Mollusc Biomphalaria Glabrata Research Articles

Toxicological evaluation of antiretroviral Tenofovir Disoproxil Fumarate on the mollusk Biomphalaria glabrata and its hemocytes

Tenofovir disoproxil fumarate (TDF) is a drug used in HIV treatment, and several studies have detected its presence in surface water. Furthermore, more information on its environmental impact is needed in the scientific literature. Thus, due to the lack of data on the impact of this drug, and its presence in different waters of the world, this work aimed to evaluate the potential toxicological effects of TDF on the mollusk Biomphalaria glabrata in vivo and in vitro. For in vitro analysis, hemocytes were exposed to different drug concentrations for 1 h and evaluated for feasibility, and phagocytic and metabolic activity. The in vivo analysis consisted of the exposure of groups of five mollusks, in triplicate, at the same drug concentrations for 72 h and 21 days, evaluating mortality, and mollusk and hemolymph behavior. Although the exposure of the mollusk to TDF did not reduce its survival, however it was toxic to its hemocytes. Even if toxicity was identified on the mollusk and its hemocytes initially, further studies should be conducted to understand the effects of this residue on the environment and different life stages of the mollusk because, per the Globally Harmonized System of Classification and Labeling of Chemicals, for aquatic ecosystems, the results obtained were classified as toxic (EC50% 2.65 [1.98; 5.29] mg/L) and could cause unfeasibility in hemocytes at concentrations below 10 mg/l.

Schiff bases complexed with iron and their relation with the life cycle and infection by Schistosoma mansoni.

The trematode Schistosoma mansoni causes schistosomiasis, and this parasite's life cycle depends on the mollusk Biomphalaria glabrata. The most effective treatment for infected people is administering a single dose of Praziquantel. However, there are naturally resistant to treatment. This work has developed, considering this parasite's complex life cycle. The synthetics compound were evaluated: i) during the infection of B. glabrata, ii) during the infection of BALB/c mice, and iii) during the treatment of mice infected with S. mansoni. For the first objective, snails infected with miracidia treated with compounds C1 and C3 at concentrations of 25% IC50 and 50% IC50, after 80 days of infection, released fewer cercariae than the infected group without treatment. For the second objective, compounds C1 and C3 did not show significant results in the infected group without treatment. For the third objective, the mice treated with C3 and C1 reduced the global and differential cell count. The results suggest that although the evaluated compounds do not present schistosomicidal properties when placed in cercariae suspension, they can stimulate an immune reaction in snails and decrease mice's inflammatory response. In general, we can conclude that compound C1 and C3 has an anti-schistosomicidal effect both in the larval phase (miracidia) and in the adult form of the parasite.

Use of Biomphalaria glabrata as a bioindicator of groundwater quality under the influence of NORM

The Brazilian northeast is known to have sedimentary areas that contain minerals with anomalous concentrations of naturally occurring radioactive material (NORM). This characteristic can contribute to the elevation of natural radiation in the air, soil, and groundwater. Due to the inefficiency of drinking water distribution in this region, the use of water from wells has become essential for the population. Therefore, the objective of this research was to monitor the concentration of 238U and 232Th associated with biomonitoring with the species of mollusc Biomphalaria glabrata in waters of residential artesian wells, used for domestic consumption, in the municipalities of Abreu e Lima and São José do Sabugi, Brazil. To check the concentration of 238U and 232Th, ICP-MS was used. For biomonitoring, ecotoxicity techniques such as embryotoxicity and genotoxicity were used. The monitoring results confirmed high concentrations of natural uranium in one of the residential artesian wells, the data being above the limit allowed by the Ministry of Health of Brazil, whose study reference is the World Health Organization (WHO). The results of the bioassays showed embryotoxicity, with malformations and deaths in the exposed organisms being observed. The comet assay showed that groundwater caused changes in the mollusc's DNA, indicating genotoxicity. The bioassays suggest that embryotoxicity and genotoxicity were caused mainly by the high concentration of natural uranium. Therefore, the bioindicator B. glabrata was shown to be sensitive to the toxic effects of anomalous concentrations of NORM present in groundwater.

Sublethal concentrations of usnic acid potassium salt impairs physiological parameters of Biomphalaria glabrata (Say, 1818) (Pulmonata: Planorbidae) infected and not infected with Schistosoma mansoni

Schistosomiasis is a public health problem in many developing countries. The mollusc Biomphalaria glabrata is the most important vector of Schistosoma mansoni in South America. The population control of this vector to prevent the spread of schistosomiasis is currently done with the application of highly toxic molluscicide to the environment. The screening of substances in sublethal concentrations that have deleterious effects on physiological parameters is very relevant for the control of schistosomiasis, since the effectiveness of disease prevention increases if it acts on population control of the vector and on reproduction and elimination in S. mansoni cercariae. The objective of this study was to evaluate the reproductive parameters (fecundity and fertility), intra-mollusk effect (sporocysts I (72 h) and II (14 days after)) on the development of cercariae of S. mansoni and the immune cell profile of B. glabrata exposed to sublethal concentrations (LC25 - 0.5 µg/mL and LC50 - 0.92 µg/mL) of the usnic acid potassium salt (potassium usnate). LC 25 and LC 50 significantly reduced (p < 0.05) the fecundity of B. glabrata when treated infected and/or not exposed to infection, while unviable embryos were not observed in sporocyst stage I, being only significant (p < 0.05) for mollusks infected and treated with LC50 on sporocyst II. LC25 and LC50 of the potassium usnate caused significant reductions (p < 0.05) in the production and cercarial shedding when evaluated on sporocysts I and II. In addition, the mortality of infected and treated B. glabrata in the sporocyst II phase was quite marked after the 9th week of infection. Regarding the immunological cell profile of uninfected B. glabrata, both concentrations led to immunomodulatory responses, with significant morphological changes predominant of hemocytes that entered programmed cell death (apoptosis). It was concluded that the application of LC25 and LC50 from the potassium usnate could be useful in the population control of B. glabrata, since it interferes both in their biology and physiology and in the reproduction of the infectious agent of schistosomiasis mansoni.

Effects of UV-B radiation on secondary metabolite production, antioxidant activity, photosynthesis and herbivory interactions in Nymphoides humboldtiana (Menyanthaceae)

Ultraviolet B-light (UV-B) can exert indirect effects on plant-herbivore interactions by inducing changes in constitutive and induced chemical defenses, since it modulates physiological aspects of plants. This study evaluated the action of UV-B radiation on photosynthesis and production of secondary metabolites in Nymphoides humboldtiana and the cascade effects on the relationship of this macrophyte with a generalist herbivore, the gastropod mollusk Biomphalaria glabrata. After 13 days of UV-B exposition under laboratory conditions, the floating macrophyte N. humboldtiana responded increasing its photosynthetic potential and the production of flavonoids with a correlated enhance in antioxidant activity. However, these changes observed in its metabolism were not enough to alter their palatability to consumption by B. glabrata verified through laboratory feeding choice experiments. Despite the known deleterious effects of exposure to UV-B on terrestrial plants, we found that N. humboldtiana does have physiological/biochemical mechanisms as a strategy or restorative response to this potencially adverse or impacting agent without changing its relationships with herbivores.

The Members of the Highly Diverse Crassostrea gigas Integrin Family Cooperate for the Generation of Various Immune Responses.

Studies on invertebrate immune receptors can provide insights into characteristics specific to innate immune system. Here, eight α and three β integrins are identified from an invertebrate, the Pacific oyster Crassostrea gigas, and their possible immune functions are studied. Oyster α/β integrins exhibit a higher degree of sequence and structural variability than the members from Homo sapiens and Drosophila melanogaster. The analysis reveals that oyster RGD- and laminin-binding receptor homologs are present in the phylogenetic tree of α integrins, but the other six oyster α integrins mainly form a species-specific branch; meanwhile, oyster β integrins are clustered with insect β integrins but distinct from a member from the mollusk Biomphalaria glabrata. Although phylogenetically lacking the important α integrin branches of LDV-binding, PS3-type, and αI-containing integrins, oyster integrins can bind to most ECM ligands, including RGDCP, LDVCP, GFOGERCP, and laminin protein in a distinct binding pattern. Besides, oyster integrins are distributed in different hemocyte subpopulations, while only specific integrins are selectively involved in hemocyte phagocytosis, migration, and encapsulation, and some of them participate in more than one immune response in a sophisticated pattern. Especially, oyster β integrins are arranged in the core to mediate complex immune responses, unlike the counterparts in humans that mainly depend on αI-containing integrins to incite immune reactions. This study represents the first comprehensive attempt to reveal the structural and evolutionary features of the integrin family and their involvement in cellular immune responses in the non-model invertebrate C. gigas and sheds light on the characteristics specific to the innate immune system in the integrin family.

Study of genotoxic and cytotoxic effects after acute and chronic exposures to industrial sewage sludge on Biomphalaria glabrata hemocytes

Industrial development has provided numerous benefits to improve quality of life in modern times, however, it has also led to the development and use of a large number of toxic chemicals which have caused damage to various ecosystems. Consequently, knowledge of techniques and organisms that can be used to monitor, identify and quantify environmental pollutants has become increasingly relevant. Therefore, the objective of this study was to use the mollusk Biomphalaria glabrata to analyze biomarker and biomonitoring parameters of industrial sewage sludge. To perform the tests, concentrations of 50, 100, 150 and 500 mg L−1 of industrial sewage sludge were standardized. All the tests were performed after the animals were exposed to the sludge in acute and chronic forms. Embryos exposure to sludge did not show a significant percentage of the animals non-viable when compared to the control group. Subsequently, hemocytes were analyzed for the presence of cytoplasmic and nuclear alterations. Finally, the comet test was performed to quantify the genotoxic damage caused by exposure to industrial sludge. Analysis hemocytes showed a significant number of cellular alterations was observed, mainly due to the high frequency of apoptosis. Moreover, during the analysis of nucleoids several degrees of nuclear damage were identified, with the groups exposed to the highest concentrations presenting the greatest genotoxic damage. Thus, we can conclude that the parameters evaluated in the mollusk Biomphalaria glabrata have proven to be a good tool, along with other techniques and complementary organisms, to assist aspects related to biomonitoring of freshwater ecosystems.

Cytotoxic and genotoxic effect of oxyfluorfen on hemocytes of Biomphalaria glabrata.

Chemicals released from anthropogenic activities such as industry and agriculture often end up in aquatic ecosystems. These substances can cause serious damage to these ecosystems, thus threatening the conservation of biodiversity. Among these substances are pesticides, such as oxyfluorfen, a herbicide used for the control of grasses and weeds. Considering its widespread use, it is important to investigate the possible toxicity of this compound to aquatic organisms, especially invertebrates. Hence, the use of biological systems able to detect such effects is of great importance. The mollusk Biomphalaria glabrata has been shown to be useful as an environmental indicator to assess the potential ecological effects of physical and chemical stressors in freshwater environments. The present study sought to detect mutagenic changes in hemocytes of B. glabrata exposed to oxyfluorfen. To perform these tests, this study used ten animals per group, exposed acutely (48h) and chronically (15days) to oxyfluorfen. The herbicide concentrations were 0.125, 0.25, and 0.5mg/L. The results showed that oxyfluorfen induced significant frequencies of micronuclei, binucleated cells, and apoptosis in hemocytes of mollusks when compared to the control group. Unlike chronic exposure, acute exposure was dose-dependent. The present study's results demonstrate the cytotoxic and genotoxic effects of oxyfluorfen on hemocytes of B. glabrata.

Laboratory assessment of divaricatic acid against Biomphalaria glabrata and Schistosoma mansoni cercariae

In this study, the molluscicidal and antiparasitic activities of divaricatic acid was evaluated, targeting the mollusc Biomphalaria glabrata and cercariae of the helminth Schistosoma mansoni. In addition, the environmental toxicity of divaricatic acid was assessed by bioassay using the microcrustacean Artemia salina. Divaricatic acid showed high toxicity against both adult snails (5μg/mL) and embryos (20μg/mL after 6h of exposure). Similar activity was observed in Schistosoma mansoni cercariae after only a short exposure time (10μg/mL after 30min of exposure). The divaricatic acid did not show toxicity in the acute test using Artemia salina at concentrations equal to or below 200μg/mL. The divaricatic acid proved to be a promising substance for the elimination of the snail Biomphalaria glabrata, an intermediate host of schistosomiasis, as well as the cercariae of the pathogen, while being non-toxic to the Artemia salina at the same concentrations. This is the first experimental observation of the molluscicidal and cercaricide activity of divaricatic acid.

Aquatic ecotoxicity of ashes from Brazilian savanna wildfires.

In a global scenario of climate change, several studies have predicted an increase in fires in different parts of the world. With the occurrence of rains following the fires in the Brazilian savanna (Cerrado biome), the compounds present in ashes may enter aquatic environments and cause adverse effects to these ecosystems. In this context, this study evaluated the potential toxicity of ashes from two areas of Cerrado and an area of pasture, through ecotoxicological bioassays and using three aquatic species from distinct trophic levels, which were exposed to different dilutions of ashes: the microcrustacean Ceriodaphnia dubia, the fish Danio rerio and the mollusc Biomphalaria glabrata. The ashes from the three sampled areas showed higher concentrations of some elements in relation to the soil samples (B, Ca, K, Mg, Mn, P, S, Si, Sr, Zn), but only a small quantity of these compounds was solubilised. Our data showed that all ash samples caused acute toxicity to C. dubia (48hs-LC50=13.4gL-1; 48hs-LC50=6.33gL-1; 48hs-LC50=9.73gL-1 respectively for transition area, pasture, typical cerrado areas), while in relation to D. rerio and B. glabrata, no acute toxicity was observed when they were exposed to ashes from native Cerrado vegetation and pasture areas. Ashes from a transition area showed toxicity for D. rerio (48hs-LC50=25.0gL-1); possibly, this was due to the combination of multiple preponderant inorganic elements of ashes with other organic compounds not analysed, such as polycyclic aromatic hydrocarbons (PAHs). In summary, these results suggest that wildfires may pose risks to zooplankton communities and emphasize the need for more studies to better understand the complexity of the ecological effects of fire on aquatic ecosystems.

Deep, multi-stage transcriptome of the schistosomiasis vector Biomphalaria glabrata provides platform for understanding molluscan disease-related pathways.

BackgroundThe gastropod mollusc Biomphalaria glabrata is well known as a vector for the tropical disease schistosomiasis, which affects nearly 200 million people worldwide. Despite intensive study, our understanding of the genetic basis of B. glabrata development, growth and disease resistance is constrained by limited genetic resources, constraints for which next-generation sequencing methods provide a ready solution.MethodsIllumina sequencing and de novo assembly using the Trinity program was used to generate a high-quality transcriptomic dataset spanning the entirety of in ovo development in schistosomiasis-free B. glabrata. This was subjected to automated (KEGG, BLAST2GO) and manual annotation efforts, allowing insight into the gene complements of this species in a number of contexts.ResultsExcellent dataset recovery was observed, with 133,084 contigs produced of mean size 2219.48 bp. 80,952 (60.8 %) returned a BLASTx hit with an E value of less than 10-3, and 74,492 (55.97 %) were either mapped or assigned a GO identity using the BLAST2GO program. The CEGMA set of core eukaryotic genes was found to be 99.6 % present, indicating exceptional transcriptome completeness. We were able to identify a wealth of disease-pathway related genes within our dataset, including the Wnt, apoptosis and Notch pathways. This provides an invaluable reference point for further work into molluscan development and evolution, for studying the impact of schistosomiasis in this species, and perhaps providing targets for the treatment of this widespread disease.ConclusionsHere we present a deep transcriptome of an embryonic sample of schistosomiasis-free B. glabrata, presenting a comprehensive dataset for comparison to disease-affected specimens and from which conclusions can be drawn about the genetics of this widespread medical model. Furthermore, the dataset provided by this sequencing provides a useful reference point for comparison to other mollusc species, which can be used to better understand the evolution of this commercially, ecologically and medically important phylum.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1944-x) contains supplementary material, which is available to authorized users.

Schistosoma mansoni Mucin Gene (SmPoMuc) Expression: Epigenetic Control to Shape Adaptation to a New Host

The digenetic trematode Schistosoma mansoni is a human parasite that uses the mollusc Biomphalaria glabrata as intermediate host. Specific S. mansoni strains can infect efficiently only certain B. glabrata strains (compatible strain) while others are incompatible. Strain-specific differences in transcription of a conserved family of polymorphic mucins (SmPoMucs) in S. mansoni are the principle determinants for this compatibility. In the present study, we investigated the bases of the control of SmPoMuc expression that evolved to evade B. glabrata diversified antigen recognition molecules. We compared the DNA sequences and chromatin structure of SmPoMuc promoters of two S. mansoni strains that are either compatible (C) or incompatible (IC) with a reference snail host. We reveal that although sequence differences are observed between active promoter regions of SmPoMuc genes, the sequences of the promoters are not diverse and are conserved between IC and C strains, suggesting that genetics alone cannot explain the evolution of compatibility polymorphism. In contrast, promoters carry epigenetic marks that are significantly different between the C and IC strains. Moreover, we show that modifications of the structure of the chromatin of the parasite modify transcription of SmPoMuc in the IC strain compared to the C strain and correlate with the presence of additional combinations of SmPoMuc transcripts only observed in the IC phenotype. Our results indicate that transcription polymorphism of a gene family that is responsible for an important adaptive trait of the parasite is epigenetically encoded. These strain-specific epigenetic marks are heritable, but can change while the underlying genetic information remains stable. This suggests that epigenetic changes may be important for the early steps in the adaptation of pathogens to new hosts, and might be an initial step in adaptive evolution in general.

A Large Repertoire of Parasite Epitopes Matched by a Large Repertoire of Host Immune Receptors in an Invertebrate Host/Parasite Model

For many decades, invertebrate immunity was believed to be non-adaptive, poorly specific, relying exclusively on sometimes multiple but germ-line encoded innate receptors and effectors. But recent studies performed in different invertebrate species have shaken this paradigm by providing evidence for various types of somatic adaptations at the level of putative immune receptors leading to an enlarged repertoire of recognition molecules. Fibrinogen Related Proteins (FREPs) from the mollusc Biomphalaria glabrata are an example of these putative immune receptors. They are known to be involved in reactions against trematode parasites. Following not yet well understood somatic mechanisms, the FREP repertoire varies considerably from one snail to another, showing a trend towards an individualization of the putative immune repertoire almost comparable to that described from vertebrate adaptive immune system. Nevertheless, their antigenic targets remain unknown. In this study, we show that a specific set of these highly variable FREPs from B. glabrata forms complexes with similarly highly polymorphic and individually variable mucin molecules from its specific trematode parasite S. mansoni (Schistosoma mansoni Polymorphic Mucins: SmPoMucs). This is the first evidence of the interaction between diversified immune receptors and antigenic variant in an invertebrate host/pathogen model. The same order of magnitude in the diversity of the parasite epitopes and the one of the FREP suggests co-evolutionary dynamics between host and parasite regarding this set of determinants that could explain population features like the compatibility polymorphism observed in B. glabrata/S. mansoni interaction. In addition, we identified a third partner associated with the FREPs/SmPoMucs in the immune complex: a Thioester containing Protein (TEP) belonging to a molecular category that plays a role in phagocytosis or encapsulation following recognition. The presence of this last partner in this immune complex argues in favor of the involvement of the formed complex in parasite recognition and elimination from the host.

In search of the origin of FREPs: Characterization of Aplysia californica fibrinogen-related proteins

All haemolymph lectins with uniquely juxtaposed N-terminal domain similar to the immunoglobulin superfamily (IgSF) and C-terminal fibrinogen (FBG) termed FBG-related proteins (FREP) are documented till now only in the pulmonate mollusc Biomphalaria glabrata. Using genomic WGS database we have found two FREP genes from marine opistobranch Aplysia californica named AcFREP1 and AcFREP2. The AcFREP1 and AcFREP2 mRNA molecules have been subsequently isolated from cDNA of sea hare larvae as well as adult mollusc tissues. These genes encode proteins (504 and 510 aa respectively) with domain architecture typical for FREPs with two N-terminal IgSF domains and C-terminal FBG domain. Although cDNA sequences of AcFREP1 and AcFREP2 are 81% identical, their genomic structure is entirely different: AcFREP1 is intronless and AcFREP2 is encoded in four exons. These genes are paralogous pair in which AcFREP2 is a parental gene and AcFREP1 is the new transposed copy that has lost the introns (retrogene). Using RT-PCR analysis, expression of AcFREP1 and AcFREP2 was shown to be developmentally and tissue-specific and no constitutive expression in haemocytes was found. The overall frequency of nucleotide substitutions in genomic DNA trace sequences of coding region of the AcFREP1 and AcFREP2 is not higher than in the sequences of control conserved genes (actin, FMRFamide). Thus, previously reported high diversification of Biomphalaria FREP gene, BgFREP3, is not detected in Aplysia FREPs. A search for FREP homologs in other available complete genome of mollusc, Lottia gigantea (Patellogastropoda), a representative of the evolutionary earliest gastropod clade, did not reveal any DNA sequences coding for similar lectins. We suggest that unique domain architecture of FREPs is an evolutionary novelty that appeared and evolved only within one branch of Protostomata species, exclusively in heterobranch molluscs (Pulmonata and Opistobranchia).

Controlled Chaos of Polymorphic Mucins in a Metazoan Parasite (Schistosoma mansoni) Interacting with Its Invertebrate Host (Biomphalaria glabrata)

Invertebrates were long thought to possess only a simple, effective and hence non-adaptive defence system against microbial and parasitic attacks. However, recent studies have shown that invertebrate immunity also relies on immune receptors that diversify (e.g. in echinoderms, insects and mollusks (Biomphalaria glabrata)). Apparently, individual or population-based polymorphism-generating mechanisms exists that permit the survival of invertebrate species exposed to parasites. Consequently, the generally accepted arms race hypothesis predicts that molecular diversity and polymorphism also exist in parasites of invertebrates. We investigated the diversity and polymorphism of parasite molecules (Schistosoma mansoni Polymorphic Mucins, SmPoMucs) that are key factors for the compatibility of schistosomes interacting with their host, the mollusc Biomphalaria glabrata. We have elucidated the complex cascade of mechanisms acting both at the genomic level and during expression that confer polymorphism to SmPoMuc. We show that SmPoMuc is coded by a multi-gene family whose members frequently recombine. We show that these genes are transcribed in an individual-specific manner, and that for each gene, multiple splice variants exist. Finally, we reveal the impact of this polymorphism on the SmPoMuc glycosylation status. Our data support the view that S. mansoni has evolved a complex hierarchical system that efficiently generates a high degree of polymorphism—a “controlled chaos”—based on a relatively low number of genes. This contrasts with protozoan parasites that generate antigenic variation from large sets of genes such as Trypanosoma cruzi, Trypanosoma brucei and Plasmodium falciparum. Our data support the view that the interaction between parasites and their invertebrate hosts are far more complex than previously thought. While most studies in this matter have focused on invertebrate host diversification, we clearly show that diversifying mechanisms also exist on the parasite side of the interaction. Our findings shed new light on how and why invertebrate immunity develops.

Molluscicidal activity of 2-hydroxy-[1,4]naphthoquinone and derivatives

The toxic profile of lawsone (2-hydroxy-[1,4]naphthoquinone) and a series of [1,4]naphthoquinone derivatives was evaluated against the brine shrimp Artemia salina and against the mollusk Biomphalaria glabrata, the main transmitting vector of schistosomiasis in Brazil. Of the seventeen compounds tested nine fell below the threshold of 100 microg/mL set for potential molluscicidal activity by the World Health Organization. As a general rule derivatives with non-polar substituents presented the highest molluscicidal activities. These substances showed significant toxicity in A. salina lethality bioassay.

Crystal Structure of a Novel Tetrameric Complex of Agonist-bound Ligand-binding Domain of Biomphalaria glabrata Retinoid X Receptor

Nuclear receptors form an important class of transcription regulators in metazoans. To learn more about the evolution of these proteins, we have initiated structural studies on nuclear receptor ligand-binding domains from various animals. Here we present the crystal structure of the ligand-binding domain (LBD) of the retinoid X receptor (RXR) from the mollusc Biomphalaria glabrata. The structure reveals a novel tetrameric association in which each monomer is complexed to the human RXR ligand 9-cis retinoic acid and to a human co-activator-derived peptide. The ligand and the co-activator peptide are bound in essentially the same manner as observed in previously reported human RXR LBD structures, suggesting that the mechanisms of RXR-mediated transcription regulation are very similar in mollusc and human. The structure shows further that binding of ligand and co-activator peptide does not necessarily lead to the typical holo-conformation in which helix 12 (H12) folds back and packs against the LBD. Within a canonical dimer, only one monomer is in this closed agonist conformation. The other monomer is in an open conformation with H12 protruding from the LBD core, occupying the H12 interaction groove of another open monomer in an adjacent dimer in a domain swapping fashion, thus resulting in a tetrameric association. Additional tetramer interfaces are formed between H11 of the closed LBD and H6 of the open LBD. This novel holo-tetramer configuration may have a biological role in activating genes whose promoters are poorly recognised by dimers but much more efficiently by the corresponding tetramers.

A conserved retinoid X receptor (RXR) from the mollusk Biomphalaria glabrata transactivates transcription in the presence of retinoids

Retinoid X receptors (RXR) are members of the nuclear receptor superfamily of ligand-activated transcription factors that have been characterized in a wide variety of metazoan phyla. They act as heterodimer partners of other nuclear receptors, and in vertebrates also activate transcription as homodimers in the presence of a ligand, 9-cis retinoic acid. In order to test the hypothesis that retinoic acid signaling pathways involving RXRs are present in the Lophotrochozoa, we have sought to isolate conserved members of this family from the platyhelminth parasite Schistosoma mansoni and its intermediate host, the mollusk Biomphalaria glabrata. Here we report that an RXR ortholog from B. glabrata (BgRXR) is better conserved, compared with mouse RXRalpha, both in the DNA-binding domain (89% identity) and in the ligand-binding domain (LBD) (81% identity), than are arthropod homologs. In EMSA, BgRXR binds to the direct repeat response element DR1 as a homodimer or as a heterodimer with mammalian RARalpha, LXR, FXR or PPARalpha. When transfected alone into mammalian cell lines, BgRXR transactivated transcription of a reporter gene from the Apo-A1 promoter in the presence of 9-cis retinoic acid or DHA. Constructs with the Gal4 DNA binding domain fused to the hinge and LBDs of BgRXR were used to show that ligand-dependent activation of transcription by BgRXR required its intact AF-2 activation domain, and that the LBD can form homodimers. Finally, the binding of 9-cis retinoic acid preferentially protected the LBD of BgRXR from degradation by trypsin in a proteolysis protection assay. Our results show that BgRXR binds and is activated by retinoids and suggest that retinoid signaling pathways are conserved in the Lophotrochozoa. The nucleotide sequence reported in this paper has been submitted to the GenBank/EBI Data Bank with accession no. AY048663.

Molecular Characterization of Brevibacillus laterosporus and Its Potential Use in Biological Control

Thirty-three strains of Brevibacillus laterosporus, including three novel strains isolated from Brazilian soil samples, were examined for genetic variability by the use of different PCR-based methods. Molecular markers that could characterize bacterial strains with regards to their pathogenic potential were investigated. In addition, toxicity was assessed by the use of insects belonging to the orders Lepidoptera and Coleoptera and the mollusk Biomphalaria glabrata. Among the targets tested, Biomphalaria glabrata demonstrated the highest degree of sensitivity to B. laterosporus, with some strains inducing 90 to 100% mortality in snails aged 3 and 12 days posteclosion. Larvae of the coleopteron Anthonomus grandis were also susceptible, presenting mortality levels of between 33 and 63%. Toxicity was also noted towards the lepidopteron Anticarsia gemmatalis. In contrast, no mortality was recorded among test populations of Tenebrio molitor or Spodoptera frugiperda. The application of intergenic transcribed spacer PCR and BOX-PCR generated 15 and 17 different genotypes, respectively. None of the molecular techniques allowed the identification of a convenient marker that was associated with any entomopathogenic phenotype. However, a 1,078-bp amplicon was detected for all strains of B. laterosporus when a primer for amplification of the BOXA1R region was used. Similarly, a 900-bp amplicon was generated from all isolates by use of the primer OPA-11 for randomly amplified polymorphic DNA analysis. These amplicons were not detected for other phenotypically related Brevibacillus species, indicating that they represent markers that are specific for B. laterosporus, which may prove useful for the isolation and identification of new strains of this species.

Representation of an immune responsive gene family encoding fibrinogen-related proteins in the freshwater mollusc Biomphalaria glabrata, an intermediate host for Schistosoma mansoni

Fibrinogen-related proteins (FREPs) are found in the hemolymph of the freshwater snail Biomphalaria glabrata, are up-regulated following exposure to digenetic trematode parasites, and bind to trematode larval surfaces, suggestive of a role in internal defense. Southern blot and degenerate-polymerase chain reaction (PCR) analyses were undertaken to better understand the diversity of the FREP-encoding gene family. Probes corresponding to the N-terminal IgSF domains of specific FREP gene subfamilies ( FREPs 2, 3, 4, 7, 12 and 13) revealed between 1 to 8 loci per subfamily on Southern blots. Probes representing the relatively conserved C-terminal fibrinogen domain of FREPs bound many sequences in Southern blots of genomic DNA from B. glabrata, and from two related gastropod species, Biomphalaria pfeifferi and Helisoma trivolvis. Using degenerate-PCR, we obtained 42 unique fibrinogen-encoding sequences from 180 clones derived from a single individual of the M-line strain of B. glabrata, further supporting the notion of their abundant representation in the B. glabrata genome. The fibrinogen-encoding sequences of FREPs encoding one or two IgSF domains tended to separate into distinct clades, but bootstrap support for this separation was low. A novel category of fibrinogen-encoding sequence was also revealed. This study provides the approximate number of gene copies in several FREP subfamilies, confirms the existence of a diverse FREP gene family, reports additional unusual sequences encoding fibrinogen-like molecules, and provides further justification to explore the functional roles of FREPs in both B. glabrata and B. pfeifferi, both important intermediate hosts of the human pathogen, Schistosoma mansoni.