Evolutionary relationships and biogeography of Biomphalaria (Gastropoda: Planorbidae) with implications regarding its role as host of the human bloodfluke, Schistosoma mansoni.

The wide geographic distribution of Schistosoma mansoni, a digenetic trematode and parasite of humans, is determined by the occurrence of its intermediate hosts, freshwater snails of the genus Biomphalaria (Preston 1910). We present phylogenetic analyses of 23 species of Biomphalaria, 16 Neotropical and seven African, including the most important schistosome hosts, using partial mitochondrial ribosomal 16S and complete nuclear ribosomal ITS1 and ITS2 nucleotide sequences. A dramatically better resolution was obtained by combining the data sets as opposed to analyzing each separately, indicating that there is additive congruent signal in each data set. Neotropical species are basal, and all African species are derived, suggesting an American origin for the genus. We confirm that a proto-Biomphalaria glabrata gave rise to all African species through a trans-Atlantic colonization of Africa. In addition, genetic distances among African species are smaller compared with those among Neotropical species, indicating a more recent origin. There are two species-rich clades, one African with B. glabrata as its base, and the other Neotropical. Within the African clade, a wide-ranging tropical savannah species, B. pfeifferi, and a Nilotic species complex, have both colonized Rift Valley lakes and produced endemic lacustrine forms. Within the Neotropical clade, two newly acquired natural hosts for S. mansoni (B. straminea and B. tenagophila) are not the closest relatives of each other, suggesting two separate acquisition events. Basal to these two species-rich clades are several Neotropical lineages with large genetic distances between them, indicating multiple lineages within the genus. Interesting patterns occur regarding schistosome susceptibility: (1) the most susceptible hosts belong to a single clade, comprising B. glabrata and the African species, (2) several susceptible Neotropical species are sister groups to apparently refractory species, and (3) some basal lineages are susceptible. These patterns suggest the existence of both inherent susceptibility and resistance, but also underscore the ability of S. mansoni to adapt to and acquire previously unsusceptible species as hosts. Biomphalaria schrammi appears to be distantly related to other Biomphalaria as well as to Helisoma, and may represent a separate or intermediate lineage.     

Neotropical schistosomiasis: African affinities of the host snail Biomphalaria glabrata (Gastropoda: Planorbidae)

Schistosoma mansoni , the blood fluke responsible for human intestinal schistosomiasis in the Neotropics, was imported repeatedly with African slaves during the period 1500–1800. This trematode, and its intermediate host snails of the genus Biomphalaria , are widely distributed across Africa and the disease is thought to have quickly become established in South America and the West Indies because of the presence of an endemic susceptible congener, B. glabrata. We compared B. glabrata with four other Neotropical and three African species of Biomphalaria using 20 allozyme loci and found that it is phenetically and phylogenetically more like the African species; both parasite and American host snail are apparently of historically or geologically recent African origin. Furthermore, genetic distances, cladistic analyses and fossil data suggest the African Biomphalaria species may themselves have evolved from Neotropical founders following an initial trans-Atlantic dispersal in the reverse direction 2.3–4.5 Mya. Interpretation of existing patterns remains problematic as few African snails have been characterized genetically and both B. glabrata and African B. pfeifferi appear to comprise several cryptic species.     

Method for the individual maintenance of mollusks infected with Schistosoma larvae in a thermostatic apparatus

A technique has been suggested for individual maintenance of intermediate hosts of Schistosoma mansoni, molluscs of the genus Biomphalaria, in thermostat water bath with thermoregulator in order to study the host-parasite relationships in the mollusc-trematode system. Advantages of the suggested technique have been shown for studying total production of Cercaria when estimating the compatibility degree of various strains of S. mansoni and races of molluscs of some species of the genus Biomphalaria.     

Total evidence: molecules, morphology, and the phylogenetics of cichlid fishes

We present a most comprehensive phylogenetic analysis of the family Cichlidae. New data analyzed include mitochondrial 16S rRNA sequences and two nuclear loci (Tmo-M27 and Tmo-4C4) for a large taxonomic sampling with emphasis on South American species. We also incorporate a published morphological data set for a total evidence analysis. Character congruence among mitochondrial (74 taxa) and nuclear data (50 taxa) was high. However, partition-homogeneity tests suggest significant heterogeneity among molecular and morphological data. In agreement with results obtained from molecular data alone, total evidence analysis (1,460 characters for 34 taxa) supports a robust phylogenetic hypothesis for the family Cichlidae that is congruent with drift-vicariance events associated with the fragmentation of Gondwana. Our analyses confirm the placement of Malagasy/Indian cichlids as the most basal lineages, with a sister-group relationship to the monophyletic African and Neotropical clades. Total evidence suggests that the controversial African genus Heterochromis is at the base of the African radiation. Among more than 50 Neotropical genera analyzed, Retroculus is identified as the basal taxon, with successive branching of Cichla, Astronotus, geophagines (including crenicichlines) + chaetobranchines, and cichlasomines + heroines. Relative rate tests applied to mitochondrial DNA suggest significantly higher rates of genetic variation in Neotropical than in African taxa, and both mitochondrial and nuclear sequences show that rate heterogeneity among Neotropical lineages is confined to the geophagine cichlids.     

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.     

Phylogeny and biogeography of African Biomphalaria (Gastropoda: Planorbidae), with emphasis on endemic species of the great East African lakes

The pulmonate snails of the genus Biomphalaria are widely distributed in the tropics, and they are intermediate hosts of the digenean trematode Schistosoma mansoni that causes intestinal schistosomiasis in humans. Recent molecular evidence suggests that Biomphalaria originated in South America, and following a recent transatlantic migration colonized Africa, where it radiated into the currently recognized 12 species. In the present study we further investigate the internal phylogenetic relationships of African Biomphalaria with emphasis on the dispersal and speciation on the continent, especially in the Great Lakes in East Africa. Our results, based on 16S ribosomal DNA, cytochrome oxidase subunit I (COI), and ribosomal internal transcribed spacer I (ITS1), support the monophyly of an African clade with two separate lineages ( Biomphalaria pfeifferi / Biomphalaria camerunensis and the Nilotic species complex/ Biomphalaria angulosa ). Following the initial colonization of Africa, Biomphalaria spread towards the east where a later radiation occurred in the Lake Victoria basin and the Albertine Rift Valley Lakes. With further dispersal along the River Nile, additional speciation took place giving origin to the North-east African species Biomphalaria alexandrina . Our results present almost no support of the species groups of Mandahl-Barth (except for the pfeifferi group), which is in accordance with other molecular appraisals. Our results suggest that Biomphalaria stanleyi , which is endemic to Lake Albert, is not an ecophenotype of the continental B. pfeifferi as previously suggested by other molecular studies. B. angulosa is sequenced for the first time and it is inferred to have an important phylogenetic position as sister group to the Albertine Rift/Lake Victoria basin radiation.  © 2007 The Linnean Society of London, Zoological Journal of the Linnean Society , 2007, 151 , 337–349.     

Studies of the relationship between Schistosoma and their intermediate hosts. III. The genus Biomphalaria and Schistosoma mansoni from Egypt, Kenya, Sudan, Uganda, West Indies (St. Lucia) and Zaire (two different strains: Katanga and Kinshasa)

The compatibility between strains of Schistosoma mansoni from Egypt, Kenya, Sudan, Uganda, the West Indies, and Zaire (two strains which came from Katanga and from Kinshasa), and various species and strains of Biomphalaria, i.e. Biomphalaria pfeifferi, B. alexandrina, B. glabrata and B. camerunensis was investigated. Data as mortality, rate of infection of the surviving snails, duration of infection, cercarial production per day per positive snail, etc., were observed. The main emphasis was placed on determining the total cercarial production per 100 exposed snails for each snail population. It was possible to infect all the tested populations of B pfeifferi with the various strains of S. mansoni, but the observation as e.g. TCP/100 exposed snails varied greatly according to the population of snail and the strain of S. mansoni. The results for the remaining species of Biomphalaria varied greatly, depending on the combination, e.g. B. alexandrina was only susceptible to the local S. mansoni from Egypt. The highest TCP/100 exposed snails was more than 1 million for the strains of S. mansoni from Egypt, Kenya and the West Indies in B. alexandrina, B. pfeifferi and B. glabrata, respectively. The next group, with a TCP/100 exposed snails on 7--800 000 consists of S. mansoni from Sudan, Uganda and Zaire (Katanga) all in B. pfeifferi. The last tested strain of S. mansoni, Zaire (Kinshasa) yielded a cercarial production on 500 000 per 100 exposed snails in B. pfeifferi and B. camerunensis. The shortest prepatent period, 19 days, was observed for S. mansoni from Kinshasa, Zaire, in B. camerunensis, and the longest prepatent period, 25 days, was found for strains from Egypt and from the West Indies in B. alexandrina and B. glabrata, respectively. In general, a very long duration of infection, lasting up to 200 days, was observed.     

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.     

Multiplex PCR for both identification of Brazilian Biomphalaria species (Gastropoda: Planorbidae) and diagnosis of infection by Schistosoma mansoni (Trematoda: Schistosomatidae)

A simple and single-step technique based on multiplex PCR (multiplex polymerase chain reaction) has been developed for simultaneous identification of Brazilian Biomphalaria species, the intermediate hosts of Schistosoma mansoni, and their diagnosis of infection by the trematode. We used species-specific primers directed both to the internal transcribed spacer 2 (ITS2) of ribosomal DNA from 3 of the S. mansoni host species and to the mitochondrial DNA (mtDNA) from the trematode. Those primers were used simultaneously in a single multiplex-PCR reaction, and template DNA was obtained from S. mansoni-infected and noninfected snails. The results were visualized in silver stained polyacrylamide gels, revealing the presence of specific bands. The methodology has shown to be efficient, fast, and reproducible for Biomphalaria species identification and diagnosis of snails infected by S. mansoni during prepatent periods.     

Geographical distribution of Biomphalaria snails in the State of Minas Gerais, Brazil

Published and unpublished observations on geographical distribution of Biomphalaria snails in the State of Minas Gerais, Brazil, were compiled. This work is aimed at knowing the present occurrence of Biomphalaria species in this region, and at contributing to the elaboration of the planorbid chart of Minas Gerais. In malacological surveys, performed by several researchers, the presence of seven species of this genus was recorded. Those planorbids were found in 12 mesoregions, in 283 (33.1%) municipalities out of 853 with the following distribution: B. glabrata (185 municipalities), B. straminea (125), B. tenagophila (58), B. peregrina (57), B. schrammi (26), B. intermedia (20) and B. occidentalis (2). B. glabrata and B. tenagophila are found naturally infected by Schistosoma mansoni in Minas Gerais. In 24 municipalities the three snail hosts of S. mansoni in Brazil, B. glabrata, B. tenagophila and B. straminea, are present.     

Phylogeography of Biomphalaria glabrata and B. pfeifferi, important intermediate hosts of Schistosoma mansoni in the New and Old World tropics

The historical phylogeography of the two most important intermediate host species of the human blood fluke Schistosoma mansoni, B. glabrata in the New World, and B. pfeifferi in the Old World, was investigated using partial 16S and ND1 sequences from the mitochondrial genome. Nuclear sequences of an actin intron and internal transcribed spacer (ITS)-1 were also obtained, but they were uninformative for the relationships among populations. Phylogenetic analyses based on mtDNA revealed six well-differentiated clades within B. glabrata: the Greater Antilles, Venezuela and the Lesser Antilles, and four geographically overlapping Brazilian clades. Application of a Biomphalaria-specific mutation rate gives an estimate of the early Pleistocene for their divergence. The Brazilian clades were inferred to be the result of fragmentation, due possibly to climate oscillations, with subsequent range expansion producing the overlapping ranges. Within the Venezuela and Lesser Antilles clade, lineages from each of these areas were estimated to have separated approximately 740 000 years ago. Compared to B. glabrata, mitochondrial sequences of B. pfeifferi are about 4x lower in diversity, reflecting a much younger age for the species, with the most recent common ancestor of all haplotypes estimated to have existed 880 000 years ago. The oldest B. pfeifferi haplotypes occurred in southern Africa, suggesting it may have been a refugium during dry periods. A recent range expansion was inferred for eastern Africa less than 100 000 years ago. Several putative species and subspecies, B. arabica, B. gaudi, B. rhodesiensis and B. stanleyi, are shown to be undifferentiated from other B. pfeifferi populations.     

A Multiplex-PCR approach to identification of the Brazilian intermediate hosts of Schistosoma mansoni

Due to difficulties concerning morphological identification of planorbid snails of the genus Biomphalaria, and given a high variation of characters and in the organs with muscular tissue, we designed specific polymerase chain reaction (PCR) primers for Brazilian snail hosts of Schistosoma mansoni from available sequences of internal transcribed spacer 2 (ITS2) of the ribosomal RNA gene. From the previous sequencing of the ITS2 region, one primer was designed to anchor in the 5.8S conserved region and three other species-specific primers in the 28S region, flanking the ITS2 region. These four primers were simultaneously used in the same reaction (Multiplex-PCR), under high stringency conditions. Amplification of the ITS2 region of Biomphalaria snails produced distinct profiles (between 280 and 350 bp) for B. glabrata, B. tenagophila and B. straminea. The present study demonstrates that Multiplex-PCR of ITS2-DNAr showed to be a promising auxiliary tool for the morphological identification of Biomphalaria snails, the intermediate hosts of S. mansoni.     

Incompatibility between miracidia of Schistosoma mansoni and Helisoma duryi occurs at two stages: penetration and intramolluscan establishment

Helisoma spp. snails are not susceptible to infection with miracidia of Schistosoma mansoni because the miracidia do not penetrate them. However, in view of the phylogenetic proximity and histocompatibility between Helisoma spp. and the normal intermediate host, Biomphalaria glabrata , schistosome miracidia conceivably could survive if experimentally introduced into the hemocoel of Helisoma spp. To test this hypothesis, schistosome-susceptible NIH albino B. glabrata, schistosome-resistant Salvador B. glabrata, and Helisoma duryi were injected with miracidia of S. mansoni, and the outcome was followed both by monitoring snails for infection for several weeks and by histological examination at 24 and 48 hr post-injection (PI). Patent infections developed in most NIH albino snails but in none of the Salvador B. glabrata or H. duryi individuals. Histological analysis showed a higher proportion of normal sporocysts in various tissues of NIH albino snails at both time periods relative to Salvador snails, which contained mostly sporocysts undergoing hemocytic encapsulation. In H. duryi , nearly all sporocysts were dead by 48 hr PI.     

Miracidia of an Egyptian strain of Schistosoma mansoni differentiate between sympatric snail species

The host-finding behavior of miracidia of 2 strains of Schistosoma mansoni from Egypt and Brazil was studied by recording their responses to snail-conditioned water (SCW) from the Egyptian sympatric snails, Biomphalaria alexandrina, Physa acuta, Lymnaea cailliudi, and Balinus truncatus, as well as from Biomphalaria arabica and Biomphalaria glabrata. Miracidia of the Egyptian strain significantly preferred SCW from their compatible hosts B. alexandrina and B. arabica and showed no or a weak response to SCW from the other sympatric species, whereas miracidia of the Brazilian strain did not differentiate between SCW from different snail species.     

Freshwater snails and Schistosomiasis mansoni in the state of Rio de Janeiro, Brazil: IV - Sul Fluminense Mesoregion

In this paper, the forth of a series dealing with the survey of freshwater gastropods of the state of Rio de Janeiro, the results of collections carried out in the Sul Fluminense Mesoregion from 2000 to 2002 are presented and revealed the occurrence of 18 species: Antillorbis nordestensis; Biomphalaria glabrata; Biomphalaria peregrina; Biomphalaria straminea; Biomphalaria tenagophila; Drepanotrema anatinum; Drepanotrema cimex; Drepanotrema lucidum; Ferrissia sp.; Gundlachia ticaga; Gundlachia sp.; Heleobia sp.; Lymnaea columella; Melanoides tuberculatus; Physa acuta; Physa marmorata; Pomacea sordida and Pomacea sp. As to the snail hosts of Schistosoma mansoni the most frequent species was B. tenagophila, found in all municipalities surveyed, except Parati. Besides new records the present study extends the distribution of B. peregrina and B. straminea in the state. No specimens were found harbouring larval forms of S. mansoni although different kinds of cercariae had been observed. An account about the current schistosomiasis transmission sites in this Mesoregion is presented as well.     

Freshwater snails and Schistosomiasis mansoni in the state of Rio de Janeiro,Brazil: III--Baixadas Mesoregion

In this paper, the third of a series dealing with the survey of freshwater gastropods of the state of Rio de Janeiro, the results of collections carried out in the Mesoregion Baixadas from 2000 to 2002 are presented. Twenty-two species, belonging to seven families, were found. As to the snail intermediate hosts of Schistosoma mansoni, the most frequent species was Biomphalaria tenagophila besides some new findings of Biomphalaria straminea. No specimens were found harboring larval forms of S. mansoni although different kinds of cercariae had been observed.     

Polymerase chain reaction and restriction fragment length polymorphism of cytocrome oxidase subunit I used for differentiation of Brazilian Biomphalaria species intermediate host of Schistosoma mansoni

The intermediate hosts of Schistosoma mansoni, in Brazil, Biomphalaria glabrata, B. tenagophila and B. straminea, were identified by restriction fragment length polymorphism analysis of the mitochondrial gene cytochrome oxidase I (COI). We performed digestions with two enzymes (AluI and RsaI), previously selected, based on sequences available in Genbank. The profiles obtained with RsaI showed to be the most informative once they were polymorphic patterns, corroborating with much morphological data. In addition, we performed COI digestion of B. straminea snails from Uruguay and Argentina.     

Schistosoma mansoni: comparison of a Caribbean and African strain and experimental crossing based on compatibility with intermediate hosts and Rattus rattus

To elucidate changes relative to compatibility with intermediate and definitive hosts affecting Schistosoma mansoni since it was introduced to the New World, the compatibility of S. mansoni from Africa (the Cameroons), from the Caribbean (Guadeloupe), and those resulting from experimental crosses with the gastropods Biomphalaria glabrata and B. pfeifferi has been studied. Results show that S. mansoni, regardless of its origin or its usual snail host, always infects B. pfeifferi more successfully than B. glabrata. The success rate with B. pfeifferi is 100% with 5 miracidia of S. mansoni from Guadeloupe and 97% with 5 miracidia from the Cameroons. On the other hand, in B. glabrata infection rate was 54% with 5 miracidia from Guadeloupe and 0% with 5 miracidia from the Cameroons (a rate of 19% is reached when using 10 miracidia). Hybrid miracidia infect B. pfeifferi and B. glabrata with a success rate of 60 and 86%, respectively, which are intermediate between those of the parent strains. Studies of S. mansoni development in Rattus rattus show that there is better infectivity and survival for the Caribbean strain than the Cameroon strain: the percentage worm recovery 4 weeks after exposure in 34% for S. mansoni from Guadeloupe, 14% for S. mansoni from the Cameroons, and 31% for the hybrids. The mortality rate between 4 and 12 weeks after exposure is 51% for S. mansoni from Guadeloupe, 87% for S. mansoni from the Cameroons, and 31% for the hybrids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molluscicidal and piscicidal activities of Venezuelan Chrysobalanaceae plants

Extracts of increasing polarity of 6 Venezuelan plants belonging to the Chrysobalanaceae family were tested for possible letal toxicity against Biomphalaria glabrata Say, a snail intermediate hosts of Schistosoma mansoni Sambon. The piscicidal toxicity of the active extracts was also evaluated with the aim of findings compounds without toxicity in nontarget organisms, principally fishes and humans.     

Origin and diversification of the human parasite Schistosoma mansoni

Schistosoma mansoni is the most widespread of the human-infecting schistosomes, present in 54 countries, predominantly in Africa, but also in Madagascar, the Arabian Peninsula, and the Neotropics. Adult-stage parasites that infect humans are also occasionally recovered from baboons, rodents, and other mammals. Larval stages of the parasite are dependent upon certain species of freshwater snails in the genus Biomphalaria, which largely determine the parasite's geographical range. How S. mansoni genetic diversity is distributed geographically and among isolates using different hosts has never been examined with DNA sequence data. Here we describe the global phylogeography of S. mansoni using more than 2500 bp of mitochondrial DNA (mtDNA) from 143 parasites collected in 53 geographically widespread localities. Considerable within-species mtDNA diversity was found, with 85 unique haplotypes grouping into five distinct lineages. Geographical separation, and not host use, appears to be the most important factor in the diversification of the parasite. East African specimens showed a remarkable amount of variation, comprising three clades and basal members of a fourth, strongly suggesting an East African origin for the parasite 0.30-0.43 million years ago, a time frame that follows the arrival of its snail host. Less but still substantial variation was found in the rest of Africa. A recent colonization of the New World is supported by finding only seven closely related New World haplotypes which have West African affinities. All Brazilian isolates have nearly identical mtDNA haplotypes, suggesting a founder effect from the establishment and spread of the parasite in this large country.