium in soil using real-time PCR. Metallothionein, transcriptionally controlled tumor protein and cyclophilin-A expression were also measured following copper exposures in soil because these genes seemed to be sensitive to copper. This work enabled to distinguish metallothionein and cyclophilin-A among the 15 selected effectors. A strong decrease of the number of transcripts was also detected for most effectors soon after the exposure to cadmium suggesting that a trade-off mechanism occurs.