Genetics of Biomphalaria glabrata and its effect on the outcome of Schistosoma mansoni infection

The genetics of the snail Biomphalaria glabrata is better characterized than that of any other intermediate host of schistosomes of humans. Using techniques of selective breeding, several snail stocks have been developed that consistently display resistant or susceptible phenotypes. Investigators using these stocks have learned that several snail and parasite genes influence the course of parasite development. Here, Charles Richards, Matty Knight and Fred Lewis discuss the importance of the snail's genetics in categorizing resistance in this complex invertebrate, some recent molecular evidence that may help us understand several of the problems that still remain, and some challenges lying ahead for investigators in this field.     

Secretory protein biosynthesis in snail hemocytes: in vitro modulation by larval schistosome excretory-secretory products

Circulating hemocytes of the snail, Biomphalaria glabrata, synthesize and secrete a variety of polypeptides when maintained in vitro in serum-free medium containing [35S] methionine. SDS-PAGE/fluorographic analysis of supernatants from resistant snail (10-R2-OK strain) hemocyte cultures revealed the presence of numerous labeled polypeptides ranging in Mr from 220 to 14 kDa. Most of these same proteins were also produced by hemocytes of a susceptible B. glabrata strain (M-line), but the overall rate of secretory protein synthesis was reduced from that of resistant snail cells. In addition, excretory-secretory (ES) products contained in supernatants from Schistosoma mansoni miracidial transformation and 1-day primary sporocyst cultures stimulated increases in the synthesis of various polypeptides. Particularly striking was a 3-fold increase in the synthesis of a 66-kDa secretory polypeptide by hemocytes of both snail strains, and a concomitant increase in M-line hemocytes and decrease in 10-R2-OK cells of a 63-kDa polypeptide. Overall, however, the level of ES product-induced secretory protein synthesis was greater in 10-R2-OK snail hemocytes than in those of the M-line strain. Exposure of a nonhemocytic B. glabrata cell line to parasite culture supernatants had no stimulatory/inhibitory effect on labeled protein ouput, suggesting that the observed hemocyte response may be snail cell-type specific. Finally, the larval ES components responsible for modulating hemocyte protein metabolism are mainly concentrated in a heat-stable fraction composed of molecules of greater than 30 kDa. However, the loss of the ability of heated parasite products to stimulate synthesis of certain hemocyte proteins and the presence of minor stimulating activity in a low molecular weight fraction (less than 10 kDa) implies the possible existence of multiple larval components affecting formation of specific hemocyte secretory polypeptides. It is concluded that snail hemocytes are capable of in vitro synthesis and secretion of a variety of methionine-containing polypeptides, and that ES products of early larval schistosomes can modulate (i.e., stimulate or inhibit) this metabolic process. A differential response of susceptible vs. resistant hemocytes to larval products suggests that the degree to which these cells can be metabolically activated may determine their cytotoxic effectiveness.     

Experimental exposure of Helisoma trivolvis and Biomphalaria glabrata (Gastropoda) to Ribeiroia ondatrae (Trematoda)

Experimental infections provide an important foundation for understanding host responses to parasites. While infections with Ribeiroia ondatrae cause mortality and malformations in a wide range of amphibian second intermediate host species, little is known about how the parasite affects its snail first intermediate hosts or even what species can support infection. In this study, we experimentally exposed Helisoma trivolvis, a commonly reported host of R. ondatrae, and Biomphalaria glabrata, a confamilial snail known to host Ribeiroia marini, to increasing concentrations of embryonated eggs of R. ondatrae obtained from surrogate definitive hosts. Over the course of 8 wk, we examined the effect of parasite exposure on infection status, time-to-cercariae release, host size, and mortality of both snail species. Helisoma trivolvis was a highly competent host for R. ondatrae infection, with over 93% infection in all exposed snails, regardless of egg exposure level. However, no infections were detected among exposed B. glabrata, despite previous accounts of this snail hosting a congener parasite. Among exposed H. trivolvis, high parasite exposure reduced growth, decreased time-to-cercariae release, and caused marginally significant increases in mortality. Interestingly, while B. glabrata snails did not become infected with R. ondatrae, individuals exposed to 650 R. ondatrae eggs grew less rapidly than unexposed snails, suggesting a sub-lethal energetic cost associated with parasite exposure. Our results highlight the importance of using experimental infections to understand the effects of parasite exposure on host- and non-host species, each of which can be affected by exposure.     

Characterization of an insulin receptor-related receptor in Biomphalaria glabrata embryonic cells

Tyrosine kinase receptors play a key role in the communication of cells with their environment. Growth hormone receptors, such as insulin receptors, are involved in the regulation of cell growth, differentiation and metabolism in multicellular organisms. Insulin-related peptides and members of the insulin receptor subfamily have been described in a wide variety of invertebrates, including freshwater molluscs. In this paper, we describe the metabolic effect of insulin on a mollusc cell line (Bge) derived from embryos of the snail Biomphalaria glabrata. Using a PCR strategy, we have cloned from Bge cells a cDNA encoding a protein (BgIR) homologous to, and exhibiting all of the typical features of insulin receptors. Northern blot analysis confirmed the expression of BgIR in B. glabrata snails and suggested its wide distribution in the snail body. Bge cells have been shown to provide the environmental conditions necessary for the in vitro development of the sporocysts of Schistosoma mansoni, a trematode parasite that uses B. glabrata as an intermediate host. The possible implication of BgIR in the activating and proliferating processes observed in Bge cells during their coculture with S. mansoni larvae is discussed.     

Schistosome/mollusk: genetic compatibility

Schistosomiasis remains one of the most prevalent parasitic infections and has significant economic and public health consequences in many developing countries. Economic development and improvement in standard of living in these countries are dependent on the elimination of this odious disease. For the control of Schistosomiasis, understanding the host/parasite association is important, since the host parasite relationship is often complex and since questions remain concerning the susceptibility of snails to infection by respective trematodes and their specificity and suitability as hosts for continued parasite development. Thus, the long term aim of this research is to learn more about the genetic basis of the snail/parasite relationship with the hope of finding novel ways to disrupt the transmission of this disease. In the current research, genetic variability among susceptible and resistant strains within and between Biomphalaria glabrata and B. tenagophila was investigated using RAPD-PCR. The results indicate great genetic variations within the two snail species using three different primers (intrapopulational variations), while specimens from the same snail species showed few individual differences between the susceptible and resistant strains (interpopulational variation).     

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.     

The mitochondrial genome of Biomphalaria glabrata (Gastropoda: Basommatophora), intermediate host of Schistosoma mansoni

The complete mitochondrial (Mt) genome of the gastropod Biomphalaria glabrata, a major intermediate host for the human parasite Schistosoma mansoni, was sequenced. The circular genome, the first determined from a basommatophoran snail, is AT rich (74.6%) and the smallest Mt genome (13,670 nucleotides [nt]) characterized from mollusks to date. Sequences from 2 B. glabrata strains, M-line and 1742, differed by only 18 nt. Phylogenetic analysis of 16S and ND1 sequences confirmed the Brazilian ancestry of both B. glabrata strains. Gene predictions indicated 22 transfer RNA, 12S and 16S ribosomal RNA (rRNA), and 13 protein-encoding genes, as is typical for metazoans. Of the mollusk Mt genomes currently known, the gene order was most similar to that of stylommatophoran gastropods, concordant with the monophyly of pulmonate gastropods. Screening of GenBank (expressed sequence tags database [dbEST]) with the Mt sequence identified 108 entries from B. glabrata as Mt-derived sequences, including 12S and 16S rRNA sequences. Moreover, 11 sequences originating from the Mt genome of B. glabrata were identified among EST entries ascribed to intramolluskan stages of S. mansoni. The availability of this Mt sequence will facilitate further molecular investigations into the biology of Biomphalaria sp. and interactions between this intermediate host and intramolluskan stages of S. mansoni.     

Differential lectin labelling of circulating hemocytes from Biomphalaria glabrata and Biomphalaria tenagophila resistant or susceptible to Schistosoma mansoni infection

Lectins/carbohydrate binding can be involved in the Schistosoma mansoni recognition and activation of the Biomphalaria hemocytes. Therefore, expression of lectin ligands on Biomphalaria hemocytes would be associated with snail resistance against S. mansoni infection. To test this hypothesis, circulating hemocytes were isolated from B. glabrata BH (snail strain highy susceptible to S. mansoni), B. tenagophila Cabo Frio (moderate susceptibility), and B. tenagophila Taim (completely resistant strains), labelled with FITC conjugated lectins (ConA, PNA, SBA, and WGA) and analyzed under fluorescence microscopy. The results demonstrated that although lectin-labelled hemocytes were detected in hemolymph of all snail species tested, circulating hemocytes from both strains of B. tenagophila showed a larger number of lectin-labelled cells than B. glabrata. Moreover, most of circulating hemocytes of B. tenagophila were intensively labelled by lectins PNA-FITC and WGA-FITC, while in B. glabrata small hemocytes were labeled mainly by ConA. Upon S. mansoni infection, lectin-labelled hemocytes almost disappeared from the hemolymph of Taim and accumulated in B. glabrata BH. The role of lectins/carbohydrate binding in resistance of B. tengophila infection to S. mansoni is still not fully understood, but the data suggest that there may be a correlation to its presence with susceptibility or resistance to the parasite.     

Distribution of schistosome infections in molluscan hosts at different levels of parasite prevalence

Biomphalaria glabrata snails infected with Schistosoma mansoni were collected during consecutive seasons from a site in Brazil known to have a very high percentage of infected snails. Schistosoma mansoni cercariae from single snails were used to infect individual mice, and the recovered adult worms were genetically assessed using a mtVNTR marker. The number of unique parasite genotypes found per snail was compared to expected abundance values, based on the infection prevalence at the site, to determine the distribution of S. mansoni infections within the snail population. The observed distributions and those from previous studies were used to examine the relationship between schistosome prevalence and aggregation across a wide range of prevalence values. Our analysis showed that prevalence was inversely related to the degree of parasite overdispersion, and at high prevalence, S. mansoni infections were randomly distributed among snails.     

Ki-67 is expressed in multiplying forms of Schistosoma mansoni, but not in snail host tissues

Ki-67 is a protein expressed in the nucleus of several species during cell-division, being absent during the GO resting phase of the cellular cycle. During attempts to disclose mitosis in the so-called " amebocyte-producing organ " in Biomphalaria glabrata infected with Schistosoma mansoni, the parasite multiplying forms appeared strongly marked for Ki-67, while the snail tissues were completely negative. These data are worth registering to complement general data on Ki-67, and to help future studies on the relationship of the parasite and of its intermediate host.     

Differential binding of hemolymph proteins from schistosome-resistant and -susceptible Biomphalaria glabrata to Schistosoma mansoni sporocysts

Humoral factors have been associated with resistance of Biomphalaria glabrata to infection by Schistosoma mansoni. The goal of this study was to determine which serum (cell-free hemolymph) proteins bind to the surface of S. mansoni sporocysts. For this, 125I-labeled serum from schistosome-resistant (10-R2) and -susceptible (M-line) B. glabrata was incubated with sporocysts, washed, and then subjected to SDS-PAGE and autoradiography. Other samples examined included radiolabeled 10-R2 and M-line serum, sporocysts incubated with unlabeled serum followed by incubation with radiolabeled serum, and radiolabeled sporocysts. Results indicated that many polypeptides in the serum from both strains of B. glabrata were radiolabeled. Dominating both profiles were bands in the 90-210-kDa range. However, some differences between the serum of the 2 snail strains were observed with M-line serum having several radiolabeled polypeptides in the 31-40- and 66-85-kDa range that were absent in serum from 10-R2 B. glabrata. When sporocysts were incubated with radiolabeled serum, 3 polypeptides (116, 180, 210 kDa) from both snail strains bound to the surface of the parasite. Further, a 55-kDa polypeptide bound to sporocysts incubated with 10-R2 serum but did not bind to those parasites incubated with M-line serum. Preincubation of sporocysts with unlabeled serum prior to incubation with radiolabeled serum significantly inhibited the uptake of radiolabeled proteins. This differential binding of serum polypeptides from different strains of B. glabrata may be important in determining resistance or susceptibility of the snail to larval schistosome infection.     

Changes induced in Biomphalaria glabrata (Say, 1818) following trials for artificial stimulation of its internal defense system

Biomphalaria glabrata can react through different pathways to Schistosoma mansoni miracidium penetration, according to the degree of resistance/susceptibility presented by different snail strains, which is a genetically determined character, resistance being the dominant feature. However, it has been observed that previous susceptible snail strain may change its reactive behavior along the course of infection, exhibiting later a pattern of cercarial shedding and histopatopathological picture compatible with high resistance. Such observation suggests the possibility of B. glabrata to develop a sort of adaptative immunity face a schistosome infection. To explore on this aspect, the present investigation looked for the behavior of S. mansoni infection in B. glabrata previously subjected to different means of artificial stimulation of its internal defense system. Snails previously inoculated with irradiated miracídia (Group I); treated with S. mansoni antigens (Group II) or with a non-related parasite antigen (Group III) were challenged with 20 viable S. mansoni miracidia, and later looked for cercarial shedding and histopathologic changes at different times from exposition. Nodules of hemocyte accumulations were found at the site of antigen injection. These nodules resembled solid granulomas, and were larger and more frequent in snails injected with S. mansoni products as compared to those injected with Capillaria hepatica. However, the presence of such granulomas did not avoid the S. mansoni challenge infection from developing in a similar way as that seen in controls. The data are indicative that hemocytes are able to proliferate locally when stimulated, such capacity also remaining localized, not being shared by the population of hemocytes located elsewhere within the snail body.     

Genome size estimates for two important freshwater molluscs, the zebra mussel (Dreissena polymorpha) and the schistosomiasis vector snail (Biomphalaria glabrata).

The haploid genome sizes of two important molluscs were assessed by Feulgen image analysis densitometry. The genome size of the zebra mussel (Dreissena polymorpha), a prolific invader of North American lakes, was estimated to be 1C = 1.70 +/- 0.03 pg, and that of the freshwater snail Biomphalaria glabrata, the predominant intermediate vector of the human parasite Schistosoma mansoni, was estimated at 0.95 +/- 0.01 pg. These estimates will be important in future efforts in molluscan genomics, which at present lags far behind work being carried out with vertebrate and arthropod models. B. glabrata in particular, which has one of the smallest known gastropod genomes, is recommended as a highly suitable target for future genome sequencing.     

Involvement of nitric oxide in killing of Schistosoma mansoni sporocysts by hemocytes from resistant Biomphalaria glabrata

In strains of the snail Biomphalaria glabrata (Gastropoda) that are resistant to the parasite Schistosoma mansoni (Trematoda), hemocytes in the hemolymph are responsible for elimination of S. mansoni sporocysts. The defensive role of reactive nitrogen species was investigated in in vitro interactions between hemocytes derived from the resistant 13-16-R1 strain of B. glabrata and the parasite. The nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methylester (L-NAME) and the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide reduced cell-mediated killing of S. mansoni sporocysts. To determine if peroxynitrite (ONOO-) is involved in killing, assays were run in the presence of the ONOO- scavengers uric acid and deferoxamine. These did not influence the rate of parasite killing, indicating that NO is directly responsible for mediating cytotoxicity, but ONOO- is not. The combination of the NOS inhibitor L-NAME and catalase, an enzyme that detoxifies hydrogen peroxide (H2O2), reduced average sporocyst mortality to a greater extent than L-NAME alone. Killing of the sporocysts was, however, not totally inhibited. It is suggested that NO and H2O2 are both involved in hemocyte-mediated toxicity of 13-16-R1 B. glabrata against S. mansoni sporocysts.     

Intraspecific competition and the evolution of virulence in a parasitic trematode

Intrahost competition between parasite genotypes has been predicted to be an important force shaping parasite ecology and evolution and has been extensively cited as a mechanism for the evolution of increased parasite virulence. However, empirical evidence demonstrating the existence and nature of intraspecific competition is lacking for many parasites. Here, we compared within-host competitiveness between genetic strains of Schistosoma mansoni with high (HIGH-V) or low (LOW-V) virulence to their intermediate snail host, Biomphalaria glabrata. Groups of snails were exposed to either one or the other of two parasite strains, or a mixed infection of both strains, and the resulting progeny were identified using a molecular marker. In two separate experiments investigating simultaneous and sequential infections, we demonstrated that the lifetime reproductive success of parasite strain HIGH-V was reduced in the presence of a faster replicating parasite genotype, LOW-V, regardless of whether it was in a majority or minority in the initial inoculum of the simultaneous exposure or of its relative position in the sequential exposure experiment. Thus, we demonstrate competition between parasite genotypes and asymmetry in competitive success between parasite strains. Moreover, since the less virulent strain investigated here had a competitive advantage, we suggest that a high frequency of multiple infections could favor the evolution of less, rather than more, virulent parasites in this system.     

Structural characterization of N-glycans from the freshwater snail Biomphalaria glabrata cross-reacting with Schistosoma mansoni glycoconjugates

The human parasitic trematode Schistosoma mansoni has a complex life cycle that includes the freshwater snail Biomphalaria glabrata as intermediate host. Within each stage, the parasite synthesizes a wide array of glycoconjugates, exhibiting, in part, unique carbohydrate structures. In addition, the parasite expresses definitive host-like sugar epitopes, such as Lewis X determinants, supporting the concept of carbohydrate-mediated molecular mimicry as an invasion and survival strategy. In the present study, we investigated whether common carbohydrate determinants occur also at the level of the intermediate host. To this end, a structural characterization of hemolymph glycoprotein-N-glycans of B. glabrata was performed. N-glycans were released from tryptic glycopeptides and labeled with 2-aminopyridine. Sugar chains serologically cross-reacting with S. mansoni glycoconjugates were isolated by immunoaffinity chromatography using a polyclonal antiserum directed against schistosomal egg antigens and fractionated by Aleuria aurantia lectin affinity chromatography and high-performance liquid chromatography. Obtained glycans were analyzed by different mass spectrometric techniques as well as by monosaccharide constituent and linkage analysis. The results revealed a highly heterogeneous oligosaccharide pattern. Cross-reacting species represented about 5% of the total glycans and exhibited a terminal Fuc(alpha1-3)GalNAc unit, a (1-2)-linked xylosyl residue, or both types of structural motifs. In conclusion, our study demonstrates the presence of common carbohydrate epitopes also at the level of S. mansoni and its intermediate host.     

Interspecific competition between freshwater snails of medical importance: a Venezuelan example

Lake Valencia is located in the centre of the endemic area of the intestinal schistosimiasis in Venezuela. The dominance of two pulmonate species, Biomphalaria glabrata and B. prona., was observed in the lake. Both species are strongly associated with two distinct types of habitats suggesting that competition is occurring between these two species. B. glabrata and B. prona play the role of intermediate hosts of schistosomes in Venezuela. At the present time, parasite transmission is not occurring in the lake but the planning of important development programmes represents a risk of creation of active schistosomiasis foci. The knowledge of the importance and distribution of the snail host populations is therefore essential and must be taken into account for developing future control strategies.     

Schistosoma mansoni: use of a cloned ribosomal RNA gene probe to detect restriction fragment length polymorphisms in the intermediate host Biomphalaria glabrata.

Adult susceptibility of Biomphalaria glabrata to Schistosoma mansoni infection is controlled by simple Mendelian genetics. In this study a molecular approach was used to determine the degree of genetic variation between well-defined lines of B. glabrata which are either resistant (10-R2) or susceptible (M-line) to S. mansoni infection. A cloned probe pSM389, which contains part of the S. mansoni small ribosomal RNA gene and a portion of the nontranscribed spacer was found to cross-hybridize with B. glabrata DNA and was used in Southern hybridizations to detect restriction fragment length polymorphisms (RFLPs) between the above snail stocks. Polymorphisms were noted with a variety of restriction enzymes, namely Bg/II, BamHI, AccI, AvaII, ClaI, EcoRI, EcoRV, KpnI, PvuII, and NcoI. Although most RFLPs were relatively minor, a significant difference was observed with EcoRV. Further analysis of the EcoRV RFLPs among other isolates of the resistant stock demonstrated that a high frequency of genetic variation exists even among isolates of the same origin, but maintained in separate laboratories. Interestingly, RFLPs in the EcoRV site were detected in DNA isolated from a single generation of selfed progeny of a single 10-R2 parent. RFLPs associated with this site were found to occur between B. glabrata and B. tenagophila, B. straminea, and B. schrammi, indicating that Southern blot analysis using ribosomal gene probes may be useful for the molecular differentiation of B. glabrata from other intermediate hosts and from morphologically similar species that are refractory to infection.     

Schistosoma mansoni: passive transfer of resistance by serum in the vector snail, Biomphalaria glabrata

Passive transfer of natural resistance to Schistosoma mansoni (PR-1 strain) has been successfully accomplished in the snail intermediate host, Biomphalaria glabrata (PR albino, M-line strain). Injection of serum (cell-free hemolymph) from a naturally schistosome-resistant strain of B. glabrata (10-R2) into PR albino snails induced a complete protection from a primary infection with the parasite in 29 of 48 snails (60.4%). In comparison, inoculation of homologous PR albino serum or heterologous proteins (fetal calf serum) had no effect. Moreover, this protection could be induced 24 hr prior to, or 24 hr after, exposure to the parasite, although heating of 10-R2 serum to 70 C for 30 min destroyed its protective ability. When in vitro transformed sporocysts were preincubated in 10-R2 or PR albino serum and then were injected into susceptible snails, a high level of infection (88.5 and 83.3%, respectively) was produced in both groups. Thus, the 10-R2 serum factor does not appear to be mediating specific parasite recognition by host hemocytes. Alternatively, our results suggest that 10-R2 serum possesses a heat-labile factor which specifically activate B. glabrata hemocytes to encapsulate and destroy sporocysts whereas PR albino serum lacks this factor.