Molecular identification of symbionts from the pulmonate snail Biomphalaria glabrata in Brazil

The icthyosporean, Capsaspora owczarzaki, a known predator of Schistosoma mansoni sporocysts in vitro, is more prevalent in laboratory-reared strains of the intermediate snail host, Biomphalaria glabrata resistant to S. mansoni, than from the susceptible M line strain. We examined whether B. glabrata resistant to the NIH-PR-1 strain of S. mansoni from 2 regions in Brazil were also host to C. owczarzaki. Symbiont presence was examined using hemolymph culturing and nested polymerase chain reaction of snail genomic DNA with primers designed to specifically amplify sequences from relatives of the Icthyosporea. All B. glabrata of the resistant Salvador strain from the laboratory of Dr. Lobato Paraense in Rio de Janeiro, Brazil (n = 46) tested negative for symbionts. Three of 18 semiresistant 10-R2 B. glabrata from the laboratory of Dr. Barbosa in Recife, Brazil tested positive for C. owczarzaki. Another icthyosporean, Anurofeca sp., was identified from 1, 10-R2 snail and from 2 of 12 field-collected B. glabrata from Praia do Forte Orange, Ilha de Itamaracá. Snails from 2 other sites, Hotel Colibri, Pontezinha and Praia do Sossego, Ilha de Itamaracá, were negative for Anurofeca. Two genera of ciliates were also identified. Paruroleptus sp. was found in 4, 10-R2 snails and Trichodina sp. was identified in 2 field-collected snails from Praia do Forte Orange and Praia do Sossego.     

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.     

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.     

Genetic markers between Biomphalaria glabrata snails susceptible and resistant to Schistosoma mansoni infection

The analysis of the genetic variability related to susceptibility to Schistosoma mansoni infection in the vector of the genus Biomphalaria is important in terms of a better understanding of the epidemiology of schistosomiasis itself, the possible pathological implications of this interaction in vertebrate hosts, and the formulation of new strategies and approaches for disease control. In the present study, the genetic variability of B. glabrata strains found to be resistant or susceptible to S. mansoni infection was investigated using DNA amplification by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). The amplification products were analyzed on 8% polyacrylamide gel and stained with silver. We selected 10 primers, since they have previously been useful to detect polymorphism among B. glabrata and/or B. tenagophila. The results showed polymorphisms with 5 primers. Polymorphic bands observed only in the susceptible strain. The RAPD-PCR methodology represents an adequate approach for the analysis of genetic polymorphisms. The understanding of the genetic polymorphisms associated to resistance may contribute to the future identification of genomic sequences related to the resistance/susceptibility of Biomphalaria to the larval forms of S. mansoni and to the development of new strategies for the control of schistosomiasis.     

Killing of Schistosoma mansoni sporocysts by hemocytes from resistant Biomphalaria glabrata: role of reactive oxygen species

The fate of Schistosoma mansoni (Trematoda) sporocysts in its molluscan host Biomphalaria glabrata (Gastropoda) is determined by circulating phagocytes (hemocytes). When the parasite invades a resistant snail, it is attacked and destroyed by hemocytes, whereas in a susceptible host it remains unaffected. We used 3 inbred strains of B. glabrata: 13-16-R1 and 10-R2, which are resistant to the PR-1 strain of S. mansoni, and M-line Oregon (MO), which is susceptible to PR-1. In an in vitro killing assay using plasma-free hemocytes from these strains, the rate of parasite killing corresponded closely to the rate by which S. mansoni sporocysts are killed in vivo. Hemocytes from resistant snails killed more than 80% of S. mansoni sporocysts within 48 hr, whereas sporocyst mortality in the presence of hemocytes from susceptible snails was <10%. Using this in vitro assay, we assessed the involvement of reactive oxygen species (ROS) produced by resistant hemocytes, during killing of S. mansoni sporocysts. Inhibition of NADPH oxidase significantly reduced sporocyst killing by 13-16-R1 hemocytes, indicating that ROS play an important role in normal killing. Reduction of hydrogen peroxide (H2O2) by including catalase in the killing assay increased parasite viability. Reduction of superoxide (O2-), however, by addition of superoxide dismutase or scavenging of hydroxyl radicals (*OH) and hypochlorous acid (HOCl) by addition of hypotaurine did not alter the rate of sporocyst killing by resistant hemocytes. We conclude that H2O2 is the ROS mainly responsible for killing.     

Restriction fragment length polymorphisms distinguish ectomycorrhizal fungi

Basidiomycetous fungi, two saprophytes and three mycorrhizal, were used to assess the specificity of DNA hybridization for distinguishing genera from one another. Interspecific comparisons were done with several isolates of mycorrhizal fungi,Laccaria bicolor andL. laccata, collected from diverse geographical sites. The DNAs were digested with four restriction nucleases and separated by gel electrophoresis into patterns of DNA fragments called restriction fragment length polymorphisms (RFLPs). The RFLPs were hybridized with a radioactively-labeled DNA probe encoding Basidiomycetous ribosomal RNA genes. The five genera were discernable using both unprobed and probed RFLPs. Hybridization of probe DNA with RFLPs was isolate-specific for all nine Laccaria isolates examined. The reclassification of aL. bicolor isolate is supported, demonstrating that hybridization of RFLPs offers an additional tool for taxonomy of ectomycorrhizal fungi. The method may have field application for distinguishing known isolates if their DNA fingerprints are previously ascertained and are distinct from RFLPs of indigenous organisms.     

Genetic diversity and recruitment pattern of Schistosoma mansoni in a Biomphalaria glabrata snail population: a field study using random-amplified polymorphic DNA markers.

Random-amplified polymorphic DNA markers have been used to assess the amount and the distribution of the genetic diversity of Schistosoma mansoni within a natural population of Biomphalaria glabrata at a transmission site of the murine schistosomiasis focus of Guadeloupe. Despite high infection rate and heavy schistosome load within the definitive hosts (Ratus rattus), prevalences within intermediate snails ranged from 0.2 to 4.8%. Whatever the transmission season may be (rainy vs. dry), most of the infected snails were spatially aggregated and 88.4% of them harbored a single parasite genotype indicative of a monomiracidial infection; 4.7% had dual sex infections and a parasite intensity not exceeding 3 miracidia per snail. A substantial resistance level toward the parasite and recruitment regulatory process within snails may explain in part the observed low parasite prevalences and intensities. Considering such a distribution pattern of larval S. mansoni genetic diversity among B. glabrata, mobility of the definitive hosts, or rapid turnover of infected snails, or both, are required to maintain genetic heterogeneity within adult schistosome populations.     

Effects of exogenous interleukin-1β on primary sporocysts of Schistosoma mansoni (Trematoda) incubated with plasma and hemocytes from schistosome-susceptible and resistant Biomphalaria glabrata (Gastropoda)

Abstract. The cytokine interleukin-1β (IL-1β) mediates interactions of immune and inflammatory cells in mammals. Previous reports also have linked plasma (cell-free hemolymph) levels of IL-1β in the snail Biomphalaria glabrata to resistance against Schistosoma mansoni . In the present study, fluorescent probes were used to study larval schistosome and snail hemocyte viability during in vitro encounters. Hemolymph (plasma and hemocytes) from schistosome-susceptible (M-line) and resistant (13–16-R1) B. glabrata was added to sporocysts of S. mansoni and the viability of hemocytes and parasites was assessed. Next, IL-1β was added to sporocyst-hemolymph samples, the viability of sporocysts and hemocytes determined and then compared to control assays. The number of live sporocysts present after incubation for 1 h with hemolymph from M-line snails was significantly greater than the number seen when hemolymph from 13–16-R1 snails was tested. Nearly all sporocysts survived the 1 h incubation with M-line hemolymph, and most of the hemocytes attached to sporocysts were dead. In contrast, nearly all sporocysts were dead when hemolymph from 13–16-R1 snails was tested, and most attached hemocytes were alive. Addition of IL-1β to M-line hemolymph resulted in a dramatic increase in sporocyst death. Addition of IL-1β to 13–16-R1 hemolymph produced a small but significant increase in the rate of sporocyst death. These results indicate that the concentration of IL-1β present in hemolymph from B. glabrata is directly related to the ability of this snail to kill S. mansoni sporocysts in vitro.     

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.     

Differentiation of mitochondrial subgroups in the Verticillium lecanii species complex

Presumptive mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) were obtained from 54 fungal isolates identified as Verticillium lecanii, V. psalliotae or ' V. lecanii -like'. Analysis of the mtDNA RFLPs showed 20 different patterns, indicating considerable genetic variation within the V. lecanii species complex. There was no direct correlation between host and mtDNA pattern, or between mtDNA patterns and previously described isoenzyme-defined specific groups. Isolates from tropical and subtropical areas showed considerable variation in genotypes, while isolates from temperate regions appeared less variable.     

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.     

Resistance of Biomphalaria occidentalis from Varzea das Flores dam, Minas Gerais, to Schistosoma mansoni infection detected by low stringency polymerase chain reaction

Biomphalaria occidentalis Paraense, 1981 from Varzea das Flores dam, MG, Brazil, was exposed to infection with Schistosoma mansoni. Individual infection was performed with 140 B. occidentalis and 100 B. glabrata snails using LE and SJ strains. Two groups of B. occidentalis were killed after seven day-miracidia exposure to detect S. mansoni DNA, through the low stringency polymerase chain reaction (LS-PCR), and were negative. The infection rates were 69.2% (LE strain) and 96.7% (SJ strain) for B. glabrata and 0% for B. occidentalis. LS-PCR enabled early resistance diagnosis.     

Isolation and characterization of DNA probes from a flow-sorted human chromosome 8 library that detect restriction fragment length polymorphism (RFLP).

We have used a recombinant DNA library constructed from flow-sorted human chromosome 8 as a source of single-copy human probes. These probes have been screened for restriction fragment length polymorphism (RFLP) by hybridization to Southern transfers of genomic DNA from five unrelated individuals. We have detected six RFLPs distributed among four probes after screening 741 base pairs for restriction site variation. These RFLPs all behave as codominant Mendelian alleles. Two of the probes detect rare variants, while the other two detect RFLPs with PIC values of .36 and .16. Informative probes will be useful for the construction of a linkage map for chromosome 8 and for the localization of mutant alleles to this chromosome.     

Manganese superoxide dismutase from Biomphalaria glabrata

The investigation of the response of Biomphalaria glabrata snails to Echinostoma paraensei (digenea) at 2 days post-exposure by suppression subtractive hybridization yielded a partial sequence of the anti-oxidant enzyme manganese superoxide dismutase (MnSOD). Full-length MnSOD (669nt) from M line and BS-90 strains of B. glabrata differed by one synonymous nucleotide replacement. B. glabrata has 1-4 MnSOD loci (Southern hybridization). Both snail strains expressed MnSOD at equal baseline levels (quantitative PCR). Susceptible snails increased expression of MnSOD following infection with E. paraensei or Schistosoma mansoni, and expression was reduced in the incompatible combination (BS-90 B. glabrata and S. mansoni). Thus, MnSOD did not determine resistance or susceptibility for these parasites, but expression of MnSOD is consistent with its involvement in a stress response of B. glabrata.     

The genetic variation of compatibility in Biomphalaria glabrata and Schistosoma mansoni

Interactions between different Biomphalaria glabrata stocks and Schistosoma mansoni strains were studied. A series of inbred stocks of B. glabrata were characterized as to genetic variations in susceptibility at different ages to a series of different S. mansoni strains. A series of inbred strains of S. mansoni were characterized as to genetic variations in infectivity for B. glabrata stocks at different ages. Also described is a process of selection for substrains from a single S. mansoni isolate that differ genetically in snail infectivity.     

DNA Polymorphisms in Lentinula edodes, the Shiitake Mushroom

DNA restriction fragment length polymorphisms (RFLPs) were examined in Lentinula edodes strains. Genomic DNA from strain 70 was cloned in plasmid vector pUC19, and 18 random clones containing low-copy DNA sequences were used to probe seven strains in Southern DNA-DNA hybridizations. Each cloned fragment revealed DNA polymorphism. An RFLP genotype was determined for each strain and the genetic relatedness was assessed. The coefficients of genetic similarity among the seven strains ranged from 0.43 to 0.90. The inheritance of RFLP markers was examined in single spore isolates. Homokaryons displayed a loss of polymorphic bands compared with the parent dikaryon. Hybrids constructed by crossing compatible homokaryons displayed the inheritance of RFLP markers from each parent homokaryon.     

Rescue of sporocysts of Schistosoma mansoni in nonsusceptible Biomphalaria by head-foot transplantation into susceptible snails

To measure the longevity of sporocysts of Schistosoma mansoni in nonsusceptible snails (13-16-R1 and Salvador strains of Biomphalaria glabrata, and Biomphalaria obstructa), the head-foot (HF) of miracidia-exposed snails was transplanted into the hemocoel of a susceptible NIH albino recipient at 1-36 days postexposure (DPE). Recipient snails which were not exposed to miracidia then were monitored for infection transferred by the implant, and infection prevalences in recipients of HF transplants from nonsusceptible donors were compared to those in snails implanted with an HF from NIH albino donors. Transplants from NIH albino snails between 1 to 15 DPE infected 98% of recipients. Similarly, at 1 DPE, 69-85% of transplants from nonsusceptible snails contained viable sporocysts, as shown by resulting patent infections in the recipients. Recipient infection prevalence, and presumably numbers of transplants containing viable sporocysts, declined as a function of DPE, and by 5-9 DPE this decrease was significant for all 3 types of nonsusceptible donors. However, viable sporocysts still occurred in B. obstructa and 13-16-R1 B. glabrata as late as 19 and 20 DPE, respectively, and in Salvador B. glabrata as late as 33 DPE. Thus, sporocysts persist in nonsusceptible snails considerably longer than suggested by results of previous histological studies.     

Genetic studies in the genusBasidiobolus: II. Phylogenetic relationships inferred from ribosomal DNA analysis

We have investigated genetic relationships among isolated of the genusBasidiobolus by examining restriction site polymorphisms in the ribosomal RNA genes. Approximately 10 kb or rDNA was cloned and employed as a probe of genomic DNA from 12 isolates, of human and saprobic origin, digested with nine restriction enzymes. While we foundB. microsporus Benjamin to be distinct, 9 human isolates designatedB. haptosporus Drechsler,B. meristosporus Drechsler, andB. ranarum Eidam were indistinguishable from one another. In contrast, two unidentified saprobic isolates could be readily differentiated from the human ones. We conclude that all human isolates examined fall within a single monophyletic group, but that significant genetic differentiation has taken place between human and saprobic isolates that is not accounted for in existing taxonomies of the genus.     

Construction of genetic linkage maps in maize and tomato using restriction fragment length polymorphisms

Summary Genetic linkage maps were constructed for both maize and tomato, utilizing restriction fragment length polymorphisms (RFLPs) as the source of genetic markers. In order to detect these RFLPs, unique DNA sequence clones were prepared from either maize or tomato tissue and hybridized to Southern blots containing restriction enzyme-digested genomic DNA from different homozygous lines. A subsequent comparison of the RFLP inheritance patterns in F2 populations from tomato and maize permitted arrangement of the loci detected by these clones into genetic linkage groups for both species.     

Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species.

Detailed restriction analyses of many samples often require substantial amounts of time and effort for DNA extraction, restriction digests, Southern blotting, and hybridization. We describe a novel approach that uses the polymerase chain reaction (PCR) for rapid simplified restriction typing and mapping of DNA from many different isolates. DNA fragments up to 2 kilobase pairs in length were efficiently amplified from crude DNA samples of several pathogenic Cryptococcus species, including C. neoformans, C. albidus, C. laurentii, and C. uniguttulatus. Digestion and electrophoresis of the PCR products by using frequent-cutting restriction enzymes produced complex restriction phenotypes (fingerprints) that were often unique for each strain or species. We used the PCR to amplify and analyze restriction pattern variation within three major portions of the ribosomal DNA (rDNA) repeats from these fungi. Detailed mapping of many restriction sites within the rDNA locus was determined by fingerprint analysis of progressively larger PCR fragments sharing a common primer site at one end. As judged by PCR fingerprints, the rDNA of 19 C. neoformans isolates showed no variation for four restriction enzymes that we surveyed. Other Cryptococcus spp. showed varying levels of restriction pattern variation within their rDNAs and were shown to be genetically distinct from C. neoformans. The PCR primers used in this study have also been successfully applied for amplification of rDNAs from other pathogenic and nonpathogenic fungi, including Candida spp., and ought to have wide applicability for clinical detection and other studies.