Characterisation of nuclear ribosomal DNA sequences from Onchocerca volvulus and Mansonella ozzardi (Nematoda: Filarioidea) and development of a PCR-based method for their detection in skin biopsies

The internal transcribed spacer region (ITS1, 5.8S gene and ITS2) of the two filarial nematodes Onchocerca volvulus and Mansonella ozzardi was sequenced, and two species-specific primers designed in the ITS2 to develop a PCR-based method for their specific detection and differentiation. When used with a universal reverse primer, the two species-specific primers gave amplification products of different size, which were readily separated in an agarose gel. The PCR was tested on skin biopsies from 51 people from three localities in Brazil where M. ozzardi is present, and results have been compared with those of parasitological examination of blood. The species-specific PCR gave a higher percentage of detection of infection by M. ozzardi than the parasitological examination of blood. No infection with O. volvulus was detected by PCR. This PCR-based assay may assist in determining the nature of infection in areas where both filarial species exist in sympatry.     

Molecular phylogenetics of blackflies of the Simulium damnosum complex and cytophylogenetic implications

The molecular phylogenetics of the Simulium damnosum complex, including vectors of the human parasite Onchocerca volvulus, from various parts of Africa was studied and compared with results of cytogenetic analyses. The sequence data of mitochondrial 16s and nuclear ITS2 rDNA revealed that the complex comprises two main clades, roughly covering the more easterly and westerly African taxa, respectively. However, striking inconsistencies in the tree topologies turned up between the DNA fragments regarding the position of certain subcomplexes and species. The cytophylogenetic relationships are better reflected in the ITS2 tree where Simulium pandanophilum and Simulium mengense constitute a basal, Central African clade of the entire complex and are therefore suggested to be the chromosomal roots too. Further divisions and the corresponding biogeographic interpretations are discussed. Several species and cytoforms are placed within the system for the first time. The phylogenetic relationships within the complex do hardly correlate with host preferences or other behavioral and ecological characteristics.     

Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.      

Onchocerca volvulus: limited heterogeneity in the nuclear and mitochondrial genomes

West African populations of Onchocerca volvulus endemic to the rain forest and savanna bioclimes of West Africa differ in their ability to induce ocular disease in infected individuals. In recent years, both clinical- and animal-model-based studies have implicated particular parasite antigens in the development of ocular onchocerciasis. To test the hypothesis that the difference in pathogenic potential of blinding and nonblinding parasites might be reflected in qualitative differences in antigens that have been implicated in the development of ocular onchocerciasis, we compared the sequences of two parasite antigens implicated in the development of ocular disease in blinding- and nonblinding-strain parasites. The results demonstrated a high level of homogeneity between the parasite strains in these genes. The study was extended to include additional nuclear genes encoding antigens that are commonly recognized by individuals infected with O. volvulus and to the mitochondrial genome of the parasite. The results demonstrate a high degree of homogeneity in both the nuclear and the mitochondrial genomes among O. volvulus isolates collected from several different sites in Africa and in the Americas. This high degree of genetic homogeneity may reflect the passage of the parasite through a recent genetic bottleneck.     

Onchocerca volvulus: genetic diversity of parasite isolates from Sudan

Onchocerciasis in Sudan exists in three distinct foci which exhibit differing clinical presentations. Previous studies have demonstrated that a tandemly repeated Onchocerca sequence family with a unit repeat length of 150 bp (the O-150 family) is a useful marker for deducing relationships among different O. volvulus populations. In the current study, the O-150 repeat families of O. volvulus from Sudan were analyzed and compared to each other and to those of parasites from West Africa. Similar to West African and American O. volvulus, the O-150 families of the Sudanese parasites could be divided into clusters within which little or no intracluster variation was evident, suggesting that the O-150 family in these parasites was subject to the forces of concerted evolution. Statistical analysis of the O-150 families from the different Sudanese parasite isolates, employing a nested algorithm based on an analysis of variance, revealed that O. volvulus endemic to the northern focus at Abu Hamed were significantly different from all other O. volvulus populations examined to date. In contrast, parasites from the southern and eastern foci of Sudan were indistinguishable from those endemic to the West African savanna. The significance of these data are discussed in light of knowledge of the biogeography and biology of transmission of O. volvulus in Africa.     

Molecular systematics of five Onchocerca species (Nematoda: Filarioidea) including the human parasite, O. volvulus, suggest sympatric speciation

The genus Onchocerca (Nematoda: Filarioidea) consists of parasites of ungulate mammals with the exception of O. volvulus, which is a human parasite. The relationship between O. volvulus, O. ochengi and O. gibsoni remains unresolved. Based on morphology of the microfilariae and infective larvae, vector transmission and geographical distribution, O. ochengi and O. volvulus have been placed as sister species. Nevertheless, the cuticle morphology and chromosomal data (O. volvulus and O. gibsoni have n=4 while O. ochengi is n=5) suggest that O. gibsoni could be more closely related to O. volvulus than O. ochengi. Sequences from the 12S rRNA, 16S rRNA and ND5 mitochondrial genes have been used to reconstruct the phylogeny of five Onchocerca species including O. volvulus. Analyses with maximum likelihood and maximum parsimony showed that O. ochengi is the sister species of O. volvulus, in accordance with the classification based on morphology and geographical location. The separate specific status of the species O. gutturosa and O. lienalis was supported, although their phylogenetic relationship was not well resolved. The analyses indicated that the basal species was O. gibsoni, a South-East Asian and Australasian species, but this result was not statistically significant. The possible involvement of sympatric speciation in the evolution of this group of parasites is discussed.     

Onchocerca volvulus: molecular cloning, primary structure, and expression of a microfilarial surface-associated antigen.

Although the filarial nematode parasite Onchocerca volvulus is an important human pathogen in large areas of Africa and Latin America, little is known of the molecular interactions that govern the clinical status of patients with this chronic, debilitating disease. As a step toward defining the parasite molecules important to the immunobiology of host-parasite interactions, we have identified and cloned a major surface-associated antigen expressed by O. volvulus microfilariae. Radiolabeling experiments demonstrated that O. volvulus microfilariae have a limited repertoire of peptides at the surface. Prominent among these labeled peptides is an 18-kDa component. Immunological cross-reactivity between a surface-associated component of Dirofilaria immitis microfilariae and the 18-kDa surface-associated molecule from O. volvulus was exploited in a strategy to clone this potentially important O. volvulus microfilarial antigen. The cross-reacting antibodies were used to immunoscreen O. volvulus cDNA expression libraries. One clone, M2f.e, contained an open reading frame of 495 bp encoding an 18.1-kDa protein (OVMS18). Antibodies produced against the expression product of M2f.e recognized an 18-kDa component in extracts of O. volvulus microfilariae and bound to the surface of intact O. volvulus and D. immitis microfilariae. Southern blot analyses showed that M2f.e-like sequences are present in the genomic DNA of a number of filarial nematode species, but not in DNAs from nonfilarial nematode species.     

Molecular relationships between populations of South African citrus thrips (Scirtothrips aurantii Faure) in South Africa and Queensland, Australia

Abstract   In response to the recent establishment of a population of South African citrus thrips ( Scirtothrips aurantii Faure) in Australia, we used DNA sequence data to examine whether this population is distinct from populations in South Africa. Mitochondrial and internal transcribed spacer (ITS2) DNA from populations from different host plants in Australia and South Africa showed no clear separation between populations that was entirely congruent with host plant or country of origin. Analysis of the ITS2 data was confounded by the presence of multiple different copies of the spacer in some populations. Neither the presence or absence, nor the sequence of these copies was clearly diagnostic for any one population. These preliminary data suggest that the Australian population is not a distinct species or subspecies from the populations of S. aurantii on either citrus or Bryophyllum in South Africa.     

Intraspecific diversity of the Cassava Green Mite Mononychellus progresivus (Acari: Tetranychidae) using comparisons of mitochondrial and nuclear ribosomal DNA sequences and cross-breeding

Intraspecific diversity in Cassava Green Mite Mononychellus progresivus Doreste was examined using individuals collected in Benin and the Congo and in Columbia and Brazil. Comparisons were based on mitochondrial and ribosomal DNA sequences and the results of several cross-breeding experiments. Sequence variation was examined in a total of 1139 base pairs (bp) constituting the ITS2 ribosomal DNA (805 bp) and a fragment of the Cytochrome Oxidase I (COI) gene (334 bp). Sequence divergence is low, ranging from 0% to 2.1% for COI and from 0% to 0.4% for ITS2. Inter-strain comparisons have shown that the two African populations appear to be identical. They were similar to the Colombian population while the Brazilian population was clearly different. The data support the hypothesis of a single introduction of the species in the two African populations. Crossing experiments have shown partial hybrid sterility, suggesting a genetic incompatibility consistent with differences detected by sequence data. The results show the usefulness of molecular markers as a tool for determining taxonomic status and dispersion paths in spider mites.     

Molecular variation and speciation in Antitrichia curtipendula s.l. (Leucodontaceae, Bryophyta)

Molecular variation in Antitrichia curtipendula (Hedw.) Brid. s.l. was studied based on the nuclear internal transcribed spacer (ITS) and the chloroplast markers trn L- trn F and rpl 16, and analysed by neighbour joining (for ITS; recombination present), maximum parsimony (chloroplast markers) and TCS (haplotype network). Old World plus E North American populations belong to a different lineage than those of W North America. These are molecularly well differentiated and are treated as A. curtipendula and A. gigantea (Sull. & Lesq.) Kindb. Two distinct groups of Old World haplotypes are separated by one 'missing' haplotype and are interpreted as cryptic species. Tropical African populations share one ITS deletion and form a lineage within one of the cryptic species. Molecular variation within A. gigantea , within each of the two cryptic Old World plus E North American (except tropical African) haplotype groups, and among tropical African populations are of similar magnitude, suggesting that analogous mechanisms and similar time spans explain the found variation. Events related to Pleistocene climatic oscillations are suggested as having caused this differentiation within each group, whereas the African lineage was probably split off before this. Identical tRNA-Gly sequences were found in 33 specimens; new primers were designed for rpl 16 and ITS 1+2.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 341–354.     

Geographic distribution of the muscle-dwelling nematode Parelaphostrongylus odocoilei in North America, using molecular identification of first-stage larvae

Molecular identification of dorsal-spined larvae (DSL) from fecal samples indicates that the protostrongylid parasite Parelaphostrongylus odocoilei occupies a broader geographic range in western North America than has been previously reported. We analyzed 2,124 fecal samples at 29 locations from thinhorn sheep (Ovis dalli dalli and O. d. stonei), bighorn sheep (Ovis canadensis canadensis and O. c. californiana), mountain goats (Oreamnos americanus), woodland caribou (Rangifer tarandus caribou), mule deer (Odocoileus hemionus hemionus), and black-tailed deer (O. h. columbianus). The DSL were recovered from populations of thinhorn sheep south, but not north, of the Arctic Circle, and they were not recovered from any of the bighorn sheep populations that we examined. In total, DSL were recovered from 20 locations in the United States and Canada (Alaska, Yukon Territory, Northwest Territories, British Columbia, Alberta, and California). The DSL were identified as P. odocoilei by comparing sequences of the second internal transcribed spacer (ITS2) region of ribosomal RNA among 9 protostrongylid species validated by adult comparative morphology. The ITS2 sequences were markedly different between Parelaphostrongylus and other protostrongylid genera. Smaller fixed differences served as diagnostic markers for the 3 species of Parelaphostrongylus. The ITS2 sequences (n = 60) of P. odocoilei were strongly conserved across its broad geographic range from California to Alaska. Polymorphism at 5 nucleotide positions was consistent with multiple copies of the ITS2 within individual specimens of P. odocoilei. This work combines extensive fecal surveys, comparative morphology, and molecular diagnostic techniques to describe comprehensively the host associations and geographic distribution of a parasitic helminth.     

Evidence for a discrete evolutionary lineage within Equatorial Guinea suggests that the tsetse fly Glossina palpalis palpalis exists as a species complex

Tsetse flies of the palpalis group are major vectors of Human African Trypanosomiasis in Africa. Accurate knowledge of species identity is essential for vector control. Here, we combine ribosomal internal transcribed spacer 1 ( ITS1 ), mitochondrial Cytochrome Oxidase 1 ( COI ) and microsatellites to determine the population structure and phylogenetic relations of Glossina p. palpalis in Equatorial Guinea. CO1 sequence data suggest that G. p. palpalis in Equatorial Guinea is a distinct subspecies from previously described G. p. palpalis in West Africa and Democratic Republic of Congo. Glossina p. palpalis in Equatorial Guinea and DRC share a common ancestor which diverged from West African G. p. palpalis around 1.9 Ma. Previous ITS1 length polymorphism data suggested the possible presence of hybrids in Equatorial Guinea. However, ITS1 showed incomplete lineage sorting compared with clearly defined COI groups, and data from 12 unlinked microsatellites provided no evidence of hybridization. Microsatellite data indicated moderate but significant differentiation between the populations analysed (Rio Campo, Mbini and Kogo). Moreover, unlike previous studies of G. p. palpalis , there was no evidence for heterozygote deficiency, presence of migrants or cryptic population structure. Variance effective population size at Rio Campo was estimated at 501–731 assuming eight generations per year. This study of the population genetics of G. p. palpalis in central Africa provides the first estimate of genetic differentiation between geographically separated G. p. palpalis populations.     

Onchocerca volvulus: application of the polymerase chain reaction to identification and strain differentiation of the parasite

Previous studies have demonstrated that the genome of Onchocerca volvulus contains a variable tandemly repeated DNA sequence family with a unit length of 150 bp. The variability of the 150-bp family has been exploited to develop O. volvulus strain and species specific DNA probes. Application of these DNA probes to the study of the epidemiologically most significant life cycle stages of the parasite has been confounded by several obstacles. These include the relative insensitivity of some of the DNA probes and the difficulty in releasing genomic DNA from infective larvae and skin microfilariae in a form that may be directly detected by hybridization to the probes. DNA sequence comparison of 18 known examples of the 150-bp repeat has been used to develop two populations of degenerate oligonucleotides. These oligonucleotides have been shown to support the amplification of the 150-bp repeat family from Onchocerca DNA, using the polymerase chain reaction. Both strain and species specific members of the repeat family are faithfully amplified, allowing characterization of a parasite on the basis of hybridization of the PCR amplification products to the previously developed DNA probes. This method is shown to be applicable to all diagnostically important forms of the parasite, including adults, infective larvae, and skin microfilariae. In addition, the method is capable of detecting O. volvulus infective larvae directly in extracts of blackfly vectors.     

Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum

Multilocus genotyping of microbial pathogens has revealed a range of population structures, with some bacteria showing extensive recombination and others showing almost complete clonality. The population structure of the protozoan parasite Plasmodium falciparum has been harder to evaluate, since most studies have used a limited number of antigen-encoding loci that are known to be under strong selection. We describe length variation at 12 microsatellite loci in 465 infections collected from 9 locations worldwide. These data reveal dramatic differences in parasite population structure in different locations. Strong linkage disequilibrium (LD) was observed in six of nine populations. Significant LD occurred in all locations with prevalence <1% and in only two of five of the populations from regions with higher transmission intensities. Where present, LD results largely from the presence of identical multilocus genotypes within populations, suggesting high levels of self-fertilization in populations with low levels of transmission. We also observed dramatic variation in diversity and geographical differentiation in different regions. Mean heterozygosities in South American countries (0.3-0.4) were less than half those observed in African locations (0. 76-0.8), with intermediate heterozygosities in the Southeast Asia/Pacific samples (0.51-0.65). Furthermore, variation was distributed among locations in South America (F:(ST) = 0.364) and within locations in Africa (F:(ST) = 0.007). The intraspecific patterns of diversity and genetic differentiation observed in P. falciparum are strikingly similar to those seen in interspecific comparisons of plants and animals with differing levels of outcrossing, suggesting that similar processes may be involved. The differences observed may also reflect the recent colonization of non-African populations from an African source, and the relative influences of epidemiology and population history are difficult to disentangle. These data reveal a range of population structures within a single pathogen species and suggest intimate links between patterns of epidemiology and genetic structure in this organism.     

Transmission mode affects the population genetic structure of Daphnia parasites

Parasite life cycle variation can shape parasite evolution, by predisposing them towards different population genetic structures. We compared the population genetic structure of two co-occurring parasite species of Daphnia, to collect evidence for their expected transmission modes. The ichthyosporean Caullerya mesnili has a direct life cycle, whereas the microsporidian Berwaldia schaefernai is hypothesized to require passage through a secondary host. The parasites were collected from three geographically isolated Daphnia populations. The nucleotide variation in the internal transcribed spacer (ITS) region was assessed at the within-individual, within-population and among-population levels, using amova. We detected significant partitioning at all levels, except for a lack of among-population variation in Berwaldia. This was confirmed by neighbour-joining and principal component analyses; Caullerya populations were distinct from each other, while there was much overlap among parasite isolates representing different populations of Berwaldia. This all implies a higher amount of gene flow for Berwaldia, consistent with the hypothesized transmission mode.     

Genetic analysis of southern African gemsbok (Oryx gazella) reveals high variability, distinct lineages and strong divergence from the East African Oryx beisa

We carried out a population genetic analysis of five southern African gemsbok (Oryx gazella) populations based on 530 bp of the mitochondrial control region and ten microsatellites in 75 individuals. Both markers show the high variability often observed in African bovids. Three of the populations which can be traced back to very small founding or current sizes do not show any signs of reduced variability compared to the remaining populations. The mitochondrial haplotypes form three distinct lineages which most likely originated in the Pleistocene when climate fluctuations led to periodical reduction and spreading of gemsbok habitat and which, today, are found throughout the distribution range. Bayesian microsatellite analyses yielded two groups, suggesting a more recent geographical differentiation following the admixture of the mtDNA lineages. Combining our sequences with available published data of the remaining oryx species allowed for a direct molecular comparison of O. gazella and O. beisa which have sometimes been considered a single species. The average genetic divergence between haplotypes from the two taxa was very high (39.9%), supporting their classification into two different species.     

Morphological Plasticity and Phylogeny in a Monogenean Parasite Transferring between Wild and Reared Fish Populations

It is widely accepted that disease interactions between cultured and wild fish occur repeatedly, although reported cases have mainly relied just on the observation of similar symptoms in affected populations. Whether there is an explicit pathogen transfer between fish stocks, or each develops its own pathogen population, has been insufficiently studied and rarely supported by molecular tools. In this study, we used population dynamics and genetic structure of the monogenean Furnestinia echeneis in reared and neighbouring wild sea bream to indicate pathogen transfer, characterized by the phenotypic plasticity of the parasite attachment apparatus and the lack of phylogenetic differentiation. The observed pattern of genetic variation inferred by nuclear DNA Internal Transcribed Spacer 1 (ITS1) and mtDNA cytochrome C oxidase 1 (COI), between parasite populations is most likely caused by a recent shared demographic history like a reduced species area in the last glacial period. In spite of such recent expansion that populations underwent, F. echeneis shows differentiation in haptor morphometry as an adaptive trait in closely related populations at the aquaculture site. This suggests that differentiation in morphology may occur relatively rapidly in this species and that adaptive forces, not the speciation process, drives this monogenean parasitation. On the other hand, the observed phylogenetic inertia suggests a low to moderate gene flow (based on F_ST) between parasites in cultured and wild fish, evidencing for the first time the transfer of pathogens at the aquaculture site inferred by a molecular tool.     

Intraspecific differentiation of Allium wallichii (Amaryllidaceae) inferred from chloroplast DNA and internal transcribed spacer fragments

In the present study, we used two chloroplast DNA (cpDNA) fragments (trnL-F and rps16) and the nuclear ribosomal internal transcribed spacer (ITS) sequence data to examine intraspecific differentiation and phylogeographical history of Allium wallichii. Based on wide scale sampling (28 populations and 174 individuals) across the entire distribution range of this species, 33 cpDNA haplotypes and 25 ITS ribotypes were detected in our investigation. These cpDNA haplotypes were divided into three major lineages, which was further supported by the ITS phylogenetic results. High haplotype/ribotype diversity and population differentiation, together with most of the haplotypes/ribotypes being exclusive to single populations, implied restricted gene flow among populations and significant geographical isolation. Nearly all populations with high haplotype/ribotype diversity were found in the Hengduan Mountain Region (HMR), whereas the populations of the Himalayas and Nanling Mountains showed a lower level, suggesting the HMR might serve as a potential divergence center for A. wallichii. The main lineages of A. wallichii diverged from each other between Mid–Late Pliocene and Early Quaternary based on two sets of molecular markers, indicating that the Quaternary climatic fluctuation could not have contributed greatly to the divergence of the main lineages of A. wallichii. Instead, the intricate topography and heterogeneous habitats resulting from the drastic uplift of the Qinghai–Tibet Plateau from the Late Pliocene could be responsible for the intraspecific differentiation of A. wallichii. The present study further highlights the importance of geographic isolation and habitat heterogeneity in shaping and maintaining high species diversity within the HMR.     

Cryptic speciation on the high seas; global phylogenetics of the copepod family Eucalanidae

Few genetic data are currently available to assess patterns of population differentiation and speciation in planktonic taxa that inhabit the open ocean. A phylogenetic study of the oceanic copepod family Eucalanidae was undertaken to develop a model zooplankton taxon in which speciation events can be confidently identified. A global survey of 20 described species (526 individuals) sampled from 88 locations worldwide found high levels of cryptic diversity at the species level. Mitochondrial (16S rRNA, CO1) and nuclear (ITS2) DNA sequence data support 12 new genetic lineages as highly distinct from other populations with which they are currently considered conspecific. Out of these 12, at least four are new species. The circumglobal, boundary current species Rhincalanus nasutus was found to be a cryptic species complex, with genetic divergence between populations unrelated to geographic distance. 'Conspecific' populations of seven species exhibited varying levels of genetic differentiation between Atlantic and Pacific basins, suggesting that continental landmasses form barriers to dispersal for a subset of circumglobal species. A molecular phylogeny of the family based on both mitochondrial (16S rRNA) and nuclear (ITS2, 18S rRNA) gene loci supports monophyly of the family Eucalanidae, all four eucalanid genera and the 'pileatus' and 'subtenuis' species groups.     

Genetic Variability of the Neogregarine Apicystis bombi,an Etiological Agent of an Emergent Bumblebee Disease

The worldwide spread of diseases is considered a major threat to biodiversity and a possible driver of the decline of pollinator populations, particularly when novel species or strains of parasites emerge. Previous studies have suggested that populations of introduced European honeybee (Apis mellifera) and bumblebee species (Bombus terrestris and Bombus ruderatus) in Argentina share the neogregarine parasite Apicystis bombi with the native bumblebee (Bombus dahlbomii). In this study we investigated whether A. bombi is acting as an emergent parasite in the non-native populations. Specifically, we asked whether A. bombi, recently identified in Argentina, was introduced by European, non-native bees. Using ITS1 and ITS2 to assess the parasite’s intraspecific genetic variation in bees from Argentina and Europe, we found a largely unstructured parasite population, with only 15% of the genetic variation being explained by geographic location. The most abundant haplotype in Argentina (found in all 9 specimens of non-native species) was identical to the most abundant haplotype in Europe (found in 6 out of 8 specimens). Similarly, there was no evidence of structuring by host species, with this factor explaining only 17% of the genetic variation. Interestingly, parasites in native Bombus ephippiatus from Mexico were genetically distant from the Argentine and European samples, suggesting that sufficient variability does exist in the ITS region to identify continent-level genetic structure in the parasite. Thus, the data suggest that A. bombi from Argentina and Europe share a common, relatively recent origin. Although our data did not provide information on the direction of transfer, the absence of genetic structure across space and host species suggests that A. bombi may be acting as an emergent infectious disease across bee taxa and continents.