Population genetics of African Schistosoma species.

Blood flukes within the genus Schistosoma (schistosomes) are responsible for the major disease, schistosomiasis, in tropical and sub-tropical areas. This disease is predominantly present on the African continent with more than 85% of the human cases. Schistosomes are also parasites of veterinary importance infecting livestock and wildlife. Schistosoma population genetic structure and diversity are important characteristics that may reflect variations in selection pressures such as those induced by host (mammalian and snail) environments, habitat change, migration and also treatment/control interventions, all of which also shape speciation and evolution of the whole Schistosoma genus. Investigations into schistosome population genetic structure, diversity and evolution has been an area of important debate and research. Supported by advances in molecular techniques with capabilities for multi-locus genetic analyses for single larvae schistosome genetic investigations have greatly progressed in the last decade. This paper aims to review the genetic studies of both animal and human infecting schistosome. Population genetic structures are reviewed at different spatial scales: local, regional or continental (i.e. phylogeography). Within species genetic diversities are discussed compared and the compounding factors discussed, including the effect of mass drug administration. Finally, the ability for intra-species hybridisation questions species integrities and poses many questions in relation to the natural epidemiology of co-endemic species. Here we review molecularly confirmed hybridisation events (in relation to human disease) and discuss the possible impact for ongoing and future control and elimination.

Diversity and host specificity of monogenean gill parasites (Platyhelminthes) of cichlid fishes in the Bangweulu-Mweru ecoregion.

This study represents the first exploration of the parasite fauna of cichlid fishes in the Mweru-Luapula subregion (Central Africa). Twelve species of cichlids and 14 species of Monogenea from three genera (Cichlidogyrus, Gyrodactylus and Scutogyrus) were collected. We present a first record of the gill parasite fauna of eight host species, Oreochromis mweruensis, Orthochromis sp. 'Mambilima', Sargochromis mellandi, Serranochromis angusticeps, S. stappersi, S. thumbergi and Tylochromis mylodon. The host range of ten parasite species was expanded. The study further includes the description of Cichlidogyrus consobrini sp. n. from S. mellandi and Orthochromis sp. 'Mambilima'. A new morphotype of C. halli is characterized, and three species - C. papernastrema, C. quaestio and C. zambezensis - are redescribed. Furthermore, the biodiversity and host specificity of these parasites is compared with that of cichlid parasites from Lake Kariba and Cameroon. Two species, including C. consobrini sp. n. and a new morphotype of C. halli, are putative endemics. The parasite fauna in Bangweulu-Mweru is highly similar in species composition to Lake Kariba, but in Bangweulu-Mweru the same parasite species are more host-specific, probably because of hydrogeographical differences between the two regions.

Inbreeding within human Schistosoma mansoni: do host-specific factors shape the genetic composition of parasite populations?

The size, structure and distribution of host populations are key determinants of the genetic composition of parasite populations. Despite the evolutionary and epidemiological merits, there has been little consideration of how host heterogeneities affect the evolutionary trajectories of parasite populations. We assessed the genetic composition of natural populations of the parasite Schistosoma mansoni in northern Senegal. A total of 1346 parasites were collected from 14 snail and 57 human hosts within three villages and individually genotyped using nine microsatellite markers. Human host demographic parameters (age, gender and village of residence) and co-infection with Schistosoma haematobium were documented, and S. mansoni infection intensities were quantified. F-statistics and clustering analyses revealed a random distribution (panmixia) of parasite genetic variation among villages and hosts, confirming the concept of human hosts as 'genetic mixing bowls' for schistosomes. Host gender and village of residence did not show any association with parasite genetics. Host age, however, was significantly correlated with parasite inbreeding and heterozygosity, with children being more infected by related parasites than adults. The patterns may be explained by (1) genotype-dependent 'concomitant immunity' that leads to selective recruitment of genetically unrelated worms with host age, and/or (2) the 'genetic mixing bowl' hypothesis, where older hosts have been exposed to a wider variety of parasite strains than children. The present study suggests that host-specific factors may shape the genetic composition of schistosome populations, revealing important insights into host-parasite interactions within a natural system.

Hybridisation between the two major African schistosome species of humans.

It is generally accepted that Schistosoma mansoni and Schistosoma haematobium, causing intestinal and urinary schistosomiasis, respectively, are not able to hybridise, due to the high phylogenetic distance between them. Cloning of nuclear internal transcribed spacer rDNA and partial mitochondrial cytochrome c oxidase 1 fragments revealed two internal transcribed spacer rDNA genotypes within single eggs and miracidia, one identical to S. mansoni and the other identical to S. haematobium, suggesting hybrid ancestry. The cytochrome c oxidase 1 clones always belonged to only one of the parental species. This demonstrates that offspring of heterologous pairing between these two species is not (always) parthenogenetic.

Regular treatments of praziquantel do not impact on the genetic make-up of Schistosoma mansoni in Northern Senegal.

The Senegal River Basin (SRB) experienced a major epidemic of intestinal schistosomiasis in the early nineties, after the construction of a dam for irrigation purposes. Exceptionally low cure rates following praziquantel (PZQ) treatment at the onset of the epidemic raised concerns about PZQ resistant strains of Schistosoma mansoni, although they could also be attributed to the intense transmission at that time. A field study in the same region more than 15 years later found cure rates for S. mansoni still to be low, whereas Schistosomahaematobium responded well to treatment. We collected S. mansoni miracidia from children at base-line prior to treatment, six months after two PZQ treatments and two years after the start of the study when they had received a total of five PZQ treatments. In total, 434 miracidia from 12 children were successfully genotyped with at least six out of nine DNA microsatellite loci. We found no significant differences in the genetic diversity of, and genetic differentiation between parasite populations before and after repeated treatment, suggesting that PZQ treatment does not have an impact on the neutral evolution of the parasite. This is in stark contrast with a similar study in Tanzania where a significant decrease in genetic diversity was observed in S. mansoni miracidia after a single round of PZQ treatment. We argue that PZQ resistance might play a role in our study area, although rapid re-infection cannot be excluded. It is important to monitor this situation carefully and conduct larger field studies with short-term follow-up after treatment. Since PZQ is the only general schistosomicide available, the possibility of PZQ resistance is of great concern both for disease control and for curative use in clinical practice.

Optimal sample storage and extraction procotols for reliable multilocus genotyping of the human parasite Schistosoma mansoni.

Genotyping individual larval stages and eggs of natural parasite populations is complicated by the difficulty of obtaining reliable genotypes from low quantity DNA template. A suitable storage and extraction protocol, together with a thorough quantification of genotyping errors are therefore crucial for molecular epidemiological studies. Here we test the robustness, handling time, ease of use, cost effectiveness and success rate of various fixation (Whatman FTA(®) Classic and Elute Cards, 70% EtOH and RNAlater(®)) and subsequent DNA extraction methods (commercial kits and proteinase K protocol). None of these methods require a cooling chain and are therefore suitable for field collection. Based on a multiplex microsatellite PCR with nine loci the success and reliability of each technique is evaluated by the proportion of samples with at least eight scored loci and the proportion of genotyping errors. If only the former is taken into account, FTA(®) Elute is recommended (83% success; 44% genotyping error; 0.2 €/sample; 1h 20 m handling time). However, when also considering the genotyping errors, handling time and ease of use, we opt for 70% EtOH with the 96-well plate technology followed by a simple proteinase K extraction (73% success; 0% genotyping error; 0.2 €/sample; 15m handling time). For eggs we suggest (1) to pool all eggs per person in 1.5 ml tubes filled with 70% EtOH for transport and (2) to identify each egg to species level prior to genotyping. To this end we extended the Rapid diagnostic PCR developed by Webster et al. (2010) with a S. mansoni-specific primer to discriminate between S. mansoni, S. haematobium and S. bovis in a single PCR reaction. The success rate of genotyping eggs was 75% (0% genotyping error). This is the first study to incorporate genotyping errors through re-amplification for the evaluation of schistosome sampling protocols and the identification of error-prone loci.

Signature of selection on the rhodopsin gene in the marine radiation of American seven-spined gobies (Gobiidae, Gobiosomatini).

In comparison with terrestrial and freshwater ecosystems, information about speciation modes and the role of selection in marine environments is scarce. Recent studies have indicated that spectral adaptation could play an important role in the diversification of marine species flocks. Natural selection influences specific amino acids (AAs) that are involved in the spectral tuning mechanism of visual pigment genes. To study the wider occurrence and the characteristics of spectral adaptation in marine radiations, a reinterpretation of the rhodopsin (RH1) data of American seven-spined gobies (genus Elacatinus; Gobiidae; Teleostei) was carried out. Reanalysis revealed that some AAs, which are well known in the literature as spectral tuning sites, are variable in Elacatinus. Those crucial AA substitutions originated polyphyletically, indicating convergent evolution within the genus Elacatinus. Moreover, statistical tests based on the d(N)/d(S) ratio detected selection in several phylogenetic lineages and at specific AAs. Many of these AAs were previously shown to be under selection in other marine radiations. Therefore, the current phylogenetic approach provided an extended list of AAs that are probably involved in spectral tuning, and which should be validated by mutagenic experiments.

Rapid diagnostic multiplex PCR (RD-PCR) to discriminate Schistosoma haematobium and S. bovis.

Schistosoma haematobium and S. bovis are widespread schistosome species causing human and cattle schistosomiasis, respectively, in Africa. The sympatric occurrence of these two species and their ability to infect the same Bulinus intermediate snail hosts necessitates precise methods of identification of the larval stages. A rapid diagnostic 'mulitplex' one-step polymerase chain reaction protocol (RD-PCR) was developed using cytochrome oxidase subunit 1 (COX1) mitochondrial DNA (mtDNA) to discriminate between S. haematobium and S. bovis. A single forward primer and two species-specific reverse primers were used to produce a polymerase chain reaction (PCR) fragment of 306 bp and 543 bp for S. bovis and S. haematobium, respectively. Serial dilutions were carried out on various lifecycle stages and species combinations to test the sensitivity and specificity of the primers. This RD-PCR proved highly sensitive, detecting a single larval stage and as little as 0.78 ng of genomic DNA (gDNA) from an adult schistosome, providing a cost-effective, rapid and robust molecular tool for high-throughput screening of S. haematobium and S. bovis populations. In areas where human and cattle schistosomiasis overlap and are transmitted in close proximity, this mitochondrial assay will be a valuable identification tool for epidemiological studies, especially when used in conjunction with other nuclear diagnostic markers.

Making the most of mitochondrial genomes--markers for phylogeny, molecular ecology and barcodes in Schistosoma (Platyhelminthes: Digenea).

An increasing number of complete sequences of mitochondrial (mt) genomes provides the opportunity to optimise the choice of molecular markers for phylogenetic and ecological studies. This is particularly the case where mt genomes from closely related taxa have been sequenced; e.g., within Schistosoma. These blood flukes include species that are the causative agents of schistosomiasis, where there has been a need to optimise markers for species and strain recognition. For many phylogenetic and population genetic studies, the choice of nucleotide sequences depends primarily on suitable PCR primers. Complete mt genomes allow individual gene or other mt markers to be assessed relative to one another for potential information content, prior to broad-scale sampling. We assess the phylogenetic utility of individual genes and identify regions that contain the greatest interspecific variation for molecular ecological and diagnostic markers. We show that variable characters are not randomly distributed along the genome and there is a positive correlation between polymorphism and divergence. The mt genomes of African and Asian schistosomes were compared with the available intraspecific dataset of Schistosoma mansoni through sliding window analyses, in order to assess whether the observed polymorphism was at a level predicted from interspecific comparisons. We found a positive correlation except for the two genes (cox1 and nad1) adjoining the putative control region in S. mansoni. The genes nad1, nad4, nad5, cox1 and cox3 resolved phylogenies that were consistent with a benchmark phylogeny and in general, longer genes performed better in phylogenetic reconstruction. Considering the information content of entire mt genome sequences, partial cox1 would not be the ideal marker for either species identification (barcoding) or population studies with Schistosoma species. Instead, we suggest the use of cox3 and nad5 for both phylogenetic and population studies. Five primer pairs designed against Schistosoma mekongi and Schistosoma malayensis were tested successfully against Schistosoma japonicum. In combination, these fragments encompass 20-27% of the variation amongst the genomes (average total length approximately 14,000bp), thus providing an efficient means of encapsulating the greatest amount of variation within the shortest sequence. Comparative mitogenomics provides the basis of a rational approach to molecular marker selection and optimisation.

The mitochondrial genome of Gyrodactylus salaris (Platyhelminthes: Monogenea), a pathogen of Atlantic salmon (Salmo salar).

In the present study, we describe the complete mitochondrial (mt) genome of the Atlantic salmon parasite Gyrodactylus salaris, the first for any monogenean species. The circular genome is 14,790 bp in size. All of the 35 genes recognized from other flatworm mitochondrial genomes were identified, and they are transcribed from the same strand. The protein-coding and ribosomal RNA (rRNA) genes share the same gene arrangement as those published previously for neodermatan mt genomes (representing cestodes and digeneans only), and the genome has an overall A+T content of 65%. Three transfer RNA (tRNA) genes overlap with other genes, whereas the secondary structure of 3 tRNA genes lack the DHU arm and 1 tRNA gene lacks the TphiC arm. Eighteen regions of non-coding DNA ranging from 4 to 112 bp in length, totalling 278 bp, were identified as well as 2 large non-coding regions (799 bp and 768 bp) that were almost identical to each other. The completion of the mt genome offers the opportunity of defining new molecular markers for studying evolutionary relationships within and among gyrodactylid species.

First report of Gyrodactylus spp. (Platyhelminthes: Monogenea) in the western Mediterranean Sea: molecular and morphological descriptions.

The Gyrodactylus spp. fauna on species of gobies, Pomatoschistus, Gobiusculus, and Knipowitschia (Gobiidae: Teleostei), from the western Mediterranean and Adriatic seas is strikingly similar to that found in the Baltic Sea and eastern Atlantic Ocean, both in morphology and in internal transcribed spacer (ITS) rRNA. The fauna consisted of Gyrodactylus branchialis, G. ostendicus, G. gondae, G. rugiensis, G. rugiensoides, and G. arcuatus. No new species have been found. A morphometric comparison between G. branchialis from the Mediterranean and Adriatic seas and its type locality in Ostend (Belgium) showed significant differences in ventral bar and marginal hook features. The morphometric variation was lower in G. rugiensis, whereas no significant differences were found in G. ostendicus. Gyrodactylus branchialis and G. ostendicus collected on P. microps were slightly different in the ITS rDNA (-0.6%) compared with specimens on the closely related P. marmoratus, probably reflecting ongoing speciation. A hybrid zone was identified in the Vaccarès lagoon complex (France) where both host species are sympatric. There was no clear geographic or host-related pattern in the variation found in the ITS2 rDNA in G. arcuatus sampled from P. microps, G. flavescens, Pungitius pungitius, K. panizzae, and its original host Gasterosteus aculeatus (2 polymorphic sites). Of all studied species, only G. arcuatus, G. rugiensis, and G. rugiensoides showed minor intraspecific variation in the ITS rDNA. Hence, the physical separation by a shoreline of more than 10,000 km is hardly reflected in the parasite ITS rDNA.

Spatio-temporal dynamics of the parasitic nematode Anguillicola crassus in Flanders, Belgium.

Despite Egusa's earlier warning of the damage that the parasitic nematode Anguillicola crassus could inflict on the European eel Anguilla anguilla, its introduction in Europe was a fact in the early 1980s. Based on an elaborate dataset on Anguillicola crassus infection of 11 river catchments, this paper presents the results of a detailed study on the dispersal of the parasite in Flanders, Belgium, and the host-parasite relationship. In addition, data from 1986 and 1997 are used for comparative purposes, providing a perspective on the temporal infection pattern over 15 yr. The presence of A. crassus in Flanders was first discovered in 1985; 2 yr later a survey revealed a prevalence of 34.1% and a mean infection intensity of 5.5, based on adult nematodes only, and 10 yr later the parasite was present at all 11 sites sampled. Prevalence had increased to 62.5 % but the mean infection intensity had decreased to 3.9 adults per infected eel. Finally, in the year 2000, a third study revealed that A. crassus was present in 139 of 140 investigated sites; a further increase in prevalence to 68.7% and a decrease in mean infection intensity to 3.4 adults per infected eel was observed. When all larval stages were taken into account, mean prevalence amounted to 88.1% and mean intensity to 5.5 adults. The high infection level in Flanders is thought to be the result of restocking with glass eel and yellow eel, both of which are susceptible to A. crassus. The general infection parameters were similar in all 11 river catchments. It is possible that in Flanders both prevalence and mean infection intensity are stabilizing due to density-dependent regulation of the parasite infrapopulation. Fibrotic swimbladder walls were observed, mainly in large eels, and 20% of the total number of nematodes consisted of encapsulated larvae in the surveys of 1997 and 2000; 8 cases of swimbladder regeneration were observed.

Deep divergence among subgenera of Gyrodactylus inferred from rDNA ITS region.

This paper adds new insight to a molecular phylogeny of Gyrodactylus, based on a complete sequence of the ITS rDNA region of 4 subgenera and a more detailed molecular analysis. We propose a hierarchical approach in elucidating the phylogeny of this species-rich genus. A total of 37 sequences (915-1239 bp) from 10 representative species from 4 out of 6 subgenera, as defined by Malmberg (1970), are included in the analysis. Genetic differences observed at the 5.8S locus provide objective criteria to separate (sub)genera, while deep genetic differences of the spacers form a sound basis for species-specific identification. We demonstrate that each Gyrodactylus subgenus possesses a unique sequence of the 5.8S gene. Thus, there is concordance between the 5.8S gene and the excretory system used by Malmberg (1970) as a diagnostic character of subgenus status. At the species level, there is a discrepancy between morphological and molecular variation. Whereas the morphological variation, expressed in the shape and size of the attachment apparatus, is very low, the molecular variation, expressed at the I