Shigehisa Habe

Chromosomal differentiation of the Schistosoma japonicum complex

The C-banding pattern, location of telomere sequence and chiasma frequency of four species of the Schistosoma japonicum complex were compared with those of two African species, Schistosoma mansoni and Schistosoma haematobium. In the six species, C-banding patterns of seven autosomes and the two sex chromosomes (Z and W) showed relatively species-specific and geographical (Asian and African) differences. Particularly, a plausible pathway of alteration of chromosome 2 revealed a direction from the A-chromosome to the M- chromosome in terms of rearrangements of pericentric inversion and elimination of constitutive heterochromatin (AM inversion). This chromosome change suggested hypothetically that the S. japonicum complex is the original type, and the African species represents the derived type. Moreover, the mosaic construct of the Asian and African types in Schistosoma sinensium chromosomes prompted us to propose that the species might have been formed by hybrid speciation of the genomes of Asian and African species. Localisation of telomeric repeats enabled Asian and African schistosomes to be distinguished clearly by simple terminal location and by terminal and interstitial locations, respectively. Change of chiasma frequency in the S. japonicum complex might be caused by the reduction of interstitial chiasmate (Xi) in the larger chromosomes, 1 and Z (or W), and the change seems to have progressed to Japan from South East Asia. These data enabled us to predict a tentative evolutionary pathway of schistosomes at the cytogenetic level.

Morphological and molecular identification of two Paragonimus spp., of which metacercariae concurrently found in a land crab, Potamiscus tannanti, collected in Yenbai Province, Vietnam

Paragonimosis is an important food-borne zoonosis especially in Asian countries. Among Paragonimus species, Paragonimus westermani followed by P. skrjabini complex are the major pathogens for human paragonimosis in Asia. In addition, P. heterotremus is an important pathogen in southern China and the Indochina Peninsula and is the only proven species to cause human paragonimosis in Vietnam. During a recent survey in Yenbai Province in northern Vietnam, we found small and large types of Paragonimus metacercariae often concurrently in mountainous crabs, Potamiscus tannanti. Adult worms from those small and large metacercariae were obtained separately by experimental infection in dogs and cats. Morphological and molecular phylogenetic study based on sequences of ITS2 and a part of CO1 genes were performed for the identification of small and large metacercariae and their adults. The results showed that small metacercariae and their adults are completely identical with P. heterotremus in morphology and molecular genetic profiles. In contrast, large metacercariae and their adults have some morphological similarities with P. skrjabini and P. harinasutai, but are unidentifiable from each other by morphology alone. Molecular phylogenetic tree analyses on ITS2 and CO1 genes revealed that large metacercariae and their adults were grouped in the same clade and different from any known Paragonimus species. Although they share the same ancestor with P. skrjabini complex, their genetic distance was considerably different from two other known subspecies, P. skrjabini skrjabini and P. skrjabini miyazakii. Our results provide a new insight on the phylogeny of the genus Paragonimus.

Relationships between Schistosoma malayensis and other Asian schistosomes deduced from DNA sequences.

At least three Schistosoma species can infect humans in South-East Asia. The most widespread of these is S. japonicum Katsurada, 1904 which may represent a species complex and occurs in many countries including China, Japan and the Philippines. The second species is S. mekongi Voge, Bruckner and Bruce, 1978 which is endemic to a small area near the junction of Laos, Cambodia and Thailand. Most recently described is S. malayensis Greer, Ow-Yang and Yong, 1988 from a restricted area of peninsular Malaysia. This is primarily a parasite of rats but has also been found in people Davis, on the basis of snail intermediate host phylogeny and biogeography, proposed that S. malayensis and S. mekongi are sister taxa relative to S. japonicum. The three studies on allozymes among these taxa also support this hypothesis.

C-banding analysis of six species of lung flukes,Paragonimus spp. (Trematoda: Platyhelminthes), from Japan and Korea

We examined C-banded karyotypes of six species of lung flukes from Japan and Korea; diploid and triploidParagonimus westermani, P. miyazakii, P. ohirai, P. iloktsuenensis andP. sadoensis, with special reference to their karyotypic diversification. C-band analysis between the diploid and the triploidwestermani revealed that two of three homologues of the triploid resembled those of the diploid in C-band pattern, while the remaining chromosome showed a different pattern from any species examined here. This karyological evidence indicates that the triploid is allotriploid probably induced by interspecific hybridization between the diploidwestermani and an unknown species; we, therefore, suggest that the triploidwestermani is an independent species and synonymous withP. pulmonalis (Miyazaki 1978). As the morphologically similar three species,ohirai, iloktsuenensis andsadoensis, had the same C-band polymorphism in chromosome No. 4, these species are classified as the local races ofP. ohirai. Paragonimus miyazakii has one common C-band (5q) with the diploidwestermani, but other bands (1q, 4q, 6q, 7p and 7q) are different. From these observations, the six species examined are phylogenetically divided into three groups: (1)westermani group containing diploid and triploid (=pulmonalis) species, (2)miyazakii and (3)ohirai including two geographic races,iloktsuenensis and sadoensis.

GENETIC VARIABILITY AND DIFFERENTIATION OF NATURAL POPULATIONS IN THREE JAPANESE LUNG FLUKES, PARAGONIMUS OHIRAI, PARAGONIMUS ILOKTSUENENSIS AND PARAGONIMUS SADOENSIS (DIGENEA: TROGLOTREMATIDAE)

The genetic differences among 3 closely related species, Paragonimus ohirai, Paragonimus iloktsuenensis and Paragonimus sadoensis, were assessed by electrophoretic analysis of allozymes. Fifteen enzymes were analyzed using starch gel electrophoresis. Seven of 18 loci were polymorphic. The most variable population (POs) was polymorphic at 6 of the loci (P = 0.333) with a heterozygosity H of 0.054. Genetic distances between P. ohirai and P. iloktsuenensis, between P. ohirai and P. sadoensis, and between P. iloktsuenensis and P. sadoensis were, on the average, 0.0375, 0.0809 and 0.0567, respectively; values which are well within the usual range for conspecific populations. An especially low estimate of genetic distance was obtained between sympatric populations of P. ohirai and P. iloktsuenensis (0.00059) in Sendai, indicating that gene flow occurred between the 2 species.

Identification of the Human Paragonimiasis Causative Agent in Lao People's Democratic Republic

To assess the species of human paragonimiasis in Lao Peoples Democratic Republic, 6 ovum samples from 6 native confirmed paragonimiasis patients were examined with polymerase chain reaction (PCR) amplifying the internal transcribed spacer 2 (ITS2). The PCR products were sequenced, and a homology search was performed using the GenBank. All 6 sequences were identical with Paragonimus heterotremus ITS2. Our work suggests that P. heterotremus may be the main etiological agent of human paragonimiasis in this locality.

Electrophoretic studies on enzymes of diploid and triploid Paragonimus westermani.

An enzyme analysis of diploid and triploid Paragonimus westermani was conducted using starch gel electrophoresis. In total, 16 enzymes, probably encoded by 18 loci, were studied for 3 populations of the diploid form sampled from 2 localities, and 4 populations of the triploid form from 4 localities. Comparison of the enzymes of the triploid and the diploid digeneans showed 5 different patterns: diaphorase (EC 1.6.2.2), glutamic-oxaloacetic transaminase (EC 2.6.1.1), hexokinase (EC 2.7.1.1), leucylglycylglycine aminopeptidase (EC 3.4.1.3), and phosphoglucomutase (EC 2.7.5.1). On the basis of the numbers of bands and their patterns, all individuals of the triploid are probably heterozygous at each of these 5 loci and homozygous at the remaining 13 loci. The occurrence of fixed heterozygotes found in triploid populations cannot be easily explained by only a single mutation. It is suggested that the variability may have been introduced by hybridization with a different sub-species or a closely related species and may, thus, have been maintained since the time of the origin of triploids.

Discovery of Paragonimus westermani in Vietnam and its molecular phylogenetic status in P. westermani complex

Paragonimus westermani is the most well-known species among the genus Paragonimus. It is widely distributed in Asia with considerable genetic diversity to form P. westermani species complex. While P. westermani distributed in Japan, Korea, China, and Taiwan are genetically homogeneous to form the East Asia group, those found in other geographic areas are heterogeneous and would be divided into several groups. Recent discoveries of P. westermani in India and Sri Lanka highlighted new insights on molecular phylogenetic relationship of geographic isolates of this species complex. Since Vietnam is located at the east end of Southeast Asia, the intermediate position between South and East Asia, it is of interest to see whether P. westermani is distributed in this country. Here, we report that P. westermani metacercariae were found in mountainous crabs, Potamiscus sp., collected in Quangtri province in the central Vietnam. Adult worms were successfully obtained by experimental infection in cats. Molecular phylogenetic analyses revealed that P. westermani of Vietnamese isolates have high similarities with those of East Asia group.

Morphological differences and molecular similarities between Paragonimus bangkokensis and P. harinasutai

Paragonimus bangkokensis and Paragonimus harinasutai were found one after another in the same crab host, Potamon smithianus, in Thailand in 1967 and 1968. Both species were also recently found in China and Lao Peoples Democratic Republic (PDR). Those two Paragonimus spp. are distinguishable from each other by morphological features of metacercariae and adults. However, recently, the DNA sequences of second internal transcribed spacer region (ITS2) and cytochrome oxidase subunit 1 (CO1) genes of those two species in Thailand were reported to be highly similar to each other. In the present study, we collected P. bangkokensis in two provinces in Vietnam (the first record in Vietnam) and both P. bangkokensis and P. harinasutai in Lao PDR for the morphological and molecular phylogenetic analyses to clarify the mutual relationship between the two species. The results show that P. bangkokensis and P. harinasutai were distinguishable from each other by morphology such as the size of metacercariae and the arrangement of cuticular spines of adult worms. However, the molecular phylogenetic analyses of ITS2 and CO1 genes clearly indicate that P. bangkokensis and P. harinasutai make a monophyletic group.

Electrophoretic evidence of a hybrid origin for tetraploidParagonimus westermani discovered in northeastern China

1 Department of Parasitology, Kochi Medical School, Nankoku City, Kochi 783, Japan z Department of Parasitology, Beijin Medical University, Beijing 100034, P. R. China 3 Department of Parasitology, School of Medicine, Fukuoka University, Fukuoka City, Fukuoka 814-01, Japan 4 Laboratory of Biology, St. Marys Junior College, Tsubuku-Honmachi, Kurume City, Fukuoka 830, Japan 5 Laboratory Animal Research Center, Tottori University School of Medicine, Yonago City, Tottori 683, Japan 6 Department of Parasitology, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo 141, Japan 7 Department of Parasitology, National Institute of Health, 5 Nokbun-Dong Eunpyung-ku, Seoul, 122-020, Korea s School of Health Sciences, Kyushu University, Fukuoka City, Fukuoka 812, Japan