Sequence variability in two mitochondrial DNA regions and internal transcribed spacer among three cestodes infecting animals and humans from China

Sequence variability in two mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 4 (nad4), and internal transcribed spacer (ITS) of rDNA among and within three cestodes, Spirometra erinaceieuropaei, Taenia multiceps and Taenia hydatigena, from different geographical origins in China was examined. A portion of the cox1 (pcox1), nad4 genes (pnad4) and the ITS (ITS1+5.8S rDNA+ITS2) were amplified separately from individual cestodes by polymerase chain reaction (PCR). Representative amplicons were subjected to sequencing in order to estimate sequence variability. While the intra-specific sequence variations within each of the tapeworm species were 0-0.7% for pcox1, 0-1.7% for pnad4 and 0.1-3.6% for ITS, the inter-specific sequence differences were significantly higher, being 12.1-17.6%, 18.7-26.2% and 31-75.5% for pcox1, pnad4 and ITS, respectively. Phylogenetic analyses based on the pcox1 sequence data revealed that T. multiceps and T. hydatigena were more closely related to the other members of the Taenia genus, and S. erinaceieuropaei was more closely related to the other members of the Spirometra genus. These findings demonstrated clearly the usefulness of mtDNA and rDNA sequences for population genetic studies of these cestodes of socio-economic importance.     

Internal transport and metabolism of cobalamin in Spirometra mansonoides (Cestoda) spargana

The anterior 2 mm of the sparganum of Spirometra mansonoides contained 35 to 48% of the total endogenous cobalamin (Cbl) with only 2 to 4% in the terminal 1 cm. [57Co]cobalamin taken up in the posterior region of spargana was transported directionally to the anterior region and concentrated there resulting in a uniform depletion of [57Co]Cbl along the remaining body length. The anterior 2 mm contained 24% of the sparganum's holo-MMCoAM activity with only 5% in the terminal 1 cm. When head and body preparations were chromatographed on Sephadex G-150 columns eluates from both regions exhibited single peaks of MMCoAM activity at an approximate molecular weight of 150,000 and two peaks of endogenous Cbl. The first Cbl peak cochromatographed with MMCoAM activity and the second Cbl peak eluted at the position of free Cbl. Spargana rapidly converted CN-[57Co]Cbl to hydroxocobalamin (OH-Cbl) and adenosylcobalamin (Ado-Cbl) in vitro. In the head region significant amounts of Ado-Cbl were present (25%) although OH-Cbl was the predominant form of Cbl (45%) after 264 hr in a Cbl-free medium. In the body Ado-Cbl was the predominant form of Cbl (44%) although significant amounts of OH-Cbl were present (27%) after 264 hr in a Cbl-free medium. No methylcobalamin (Me-Cbl) was detected, but an unidentified cobalamin-containing entity (Fraction 4) was present. Based on these results we propose that the sparganum takes up cobalamin, concentrates it in the anterior, "head" region, sequentially metabolizes it to the hydroxyl then the adenosyl forms, and that the adenosylated form is used in this region of high, anaerobic metabolism as an obligatory coenzyme for MMCoAM, in coordination with the differentiation of the sparganum to the adult cestode.     

Genetic Structure Analysis of Spirometra erinaceieuropaei Isolates from Central and Southern China

Background

Sparganosis caused by invasion of the plerocercoid larvae (spargana) of Spirometra erinaceieuropaei have increased in recent years in China. However, the population genetic structure regarding this parasite is still unclear. In this study, we used the sequences of two mitochondrial genes cytochrome b (cytb) and cytochrome c oxidase subunit I (cox1) to analyze genetic variation and phylogeographic structure of the S. erinaceieuropaei populations.

Methodology/Principal Findings

A total of 88 S. erinaceieuropaei isolates were collected from naturally infected frogs in 14 geographical locations of China. The complete cytb and cox1 genes of each sample was amplified and sequenced. Total 61 haplotypes were found in these 88 concatenated sequences. Each sampled population and the total population have high haplotype diversity (Hd), accompanied by very low nucleotide diversity (Pi). Phylogenetic analyses of haplotypes revealed two distinct clades (HeN+HuN+GZ-AS clade and GX+HN+GZ-GY clade) corresponding two sub-networks yielded by the median-joining network. Pairwise F _ST values supported great genetic differentiation between S. erinaceieuropaei populations. Both negative Fu’s F _S value of neutrality tests and unimodal curve of mismatch distribution analyses supported demographic population expansion in the HeN+HuN+GZ-AS clade. The BEAST analysis showed that the divergence time between the two clades took place in the early Pleistocene (1.16 Myr), and by Bayesian skyline plot (BSP) an expansion occurred after about 0.3 Myr ago.

Conclusions

S. erinaceieuropaei from central and southern China has significant phylogeographic structure, and climatic oscillations during glacial periods in the Quaternary may affect the demography and diversification of this species.     

Survey of Spirometra erinaceieuropaei in frogs in Taiwan and its experimental infection in cats

Eighteen of 56 (32.1%) wild Rana limnocharis from central and south Taiwan were found to contain plerocercoids of Spirometra erinaceieuropaei. This is the first report of S. erinaceieuropaei infections in frogs in Taiwan, with the plerocercoids being recovered from the thigh and back muscles or under the skin. Other species of frogs examined, including nine wild R. latouchii, one wild Buergeria robustus and 110 cultured R. rugulosa were free of infection. The plerocercoids were orally inoculated into four cats; three of which were each given a single plerocercoid and one a dose of three plerocercoids. Daily faecal examination showed that two cats started shedding eggs of S. erinaceieuropaei on day 8 postinfection (PI) and the other two on day 10 PI. The highest eggs per gram and eggs per day for a single worm was found to be 428,000 and 14,416,000 respectively. Only the cat inoculated with three plerocercoids shed proglottids in its faeces during the 2 month observation period.     

Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA.

The sequences of the displacement-loop (D-loop) regions of mitochondrial DNA (mtDNA) from mouse L cells and human KB cells have been determined and provide physical maps to aid in the identification of sequences involved in the regulation of replication and expression of mammalian mtDNA. Both D-loop regions are bounded by the genes for tRNAPhe and tRNAPro. This region contains the most highly divergent sequences in mtDNA with the exceptions of three small conserved sequence blocks near the 5' ends of D-loop strands, a 225 nucleotide conserved sequence block in the center of the D-loop strand template region, and a short sequence associated with the 3' ends of D-loop strands. A sequence similar to that associated with the 3' termini of D-loop strands overlaps one of the conserved sequence blocks near the 5' ends of D-loop strands. The large, central conserved sequence probably does not code for a protein since no open reading frames are discretely conserved. Numerous symmetric sequences and potential secondary structures exist in these sequences, but none appear to be clearly conserved between species.     

Characterization of Spirometra erinaceieuropaei Plerocercoid Cysteine Protease and Potential Application for Serodiagnosis of Sparganosis

BackgroundSparganosis is a neglected but important food-borne parasitic zoonosis. Clinical diagnosis of sparganosis is difficult because there are no specific manifestations. ELISA using plerocercoid crude or excretory–secretory (ES) antigens has high sensitivity but has cross-reactions with other helminthiases. The aim of this study was to characterize Spirometra erinaceieuropaei cysteine protease (SeCP) and to evaluate its potential application for serodiagnosis of sparganosis.ConclusionsThe rSeCP had the CP enzymatic activity and SeCP seems to be important for the survival of plerocercoids in host. The rSeCP is a potential diagnostic antigen for sparganosis.     

Effect of temperature on fatty acid composition in each lipid fraction of Spirometra erinaceieuropaei plerocercoids

The effects of temperature and host fatty acids on the fatty acid contents of Spirometra erinaceieuropaei plerocercoids were investigated to clarify their role in sparganosis. After 24 hr incubation at 18 C in host snake serum, omega6 series fatty acids, especially arachidonic acid in the phospholipid fraction of the plerocercoids, increased compared with those of plerocercoids incubated at 37 C. The changes in the ratio of polyunsaturated to saturated fatty acids in the phospholipid fraction of plerocercoids incubated in physiological saline for 6 hr at 10 C were almost the same as the changes at 37 C. The ratio of polyunsaturated to saturated fatty acids of the triglyceride fraction showed almost opposite change versus the phospholipid fraction. The percentage of arachidonic acid in the phospholipid fraction of plerocercoids increased during the first 3 hr of incubation and then decreased, regardless of temperature. At 37 C, the percentage of arachidonic acid in the free fatty acid fraction fell for the first 3 hr of incubation and was significantly elevated at the end of the 6-hr incubation. At 10 C, however, arachidonic acid in the free fatty acid fraction decreased for the first hour of incubation, increased at 3 hr of incubation, then decreased again. These results suggest that fatty acids of the plerocercoids are frequently exchanged between fractions. Plerocercoids can mobilize arachidonic acid to the free fatty acid fraction more quickly at lower temperature than at higher temperature. They may utilize mobilized arachidonic acid early in the infection stage to produce prostaglandins. Alternatively, they can incorporate arachidonic acid into the phospholipid fraction again when arachidonic acid is readily available in the environment.     

Sequence variability in three mitochondrial genes between the two pig nodule worms Oesophagostomum dentatum and O. quadrispinulatum

In this study, sequence variation in three mitochondrial DNA regions, namely cytochrome c oxidase subunit (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), between Oesophagostomum dentatum and O. quadrispinulatum isolated from pigs in different geographical origins in Mainland China was examined, and their phylogenetic relationships were reconstructed. A partial of the cox1 (pcox1), nad1, and nad4 genes (pnad1 and pnad4) were amplified separately from individual nodule worms by PCR and were subjected to direct sequencing in order to define sequence variations. While the intraspecific sequence variations within each of the two species were 0.3-5.2% for pcox1, 0-4.9% for pnad1, and 0-7.1% for pnad4, the interspecific sequence differences were significantly higher, being 10.7-13.4% for pcox1, 11-14.6% for pnad1, and 14.9-18% for pnad4, respectively. There were a number of nucleotide positions in the pcox1, pnad1, and pnad4 sequences with no apparent intraspecific variation but distinct interspecific differences among those samples of Oesophagostomum spp. examined, which may be used as genetic makers for the identification and differentiation of the Oesophagostomum spp. Phylogenetic analyses using three inference methods, namely Bayesian inference, maximum likelihood, and maximum parsimony based on the combined sequences of pcox1, pnad1, and pnad4 revealed that the O. dentatum and O. quadrispinulatum form monophyletic groups, respectively. These findings demonstrated clearly the usefulness of the three mitochondrial sequences for studying systematics, population genetic structures, and the molecular ecology of Oesophagostomum spp.     

Rapid concerted evolution in animal mitochondrial DNA

Recombinational genetic processes are thought to be rare in the uniparentally inherited mitochondrial (mt) DNA molecules of vertebrates and other animals. Here, however, we document extremely rapid concerted microevolution, probably mediated by frequent gene conversion events, of duplicated sequences in the mtDNA control region of mangrove killifishes (Kryptolebias marmoratus). In local populations, genetic distances between paralogous loci within an individual were typically smaller (and often zero) than those between orthologous loci in different specimens. These findings call for the recognition of concerted evolution as a microevolutionary process and gene conversion as a likely recombinational force in animal mtDNA. The previously unsuspected power of these molecular phenomena could greatly impact mtDNA dynamics within germ cell lineages and in local animal populations.     

Sequence evolution of mitochondrial tRNA genes and deep-branch animal phylogenetics

Mitochondrial DNA sequences are often used to construct molecular phylogenetic trees among closely related animals. In order to examine the usefulness of mtDNA sequences for deep-branch phylogenetics, genes in previously reported mtDNA sequences were analyzed among several animals that diverged 20–600 million years ago. Unambiguous alignment was achieved for stem-forming regions of mitochondrial tRNA genes by virtue of their conservative secondary structures. Sequences derived from stem parts of the mitochondrial tRNA genes appeared to accumulate much variation linearly for a long period of time: nearly 100 Myr for transition differences and more than 350 Myr for transversion differences. This characteristic could be attributed, in part, to the structural variability of mitochondrial tRNAs, which have fewer restrictions on their tertiary structure than do nonmitochondrial tRNAs. The tRNA sequence data served to reconstruct a well-established phylogeny of the animals with 100% bootstrap probabilities by both maximum parsimony and neighbor joining methods. By contrast, mitochondrial protein genes coding for cytochrome b and cytochrome oxidase subunit I did not reconstruct the established phylogeny or did so only weakly, although a variety of fractions of the protein gene sequences were subjected to tree-building. This discouraging phylogenetic performance of mitochondrial protein genes, especially with respect to branches originating over 300 Myr ago, was not simply due to high randomness in the data. It may have been due to the relative susceptibility of the protein genes to natural selection as compared with the stem parts of mitochondrial tRNA genes. On the basis of these results, it is proposed that mitochondrial tRNA genes may be useful in resolving deep branches in animal phylogenies with divergences that occurred some hundreds of Myr ago. For this purpose, we designed a set of primers with which mtDNA fragments encompassing clustered tRNA genes were successfully amplified from various vertebrates by the polymerase chain reaction.Abbreviations AA stem amino acid-acceptor stem - AC stem anticodon stem - COI cytochrome oxidase subunit I - cytb cytochrome b - D stem dihydrouridine stem - MP maximum parsimony - mtDNA mitochondrial DNA - Myr million years - NJ neighbor joining - PCR polymerase chain reaction - Ti transition - T stem tC stem - Tv transversion Correspondence to: Y. Kumazawa     

Mitochondrial DNA variation in human populations and implications for detection of mitochondrial DNA mutations of pathological significance

Haplotype and phylogenetic analyses of normal mitochondrial DNAs (mtDNAs) have allowed a clarification of several controversial issues concerning the origin of humans, the time and colonization pattern of the various regions of the world, and the genetic relationships of modern human populations. More recently, the same type of analyses has also been applied to mtDNA disease studies. A review of these studies indicates that exhaustive screenings of normal mtDNA variation in all human populations associated with haplotype and phylogenetic analyses are essential if we are to understand the etiology of mitochondrial pathologies.     

Sequence homologies of (guanosine + cytidine)-rich regions of mitochondrial DNA of Saccharomyces cerevisiae.

The nucleotide sequences of two distinct regions of mitochrondrial DNA of Saccharomyces cerevisiae are reported. The regions studied have a high content of G + C (45%) and contain closely spaced Hpa II and Hae III restriction sites. Both regions have sequences that are homologous over a lenght of 47 base-pairs. In addition, the two regions are highly palindromic. These data support certain aspects of the organization of mitochondrial DNA proposed by Prunell and Bernardi (Prunell, A., and Bernardi, G. (1977) J. Mol. Biol. 110, 53--74).