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PubMed1995

A molecular phylogeny of the genus Echinococcus.

Three nucleotide data sets, two mitochondrial (COI and ND1) and one nuclear (ribosomal ITS1), have been investigated in order to resolve relationships among species and strains of the genus Echinococcus. The data have some unusual properties in that mitochondrial heteroplasmy was detected in one strain of E. granulosus, and more than one class of ITS1 sequence variant can occur in a single isolate. The data failed to support the hypothesis that E. granulosus, as it is currently viewed, is a single valid species. Rather, the strains of E. granulosus seem to comprise at least three evolutionarily diverse groups, the sheep strain group, bovine strain group and horse strain group. Molecular distances between them are comparable to, or greater than, molecular evolutionary distances observed between recognized species. The affinities of the cervid strain of E. granulosus are unclear because of ambiguous data, but this strain does not appear to be ancestral to others. E. multilocularis may not be distinct from E. granulosus. However, the remaining two species. E. vogeli and E. oligarthrus appear distinct and rather distant from the first two. Based on the results presented here, taxonomic revision of the genus is clearly warranted.

Animals↗

PubMed2001

Molecular approaches to the taxonomic position of Peruvian potato cyst nematodes and gene pool similarities in indigenous and imported populations of Globodera.

Peruvian potato cyst nematode populations were analysed to assess both their inter- and intraspecific similarities. ITS--RFLP and two satellite DNA sequences were used as taxonomic tools. Both techniques have confirmed that the Peruvian populations have as their closest relatives the European Globodera pallida, despite the detection of clear differences that prevents us from assigning these South American populations unambiguously to any Globodera species. A more precise study of the variability of these Peruvian populations was investigated and they were compared with the imported European populations using protein (2-DGE) and DNA (RAPD) datasets. The clear distinction between the Peruvian and the European populations was confirmed and, inside each group, no correlation was found between the pathotype classification and the observed clustering of the populations. Surprisingly, while RAPDs revealed a higher variability in the Peruvian group than in the European one, some characteristic proteins were found by 2-DGE in some European populations, whereas it was impossible to find any in the Peruvian populations. It is concluded that the primary founders of the European populations may have an origin other than that of the Peruvian populations involved in this study.

Animals↗

PubMed1996

Genetic markers in ribosomal DNA for hookworm identification.

Polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) analysis of ribosomal (r) DNA was conducted on Uncinaria stenocephala, Ancylostoma caninum, A. tubaeforme and A. ceylanicum. The rDNA region spanning the first and second internal transcribed spacers (ITS1 and ITS2) plus the 5.8S (ITS+) gene was amplified by PCR from each of the species, digested separately with 9 restriction endonucleases and the fragments separated by agarose gel electrophoresis. Characteristic PCR-RFLP patterns were produced for each morphologically defined species using some of the endonucleases. The present study demonstrated that the ITS+ provides genetic markers for the delineation of each species examined and suggests that this region of rDNA will be useful for the identification of other hookworms from a range of hosts. The results are likely to have important implications for studying the genetic structure of hookworm populations, the systematics and the epidemiology of hookworm infections.

Ancylostomatoidea↗

PubMed1997

Species-specific polymerase chain reaction for the differentiation of larvae from Dictyocaulus viviparus and Dictyocaulus eckerti.

Using substantial interspecific differences between the second internal transcribed spacer (ITS2) region within the rDNA gene of Dictyocaulus eckerti and Dictyocaulus viviparus a species-specific PCR was developed to distinguish between lungworm larvae of the two species from fallow deer and cattle. It was found that the method of DNA extraction was crucial for the sensitivity of the PCR. With serial dilutions of DNA extracted from 10,000 larvae the ITS2 fragment could be amplified from all dilutions down to a calculated amount of DNA equivalent to one larva. Using lower numbers of larvae, DNA from at least 100 larvae was necessary for a successful amplification. From this extraction a species-specific polymerase chain reaction (PCR) product was generated with a calculated amount of DNA equivalent to 33 larvae, whereas amplification of further diluted DNA was not successful. However, in a direct PCR single larvae could be detected after direct PCR amplification without preceding DNA extraction.

Animals↗

PubMed1998

5'-flanking sequences required for efficient transcription in vitro of 5S RNA genes, in the related nematodes Caenorhabditis elegans and Caenorhabditis briggsae.

In the nematode C. elegans, we had previously observed apparent species specificity in 5S RNA transcription. We have now undertaken a further study of 5S RNA gene transcription in this organism and in the related nematode, C. briggsae; the latter was chosen because it might show evolutionarily conserved, functionally important features. Deletion mutagenesis and transcription in vitro, followed by more precise replacements of short blocks of 5' sequence, show that a short, TATA-like sequence at -25 is essential for efficient transcription in vitro of the 1.0-kb C. elegans 5S DNA repeat, and of both C. briggsae 0.7- and 1.0-kb 5S DNA repeats. Internal sequences within the 5S RNA gene appear to be required and can compete for limiting transcription components, whereas 5' flanking sequences do not. These observations suggest that the process of 5S RNA transcription is similar in these nematodes and other higher eukaryotes.

Animals↗

PubMed1997

Genetic diversity among a complex of cereal cyst nematodes inferred from RFLP analysis of the ribosomal internal transcribed spacer region.

This study examined the restriction polymorphism (RFLP) of the nuclear ribosomal DNA in Heterodera avenae, H. filipjevi, H. mani, H. latipons, and the taxonomically unclear Gotland strain in order to establish a molecular characterization and phylogenetic relationships in the complex of cereal cyst nematodes (CCN). The internal transcribed spacer (ITS) and 5.8S rDNA were amplified by PCR from a single female or a cyst of 27 different geographic isolates of the CCN complex and one population of H. schachtii, used as outgroup. The amplified product was 1.2 kb long and 14 of 15 enzymes produced restriction fragments for each isolate. Relationships between populations were determined from UPGMA analysis based on distance values calculated from RFLP data. Digestions with TaqI clearly differentiated H. avenae, H. latipons, and a group composed of H. filipjevi and the Gotland strain. Six endonucleases (HaeIII, HinfI, ItaI, PstI, TaqI, and Tru9I) produced the same restriction pattern with H. filipjevi and the Gotland strain, and both were clearly separated from H. avenae with PstI. Restriction sites have revealed a mixture of the species H. latipons and H. avenae, and possible infraspecific variation in H. avenae. The inferred phylogenetic relationships of species in the CCN complex are in agreement with their morphological characterization.

Animals↗

PubMed2003

Molecular phylogeny of Haematoloechus Looss, 1899 (Digenea: Plagiorchiidae), with emphasis on North American species.

Phylogenetic hypothesis of 23 populations corresponding to 18 species of the digenean Haematoloechus from America, Europe, and Africa, based on ribosomal DNA 28S partial sequences (approximately 890 bp), is presented. Genetic divergence between the in-group and the out-groups ranged from 9.7 to 14.5% and within the in-group, from 0.9 to 12.2%. Eight most parsimonious trees 569 steps long were obtained, with a consistency index of 72%. Groups in the tree are not congruent with those in previous classification schemes of species in the genus, based on a small number of morphological characters. For this subset of Haematoloechus species, plesiomorphic hosts are species of Rana, with 2 colonizations to other amphibian groups. African species appear to have diverged after the separation of Gondwana and Laurasia. Therefore, South American species should appear as the closest relatives of African species when included in the analysis. The evidence presented suggests an ancestral wide distribution of North American representatives of the group, followed by successive contraction, amplification, and fragmentation of ranges and speciation events as a result of the intense volcanic activity in the central part of Mexico since the late Tertiary, the drying climate of western and central United States and northwestern Mexico from the early Eocene to the Pleistocene, and the glaciation during the Pleistocene.

Animals↗

PubMed2006

Mitochondrial cytochrome c oxidase subunit 1 gene and nuclear rDNA regions of Enterobius vermicularis parasitic in captive chimpanzees with special reference to its relationship with pinworms in humans.

Sequences of mitochondrial cytochrome c oxidase subunit 1 (CO1) gene, nuclear internal transcribed spacer 2 (ITS2) region of ribosomal DNA (rDNA), and 5S rDNA of Enterobius vermicularis from captive chimpanzees in five zoos/institutions in Japan were analyzed and compared with those of pinworm eggs from humans in Japan. Three major types of variants appearing in both CO1 and ITS2 sequences, but showing no apparent connection, were observed among materials collected from the chimpanzees. Each one of them was also observed in pinworms in humans. Sequences of 5S rDNA were identical in the materials from chimpanzees and humans. Phylogenetic analysis of CO1 gene revealed three clusters with high bootstrap value, suggesting considerable divergence, presumably correlated with human evolution, has occurred in the human pinworms. The synonymy of E. gregorii with E. vermicularis is supported by the molecular evidence.

Animals↗

PubMed2005

The complete mitochondrial genome of Anisakis simplex (Ascaridida: Nematoda) and phylogenetic implications.

We determined the nucleotide sequence of the complete mitochondrial genome of the nematode species Anisakis simplex. The genome is circular, 13,916 bp in size and conforms to the general characteristics of nematode mitochondrial DNAs. The gene arrangement of A. simplex is the same as that of Ascaris suum and almost identical to those of rhabditid species with a minor exception concerning the relative position of the AT-rich and non-coding regions and radically different from those of spirurid species. Along with comparisons of gene arrangement, phylogenetic analyses (maximum parsimony, neighbour joining and maximum likelihood methods) based on concatenated amino acid sequences of 12 protein-coding genes from 13 nematode species provided strong support for the sister-group relationship between Ascaridida and Rhabditida. The Shimodaira-Hasegawa and Templeton's tests both rejected the alternative hypothesis of a closer relationship between Ascaridida and Spirurida. These results contradicted the traditional view of nematode classification and a recent molecular phylogenetic study of 18S rDNA data that assigned Ascaridida and Spirurida as being a sister-group. Mapping of gene arrangement across the phylogenetic tree lead to the assumption that the conserved gene arrangement found in Ascaridida-Rhabditida members might have been acquired after the most recent common ancestor of ascaridid/rhabditid members branched off from the basal stock of the rhabditid lineage.

Amino Acid Sequence↗

PubMed2001

Phylogenetic identification of Sparganum proliferum as a pseudophyllidean cestode by the sequence analyses on mitochondrial COI and nuclear sdhB genes.

Sparganum proliferum is a larval cestode for which the adult stage is unknown. It is characterized by the continuous branching and budding when parasitized to humans, and causes fatal human sparganosis. However, the biological features of S. proliferum, including its taxonomic status, still remain obscure. Our previous investigation suggested that S. proliferum might be phylogenetically distinct from Spirometra erinaceieuropaei, by the analysis on mitochondrial NADH dehydrogenase subunit 3 (ND3) gene. However, mitochondrial DNA sequence in Platyhelminth is known to have heteroplasmy within a species. Therefore, in the present study, we have investigated the complete nucleotide sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene and the partial nucleotide sequences of nuclear coded succinate dehydrogenase iron-sulfur protein subunit gene (sdhB). The results clearly demonstrated that S. proliferum is a distinct species from S. erinaceieuropaei, and that S. proliferum belongs to the order Pseudophyllidea.

Amino Acid Sequence↗

PubMed2004

A single nucleotide polymorphism map of the mitochondrial genome of the parasitic nematode Cooperia oncophora.

The 13,636 bp mitochondrial (mt) genome sequence of the trichostrongylid nematode Cooperia oncophora was determined. Like the mt genomes of other nematodes it is AT rich (76.75%) and cytidine is the least common nucleoside in the coding strand. There are 2 ribosomal RNA (rrn) genes, 22 transfer RNA (trn) genes and 12 protein coding genes. The relatively short AT-rich region (304 bp) and the lack of a non-coding region between two of the NADH dehydrogenase genes, nad3 and nad5, makes the mt genome of C. oncophora one of the smallest known to date, having only 525 bp of non-coding regions in total. The majority of the C. oncophora protein encoded genes are predicted to end in an abbreviated stop codon like T or TA. In total, 426 single nucleotide polymorphisms (SNP) were mapped on the mt genome of C. oncophora, which is an average of 1 polymorphism per 32 bp. The most common SNPs in the mt genome of C. oncophora were G/A (59.2%) and C/T (28.4%) transitions. Synonymous substitutions (86.4%) were favoured over non-synonymous substitutions. However, the degree of sequence conservation between individual protein genes of different parasitic nematode species did not always correspond to the relative number of non-synonymous SNPs. The mt genome sequence of C. oncophora presents the first mt genome of a member of the Trichostrongyloidea and will be of importance in refining phylogenetic relationships between nematodes. The, still limited, SNP map presented here provides a basis for obtaining insight into the genetic diversity present in the different protein coding genes, trn, rrn and non-coding regions. A more detailed study of the more variable regions will be of use in determining the population genetic structure of C. oncophora. Ultimately this knowledge will add to the understanding of the host-parasite relationship.

Amino Acid Sequence↗

PubMed2006

A molecular phylogeny of the genus Echinococcus inferred from complete mitochondrial genomes.

Taxonomic revision by molecular phylogeny is needed to categorize members of the genus Echinococcus (Cestoda: Taeniidae). We have reconstructed the phylogenetic relationships of E. oligarthrus, E. vogeli, E. multilocularis, E. shiquicus, E. equinus, E. ortleppi, E. granulosus sensu stricto and 3 genotypes of E. granulosus sensu lato (G6, G7 and G8) from their complete mitochondrial genomes. Maximum likelihood and partitioned Bayesian analyses using concatenated data sets of nucleotide and amino acid sequences depicted phylogenetic trees with the same topology. The 3 E. granulosus genotypes corresponding to the camel, pig, and cervid strains were monophyletic, and their high level of genetic similarity supported taxonomic species unification of these genotypes into E. canadensis. Sister species relationships were confirmed between E. ortleppi and E. canadensis, and between E. multilocularis and E. shiquicus, regardless of the analytical approach employed. The basal positions of the phylogenetic tree were occupied by the neotropical endemic species, E. oligarthrus and E. vogeli, whose definitive hosts are derived from carnivores that immigrated from North America after the formation of the Panamanian land bridge. Host-parasite co-evolution comparisons suggest that the ancestral homeland of Echinococcus was North America or Asia, depending on whether the ancestral definitive hosts were canids or felids.

Animals↗

PubMed1998

A robust molecular phylogeny of the Tricladida (Platyhelminthes: Seriata) with a discussion on morphological synapomorphies.

The suborder Tricladida (Platyhelminthes: Turbellaria, Seriata) comprises most well-known species of free-living flatworms. Four infraorders are recognized: (i) the Maricola (marine planarians); (ii) the Cavernicola (a group of primarily cavernicolan planarians); (iii) the Paludicola (freshwater planarians); and (iv) the Terricola (land planarians). The phylogenetic relationships among these infraorders have been analysed using morphological characters, but they remain uncertain. Here we analyse the phylogeny and classification of the Tricladida, with additional, independent, molecular data from complete sequences of 18S rDNA and 18S rRNA. We use maximum parsimony and neighbour-joining methods and the characterization of a unique gene duplication event involving the Terricola and the dugesiids to reconstruct the phylogeny. The results show that the Maricola is monophyletic and is the primitive sister group to the rest of the Tricladida (the Paludicola plus the Terricola). The Paludicola are paraphyletic since the Terricola and one paludicolan family, the Dugesiidae, share a more recent common ancestor than the dugesiids with other paludicolans (dendrocoelids and planariids). A reassessment of morphological evidence may confirm the apparent redundancy of the existing infraorders Paludicola and Terricola. In the meantime, we suggest replacing the Paludicola and Terricola with a new clade, the Continenticola, which comprises the families Dugesiidae, Planariidae, Dendrocoelidae and the Terricola.

Animals↗

PubMed2003

Enterobius vermicularis: ancient DNA from North and South American human coprolites.

A molecular paleoparasitological diagnostic approach was developed for Enterobius vermicularis. Ancient DNA was extracted from 27 coprolites from archaeological sites in Chile and USA. Enzymatic amplification of human mtDNA sequences confirmed the human origin. We designed primers specific to the E. vermicularis 5S ribosomal RNA spacer region and they allowed reproducible polymerase chain reaction identification of ancient material. We suggested that the paleoparasitological microscopic identification could accompany molecular diagnosis, which also opens the possibility of sequence analysis to understand parasite-host evolution.

Animals↗

PubMed1999

PCR-based technology in veterinary parasitology.

DNA technology is having a major impact in many areas of veterinary parasitology. In particular, the polymerase chain reaction (PCR) has found broad applicability because its sensitivity permits enzymatic amplification of gene fragments from minute quantities of nucleic acids derived from limited amounts of parasite material. This paper discusses some recent applications of PCR-based methods to parasites and highlights their usefulness or potential for those of veterinary importance. The focus is on PCR tools for the accurate identification of parasites and their genetic characterisation, the diagnosis of infections, the isolation and characterisation of expressed genes, the detection of anthelmintic resistance, and mutation scanning approaches for the high resolution analysis of PCR products.

Animals↗

PubMed1994

The phylogenetic relationships of Kronborgia (Platyhelminthes, Fecampiida) based on comparison of 18S ribosomal DNA sequences.

Approximately 580 bp at the 5' end of the small subunit RNA gene were amplified by PCR for 19 platyhelminth taxa, and Homo and Artemia were used as outgroups. These were analysed to test the hypothesis that fecampiids and Neodermata are sister groups. No evidence was found that the fecampiid Kronborgia isopodicola is closely related to the Neodermata or to the Rhabdocoela (in which the fecampiids are usually included). Morphological, including ultrastructural, characters and DNA data do not support a close relationship of fecampiids with any other platyhelminth taxon, although the DNA sequence analysis provides some evidence that the Acoela and Tricladida are closest. Fecampiids are sufficiently different from any other platyhelminth group to warrant establishment of a class, Fecampiida, for them. A diagnosis of the new class is given.

Animals↗

PubMed2006

A novel non-coding DNA family in Caenorhabditis elegans.

Many repetitive elements, for example, SINEs, LINEs, LTR-retrotransposons and other SSRs are dispersed throughout eukaryotic genomes. To understand the biological function of these repetitive elements is of great current research interest. In this study, we report on the identification of a novel non-coding DNA family, designated CE1 family, in the nematode C. elegans genome. Some CE1 elements constituted a large palindrome sequence. The CE1 elements were interspersed at 95 sites in the C. elegans genome. Most of the CE1 elements were associated with, or were within, protein-coding genes. The sequence of the CE1 elements indicated that some could form a hairpin structure. One of the CE1 family, CE1(bs258), is located in the first intron of a novel gene, C46H11.6 which encodes a PDZ/DHR/GLGF domain protein. In gfp and lacZ reporter gene assays the CE1(bs258) element appeared to behave as an enhancer element for the expression of C46H11.6 but no effect on the expression of the opposite direction gene, pat-10 which encodes the body-wall muscle troponin C. The CE1(bs258) RNA transcript was detected by RT-PCR even when CE1(bs258) was located in an intron. We conclude that CE1 elements are involved in the expression of adjacent genes and are therefore selectively retained in the C. elegans genome. We discussed a biological function of the CE1(bs258) having many transcription factor-binding sites.

Animals↗

PubMed2002

On the systematic position of Ophiosacculus Macy, 1935 (digenea: Lecithodendriidae), with the erection of the Ophiosacculinae n. subfam.

The phylogenetic relationships and systematic position of the digenean genus Ophiosacculus Macy, 1935 has been controversial and opinions of different authors on its systematic position and content are contradictory. Molecular analysis based on the partial sequences of the large subunit ribosomal DNA gene of the type and only valid species of the genus, Ophiosacculus mehelyi (Mödlinger, 1930), as well as previously published sequences of members of several families of Plagiorchiata (including the Allassogonoporidae, Lecithodendriidae and Pleurogenidae as potential relatives of Ophiosacculus) has shown that Ophiosacculus forms a clade with the typical representatives of the Lecithodendriidae from bats. Ophiosacculus is basal to the cluster containing Lecithodendrium, Prosthodendrium and Pycnoporus and has quite pronounced differences in the sequenced fragment compared to these genera. Based on the results of the molecular study, morphological characteristics of Ophiosacculus (in particular, possession of a seminal vesicle lying freely in parenchyma) and the fact that the type-specimen of Gyrabascus brevigastrus Macy, 1835 (type-species of the monotypic genus Gyrabascus and type-genus of the subfamily Gyrabascinae) belongs to Allassogonoporus, a new subfamily, the Ophiosacculinae, with Ophiosacculus as the type-genus, is established within the Lecithodendriidae. Molecular study did not support a close phylogenetic relationship between Allassogonoporus and Ophiosacculus, although several authors previously allocated both these genera to the Allassogonoporidae. Morphological study revealed the position of the genital pore in O. mehelyi to be at the posterior margin of the ventral sucker. An amended diagnosis of Ophiosacculus and a diagnosis of Ophiosacculinae n. subfam. are given.

Animals↗