Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae)

Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.     

Comparative cytogenetics of opisthorchid species (Trematoda, Opisthorchiidae)

In the present study karyotypes and chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus (Rivolta, 1884), O. viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), M. bilis (Braun, 1893), and Clonorchis sinensis (Cobbold, 1875)) were compared. Karyotypes of O. felineus, M. xanthosomus, M. bilis and C. sinensis consist of two pairs of large meta- and submetacentrics and five pairs of small chromosomes (2n = 14). The karyotype of O. viverrini is 2n = 12, which indicates a fusion of two chromosomes of opisthorchid ancestral karyotype. Analysis of mitotic and meiotic chromosomes was performed by heterologous in situ hybridization of microdissected DNA probes obtained from chromosomes 1 and 2 of O. felineus and chromosomes 1 and 2 of M. xanthosomus. Results of chromosome staining (C- and AgNOR-banding) and FISH of telomeric probes and ribosomal DNA probe on opisthorchid chromosomes were used for chromosome comparison. Data on chromosome number in opisthorchid species were also discussed.     

Conservation and characterization of unique porcine interstitial telomeric sequences

Telomeres are composed of TTAGGG repeats and located at the ends of chromosomes. Telomeres protect chromosomes from instability in mammals, including mice and humans. Repetitive TTAGGG sequences are also found at intrachromosomal sites, where they are named as interstitial telomeric sequences (ITSs). Aberrant ITSs are implicated in chromosomal instability and found in cancer cells. Interestingly, in pigs, vertebrate telomere sequences TTAGGG (vITSs) are also localized at the centromeric region of chromosome 6, in addition to the end of all chromosomes. Surprisingly, we found that botanic telomere sequences, TTTAGGG (bITSs), also localize with vITSs at the centromeric regions of pig chromosome 6 using telomere fluorescence in situ hybridization (FISH) and by comparisons between several species. Furthermore, the average lengths of vITSs are highly correlated with those of the terminal telomeres (TTS). Also, pig ITSs show a high incidence of telomere doublets, suggesting that pig ITSs might be unstable and dynamic. Together, our results show that pig cells maintain the conserved telomere sequences that are found at the ITSs from of plants and other vertebrates. Further understanding of the function and regulation of pig ITSs may provide new clues for evolution and chromosomal instability.     

Repetitive sequences associated with differentiation of W chromosome in Semaprochilodus taeniurus

The possible origins and differentiation of a ZZ/ZW sex chromosome system in Semaprochilodus taeniurus, the only species of the family Prochilodontidae known to possess heteromorphic sex chromosomes, were examined by conventional (C-banding) and molecular (cross-species hybridization of W-specific WCP, Fluorescence in situ hybridization (FISH) with telomere (TTAGGG)n, and Rex1 probes) cytogenetic protocols. Several segments obtained by W-specific probe were cloned, and the sequences localized on the W chromosome were identified by DNA sequencing and search of nucleotide collections of the NCBI and GIRI using BLAST and CENSOR, respectively. Blocks of constitutive heterochromatin in chromosomes of S. taeniurus were observed in the centromere of all autosomal chromosomes and in the terminal, interstitial, and pericentromeric regions of the W chromosome, which did not demonstrate interstitial telomeric sites with FISH of the telomere probe. The Rex1 probe displayed a compartmentalized distribution pattern in some chromosomes and showed signs of invasion of the pericentromeric region in the W chromosome. Chromosomal painting with the W-specific WCP of S. taeniurus onto its own chromosomes showed complete staining of the W chromosome, centromeric sites, and the ends of the Z chromosome, as well as other autosomes. However, cross-species painting using this WCP on chromosomes of S. insignis, Prochilodus lineatus, and P. nigricans did not reveal a proto-W element, but instead demonstrated scattered positive signals of repetitive DNAs. Identification of the W-specific repetitive sequences showed high similarity to microsatellites and transposable elements. Classes of repetitive DNA identified in the W chromosome suggested that the genetic degeneration of this chromosome in S. taeniurus occurred through accumulation of these repetitive DNAs.     

Zoonotic Trematode Metacercariae in Fish from Phnom Penh and Pursat,Cambodia

A survey was performed to investigate the infection status of freshwater fish with zoonotic trematode metacercariae in Phnom Penh and Pursat Province, Cambodia. All collected fish with ice were transferred to our laboratory and examined using the artificial digestion method. In fish from Phnom Penh, 2 kinds of metacercariae (Opisthorchis viverrini and Haplorchis yokogawai) were detected. O. viverrini metacercariae were positive in 37 (50.0%) of 74 fish in 11 species (average no. metacercariae/fish, 18.6). H. yokogawai metacercariae were detected in 23 (57.5%) of 40 fish in 5 species (average no. metacercariae/fish, 21.0). In fish from Pursat Province, 5 kinds of metacercariae (O. viverrini, H. yokogawai, Haplorchis pumilio, Centrocestus formosanus, and Procerovum sp.) were detected; O. viverrini metacercariae (n=3) in 2 fish species (Henicorhynchus lineatus and Puntioplites falcifer), H. yokogawai metacercariae (n=51) in 1 species (P. falcifer), H. pumilio metacercariae (n=476) in 2 species (H. lineatus and Pristolepis fasciata), C. formosanus metacercariae (n=1) in 1 species (H. lineatus), and Procerovum sp. metacercariae (n=63) in 1 species (Anabas testudineus). From the above results, it has been confirmed that various freshwater fish play the role of a second intermediate host for zoonotic trematodes (O. viverrini, H. yokogawai, H. pumilio, C. formosanus, and Procerovum sp.) in Cambodia.     

Three-dimensional organization of interphase fibroblast nuclei in two closely related shrew species (<Emphasis Type="Italic">Sorex granarius</Emphasis> and <Emphasis Type="Italic">Sorex araneus</Emphasis>) differing in the structures of their chromosome termini

A FISH with a probe for telomeric and rDNA repeats and immunofluorescence with ANA CREST and antibodies to nucleolae protein B23 were used to study the three-dimensional (3D) organization of fibroblast interphase nuclei in two shrew twin species, Sorex granarius and Sorex araneus, of the Cordon race. Karyotypes of these species are composed of nearly identical chromosomal arms and differ in the number of their metacentrics and the structures of their terminal chromosome regions. In the short arms of S. granarius, 32 of the acrocentrics have large telomeres that contain an average of 218 kb telomere repeats, which alternate with ribosomal repeats. These regions also contain active nucleolar organizing regions (NORs). In contrast, in active NORs in S. araneus are localized at the terminal regions of 8 chromosomal arms (Zhdanova et al., 2005; 2007b). Here, we show that associations of chromosomes by telomeres and the contact of a part of the telomere clusters with the inner nuclear membrane and nucleolus characterize the interphase nuclei of both Sorex granarius and Sorex araneus. We also reveal the partial colocalization of telomere and ribosomal clusters and the spatial proximity of centomeric and telomeric regions in the interphase nuclei of S. granarius. It appears that only ribosomal clusters containing a sufficient number of active ribosomal genes exhibit a connection with the nucleolus. Nucleolus disassembly during the fibroblastís transition to mitosis and the role of the B23 protein in this process have been studied.     

Fishborne Trematode Metacercariae in Luang Prabang,Khammouane, and Saravane Province,Lao PDR

Fishborne trematode (FBT) metacercariae were investigated in fish from 3 Provinces of Lao PDR. Total 242 freshwater fish of 40 species were collected in local markets of Luang Prabang (59 fish of 16 species), Khammouane (81 fish of 19 species), and Saravane (97 fish of 14 species), and each of them was examined by artificial digestion method. Four species of metacercariae (Opisthorchis viverrini, Haplorchis taichui, Haplorchis yokogawai, and Centrocestus formosanus) were detected. O. viverrini was detected in 35 fish (14.5%), and their density was 252 per infected fish (Luang Prabang, 88 metacercariae in 5 fish; Khammouane, 187 in 6 fish; Saravane, 303 in 24 fish). H. taichui was found in 102 fish (42.1%), and their density was 485 per infected fish (Luang Prabang, 260 metacercariae in 38 fish; Khammouane, 1,084 in 23 fish; Saravane, 359 in 41 fish). H. yokogawai was detected in 92 fish (38.0%), and their density was 222 per infected fish (Luang Prabang, 362 metacercariae in 17 fish; Khammouane, 126 in 20 fish; Saravane, 214 in 55 fish). Metacercariae of C. formosanus were found in 8 fish (3.3%), and their density was 3 per infected fish. In the present study, it has been confirmed that FBT metacercariae, in particular, H. taichui, H. yokogawai, and O. viverrini, are highly prevalent in fish from Luang Prabang, Khammouane, and Saravane Province, Lao PDR.     

TaqMan real-time PCR assay for specific detection of Opisthorchis viverrini DNA in Thai patients with hepatocellular carcinoma and cholangiocarcinoma

The aim of this study was to develop TaqMan real-time PCR assay that detected Opisthorchis viverrini DNA from 18 normal and 18 tumor tissue specimens from Thai patients with hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), who underwent liver resection from October 2005 to May 2006. Control liver specimens were seven non-primary liver cancers. A conserved probe representing 100% sequence homology was used as a reference for O. viverrini-specific probe. Five of six tumors (83%) and all six normal tissues from CCA group; and seven of twelve tumors (58%) and ten of twelve normal tissues (83%) from HCC group were found to have O. viverrini DNA. The O. viverrini DNA detection among HCC and CCA patients were not associated (p = 0.193; 90%CI). This RT-PCR will be a useful tool for investigating the relationship between cancer type and presence of the parasite and also for conducting epidemiological surveys.     

Characterization of the telomere regions of scleractinian coral, <Emphasis Type="Italic">Acropora surculosa</Emphasis>

Terminal ends of vertebrate chromosomes are protected by tandem repeats of the sequence (TTAGGG). First thought to be vertebrate specific, (TTAGGG) _n has recently been identified in several aquatic invertebrates including sea urchin (Strongylocentrotus purpuratus), bay scallop (Argopecten irradians), and wedgeshell clam (Donax trunculus). We analyzed genomic DNA from scleractinian corals, Acropora surculosa, Favia pallida, Leptoria phrygia, and Goniastrea retiformis to determine the telomere sequence. Southern blot analysis suggests the presence of the vertebrate telomere repeats in all four species. Treatment of A. surculosa sperm DNA with Bal31 exonuclease revealed progressive shortening of the DNA fragments positive for the (TTAGGG)₂₂ sequence, supporting location of the repeats at the chromosome ends. The presence of the vertebrate telomere repeats in corals is evidence that the (TTAGGG) _n sequence is highly conserved among a divergent group of vertebrate and invertebrate species. Corals are members of the Lower Metazoans, the group of organisms that span the gap between the fungi and higher metazoans. Corals are the most basal organism reported to have the (TTAGGG) _n sequence to date, which suggests that the vertebrate telomere sequence may be much older than previously thought and that corals may share a number of genes with their higher relatives.     

Hyperendemicity of Haplorchis taichui Infection among Riparian People in Saravane and Champasak Province,Lao PDR

In this study, we found that Haplorchis taichui, a heterophyid intestinal fluke, is highly prevalent, with heavy worm loads, among riparian people in Saravane and Champasak province, Lao PDR. Fecal specimens were collected from 1,460 people (717 men and 743 women) in 12 riparian (Mekong river) districts and were examined by the Kato-Katz fecal smear technique. The overall helminth egg positive rate was 78.8% and 66.4% in Saravane and Champasak province, respectively. The positive rate for small trematode eggs (STE), which included H. taichui and other heterophyids, Opisthorchis viverrini, and lecithodendriids, was 69.9% and 46.3% in Saravane and Champasak province, respectively. To obtain adult flukes, 30 STE-positive people were treated with 40 mg/kg praziquantel and then purged. Whole diarrheic stools were collected 4-5 times for each person and searched for fluke specimens using a stereomicroscope. Mixed infections with various species of trematodes (H. taichui, Haplorchis pumilio, O. viverrini, Prosthodendrium molenkampi, Centrocestus formosanus, and Echinochasmus japonicus) and a species of cestode (Taenia saginata) were found. However, the worm load was exceptionally high for H. taichui compared with other trematode species, with an average of 21,565 and 12,079 specimens per infected person in Saravane and Champasak province, respectively, followed by H. pumilio (41.9 and 22.5, respectively) and O. viverrini (9.4 and 1.5, respectively). These results show that diverse species of intestinal and liver flukes are prevalent among riparian people in Saravane and Champasak province, Lao PDR, with H. taichui being the exceptionally dominant species.     

Distribution of telomeric (TTAGGG)<Subscript>n</Subscript> sequences in avian chromosomes

The physical ends of mammalian and other vertebrate chromosomes consist of tandemly repeated (TTAGGG)(n) hexamers, nucleating a specialized telomeric structure. However, (TTAGGG)(n) sequences can also occur at non-telomeric sites, providing important insights into karyotypic evolution. By fluorescence in situ hybridization (FISH) we studied the chromosomal distribution of (TTAGGG)(n) sequences in 16 bird species, representing seven different orders. Many species, in particular the ratites, display (TTAGGG)(n) hybridization signals in interstitial and centromeric regions of their macrochromosomes in addition to the typical telomeric signals. In some but not all species these non-telomeric sites coincide with C-band-positive heterochromatin. The retention and/or amplification of telomeric (TTAGGG)(n) repeats at interstitial and centromeric sites may indicate the fusion of ancestral chromosomes. Compared with the macrochromosomes, the microchromosomes of most species are enriched with (TTAGGG)(n) sequences, displaying heterogeneous hybridization patterns. We propose that this high density of (TTAGGG)(n) repeats contributes to the exceptionally high meiotic recombination rate of avian microchromosomes.     

Infection Status of Zoonotic Trematode Metacercariae in Fishes from Vientiane Municipality and Champasak Province in Lao PDR

The infection status of fishborne zoonotic trematode (FZT) metacercariae was investigated in fishes from 2 localities of Lao PDR. Total 157 freshwater fishes (17 species) were collected in local markets of Vientiane Municipality and Champasak Province in December 2010 and July 2011, and each fish was examined by the artificial digestion method. Total 6 species of FZT metacercariae, i.e., Opisthorchis viverrini, Haplorchis taichui, Haplorchis yokogawai, Haplorchis pumilio, Centrocestus formosanus, and Procerovum varium, were detected in fishes from Vientiane Municipality. The metacercariae of O. viverrini were detected in 50 (49.5%) out of 101 fishes (6 species), and their average number was 154 per fish infected. The remaining 5 species of heterophyid metacercariae were detected in 36.8%, 65.8%, 9.4%, 23.9%, and 5.1% fishes examined, and their average densities were 12, 1,038, 4, 15, and 13 per infected fish, respectively. In fishes from Champasak Province, 3 species of FZT metacercariae, i.e., O. viverrini, H. taichui, and H. yokogawai, were detected. Only 2 O. viverrini metacercariae were found in only 1 Barbonymus schwanefeldi. The metacercariae of H. taichui and H. yokogawai were detected in 60.0% and 50.0% of fishes examined, and their average densities were 47 and 28 per fish infected. By the present study, it has been confirmed that several species of FZT metacercariae are prevalent in fishes from Vientiane Municipality, with P. varium being a new member of FZT in Lao PDR. In comparison, FZT metacercariae are less prevalent in fishes from Champasak Province.     

Chronic Opisthorchis viverrini Infection and Associated Hepatobiliary Disease Is Associated with Iron Loaded M2-like Macrophages

Chronic Opisthorchis viverrini-induced hepatobiliary disease is associated with significant leukocyte infiltration, including activated macrophages; however, the polarization of infiltrating macrophages remains to be fully characterized. In this study, we characterized macrophage polarization and phenotype in chronic O. viverrini-induced hepatobiliary disease in humans and hamsters using gene expression and histochemical analysis. Chronic O. viverrini infection and associated hepatobiliary diseases were associated with iron loaded M2-like macrophages in both humans and hamsters. This study provides suggestive evidence that iron loaded M2-like macrophages promote hepatobiliary disease in chronic O. viverrini infection.     

Chromosomal localization of the telomeric (TTAGGG)n sequence in eight species of New World Primates (Neotropical Primates, Platyrrhini)

Chromosomal localization of the telomeric sequence (TTAGGG)(n) in eight New World Primates (Platyrrhini) (Alouatta caraya, Alouatta palliata, Alouatta guariba clamitans, Aotus azarae, Ateles chamek, Cebus nigritus, Cebus paraguayanus, and Saimiri boliviensis) using Fluorescence In Situ Hybridization (FISH) with a peptide nucleic acid (PNA) pantelomeric probe and their possible relationship with the C-banding pattern were analyzed. FISH showed telomeric signals only at the terminal regions of chromosomes from all the species analyzed. Although all of them showed centromeric C+ bands and different size and location of extracentromeric C+ bands, none, except Aotus azarae exhibited (peri)centromeric interstitial telomere-like sequences (ITS). The presence of ITS in Aotus azarae was limited to one pair of submetacentric chromosomes and very likely represents telomeric sequences remaining after a fusion event of ancestral chromosomes during karyotype evolution. Therefore, our data indicate that the distribution of heterochromatin blocks do not correlate with the presence of ITS. However, we cannot rule out the possibility that simple ITS arrays with a few copies of the (TTAGGG)(n) sequence, not detectable by conventional FISH, might play a role in the karyotypic evolution of Ceboidea. Further FISH and molecular studies will be needed to confirm this hypothesis.     

Prevalence of Opisthorchis viverrini-Like Fluke Infection in Ducks in Binh Dinh Province,Central Vietnam

Following the first report of Opisthorchis viverrini infection in a domestic duck in Phu My District of Binh Dinh Province, Central Vietnam, many other cases were observed in the province. We determined the infection rate and intensity of O. viverrini infection in ducks in 4 districts of the province. A total of 178 ducks were randomly selected from 34 farms for examination of flukes in the liver and gall bladder. An infection rate of 34.3% (range 20.7-40.4% among districts) was found; the intensity of infection was 13.8 worms per infected duck (range 1-100). These findings show the role of ducks as a host for O. viverrini, duck genotype, which is sympatric with the human O. viverrini genotype in this province. It also stresses the need for investigations on the zoonotic potential and the life cycle of this parasite.     

Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.     

Fluorescence in situ hybridization of rDNA, telomeric (TTAGGG)n and (GATA)n repeats in the red abalone Haliotis rufescens (Archaeogastropoda: Haliotidae)

The physical location of 18S-5.8S-28S rDNA, telomeric sequences with (TTAGGG)n DNA probe and (GATA)n microsatellites were performed by fluorescence in situ hybridization in chromosomes of red abalone Haliotis rufescens. The karyotype of red abalone showed a diploid number of 36 (8M+9SM+1ST). FISH performed with rDNA probe, showed the location of major ribosomal clusters in the terminal region of the large arms of two submetacentric pairs (chromosome 4 and 5). Localization of heteromorphisms of FISH-rDNA was found between chromosome homologues and sister chromatids in all metaphases analyzed. This indicates that rDNA clusters are variable within the red abalone genome. The variability in the NOR-bearing reported using silver staining in other gastropods and our result are discussed. In addition, the presence of microsatellite (TTAGGG)n and (GATA)n was demonstrated after FISH treatment by DNA probes. The telomeric sequence occurred at the ends of all mitotic chromosomes, while the (GATA)n repetitive was found on chromosomal interstitial zones as well as at the telomeres in abalone chromosomes.     

Establishment of an Allo-Transplantable Hamster Cholangiocarcinoma Cell Line and Its Application for In Vivo Screening of Anti-Cancer Drugs

Opisthorchis viverrini (O. viverrini) is a well-known causative agent of cholangiocarcinoma (CCA) in humans. CCA is very resistant to chemotherapy and is frequently fatal. To understand the pathogenesis of CCA in humans, a rodent model was developed. However, the development of CCA in rodents is time-consuming and the xenograft-transplantation model of human CCA in immunodeficient mice is costly. Therefore, the establishment of an in vivo screening model for O. viverrini-associated CCA treatment was of interest. We developed a hamster CCA cell line, Ham-1, derived from the CCA tissue of O. viverrini-infected and N-nitrosodimethylamine-treated Syrian golden hamsters. Ham-1 has been maintained in Dulbecco''s Modified Essential Medium supplemented with 10% fetal bovine serum for more than 30 subcultures. These cells are mostly diploid (2n=44) with some being polyploid. Tumorigenic properties of Ham-1 were demonstrated by allograft transplantation in hamsters. The transplanted tissues were highly proliferative and exhibited a glandular-like structure retaining a bile duct marker, cytokeratin 19. The usefulness of this for in vivo model was demonstrated by berberine treatment, a traditional medicine that is active against various cancers. Growth inhibitory effects of berberine, mainly by an induction of G1 cell cycle arrest, were observed in vitro and in vivo. In summary, we developed the allo-transplantable hamster CCA cell line, which can be used for chemotherapeutic drug testing in vitro and in vivo.     

Strand-specific fluorescence in situ hybridization: the CO-FISH family

The ability to prepare single-stranded chromosomal target DNA allows innovative uses of FISH technology for studies of chromosome organization. Standard FISH methodologies require functionally single-stranded DNAs in order to facilitate hybridization between the probe and the complementary chromosomal target sequence. This usually involves denaturation of double-stranded probes to induce temporary separation of the DNA strands. Strand-specific FISH (CO-FISH; Chromosome Orientation-FISH) involves selective removal of newly replicated strands from DNA of metaphase chromosomes which results in single-stranded target DNA. When single-stranded probes are then hybridized to such targets, the resulting strand-specific hybridization is capable of revealing a level of information previously unattainable at the cytogenetic level. Mammalian telomeric DNA consists of tandem repeats of the (TTAGGG) sequence, oriented 5'-->3' towards the termini of all vertebrate chromosomes. Based on this conserved structural organization, CO-FISH with a telomere probe reveals the absolute 5'-->3' orientation of DNA sequences with respect to the pter-->qter direction of chromosomes. Development and various applications of CO-FISH will be discussed: detection of cryptic inversions, discrimination between telomeres produced by leading- versus lagging-strand synthesis, and replication timing of mammalian telomeres.     

The ecology of the Bithynia first intermediate hosts of Opisthorchis viverrini

Opisthorchiasis, together with its associated cholangiocarcinoma, is one of the most important human parasitic diseases on continental Southeast Asia. A great deal of epidemiological data from humans is available on this disease, particularly from the northeast of Thailand, however, only limited information is available on those aspects of the life cycle relating to its Bithynia (Gastropoda) and cyprinid fish intermediate hosts. Here we review the information which is available on the Bithynia hosts of Opisthorchis viverrini. Only one major ecological study has been carried out at one site on a single species of Bithynia. We show not only that detailed ecological studies are required to clarify the epidemiology of opisthorchiasis, but also that the taxonomic status of the Bithynia species transmitting O. viverrini requires clarification.