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.

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.

Effects of laboratory culture on compatibility between snails and schistosomes.

The genetic control of compatibility between laboratory strains of schistosomes and their snail hosts has been studied intensively since the 1970s. These studies show (1) a bewildering array of genotype-by-genotype interactions - compatibility between one pair of strains rarely predicts compatibility with other strains, and (2) evidence for a variety of (sometimes conflicting) genetic mechanisms. Why do we observe such variable and conflicting results? One possibility is that it is partly an artifact of the use of laboratory strains that have been in culture for many years and are often inbred. Here we show that results of compatibility trials between snails and schistosomes - all derived from the same natural population - depend very much on whether one uses laboratory-cultured or field-collected individuals. Explanations include environmental effects of the lab on either host or parasite, and genetic changes in either host or parasite during laboratory culture. One intriguing possibility is that genetic bottlenecks during laboratory culture cause the random fixation of alleles at highly polymorphic loci that control the matched/mismatched status of hosts and parasites. We show that a simple model of phenotype matching could produce dose response curves that look very similar to empirical observations. Such a model would explain much of the genotype-by-genotype interaction in compatibility observed among strains.

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.

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.

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.

Are Biomphalaria snails resistant to Schistosoma mansoni?

Among Biomphalaria glabrata/Schistosoma mansoni snail-trematode combinations, it appears that some parasites succeed whilst others fail to infect snails. Snails that become infected are termed susceptible hosts. Those which are not infected are traditionally determined as 'resistant'. Here the concept of B. glabrata resistance to S. mansoni is re-examined in the light of additional observations. It is suggested that, in B. glabrata/S. mansoni, compatibility is tested independently for each individual miracidium and host, and that the success or failure of an infection does not depend on the snail susceptibility/resistance status, but on the 'matched' or 'mismatched' status of the host and parasite phenotypes.

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.

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.

Schistosoma mansoni and Echinostoma caproni excretory-secretory products differentially affect gene expression in Biomphalaria glabrata embryonic cells.

Biomphalaria glabrata embryonic (Bge) cells have been shown to be a valuable in vitro cellular model for the study of snail host-parasite interactions. They both promote the growth and differentiation of various trematode species including Schistosoma mansoni, and Echinostoma caproni and share some morphological and functional features with circulating haemocytes. As an approach to investigate snail genes potentially regulated following exposure to trematode excretory-secretory (ES) products, we compared gene expression profiles of Bge cells exposed to saline solution, or saline solution containing ES products from S. mansoni or E. caproni, two trematode species parasitizing B. glabrata. Following differential display RT-PCR analysis we characterized 23 differentially displayed cDNAs and we focussed on the 5 cDNAs showing sequence similarity to known genes for expression validation. Using RT-PCR, we confirmed that ES products from S. mansoni and E. caproni differentially affect the expression levels of 4 out of the 5 transcripts. These partial transcripts corresponded to novel B. glabrata sequences, and showed significant sequence similarity to genes coding for (i) cytochrome C, (ii) methyl-binding proteins, (iii) glutamine synthetases, and (iv) protease inhibitors from the Kunitz family. The possible significance of these gene expression changes in host-parasite molecular interactions is discussed.

Needle in a haystack: involvement of the copepod PARACARTIA grani in the life-cycle of the oyster pathogen Marteilia refringens.

Marteilia refringens is a major pathogen of the European flat oyster, Ostrea edulis Linnaeus. Since its description, the life-cycle of this protozoan parasite has eluded discovery. Attempts to infect oysters experimentally have been unsuccessful and led to the hypothesis of a complex life-cycle involving several hosts. Knowledge of this life-cycle is of central importance in order to manage oyster disease. However, the exploration of M. refringens life-cycle has been previously limited by the detection tools available and the tremendous number of species to be screened in enzootic areas. In this study, these two restrictions were circumvented by the use of both molecular detection tools and a mesocosm with low biodiversity. Screening of the entire fauna of the pond for M. refringens DNA was systematically undertaken using PCR. Here, we show that the copepod Paracartia (Acartia) grani is a host of M. refringens. Not only was DNA of M. refringens consistently detected in P. grani but also the presence of the parasite in the ovarian tissues was demonstrated using in situ hybridization. Finally, successful experimental transmissions provided evidence that P. grani can be infected from infected flat oysters.

Characterization of cDNA encoding a L37a ribosomal protein from Taenia crassiceps and its potential use in phylogenetic reconstructions.

In this study, we characterized for the first time the complete sequence of a L37a cDNA from a cestode specie: Taenia crassiceps. A phylogenetic analysis of L37a ribosomal proteins from distant animal species is presented and the potential use of such proteins in molecule-based phylogeny is discussed.

Refractoriness of host haemocytes to parasite immunosuppressive factors as a putative resistance mechanism in the Biomphalaria glabrata-Echinostoma caproni system.

In contrast to the growing knowledge accumulated on plant resistance to pathogens, mechanisms of parasite resistance largely remain to be elucidated in animal species. In the present study we investigated mechanisms underlying resistance/susceptibility in the snail-trematode system Biomphalaria glabrata-Echinostoma caproni. In particular, we compared the effect of the parasite excretory-secretory (E-S) products on the defence functions of haemocytes from 2 susceptible and 2 resistant snail strains. In vitro experiments showed that E. caproni E-S products inhibit adhesion and phagocytosis of haemocytes from susceptible snails. A partial biochemical characterization also suggested that the interfering factor(s) is (are) heat-labile glycosylated polypeptides of molecular mass between 10 and 30 kDa. Interestingly, haemocytes from resistant snails remained unaffected by the parasite E-S products, suggesting that a constitutive difference results in their refractoriness to the parasite's immunosuppressive factor(s).

Claire ponds as an experimental model for Marteilia refringens life-cycle studies: new perspectives.

Since its first description, the paramyxean parasite Marteilia refringens (Grizel et al.) has been recognised as a significant pathogen of the European flat oyster Ostrea edulis L. The existence of a complex life-cycle involving several hosts was postulated early on by many authors, although it remains unsolved. Recent developments in the DNA-based diagnosis of M. refringens provides new prospects for the detection of the parasite in potential hosts. However, this screening remains impeded by the number of species living in the vicinity of oyster beds. We report here on the use of semi-closed oyster ponds (so called 'claire' in Marennes-Oléron Bay) as a study model for the life-cycle of M. refringens. Claires are located in an endemic area for M. refringens and transmission of the disease to healthy oysters has been shown to be effective during the course of this study. The environmental characteristics of the claires strongly limit the number of species compared with intertidal areas and oyster beds. Consequently, extensive sampling of a limited number of species cohabiting with oysters was possible. These were preserved for future screening of M. refringens. The experimental model should bring new insights to the life-cycle of M. refringens, as it enables us to propose new conceptual schemes of M. refringens transmission. The role of species as potential hosts is discussed regarding their biology and geographical distribution.

Resistance to xenobiotics and parasites: can we count the cost?

The nature and cost of single genes of major effect is one of the longest running controversies in biology. Resistance, whether to xenobiotics or to parasites, is often paraded as an obvious example of a single gene effect that must carry an associated fitness 'cost'. However, a review of the xenobiotic resistance literature shows that empirical evidence for this hypothesis is, in fact, scarce. We postulate that such fitness costs can only be fully interpreted in the light of the molecular mutations that might underlie them. We also derive a theoretical framework both to encompass our current understanding of xenobiotic resistance and to begin to dissect the probable cost of parasite resistance.

Insect immunity: a genetic factor (hrtp) is essential for antibacterial peptide expression in Drosophila after infection by parasitoid wasps.

We have used a parasitoid wasp Drosophila melanogaster system to investigate the relationship between the humoral and cellular immune responses in insects. Expression of the gene encoding diptericin, an antibacterial peptide in various D. melanogaster strains parasitized by several species of parasitoid wasps, was studied by Northern blot. These strains have the capacity to encapsulate parasitoid eggs. Two strains appeared to produce diptericin mRNA after parasitoid challenge, regardless of their cellular immune reaction to the wasp species. This suggests that a specific genetic factor, or factors, here designated humoral response to parasitoid (hrtp), is present in these two strains of D. melanogaster and is implicated in the expression of the antibacterial gene after parasite infection. This hrtp genetic factor is recessively expressed and located on the second chromosome, suggesting that it is monofactorial. The transgenic strain Dipt.2.2-lacZ:1, in which the transgene is present on the first chromosome, is normally susceptible to the parasitoid wasp. The chromosome bearing the hrtp factor was transferred to this transgenic strain, which then became reactive when triggered by wasp infection. The hrtp factor appears necessary for the activation of diptericin by the parasitoid wasp. No correlation between the cellular immune capacity and the humoral response was observed, suggesting that the two components of insect immunity are regulated independently. Arch.

Flukes without snails: advances in the in vitro cultivation of intramolluscan stages of trematodes.

In vitro cultivation of parasitic helminths, including the digenetic trematodes, has long been a valuable tool in medical and veterinary parasitology, permitting and/or facilitating the development of diagnostic reagents, chemotherapeutic agents, and vaccines and providing insights into naturally complex host-parasite interactions. In vitro cultivation of the intramolluscan stages of trematodes has been particularly challenging, given the ontogenic complexities involved in the production of multiple larval generations from germinal tissues through an asexual "budding" process. Recently, however, advanced larval development has been achieved by incorporating the Biomphalaria glabrata embryonic (Bge) cell line into cocultivation systems. Most notably, the entire intramolluscan cycle (from miracidium to cercaria) has been completed for the human blood fluke Schistosoma mansoni, while significant primary sporocyst development has been attained for several other digeneans including S. japonicum and Fascioloides magna. Here we review recent advances in the cultivation of several larval trematode species and discuss the potential use of this culture system for addressing fundamental questions of host-parasite compatibility.

Chemical communication and mate attraction in echinostomes.

Mate attraction is widespread among animals and appears to facilitate mating and to prevent hybridisation between closely related species. In this study we investigated mate preference between two geographical isolates of Echinostoma caproni (Trematoda, Platyhelminth) and another species of the genus Echinostoma E. sp. Because previous experiments showed a partial reproductive isolation between echinostome isolates, we examined the possibility that such isolation resulted from differential mate attraction. We compared intra-isolate, inter-isolate and interspecific pairings using two in vitro experimental designs. In the first experiment we compared mate attraction of two individuals belonging to or not belonging to the same isolate, while in the second experiment we examined mate choice when individuals were in the presence of individuals from both the same isolate and from a different isolate or a different species. Distances between worms were measured over a period of 90 min. Results from both experiments suggested that mate attraction was similar for intra-isolate, inter-isolate or interspecific combinations. This lack of mate preference in vitro would therefore support an alternative hypothesis of a reproductive isolation through sperm selection.

In vitro culture of rediae of Echinostoma caproni.

Rediae of Echinostoma caproni (Egyptian strain) were dissected from Biomphalaria glabrata snails at intervals from 13-34 days post-exposure and co-cultured for up to 51 days with cells of the B. glabrata embryonic (Bge) cell line. Rediae readily ingested Bge cells and survived longer when co-cultured with cells than in cell-free cultures. Rediae released mostly motile cercariae throughout the observation period when in Bge medium and cells. Rediae cultured in 199 medium with Bge cells also produced progeny throughout most of the observation period. In the latter medium, progeny were much more likely to include rediae as well as cercariae. Some cercariae produced in vitro encysted as metacercariae. Rediae consumed cercariae released into culture but were not observed to attack one another or rediae of a different echinostome species.