The interaction of Schistosoma haematobium and S. guineensis in Cameroon.

Interactions between schistosomes are complex with some different species being able to mate and hybridize. The epidemiology of schistosomiasis in specific areas of South West Cameroon has evolved remarkably over 30 years as a result of hybridization between Schistosoma guineensis and S. haematobium. Morphological and biological data suggest that S. haematobium replaced S. guineensis in areas of Cameroon through introgressive hybridization. Data are reported on the use of single stranded conformational polymorphism (SSCP) analysis of the nuclear ribosomal second internal transcribed spacer (ITS2) of individual schistosomes from hybrid zones of Cameroon. The data show that since 1990 S. haematobium has completely replaced S. guineensis in Loum, with S. haematobium and the recombinants still present in 2000. This study illustrates the complexities of the dynamics between S. haematobium and S. guineensis in South West Cameroon.

A phylogeny based on three mitochondrial genes supports the division of Schistosoma intercalatum into two separate species.

Two recognized strains of Schistosoma intercalatum, one from the Democratic Republic of Congo (DRC), formerly Zaire, and the other from Cameroon, have been investigated using DNA sequences from 3 mitochondrial genes, cytochrome oxidase subunit 1 (cox1), NADH dehydrogenase subunit 6 (nad6) and the small ribosomal RNA gene (rrnS). In addition, partial DNA sequences from the nuclear large subunit ribosomal RNA gene (lsrDNA) were included within the study. Although partial lsrDNA alone reveals little taxonomic information, phylogenetic analysis of the mitochondrial data demonstrates a clear dichotomy between the 2 purported strains and it is proposed that they should be treated as distinct taxa. The 'original' S. intercalatum now falls relatively basal in the S. haematobium group, while the proposed new species is more derived and sister taxon to S. bovis and S. curassoni.

A model to explain the replacement of Schistosoma intercalatum by Schistosoma haematobium and the hybrid S. intercalatum x S. haematobium in areas of sympatry.

Numerous hypotheses have been postulated to explain the rapidly changing parasitological situation in Loum, Cameroon as a result of the interaction between Schistosoma haematobium and S. intercalatum. The aim of this study is to test the various hypotheses using a simple mathematical model, incorporating equal and unequal sex ratios of adult schistosomes, recombinations, and levels of compatibility with the intermediate molluscan hosts, B. forskalii and B. truncatus. The model assuming an equal sex ratio does not fit with the existing field data in that it predicts a continued presence of S. intercalatum, S. haematobium and the hybrids. The model assuming a sex bias in favour of males, which reflects the situation usually observed in schistosome populations, predicts the loss S. intercalatum which indeed concurs with the most recent data.

Experimental evidence of hybrid breakdown between the two geographical strains of Schistosoma intercalatum.

Reciprocal crosses (Schistosoma intercalatum male Zaire x S. intercalatum female Cameroon: S. intercalatum female Zaire x S. intercalatum male Cameroon) were produced in 10 mice by exposing each mouse to 60 male cercariae of one isolate and 60 female cercariae of the other isolate, and vice versa. Hybrid generations originating from the two crosses were established. The infectivity of the F1, F2, F3 and F4 hybrid generations were evaluated after exposing snails individually to 5 miracidia. A comparative histological study of snails infected with F2 and F4 hybrid sporocysts from both crosses was made to assess abnormalities in the intramolluscan development of the hybrids. The worm recovery rate and fecundity were measured by comparing the fitness of the mid-parents with that of the hybrids. S. intercalatum Cameroon was compatible with Bulinus forskalii and incompatible with B. globosus whereas S. intercalatum Zaire was compatible with B. globosus and incompatible with B. forskalii. In the case of S. intercalatum male Cameroon x S. intercalatum female Zaire, hybrid miracidia develops in both B: forskalii and B. globosus in F1, F2 and F3 generations. The infection rate was much lower for B. globosus and F2 and F3 generations produced few cercariae (less than 30 cercariae in overall per snail) and F4 miracidia were only infective to B. forskalii again producing few cercariae. At 40 days post-infection the sporocyst contains masses of acidophilic granules originating from the breakup of pycnotic nuclei. Similarly the F1, F2 and F3 miracidia of the reciprocal cross (S. intercalatum male Zaire x S. intercalatum female Cameroon) exhibited a dual infectivity for B. forskalii and B. globosus, but cercarial productivity was low (less than 30 cercariae overall per snail for F2 and F3 generations). Histological studies demonstrated sporocyst degeneration in snails infected with F4 generation. In the definitive host, the F, generation (both crosses) exhibited hybrid vigour in that the worm return was greater than that of the mid-parent, F2 and F3 generations. No significant difference in fecundity was demonstrated between the parental strains and the F1 and F2 generations, yet egg production of the F3 generation was significantly lower. It is apparent that there is a post-zygotic barrier in the crosses of S. intercalatum Zaire and S. intercalatum Cameroon from the F2 generations onwards, and sterility of the F4 hybrid sporocyst supports the concept of the existence of 2 distinct taxa.

Genetic structure of Oestrus ovis populations in sheep and goats.

A genetic analysis using RAPD markers was performed on 12 natural populations of Oestrus ovis (Linné, 1761). Three-hundred and six O. ovis larvae (first, second and third instars) were randomly recovered in nasal cavities of sheep and goats naturally infected in Algeria, Ethiopia, France, Mauritania, Rumania and Tunisia and were analysed by 56 RAPD fragments. The results showed a high diversity within all samples. A significant genetic divergence was showed by discriminant analyses among the 12 populations sampled (p<0.0001). Moreover, discriminant analyses showed significant differentiation (p<0.0001) between O. ovis larva populations of sheep and goats and also among samples collected in the same region.

[Africa or Asia, which is the evolutionary origin of human schistosomes?]. / Afrique ou Asie, quelle est l'origine évolutive des schistosomes humains?

The origin and the evolution of Schistosomatidae species, due to their medical importance (responsible of the second most important human parasitosis after malaria), arouse a great interest. A combination of phylogenetic studies using several molecular markers has provided support for the traditional grouping and evolutionary inferences derived from morphological and biological data. The genus Schistosoma, which comprises all species parasitizing Man, is generally split into four evolutionary lineages (mansoni, haematobium, indicum and japonicum lineages). The group of African schistosomes (including mansoni and haematobium lineages) appears very divergent from the japonicum lineage. Recent phylogenetic studies using partial 28S rDNA sequencing and including Orientobilharzia turkestanicum from Iran, an Asian parasite of livestock, found, unexpectedly, that this species nested among Schistosoma species, thus rendering the latter paraphyletic, and suggested an Asian origin for the Schistosoma genus. The present work re-examines the question of the geographical origin of human schistosomes by analysing a new genomic marker (ITS2) as well as by including the use of O. turkestanicum originating from northeastern China. Our results are in agreement with previous work using 28S, in demonstrating that Schistosoma is not monophyletic. However, O. turkestanicum, whatever the method of analysis used (distance or parsimony), was grouped with members of the japonicum group to the exclusion of African Schistosoma species. Then, our data argue strongly for the need for further phylogenetic study including new taxa and new genomic sequences before definitely concluding either an Asian or African origin for the genus Schistosoma.

Lack of prezygotic isolation by assortative mating between the two cryptic species of the polytypic Schistosoma intercalatum taxon.

Ten mice were individually exposed to 30 male and 30 female cercariae of Schistosoma intercalatum from Cameroon and to 30 male and 30 female cercariae of S. intercalatum from Zaire. Ten weeks later, the nine surviving mice were killed, and a total of 263 adult schistosomes (228 pairs, 7 unpaired males, 28 unpaired females) were perfused. All worms were identified utilising the random amplified polymorphic DNA technique using primer OPG06 which enables the differentiation of the Cameroon and Zaire genotypes. The data demonstrate that there were no significant differences between the number of homospecific pairs observed compared with the expected number under the null hypothesis. Matings occurred in a random manner indicating that mate choice did not exist for the two genotypes of S. intercalatum. Hence there is no prezygotic isolation mechanism which would prevent the interaction of the two genotypes if they were sympatric. However, as they are allopatric, the possibility of the production of unfit hybrids does not arise. Consequently, the reinforcement of a prezygotic isolating mechanism does not play a significant role in the speciation process.

Recent studies on Schistosoma intercalatum: taxonomic status, puzzling distribution and transmission foci revisited.

Schistosoma intercalatum, which causes human rectal schistosomiasis in Africa, still presents a great interest for its imprecise taxonomic status and its puzzling distribution in Africa. Two geographically isolated strains of S. intercalatum are recognized, the Lower Guinea strain and the Congo strain, which differ from each other in a number of morphological, biological and biochemical characteristics. Recent molecular data using RAPD markers indicate high divergence between the two strains, with values of Nei and Li's similarity index allowing recognition of two genetically distinct taxa: experiments on pre- and post-isolating mechanisms are in progress in order to re-evaluate the taxonomic status of this polytypic species. With regard to its geographical distribution, S. intercalatum is characterized by the existence of two stable endemic areas (localized in Lower Guinea and North East of Democratic Republic of Congo) which correspond to the historical areas of species discovery, and the emergence during the last 15 years of new foci of the Lower Guinea strain outside previously known endemic areas. The absence of local adaptation of the Lower Guinea strain to its intermediate host, supported by experimental studies, may help to facilitate the spread of this strain. Nevertheless, the present restricted distribution of this species remains puzzling, because its potential snail hosts (bulinids) are widely distributed throughout much of Africa. Recent experimental and epidemiological studies suggest that interspecific sexual interactions between human schistosomes could have a role in limiting the distribution of S. intercalatum: the competitive sexual processes acting among human schistosomes show that S. haematobium and S. mansoni are always competitively dominant over S. intercalatum. These epidemiological observations lead the authors to distinguish three kinds of transmission foci for S. intercalatum.

Studies on the biology of schistosomiasis with emphasis on the Senegal river basin.

The construction of the Diama dam on the Senegal river, the Manantali dam on the Bafing river, Mali and the ensuing ecological changes have led to a massive outbreak of Schistosoma mansoni in Northern Senegal, associated with high intensity of infections, due to intense transmission, and the creation of new foci of S. haematobium. Data on the vectorial capacity of Biomphalaria pfeifferi from Ndombo, near Richard Toll, Senegal are presented with sympatric and allopatric (Cameroon) S. mansoni. Comparisons are made on infectivity, cercarial production, chronobiology of cercarial emergence and longevity of infected snails. Recent data on the intermediate host specificity of different isolates of S. haematobium from the Lower and Middle Valley of the Senegal river basin (SRB) demonstrate the existence of at least two strains of S. haematobium. The role of Bulinus truncatus in the transmission of S. haematobium in the Lower and Middle Valleys of the SRB is reviewed. Both S. haematobium and S. mansoni are transmitted in the same foci in some areas of the SRB.

Studies on the biology of schistosomiasis with emphasis on the Senegal river basin

The construction of the Diama dam on the Senegal river, the Manantali dam on the Bafing river, Mali and the ensuing ecological changes have led to a massive outbreak of Schistosoma mansoni in Northern Senegal, associated with high intensity of infections, due to intense transmission, and the creation of new foci of S. haematobium. Data on the vectorial capacity of Biomphalaria pfeifferi from Ndombo, near Richard Toll, Senegal are presented with sympatric and allopatric (Cameroon) S. mansoni. Comparisons are made on infectivity, cercarial production, chronobiology of cercarial emergence and longevity of infected snails. Recent data on the intermediate host specificity of different isolates of S. haematobium from the Lower and Middle Valley of the Senegal river basin (SRB) demonstrate the existence of at least two strains of S. haematobium. The role of Bulinus truncatus in the transmission of S. haematobium in the Lower and Middle Valleys of the SRB is reviewed. Both S. haematobium and S. mansoni are transmitted in the same foci in some areas of the SRB

Lack of molluscan host diversity and the transmission of an emerging parasitic disease in Bolivia.

Fasciolosis is a re-emerging parasitic disease that affects an increasing number of people in developing countries. The most severe endemic affects the Bolivian Altiplano, where the liver fluke (Fasciola hepatica) and its hermaphroditic snail host, Lymnaea truncatula, have been introduced from Europe. To achieve a better understanding of the epidemiological situation and the consequences of the colonization event of this invasive species, genetic analysis of Bolivian snail populations was needed. Here we compare the genetic diversity and population structure of snail samples from the Bolivian Altiplano with samples from the Old World at six polymorphic microsatellite loci. Whereas some variability exists in the snail populations from the Old World, we observe only a single genotype of L. truncatula in the Bolivian Altiplano. We discuss the possible explanations for such a reduction in genetic variability, and, given the high natural parasitism pressures exerted on the snail populations, we discuss the relevance of this result for host-parasite interactions.

Molecular arguments for splitting of Schistosoma intercalatum, into two distinct species.

The taxonomic status of the two known strains of Schistosoma intercalatum, the Lower Guinea strain (originating from Edea, Cameroon) and the Zaire strain (originating from Kinshasa, Democratic Republic of Congo, formerly Zaire) was examined using random amplified polymorphic DNA (RAPD) markers. Two additional species within the S. haematobium group, S. haematobium and S. mattheei, were included in the study. DNA was extracted from four male and four female worms of each species and strain under investigation. In all, 13 primers gave reproducible and informative marker patterns; the monomorphic bands in all the males and females of each sample were scored, and 138 bands were included in the final analysis. Overall, 14 RAPD fragments were shared by all the schistosomes studied, and 19 RAPD fragments were considered to be sex markers. Only 22% (20/91) of the RAPD fragments were shared between S. intercalatum Zaire and S. intercalatum Cameroon. The mean values recorded for the Nei and Li's genetic distances between S. haematobium and S. mattheei and between S. intercalatum Zaire and S. intercalatum Cameroon were 0.546 and 0.596, respectively. A principal component analysis and one-way analysis of variance (ANOVA/MANOVA) showed a significant separation between S. intercalatum Zaire and S. intercalatum Cameroon. The data support the hypothesis that S. intercalatum Zaire and S. intercalatum Cameroon are distinct species. Additional molecular-biology studies are in progress that involve the use of nuclear and mitochondrial markers to confirm the extent of the genetic divergence prior to the establishment of final decision on the taxonomic status of the two strains of S. intercalatum.

Polymerase chain reaction assay based on a highly repeated sequence of Schistosoma haematobium: a potential tool for monitoring schistosome-infested water.

We have cloned from Schistosoma haematobium genome a repeated sequence, the DraI repeated sequence, which consists of tandemly arranged 121-bp-long units and which is highly abundant (approximately 15% of the S. haematobium genome). By these features, the DraI repeat is similar to the Sm1-7 sequence of Schistosoma mansoni previously described by us. However, their nucleotide sequences are profoundly different. Polymerase chain reaction (PCR) primers were designed on the basis of the DraI sequence information and were used in a PCR assay by which as little as 10 fg of schistosomal DNA as well as individual cercariae were detected. The DraI repeat cross-hybridized with DNA from Schistosoma bovis, Schistosoma magrebowiei, Schistosoma mattheei, Schistosoma curassoni, and Schistosoma intercalatum, but not with DNA from S. mansoni nor from Trichobilharzia ocellata and Echinostoma sp. A potential value of this PCR assay is suggested for monitoring free-living cercariae and infected snails only in bodies free of cross-hybridizing species.

Compatibility of Schistosoma mansoni Cameroon and Biomphalaria pfeifferi Senegal.

The vectorial capacity of Biomphalaria pfeifferi from Ndiangue, Senegal, was investigated with an allopatric isolate of Schistosoma mansoni from Nkolbisson, Cameroon. The snail infection rate after exposure to a single miracidium per snail (MD1) was 56. 3 %, and 91.6%, for snails exposed to 5 miracidia per snail (MD5). The minimum pre-patent period was 21 days. The mean total cercarial production for the MDI group was 18,511 cercariae per snail, and 9757 cercariae for the MD5 group. The maximum production of cercariae for 1 day was 4892 observed in a snail from the MDI group at day 43 post-infection. The mean longevity of snails was higher in group MD1 (88 days p.i.) than in group MD5 (65 days p.i.). The chronobiological emergence pattern revealed a circadian rhythm with one shedding peak at mid-day. Comparisons are made with the vectorial capacity of the sympatric combination of B. pfeifferi Senegal/S. mansoni Senegal.

Biological control of the snail hosts of schistosomiasis in areas of low transmission: the example of the Caribbean area.

The biological control of schistosomiasis has already proven its efficiency in several habitats in the Caribbean area. Two main types of biological control agents, either trematode parasites or competitor snails have been studied and tested against the snail hosts of schistosomiasis in this region. The first one, Ribeiroia guadeloupensis, a trematode sterilizing Biomphalaria glabrata was successfully tested in a Guadeloupean pond housing a natural population of B. glabrata. The second agent involves several species of competitor snails belonging to the Ampullariidae (Pomacea glauca, Marisa cornuarietis) and Thiaridae (Tarebia granifera, Melanoides tuberculata) families. Ampullarid snails were tested with success in several West Indian islands such as Guadeloupe. Thiarid snails have also proven their efficiency but also their limits in several types of habitats in Guadeloupe, Martinique, St. Lucia and Venezuela. Competitor snails have also proven to be useful in preventing the recolonization by the snail hosts after molluscicide treatments. The case of the rivers of the littoral central part of Venezuela is particularly relevant to this issue. The island of Martinique also constitutes a good example of the importance of competitor snails in a post-transmission phase of schistosomiasis control. This island is a well-developed country where schistosomiasis transmission was interrupted in the 1970s. However, the reactivation of some transmission sites was observed in the 1980s. The introduction of M. tuberculata into these sites resulted in the interruption of transmission and the near total disappearance of the snail hosts. Presently, the thiarid snails have colonized the whole Martinican hydrographic system and maintain dense populations preventing an eventual recolonization by the planorbid snails and thus are maintaining a sustainable control.

Reproductive and mate choice strategies in the hermaphroditic flatworm Echinostoma caproni.

Due to the important role that mating systems play in the evolution of species, we investigate the selfing rate and mate choice in the simultaneous hermaphroditic parasite Echinostoma caproni (Trematoda). The echinostomes were maintained in two situations in mice: (1) double infections where the two individuals do or do not belong to the same geographic area isolate, and (2) triple infections where two of the three individuals originate from the same isolate and the third one originates from a different isolate. This experimental design permits analysis of intra- and interisolate selfing rates and of mate preference. We predict, in the first experiment, no difference between intra- and interisolate selfing rates. In the second experiment we expect a preferential outcrossing between individuals originating from the same isolate in order to avoid hybrid breakdown. The results obtained corroborate our predictions and emphasize the important and synergistic roles of selfing, inbreeding depression, and hybrid breakdown in the evolution of echinostome reproductive strategies.

In vitro culture of rediae of Echinostoma caproni.

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

Compatibility of Schistosoma mansoni and Biomphalaria pfeifferi in northern Senegal.

The construction of the Diama dam on the Senegal River and the ensuing ecological changes have led to a massive outbreak of Schistosoma mansoni infection in Northern Senegal, associated with very high intensity of infections, due to extremely intense transmission. The vectorial capacity of Biomphalaria pfeifferi from Ndombo, near Richard-Toll was investigated in order to assess the role of the snail-parasite relationship in this particular epidemiological situation. The results revealed an unusually high compatibility between the Senegalese S. mansoni strain and its local snail intermediate host, B. pfeifferi. The snail infection rate after exposure to a single miracidium per snail was 87%. The cercarial production of infected snails was very high, with a mean total production of 50,456 cercariae per snail. No significant difference was found in the total cercarial output between snails exposed to 1 miracidium and those exposed to 5 miracidia. The increase in the rate of cercarial output was significantly greater in snails exposed to 5 miracidia, but there was a higher mortality in this group. The chronobiological cercarial production pattern showed a peak around mid-day. The implications of these findings on the epidemiology of schistosomiasis in Northern Senegal are discussed.

Experimental evidence of hybrid breakdown between genetically distinct populations of Echinostoma caproni.

In this paper we investigate whether the assortative mating between individuals of the same genetic entity (i.e. coming from the same geographical area) of Echinostoma caproni, reported in a previous paper, may be explained by a post-zygotic isolating mechanism. The fecundity of the adults of 2 parental genetic entities and of their hybrids (i.e. F1, F2, F3) was quantified through 3 successive generations. Whereas the number of eggs released by F1 hybrids is similar to that of the mid-parent, that of recombinant hybrids (F2-3 hybrids) is significantly lower than that of F1 and that of the mid-parent. Since these results seem to demonstrate hybrid breakdown, 2 important factors maintaining reproductive isolation, i.e. pre-mating reproductive isolation and low hybrid fecundity, may influence the evolution of E. caproni.

Recent studies on the reproductive biology of the schistosomes and their relevance to speciation in the Digenea.

The members of the family Schistosomatidae, dioecious Digenea, are discussed with regard to their distribution, intermediate and definitive host-parasite relationships. The biological species concept is considered together with the difficulties of its application to Schistosoma spp. and the Digenea. The correlation between pairing of adult schistosomes, physical and sexual development and the maintenance of reproductive potential is emphasised. Development of the female reproductive system does not depend upon species-specific pairing. In some combinations, e.g., Schistosoma haematobium/Schistosoma intercalatum and Schistosoma bovis/Schistosoma curassoni, a specific mate choice system apparently does not exist, whereas it does in other combinations, e.g., Schistosoma mansoni/Schistosoma intercalatum. In mixed infections change of mate may occur and when the opportunity arises heterospecific pairs of worms will change partners to conspecific pairs. Interspecific pairing in adult schistosomes will lead to either hybridisation or parthenogenesis. Yet the majority of schistosomes that inhabit the same definitive host maintain their genetic identity: specific mate recognition, site selection within the host and heterologous immunity have been suggested as isolating mechanisms. Experimental intraspecific crosses have enabled evaluation of the degree to which some populations separated and became reproductively isolated through pre-mating isolating mechanisms, indicative of incipient speciation, e.g., the Lower Guinea and Zaire strains of S. intercalatum. The occurrence and significance of parthenogenesis in schistosomes and other species of Digenea are discussed. The consequences of interspecific mating interactions in schistosomes with regard to parasite epidemiology, interspecific competition and genetic heterogeneity are debated. Geographical isolation and host specificity represent important pre-zygotic isolating mechanisms. It is suggested that site selection within the host and heterologous immunity may both reduce interspecific genetic interchange when digenean parasites utilise the same definitive host.