First report of human infection with Gynaecotyla squatarolae and first Korean record of Haplorchis pumilio in a patient

Gynaecotyla squatarolae (Digenea: Microphallidae) is a minute intestinal trematode whose natural hosts are aves. We conducted a feces screening survey in a coastal village of Muan-gun, where the residents routinely consume brackish water crabs as a food. Through this survey, a 50-year-old female was found to shed gymnophallid and heterophyid eggs in her stool, and 845 adult flukes were collected from her purged stool. The adult worms were morphologically grouped into three species. A total of 841 worms were Gymnophalloides seoi. Three worms were identified as G. squatarolae, and the last one proven to be Haplorchis pumilio. This is the first worldwide report of G. squatarolae infection in humans, and the first H. pumilio infection in Korean people.     

Fluorescent probes for imaging endogenous β-actin mRNA in living cells using fluorescent protein-tagged pumilio

Subcellular localization and dynamics of mRNAs control various physiological functions in living cells. A novel technique for visualizing endogenous mRNAs in living cells is necessary for investigation of the spatiotemporal movement of mRNAs. A pumilio homology domain of human pumilio 1 (PUM-HD) is a useful RNA binding protein as a tool for mRNA recognition because the domain can be modified to bind a specific 8-base sequence of target mRNA. In this study, we designed PUM-HD to match the sequence of β-actin mRNA and developed an mRNA probe consisting of two PUM-HD mutants flanking full-length enhanced green fluorescent protein (EGFP). Fluorescence microscopy with the probe in living cells revealed that the probe was labeled precisely with the β-actin mRNA in cytosol. Fluorescent spots from the probe were colocalized with microtubules and moved directionally in living cells. The PUM-HD mutants conjugated with full-length EGFP can enable visualization of β-actin mRNA localization and dynamics in living cells.     

A pumilio homolog in Polycelis sp.

Pumilio proteins (PUMs), members of the pumilio/fem-3 mRNA-binding factor (PUF) family, are eukaryote-specific RNA-binding proteins. We isolated a 2,048-basepair cDNA fragment of a pumilio homolog from the planarian flatworm Polycelis sp. This pumilio protein (PyPUM) contains a conserved pumilio homology domain (PUM-HD) consisting of eight repeats and two flanking half repeats. PyPUM shows high similarity to Dugesia japonica pumilio (DjPUM) from another planarian D. japonica, and their PUM-HD also shows high similarity to each other. Furthermore, our data showed that there is a flatworm-specific spacer between repeats 7 and 8. Phylogenetic analysis showed that PyPUM has a closer relationship to other PUM homologs from flatworms. These results provide a foundation for future functional studies of pumilio gene in Polycelis sp.     

Identification of Two Subfamilies of micropia Transposable Element in Species of the repleta Group of Drosophila

The occurrence, number of insertion sites and antisense RNA expression of micropia transposable element were studied in 26 species that belong to three subgroups (mercatorum, mulleri and hydei) of repleta group of Drosophila. Under high specific PCR, micropia sequences were detected in 11 species, but under less stringent condition, this retrotransposon was detected in all species. The widespread distribution of micropia suggests that this element was already present at the common ancestor of the repleta group of Drosophila. Southern blot analysis showed a variation from 0 to 17 different insertion sites and the occurrence of male-specific sequences. We found that the expression of the 1.0 kb micropia antisense RNA is variable among the species and tissues (soma and testis), which suggests that more than one mechanism regulates transposition in these species. Variation of amplification by PCR and of antisense RNA expression, as well as divergence of nucleotide sequences among the species allow us to suggest that at least two subfamilies of micropia transposable element are harbored by the genome of this species group.     

Molecular genetics of Rhabdomys pumilio subspecies boundaries: mtDNA phylogeography and karyotypic analysis by fluorescence in situ hybridization

The phylogeography of the African four-striped mouse, Rhabdomys pumilio, was investigated using complete sequences of the mtDNA cytochrome b gene (1140 bp) and a combination of fluorescence in situ hybridization (FISH) and conventional cytogenetic banding techniques (G- and C-banding). Two cytotypes (2n=46 and 2n=48) were identified by cytogenetic analysis. There is no evidence of diploid number variation within populations, difference in gross chromosome morphology or of subtle interchromosomal rearrangements at levels detected by ZOO-FISH. Analysis of the mtDNA cytochrome b resulted in two major lineages that correspond roughly to the xeric and mesic biotic zones of southern Africa. One mtDNA clade comprises specimens with 2n=48 and the other representatives of two cytotypes (2n=48 and 2n=46). The mean sequence divergence (12%, range 8.3–15.6%) separating the two mtDNA clades is comparable to among-species variation within murid genera suggesting their recognition as distinct species, the prior names for which would be R. dilectus and R. pumilio. Low sequence divergences and the diploid number dichotomy within the mesic lineage support the recognition of two subspecies corresponding to R. d. dilectus (2n=46) and R. d. chakae (2n=48). Our data do not support subspecific delimitation within the nominate, R. pumilio. Molecular dating places cladogenesis of the two putative species at less than five million years, a period characterised by extensive climatic oscillations which are thought to have resulted in habitat fragmentation throughout much of the species range.     

Taxonomical classification of yeasts isolated from kefir based on the sequence of their ribosomal RNA genes

Yeast strains present in 10 samples of kefir of different commercial and domestic origins have been isolated and classified taxonomically on the basis of the internal transcribed sequences (ITS) of their ribosomal RNA genes. A total of 18 yeast strains representing 10 different species have been characterized. Of the three commercial kefir samples analyed, two contained the well characterized yeast Kluyveromyces lactis while no yeast was found in the other one. A broader spectrum of yeast species was found among the home-made kefir samples, of which Issatchenkia orientalis, Saccharomyces unisporus, Saccharomyces exiguus and Saccharomyces humaticus were the most representative species.     

Characterization of Charr Chromosomes Using Fluorescence in Situ Hybridization

The chromosomal locations of several families of tandem repetitive DNA sequences and the 5S rDNA were determined using fluorescence in situ hybridization (FISH) in the five North American charr species: Salvelinus namaycush, S. fontinalis, S. alpinus, S. malma, and S. confluentus. The pattern of hybridization of three centromeric repetitive sequences previously isolated from S. namaycush and S. alpinus was unique in each species. Dual-color FISH experiments showed that in several species many of the centromeres had the EcoRI-DraI family in addition to either the AluI-RsaI type A or type B families. The EcoRI-DraI family which was found only at the centromeres of acrocentric chromosomes in S. namaycush, S. fontinalis and S. malma was also found at centromeres of selected metacentrics in S. alpinus (one pair) and S. confluentus (four pairs) whose chromosomes have undergone additional centric fusions compared to the other species. The locations of 5S rDNA sequences were different in each species except for the two most closely related (S. alpinus and S. malma). Two whole-arm chromosome paint probes, one specific for the short and the other for the long arm of the lake charr sex chromosomes, hybridize to the same chromosome pair in all species. Results with other paint probes suggest that independent centric fusions have occurred in S. alpinus and S. confluentus which is consistent with the phylogenetic tree obtained previously for Salvelinus with cytogenetic and DNA data.     

Kunitz-type protease inhibitors from acrorhagi of three species of sea anemones

Sea anemones are rich in biologically active polypeptides such as toxins and protease inhibitors. These polypeptides have so far been isolated from whole bodies, tentacles or secreted mucus. Recently, two novel peptide toxins with crab lethality have been isolated from acrorhagi (specialized aggressive organs elaborated by only certain species of sea anemones belonging to the family Actiniidae) of Actinia equina. This prompted us to survey biologically active polypeptides in the acrorhagi of two species of sea anemones, Anthopleura aff. xanthogrammica and Anthopleura fuscoviridis. No potent crab lethality was displayed by the acrorhagial extracts of both species. However, significantly high protease inhibitory activity was instead detected in the acrorhagial extracts of the two species and also in that of A. equina. From the acrorhagi of A. equina, A. aff. xanthogrammica and A. fuscoviridis, one (AEAPI), one (AXAPI) and two (AFAPI-I and AFAPI-III) protease inhibitors were isolated, respectively. The complete amino acid sequences of the four inhibitors were elucidated by N-terminal sequencing and sequencing of the C-terminal peptide fragment produced upon asparaginylendopeptidase digestion. The determined amino acid sequences revealed that all the four inhibitors are new members of the Kunitz-type protease inhibitor family.     

Taxonomic studies of predatory bdellovibrios based on 16S rRNA analysis, ribotyping and the hit locus and characterization of isolates from the gut of animals

The aim of our study was to obtain data for the molecular characterization of bdellovibrio bacteria, which were recently split into the genus Bdellovibrio and the newly designated genus Bacteriovorax. We determined the 16S rDNA sequences of five reference strains and performed a phylogenetic analysis including published 16S rRNA sequences of bdellovibrios. A comparison of the secondary structure showed significant differences in two regions of the 16S rRNAs of the species Bdellovibrio bacteriovorus, Bacteriovorax starrii, and Bacteriovorax stolpii. In addition, ribotyping techniques gave specific hybridization patterns and revealed that two rRNA operons are present in the investigated strains. A hybridization probe derived from the genetic locus hit, associated with the host independent (HI) phenotype of B. bacteriovorus, was found to be specific for this species. Sequence comparison of the hit locus revealed few base pair changes between host independent (HI) and host dependent (HD) strains. Ribotyping and hybridization experiments using the hit probe were applied to characterize bdellovibrio strains isolated from the gut of animals and humans and one isolate from sewage.     

Phylogenetic Distribution of Unusual Triheme to Tetraheme Cytochrome Subunit in the Reaction Center Complex of Purple Photosynthetic Bacteria

To understand the evolutionary relationship between triheme and tetraheme cytochrome subunits in the reaction center complex, genes located downstream of that coding for the M subunit of the reaction center complex (pufM) were amplified by PCR and analyzed in six established and two unidentified species of the genus Rhodovulum and five species of the genus Rhodobacter. All the Rhodovulum species tested had the pufC gene coding for the reaction-center-bound cytochrome subunit, while all the Rhodobacter species were found to have the pufX gene at the corresponding position. Analyses of the amino acid sequences of the pufC gene products showed that the cytochrome subunits of all the Rhodovulum species have three heme-binding-motifs and lack a methionine residue probably working as the sixth axial-ligand to one of the three hemes. Phylogenetic relationships among Rhodovulum species based on the pufC gene products were basically consistent with those based on 16S rRNA sequences, suggesting that the basic characteristics of the triheme cytochrome subunit have been conserved during the evolutionary process of the Rhodovulum species.     

Speciation in the Artemia genus: Mitochondrial DNA analysis of bisexual and parthenogenetic brine shrimps

From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the CT substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.On sabbatical leave from Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madridearly Italian artemiologists to designate the Medi-Beatriz Batuecas died in an accident during the Christmas holy days of 1988 after she had initiated this workCorrespondence to: R. Garesse     

Survey for zoonotic liver and intestinal trematode metacercariae in cultured and wild fish in An Giang Province, Vietnam

Although Vietnam has a high risk of fishborne zoonotic trematode (FZT) infections for humans, little information exists on the epidemiology of these infections in the country's fish. Because of the importance of cultured catfish and snakehead production in An Giang province, a major production area in the Mekong Delta of Vietnam, a survey for FZTs was carried out in randomly selected fish farms between June 2005 and March 2006. For comparison, wild fish from the same area were also surveyed. A total of 852 cultured fish from 4 districts were collected and examined by pepsin digestion to determine their FZT infection status. In Tra catfish, the prevalence of all types of metacercariae was 2.6%, of which the prevalence of Haplorchis pumilio was 0.7%. The overall prevalence of metacercariae in wild fish was 30.6%, of which 10.3% harbored zoonotic species: H. pumilio (2.8%) and Procerovum sp. (5.6%). The prevalence of Opisthorchis metacercariae, which were diagnosed as O. viverrini, was 1.9%. No metacercariae were found in cultured snakehead fish, although wild-caught snakehead fish had a FZT prevalence of 10.3%: 5.1% were O. viverrini 2.6% H. pumilio and 2.6% were Procerovum sp. These are the first reports of H. pumilio, Procerovum sp., and O. viverrini metacercariae in Vietnamese fish. These results indicate that consumption of improperly prepared fish represents a significant risk of acquiring FZTs in this south Vietnam region.     

Crypthecodinium and Tetrahymena: An exercise in comparative evolution

Summary Nucleotide sequences have been determined for the highly variable D2 region of the large rRNA molecule for over 60 strains of dinoflagellates. These strains were selected from a worldwide collection that represents all the known sibling species (compatibility groups, Mendelian species) in the sibling swarm referred to as Crypthecodinium cohnii. A phylogenetic tree has been constructed from an analysis of the variations in a length of about 180 bases, using PHYLOGEN string analysis programs. The Crypthecodinium tree is compared with the previously published but here augmented tree constructed upon the same rRNA region for the sibling species of a worldwide collection of ciliated protozoa related to the genus Tetrahymena. The first reported sequence of Lambornella clarki, the parasite of tree-hole mosquitoes, is included.The dinoflagellate species complex is much more homogeneous with respect to ribosomal variation. The mean number of differences among sequences from different Crypthecodinium species is about 7, in comparison with 22 differences among the ciliate species examined. Moreover, all the diversity in the dinoflagellates can be explained by base substitutions, whereas insertions and deletions are common in the ciliates. The dinoflagellates are also much more uniform with respect to nutritional and genetic economies.The two complexes differ also in the relationship between molecular variations and breeding compatibility. All tetrahymenine sibling species thus far examined are monomorphic in the D2 region, but several dinoflagellate species are polymorphic. Several different dinoflagellate species, moreover, have identical D2 regions. This kind of ribosomal identity of incompatible strains is found in these ciliates only in one tight cluster of species—Group C.The tetrahymenine swarm is apparently much older than the Crypthecodinium swarm, and the dinoflagellate species produce incompatible progeny species much more readily than do the ciliates, perhaps by the acquisition of mutations that potentiate incompatibility in sympatric populations.Offprint requests to: D. L. Nanney     

A Comparative Analysis of Regulatory Regions of the Transthyretin Gene in the Mouse, Human, and Chimpanzee Genomes

A full genome analysis of differences between the gene expression in the human and chimpanzee brains revealed that the gene for transthyretin, the carrier of thyroid hormones, is differently transcribed in the cerebella of these species. A 7-kbp DNA fragment of chimpanzee was sequenced to identify possible regulatory sequences responsible for the differences in expression. One hundred and thirteen substitutions were found in the chimpanzee sequence in comparison with the human sequence. About 40% of the substitutions were revealed within the repeating elements of the genome; their location and sizes did not differ from those in the corresponding fragments of the human genome, and the nucleotide sequences had a high degree of identity. A comparison of nucleotide sequences of the transthyretin region of human, chimpanzee, and mouse genes revealed substantial differences in the distribution of G + C content along the examined fragment in the human (chimpanzee) and mouse genes and allowed us to localize three sequence tracts with a higher degree of identity in the three species. One of these tracts was located in the promoter region of the gene, and the other two probably determine the specificity of transthyretin gene expression in the liver and brain. One of the conserved tracts of the chimpanzee genome was found to have a single and a triple nucleotide substitution. The triple substitution distinguishes chimpanzees from humans and mice, which have identical sequences of this site. It is likely that these substitutions are responsible for the differences in the expression levels of the transthyretin gene in the human and chimpanzee brains.     

Fourfold polyphyly of the genus formerly known as Upucerthia, with notes on the systematics and evolution of the avian subfamily Furnariinae

The traditional avian subfamily Furnariinae, a group of terrestrial ovenbirds typical of the Andean and Patagonian arid zones, consists of the genera Furnarius, Cinclodes, Geositta, Upucerthia, Chilia, and Eremobius. We investigated phylogenetic relationships within the Furnariinae, with particular attention to the nine species of the genus Upucerthia, using nuclear and mitochondrial DNA sequences from all genera in the subfamily. Upucerthia was found to be highly polyphyletic, its constituent species forming four non-sister clades: (1) a basal lineage consisting of two Upucerthia species, U. ruficaudus and U. andaecola, as well as the monotypic genera Eremobius and Chilia; (2) a lineage consisting of U. harterti and U. certhioides, two species behaviorally divergent from other Upucerthia species; (3) a lineage consisting of U. serrana, which is not closely related to any other Upucerthia species; and (4) a lineage, sister to Cinclodes, consisting of the four Upucerthia species U. dumetaria, U. albigula, U. validirostris, and U. jelskii. The larger Furnariinae was also found to be highly polyphyletic; the terrestrial open country ecotype characteristic of this subfamily occurs in four unrelated clades in the family Furnariidae, including a basal lineage as well as derived lineages. Although the large degree of divergence among Upucerthia clades was not previously recognized, owing to ecological, behavioral, and morphological similarities, the groupings correspond closely to relationships suggested by plumage. This is in contrast to studies of other avian genera in which plumage patterns have been shown to be extensively convergent. The generic names Upucerthia and Ochetorhynchus are available for two of the former Upucerthia clades; new generic names may be warranted for the other two.     

Identification and gene expression profiling of the Pum1 and Pum2 members of the Pumilio family in the chicken

Members of the Pumilio (Pum) family of RNA-binding proteins act as translational repressors and are required for germ cell development and asymmetric division. We identified the chicken Pum1 and Pum2 genes and analyzed their expression patterns in various tissues. Comparative sequence analysis of the Pum1 and Pum2 proteins from the drosophila, chicken, mouse, and human revealed a high degree of evolutionary conservation in terms of the levels of homology of the peptide sequences and the structure of Pumilio homology domain (PUM-HD), C-terminal RNA-binding domain, with similar spacing between the adjacent Pum eight tandem repeats. In addition, phylogenetic patterns of pumilio family showed that Pum 1 and 2 of chicken are more closely related to those of mouse and human than other species and Pum1 is more conserved than Pum2. Using real-time RT-PCR, the expression levels of the Pum1 and Pum2 genes were found to be highest in hatched female gonads, and high-level expression of Pum2 was detected in 12-day and hatched gonads among the various chicken embryonic tissues tested. In adult tissues, the expression levels of Pum1 and Pum2 were expressed at higher levels in the testis and muscle than in any other tissue. The characteristics of the tissue-specific expression of Pum genes suggest that Pum1 and Pum2 have effects crucially in particular stage during development of chicken gonads depending on sexual maturation.