Differentiation of Species and Populations of Aphelenchoides and of Ditylenchus angustus Using a Fragment of Ribosomal DNA

The polymerase chain reaction (PCR) was used to amplify a fragment of the ribosomal DNA (rDNA) from species and undescribed populations of Aphelenchoides and Ditylenchus angustus. The PCR primers used were based on conserved sequences in the 18S and 26S ribosomal RNA genes of Caenorhabditis elegans. In C. elegans, these primers amplify a 1,292 base pair (bp) fragment, which consists of the two internal transcribed spacers and the entire 5.8S gene. Amplification products from crude DNA preparations of 12 species and populations of Aphelenchoides and from D. angustus ranged in size from approximately 860-1,100bp. Southern blots probed with a cloned ribosomal repeat from C. elegans confirmed the identity of these amplified bands as ribosomal fragments. In addition to the differing sizes of the amplified rDNA fragments, the relative intensity of hybridization with the C. elegans probe indicated varying degrees of sequence divergence between species and populations. In some cases, amplified rDNA from the fungal host was evident. Storage of A. composticola at - 45 C for 2 years did not affect the ability to obtain appropriate amplified products from crude DNA preparations. Amplified rDNA fragments were cut with six restriction enzymes, and the restriction fragments produced revealed useful diagnostic differences between species and some undescribed populations. These results were consistent with previous studies based on morphology and isoenzymes. Three undescribed populations of Aphelenchoides were found to be different from all the species examined and from each other.     

Substantial genetic divergence between morphologically indistinguishable populations of Fasciola suggests the possibility of cryptic speciation

The liver flukes, Fasciola hepatica and Fasciola gigantica, are considered to be sister species and between them present a major threat worldwide to livestock production. In this study sequence data have been employed from informative regions of the nuclear and mitochondrial genomes of over 200 morphologically F. hepatica-like or F. gigantica-like flukes from Europe, sub-Saharan Africa and South Asia to assess genetic diversity. Evidence is presented for the existence of four well-separated clades: African gigantica-like flukes, Indian gigantica-like flukes, European hepatica-like flukes and African high-altitude hepatica-like flukes. Application of the Biological Species Concept to trematodes is problematic; however, the degree of separation between these groups was sufficient for them to be considered as distinct species using the four times rule for speciation.     

Inter- and intra-specific characterization of tapeworms of the genus Diphyllobothrium (Cestoda: Diphyllobothriidea) from Switzerland,using nuclear and mitochondrial DNA targets

Human diphyllobothriosis is caused by at least 14 species of cestodes belonging to the genus Diphyllobothrium. Molecular analysis by sequencing of nuclear and mitochondrial targets identifies some species at inter- and intra-specific level, and helps to reconstruct their phylogenetic relationships. Nevertheless, the suitability of further molecular targets deserves to be widened, and the comparison of samples of different geographical origin could allow their intra-specific characterization, which could also be useful for epidemiological purposes. In this study, we investigated inter- and intra-specific variability among tapeworms of the genus Diphyllobothrium, with focus on Diphyllobothrium latum, originated from Switzerland. Samples were analyzed by comparing the sequences of two nuclear and two mitochondrial DNA targets. We analyzed 27 samples belonging to 4 species (D. latum, Diphyllobothrium nihonkaiense, Diphyllobothrium dendriticum and Diphyllobothrium ditremum), 15 of which isolated from clinical cases (adults and eggs), 2 from wild canines, and 2 from fish of Swiss lakes (plerocercoid larvae); 8 samples of homologous species from other geographic origins were also sequenced and compared with the Swiss ones. Sequences of partial small subunit ribosomal RNA (18S rRNA) gene and partial internal transcribed spacers 1 and 2 (ITS1-2) were not useful even in inter-specific identification, whereas sequences of complete cytochrome c oxidase subunit 1 (cox1) and cytochrome b (cob) genes allowed us to assess inter- and intra-specific variations among the samples. Cox1 and cob could differentiate 3 and 5 haplotypes within the species D. latum. The results are discussed in the light of the anamneses provided by part of the patients.     

PCR PRIMERS FOR THE AMPLIFICATION OF MITOCHONDRIAL SMALL SUBUNIT RIBOSOMAL DNA OF LICHEN-FORMING ASCOMYCETES

Abstract: Four primers for the amplification of mitochondrial DNA of lichen-forming ascomycetes are presented. The primers match the conserved regions U2, U4, and U6, respectively, of mitochondrial small subunit (SSU) ribosomal DNA (rDNA). Polymerase chain reaction using different combinations of the primers produced single amplification products from DNA of eight lichen-forming fungal species but did not amplify DNA of two axenic cultured algal species. The amplification product obtained from Lobaria pulmonaria was sequenced and the 894-bp sequence was compared with the mitochondrial SSU rDNA sequence of Podospora anserina. The two sequences revealed more than 76% identity in the conserved regions U3 to U5 demonstrating that we amplified mitochondrial DNA. The primers matching U2 and U6 yielded amplification products of 800–1000 bp depending on the species examined. The variation observed suggests that mitochondrial SSU rDNA may be useful for phylogenetic analyses of lichen-forming ascomycetes.     

Low Genetic Diversity in Wide-Spread Eurasian Liver Fluke Opisthorchis felineus Suggests Special Demographic History of This Trematode Species

Opisthorchis felineus or Siberian liver fluke is a trematode parasite (Opisthorchiidae) that infects the hepato-biliary system of humans and other mammals. Despite its public health significance, this wide-spread Eurasian species is one of the most poorly studied human liver flukes and nothing is known about its population genetic structure and demographic history. In this paper, we attempt to fill this gap for the first time and to explore the genetic diversity in O. felineus populations from Eastern Europe (Ukraine, European part of Russia), Northern Asia (Siberia) and Central Asia (Northern Kazakhstan). Analysis of marker DNA fragments from O. felineus mitochondrial cytochrome c oxidase subunit 1 and 3 (cox1, cox3) and nuclear rDNA internal transcribed spacer 1 (ITS1) sequences revealed that genetic diversity is very low across the large geographic range of this species. Microevolutionary processes in populations of trematodes may well be influenced by their peculiar biology. Nevertheless, we suggest that lack of population genetics structure observed in O. felineus can be primarily explained by the Pleistocene glacial events and subsequent sudden population growth from a very limited group of founders. Rapid range expansion of O. felineus through Asian and European territories after severe bottleneck points to a high dispersal potential of this trematode species.     

Differentiation between Diphyllobothrium dendriticum and D. latum using isozymes,restriction profiles and ribosomal gene probes

Plerocercoids of Diphyllobothrium dendriticum from cisco (Coregonus artedii) and whitefish (C. clupeaformis) and of Diphyllobothrium latum from pike (Esox lucius) and walleye (Stizostedion vitreum) were collected at Quigly Lake, Manitoba. Adult tapeworms were obtained by experimental infection of hamsters (Mesocricetus auratus) and were used to verify species identity. Isozyme analysis showed no differences between adult and plerocercoid stages. The enzymes esterase and malate dehydrogenase differed between species. Comparison of DNA restriction fragment profiles showed species specific differences, as did Southern hybridization analysis using nematode ribosomal gene probes.     

Evaluation of the Extent of Homologous Chloroplast DNA Sequences in the Mitochondrial Genome of Cowpea (Vigna unguiculata L.)

Southern blot hybridization techniques were used to estimate the extent of chloroplast DNA sequences present in the mitochondrial genome of cowpea (Vigna unguiculata L.) The entire mitochondrial chromosome was homogeneously labeled and used to probe blotted DNA fragments obtained by extensive restriction of the tobacco chloroplast genome. The strongest cross-homologies were obtained with fragments derived from the inverted repeat and the atpBE cluster regions, although most of the clones tested (spanning 85% of the tobacco plastid genome) hybridized to mitochondrial DNA. Homologous chloroplast DNA restriction fragments represent a total of 30 to 68 kilobase pairs, depending upon the presence or absence of tRNA-encoding fragments. Plastid genes showing homology with mitochondrial DNA include those encoding ribosomal proteins, RNA polymerase, subunits of photosynthetic complexes, and the two major rRNAs.     

Molecular phylogenetic identification of Fasciola flukes in Nepal

Eighty-one Fasciola flukes collected from 8 districts in Nepal were analyzed for their species identification on the basis of their spermatogenic status and nuclear ribosomal internal transcribed spacer 1 (ITS1) and for their phylogenetic relation with Fasciola flukes from other Asian countries on the basis of the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene. Sixty-one flukes (75.3%) were aspermic Fasciola sp., and 20 flukes (24.7%) were identified as Fasciola gigantica. All of the aspermic flukes displayed the Fh/Fg type in ITS1, which was predominant in aspermic Fasciola sp. from China, and most (60 flukes) displayed the Fsp-ND1-N1 haplotype in the nad1, which had an identical nucleotide sequence to the major haplotype (Fg-C2) of the aspermic flukes from China. These results suggest that aspermic Fasciola sp. was introduced into Nepal from China. Furthermore, the results of the diversity indices, neutrality indices, and median-joining network analysis with reference haplotypes from Asian countries suggest that aspermic Fasciola sp. rapidly expanded its distribution. In contrasts, F. gigantica displayed 10 nad1 haplotypes, which showed higher population diversity indices than the haplotypes of aspermic flukes, indicating that the F. gigantica population was clearly distributed in Nepal earlier than the aspermic Fasciola population. Although the F. gigantica haplotypes from Nepal formed a star-like phylogeny consisting of a main founder haplotype (Fg-ND1-N1), together with some F. gigantica haplotypes from Myanmar and Thailand, the Nepal population differed genetically from F. gigantica populations of neighboring countries as each country had distinct founder haplotype(s).     

I-CeuI fragment analysis of the Shigella species: evidence for large-scale chromosome rearrangement in S. dysenteriae and S. flexneri

I-CeuI fragments of four Shigella species were analyzed to investigate their taxonomic distance from Escherichia coli and to collect substantiated evidence of their genetic relatedness because their ribosomal RNA sequences and similarity values of their chromosomal DNA/DNA hybridization had proved their taxonomic identity. I-CeuI digestion of genomic DNAs yielded seven fragments in every species, indicating that all the Shigella species contained seven sets of ribosome RNA operons. To determine the fragment identities, seven genes were selected from each I-CeuI fragment of E. coli strain K-12 and used as hybridization probes. Among the four Shigella species, S. boydii and S. sonnei showed hybridization patterns similar to those observed for E. coli strains; each gene probe hybridized to the I-CeuI fragments with sizes similar to that of the corresponding E. coli fragment. In contrast, S. dysenteriae and S. flexneri showed distinct patterns; rcsF and rbsR genes that located on different I-CeuI fragments in E. coli, fragments D and E, were found to co-locate on a fragment. Further analysis using an additional three genes that located on fragment D in K-12 revealed that some chromosome rearrangements involving the fragments corresponding to fragments D and E of K-12 took place in S. dysenteriae and S. flexneri.     

Mitochondrial ribosomal proteins in Xenopus laevis/X. mulleri interspecific hybrids.

The large subunits of mitochondrial ribosomes were isolated from two related frog species, Xenopus laevis and X. mulleri, and their proteins were compared by two-dimensional polyacrylamide gel electrophoresis. Three of the proteins observed in X. laevis are absent from X. mulleri, and four of the proteins observed in X. mulleri are absent from X. laevis. More than these seven such species-specific proteins may occur.Reciprocal crosses between frogs of the two species gave two groups of F1 hybrids. Nuclear genes in these hybrids derive equally from both species, while mitochondrial DNA (and therefore mitochondrial rRNA) derived exclusively from the maternal species. Electrophoretic analyses of the large subunit proteins of these F1 animals revealed that four of the species-specific proteins are present only when their corresponding species was the mother. While this result is consistent with the coding of these four proteins by mitochondrial DNA, it does not provide evidence against nuclear coding of these proteins. A fifth protein is absent from both F1 hybrids. A sixth is present in both F1 hybrids, and a seventh is present only when its corresponding species was the father. We conclude that at least these latter two mitochondrial ribosomal proteins are encoded by nuclear genes.     

Transfer of mitochondrial DNA to nuclear genome of cells of passaged cell line of Drosophila virilis

The variability of the chromosomal fragments of the atp6 mitochondrial gene, which is integrated into chromosomal DNA in the lines of flies of different geographic origins and in the passaged cell lines of D. virilis has been analyzed. We did not reveal any nucleotide variability in this DNA marker among the studied fly lines. This result is consistent with the proposition that the D. virilis species is monomorphic. The new fragments of the atp6 gene that are associated with the insertions of the Tv1 retrotransposon and are absent in the fly genome are revealed in the genome of the passaged cell line of D. virilis. This fact is evidence of the activation of the mitochondrial DNA transfer into the nuclear genome of the cells of passaged cell culture.     

Molecular Characterization of Meloidogyne christiei Golden and Kaplan, 1986 (Nematoda,Meloidogynidae) Topotype Population Infecting Turkey Oak (Quercus laevies) in Florida

Meloidogyne christiei isolated from turkey oak, Quercus laevies, from the type locality in Florida was characterized using isozyme profiles and ribosomal and mitochondrial gene sequences. The phenotype N1a detected from a single egg-laying female of M. christiei showed one very strong band of malate dehydrogenase (MDH) activity; however, no esterase (EST) activity was identified from macerate of one or even 20 females per well. Phylogenetic relationships within the genus Meloidogyne as inferred from Bayesian analysis of partial 18S ribosomal RNA (rRNA), D2-D3 of 28S rRNA, internal transcribed spacer (ITS) rRNA, and cytochrome oxidase subunit II (COII)-16S rRNA of mitochondrial DNA (mtDNA) gene fragments showed that M. christiei formed a separate lineage within the crown group of Meloidogyne and its relationships with any of three Meloidogyne clades were not resolved.     

The complete mitochondrial genomes of the liver flukes Opisthorchis felineus and Clonorchis sinensis (Trematoda)

The complete mitochondrial genomes of the parasitic trematodes Opisthorchis felineus and Clonorchis sinensis (family Opisthorchiidae) were fully sequenced in order to develop markers for DNA diagnostics of the liver flukes infection, molecular ecology, population and phylogenetic studies. The complete sequences of mitochondrial genomes of these species comprise 14,277 and 13,875 bp, respectively, and are thus the shortest trematode mitochondrial genomes sequenced to date. The gene content and arrangement are identical to that of Fasciola hepatica. ATG and GTG are used as the start-codons and TAG and TAA are used as the stop-codons. The stop-codon TAG of the C. sinensis nad1 gene overlap by 1 nt with the downstream tRNA-Asn gene. Alternative structures for the Ser(UCN) tRNAs were found for both species. The noncoding control regions are separated into two parts by the tRNA-Gly gene and contain neither tandem repeats, which are characteristic for trematode control regions, nor secondary structures. In conclusion, the complete mitochondrial DNA sequences of O. felineus and C. sinensis will serve as a resource for comparative mitochondrial genomics and systematic studies of parasitic trematodes.     

A comparison of the structural organization of amplified ribosomal DNA from Xenopus mulleri and Xenopus laevis.

Secondary structure maps of long single strands of amplified ribosomal DNA from two closely related species of frogs, Xenopus laevis and X. mulleri, have been compared. The secondary structure pattern of the gene region is identical in both ribosomal DNAs while the patterns in the non-transcribed spacers² differ. In X. mulleri, the spacer shows an extended region without any secondary structure adjacent to the 28 S ribosomal RNA sequence. In contrast, the same region in the X. laevis spacer has extensive secondary structure. A comparison of secondary structure maps and denaturation maps of these two ribosomal DNAs (Brown et al., 1972) reveals that the portion without secondary structure in the X. mulleri spacer corresponds to an early melting A + T-rich region. As in X. laevis ribosomal DNA, Escherichia coli restriction endonuclease (EcoRI) makes two cuts in each repeating unit of amplified ribosomal DNA from X. mulleri. The position of the cleavage sites is identical in the two species as judged from secondary structure mapping of the two classes of EcoRI fragments generated. The small fragments of X. mulleri ribosomal DNA are homogeneous in size with a duplex molecular weight of 3.0 × 10⁶, and contain about 85% of the 28 S ribosomal RNA gene and about 17% of the 18 S ribosomal RNA gene. The large fragments are heterogeneous in size with molecular weights ranging from 4.2 to 4.9 × 10⁶, and contain the remaining portions of the gene regions and the nontranscribed spacer. Heteroduplexes made between large fragments of different lengths show only deletion loops. The position of these loops indicates that the length heterogeneity resides in the non-transcribed spacer region. Electrophoretic analysis of EcoRI digests of chromosomal ribosomal DNA from X. mulleri demonstrates that this DNA is heterogeneous in length as well.     

Genetic diversity of Fasciola hepatica in Spain and Peru

In the present study, molecular characterization of Fasciola flukes from Spain was performed to reveal the relation with the previously reported Peruvian F. hepatica population. The nuclear DNA markers, phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold), were used for species identification of Fasciola flukes. A total of 196 Fasciola flukes were identified as F. hepatica by pepck and pold, and 26 haplotypes were detected in mitochondrial NADH dehydrogenase subunit 1 (nad1). Only one of them was previously found in Spanish samples; which indicates the existence of high genetic diversity and population structure in F. hepatica from Spain. Three haplotypes were identical to those from Peruvian F. hepatica. The pairwise fixation index value confirmed a relatively close relationship between the Spanish and Peruvian F. hepatica samples. The Spanish samples showed clearly higher genetic variability than the Peruvian population. These results are discussed in relation with the hypothesis of the introduction of the parasite in America from Europe and recent evidence of pre-Hispanic F. hepatica from Argentina revealed by ancient DNA.     

Comparison of surface topography of three species of Diphyllobothrium (Cestoda,pseudophyllidea) by scanning electron microscopy

Plerocercoids of different sizes as well as adult worms of D. dendriticum, D. latum and D. ditremum were studied using scanning electron microscopy (S.E.M.). In the plerocercoids there were found distinct differences in appearance and length of microtriches between these three species, while the microtriches of adult worms were more similar. A regional difference in microtrix appearance was found in the larvae of D. ditremum and D. dendriticum. This was not apparent with S.E.M. in adult worms. The length of ‘body’ microtriches in D. dendriticum varied with the length of the larvae. The topography of the genital atrium of mature and gravid proglottids in adult worms of these three species is also described.     

Separation of Three Species of Ditylenchus and Some Host Races of D. dipsaci by Restriction Fragment Length Polymorphism

This study examined the ribosomal cistron of Ditylenchus destructor, D. myceliophagus and seven host races of D. dipsaci from different geographic locations. The three species showed restriction fragment length polymorphisms (RFLPs) in the ribosomal cistron, the 18S rDNA gene, and the ribosomal internal transcribed spacer (ITS). Southern blot analysis with a 7.5-kb ribosomal cistron probe differentiated the five host races of D. dipsaci examined. Polymerase chain reaction (PCR) amplification of the ITS, followed by digestion with some restriction endonucleases (but not others), produced restriction fragments diagnostic of the giant race. Because the PCR product from D. myceliophagus and the host races of D. dipsaci was about 900 base pairs and the ITS size in D. destructor populations was 1,200 base pairs, mixtures of populations could be detected by PCR amplification. ITS fragments differentiated between D. dipsaci and Aphelenchoides rhyntium in mixed populations. This study establishes the feasibility of differentiation of the host races of D. dipsaci by probing Southern blots with the whole ribosomal cistron.     

Molecular identification and genetic-polymorphism analysis of Fasciola flukes in Dali Prefecture,Yunnan Province,China

This study aimed to identify species of Fasciola flukes in Dali Prefecture (Yunnan Province, China) and analyze their genetic diversity. Fasciola flukes (n = 122) were collected from cattle livers in a farmers' market in Xiaguan Town, Dali Prefecture. Nucleotide sequences of ribosomal internal transcribed spacer (ITS) as well as nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) and mitochondrial cytochrome c oxidase subunit 1 (CO1) were amplified, sequenced, and subjected to homology analysis. The heterozygosity ratios of different ITS alleles were determined using the peak-height ratio of heterozygous loci. Multiplex PCR analysis of the nuclear protein coding gene, phosphoenolpyruvate carboxykinase (pepck), was used to identify Fasciola species. Multiple ND1 sequence alignments enabled further genetic diversity analysis of regional Fasciola flukes. Seven ITS sequences belonged to F. hepatica and 115 belonged to Fh/Fg heterozygous flukes. Sequencing analysis of heterozygous flukes revealed 11 heterozygous loci with double peaks, with significantly variable ratios among individuals. ND1 and CO1 results indicated that one specimen was identical to F. hepatica, while 121 specimens were identical to F. gigantica or contained one variable site. Multiplex PCR results for pepck showed that double bands for F. hepatica and F. gigantica were amplified from Dali Fasciola specimens; hence, they were all heterozygous. By combining ITS, ND1, and CO1 sequences with multiplex pepck PCR results, all 122 specimens were identified as Fh/Fg heterozygous Fasciola flukes. Our experimental results preliminarily confirmed a high degree of Fh/Fg heterozygosity among Fasciola flukes in the Dali area. Selecting multiple molecular markers for concurrent analysis will provide more comprehensive and accurate genetic information.     

Estimation of Genetic Divergence in Meloidogyne Mitochondrial DNA

Restriction fragments from purified mitochondrial DNA can be readily detected following rapid end-labeling with [α-³²]nucleoside triphosphates and separation by gel electrophoresis. Mitochondrial DNA from 12 populations of Meloidogyne species was digested with 12 restriction enzymes producing more than 60 restriction fragments for each species. The mitochondrial genome of M. arenaria is the most genetically distinct of the four species compared. M. arenaria shows approximately 2.1-3.1% nucleotide sequence divergence from the mitochondrial genomes of M. javanica, M. incognita, and M. hapla. Among the latter three species, interspecific estimates of sequence divergence range from 0.7 to 2.3%. Relatively high intraspecific variation in mitochondrial restriction fragment patterns was observed in M. hapla. Intraspecific variation in M. incognita resulted in sequence divergence estimates of 0.5-1.0%. Such polymorphisms can serve as genetic markers for discerning mitochondrial DNA genotypes in nematode populations in the same way that allozymes have been used to discern nuclear DNA genotypes.     

Analysis of an expressed sequence tag library from Dicrocoelium dentriticum

Dicrocoeliosis caused by Dicrocoelium dendriticum is an important liver disease, which affects ruminants all around the world. Despite the significant economic losses caused by this trematode, molecular knowledge is very scarce. In fact, there is no information in the expressed sequence tag (EST) database about the parasite. Furthermore, the immunological diagnosis of dicrocoeliosis remains unsatisfactory, and there aren’t available recombinant proteins that could be tested in the diagnosis. For this reason a cDNA library was constructed with mRNA extracted from D. dendriticum adults for first time. A random preliminary screening of 230 phage plaques from the library resulted in the identification of 173 new EST. The deduced proteins expressed by these genes have been described as possible vaccine targets in other trematodes, and/or as relevant diagnosis antigens. Then, our goal was to identify D. dentriticum diagnosis genes to be used as recombinant antigens in the specific immunological diagnosis of the trematodoses. A D. dendriticum cDNA encoding an 8-kDa recombinant protein has been cloned, expressed in Escherichia coli and evaluated in dicrocoeliosis diagnosis using both Western Blot and enzyme-linked immunosorbent assay (ELISA). The recombinant expression molecule has demonstrated its value as a diagnosis antigen of dicrocoeliosis, able to discriminate between positive and controls on day 30 post infection. This is the first research conducted for identification and characterization of D. dendriticum ESTs, which can serve as a starting point for future research on immunodiagnosis and immunoprofilaxis of dicrocoeliosis.