Evidence of late root formation of molars in Anderson's red-backed vole, Eothenomysandersoni (Thomas, 1905) (Cricetidae, Rodentia), and arguments for its generic allocation.

We evaluated the molars in Anderson's red-backed vole (n = 114) from the Kii Peninsula of Honshu, Japan. Two of the specimens are considered extremely old aged based on their dimensions and on the loss of alveolar capsules of M2, and a third one is also old based on its strongly worn left M3 and M1. Of the former two individuals, one showed an incipient closure of re-entrant angles at its basal end, as estimated from the difference between the occlusal patterns of the occlusal and basal surfaces of the left M2. The latter individual also showed a complete closure of the basal end in the left M3. These patterns differ from incipient roots observed in other vole taxa but were similar to a previous example of incipient roots in Anderson's red-backed vole. Therefore, we suggest that molar roots in this species form at an extremely late age or by strong wear. Root formation in molars is considered an important diagnostic character, as Eothenomys molars lack roots, while Craseomys molars develop roots at a late age. However, this dental character may be particularly difficult to assess in voles under natural conditions. Considering previous phylogenetic findings based on molecular analyses, Craseomys is the most appropriate genus for Anderson's and other Asiatic red-backed voles.

A genetic typing method for albino mutation in the Mongolian gerbil by PCR-RFLP analysis.

1. We established a PCR-RFLP analysis targeting R77H mutation in the Tyr gene as a more effective genotyping to identify carrier (C/c) with the albino allele and the agouti phenotypes. 2. Our breeding system, which targets the R77H site, is a useful cue for detecting C/c carriers with the agouti-phenotype and helps us to obtain albinos by mating agouti-phenotype carriers.

Colonization and Differentiation Traits of the Japanese House Mouse, Mus musculus (Rodentia, Muridae), Inferred from Mitochondrial Haplotypes and External Body Characteristics.

To evaluate the colonization histories of the Japanese house mice (Mus musculus), phenotypic and genotypic admixtures of the subspecific traits were studied by evaluation of external body characteristics and mitochondrial gene elements. We analyzed mitochondrial Cytb gene and coat colorations and body dimensions as subspecific characteristics in mice from four areas of the Japanese Islands, the Sorachi, Ishikari and Iburi areas of Hokkaido, the Hidaka area of Hokkaido, and northeastern and central Honshu. Three occurrence patterns of the subspecific haplotypes of Cytb-the castaneus type only, the musculus type only, and the castaneus, musculus, and domesticus types together-were observed in the study areas. In central Honshu, the properties of haplotypes were in accord with the external characteristics as reported in previous findings. In contrast, complicated external characteristics were observed in the Hidaka area, where mice showed multiple haplotype properties. In addition, in northeastern Honshu, coat colorations were not in accord with haplotype properties and such discordance was also observed in most mice in the Sorachi, Ishikari and Iburi areas of Hokkaido. These complexities and discordances suggest that the genetic and phenotypic properties have been caused by different processes, not only through founder effects by migrations and subsequent subspecific hybridizations but also through differentiation in each study area.

Molecular analyses of the agouti allele in the Japanese house mouse identify a novel variant of the agouti gene.

It has been thought that the Japanese house mouse carries the Aw allele at the agouti locus causing light-colored bellies, but they do not always show this coloration. Thus, the presence of the Aw allele seems to be doubtful in them. To ascertain whether the Aw allele is present, a two-pronged approach was used. First, we compared lengths of DNA fragments obtained from three PCRs conducted on them to the known fragment sizes generated from mouse strains exhibiting homozygosities of either a/a, A/A, or Aw/Aw. PCR I, PCR II, and PCR III amplify only in the A and Aw alleles, the a and Aw alleles, and the a allele, respectively, and we detected amplifications in strains with A/A and Aw/Aw by PCR I, in those with a/a and the Japanese house mouse by PCR II, and in those with a/a by PCR III. Second, we sequenced the exon 1A region of the agouti gene and obtained sequences corresponding to the above strains and the Japanese house mouse, but their sequences were similar to those of the a allele. We concluded that their agouti allele is not identical to the Aw allele and seems to be a novel type similar to the a allele.

Cross-experimental analysis of coat color variations and morphological characteristics of the japanese wild mouse, Mus musculus.

There are many coat colors in the laboratory mouse, Mus musculus. On the basis of traditional genetics, there are four loci, A-D, related to coat color expressions. As shown by previous studies, Japanese wild mice have gray backs and white bellies and are assumed to carry the A(w) allele at the A (agouti) locus, which is dominant over any other alleles. However, we collected Japanese wild mice from central Honshu with black coats. To understand this black coat expression, we performed cross experiments concerning the four loci using wild-caught mice and DBA/2 laboratory mice from the standpoint of traditional genetics. The offspring of the current crosses showed the wild type, the blackish type, and the intermediate type from some combinations of parents. Considering the coat colors of the offspring, we did not obtain any evidence that the Japanese wild mice always carry the A(w) allele at the A locus. Furthermore, we were not able to explain the current coat color expressions using the traditional logic with regard to the A-D loci and concluded that it is possible for another locus (loci) to be related to the coat color expressions. On the other hand, skull characteristics and external body measurements of the captured wild mice were fundamentally different from those of DBA/2 and offspring from captured wild mice and DBA/2 combinations. Thus, we concluded that the Japanese wild mice had original criteria from a morphological viewpoint.

Establishment of gerbil-mouse heterohybridoma secreting immunoglobulin of Mongolian gerbil (Meriones unguiculatus) establishment of gerbil-mouse heterohybridoma.

Hybridomas, generated by fusing myeloma cells with B lymphocytes, secrete monoclonal antibodies (mAbs) specific for an antigen. To establish hybridomas that secrete Mongolian gerbil (Meriones unguiculatus) mAbs, we immunized gerbils with keyhole limpet hemocyanin (KLH) and fused splenocytes from them with a non-secreting mouse myeloma cell line (P3-X63-Ag8.653). We obtained hybridomas that secreted immunoglobulin (Ig) against KLH. These hybridomas had more chromosomes than either parent and retained several gerbil chromosomes. Therefore, these cells were heterohybridomas with both gerbil and mouse chromosomes. They expressed gerbil Ig heavy-chain constant (IGHC) region mRNA, but not mouse gamma (γ) 1 IGHC. SDS-PAGE and Western blot analyses indicated that the cells secreted whole Ig molecules of gerbil origin. Moreover, the cells continued secreting Ig for several months.

Intra- and interspecific nuclear ribosomal gene variation in the two Japanese Eothenomys species, E. andersoni and E. smithii.

We analyzed partial sequences (approx. 630 bp) of the 18S nuclear ribosomal RNA gene (rDNA) in the Japanese Eothenomys voles, E. andersoni and E. smithii, to evaluate their evolutionary complexity. There were two rDNA types of sequences with a few substitutions and an indel, and both species alternatively carried one of the two rDNA types, irrespective of their classifications. One of the rDNA types was mainly distributed in Eastern Japan, and the other in Western Japan. Such rDNA type distributions were not related to the species classifications. The complex genomic elements across both species were confirmed by previous studies of nuclear, mitochondrial, and Y chromosomal genes, and our current data agree with previous findings. Previous studies indicated that the genome constitutions of the Japanese Eothenomys species may have been caused by interspecific genetic exchanges at an early period after the speciation into E. andersoni and E. smithii from their common ancestor, as neither species crosses at present. In addition, the present results also suggest the additional hypothesis that their common ancestor might have two (or more) rDNA gene types, and that one of the rDNA types was fixed in each population of both species during their colonization processes.

Intraspecific differentiation in the lesser Japanese mole in eastern Honshu, Japan, indicated by nuclear and mitochondrial gene analyses.

The lesser Japanese mole, Mogera imaizumii, recognized by Motokawa and Abe (1996), occurs in eastern Honshu, western Honshu and Shikoku. Mitochondrial and nuclear DNA were analyzed for mole samples from eastern Honshu to elucidate intraspecific differentiation. Analyses of sequences of the mitochondrial cytochrome b gene (Cytb) and of a restriction fragment length polymorphism of the nuclear 28S ribosomal RNA gene spacer (rDNA-RFLP) revealed two genetic types, partially corresponding to Hutterer's (1993) taxa, M. wogura (= M. imaizumii) and M. minor. Most samples showed either of two combinations of mitochondrial / nuclear gene types. However, two specimens showed a different combination. This incongruent combination of mitochondrial and nuclear genes might have derived, in part, from an introgression event between genetically differentiated populations after secondary contact during the evolutionary history of the lesser Japanese mole in eastern Honshu.

Epizootiological and epidemiological study of hantavirus infection in Japan.

Epizootiological surveys on hantavirus infections in rodents were carried out in various areas of Japan, including the four major islands of Hokkaido, Honshu, Shikoku, and Kyushu from 2000 to 2003. A total of 1,221 rodents and insectivores were captured. Seropositive animals were found in Apodemus (A.) speciosus (5/482, 1.0%), Rattus (R.) norvegicus (4/364, 1.1%), R. rattus (3/45, 6.7%), and Clethrionomys (C.) rufocanus (7/197, 3.6%). The partial S segment was amplified from one seropositive R. rattus captured at Hakodate. The nucleotide sequence showed 96% identity with the Seoul virus (SEOV) prototype strain SR-11. In addition, we conducted an epidemiological survey on human hantavirus infection in a high-risk population, the personnel of the Japan Ground Self-defense Force on Hokkaido. One out of 207 human blood samples was positive for anti-hantavirus antibody by IFA, ELISA, and WB analysis. The result of the serotype specific ELISA indicates that this individual acquired SEOV infection. This study indicates that A. speciosus, R. norvegicus, R. rattus, and C. rufocanus carry hantaviruses as the reservoir animals in Japan. Infected R. rattus and R. norvegicus in port areas could be the sources of human SEOV infection and a threat to travelers and individuals working in seaports.

Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of Mongolian gerbils.

Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10 (5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C.) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.

Intra- and interspecific genetic complexities of two Eothenomys species in Honshu, Japan.

Differences in the nuclear ribosomal DNA (rDNA), mitochondrial DNA (mtDNA), cytochrome b (Cytb), and Y chromosomal Sry genes were used to assess intra- and interspecific relationships in two Japanese red-backed voles, Eothenomys andersoni and E. smithii, focusing on areas where the two species might come into contact. In the Kii Peninsula, southwestern Honshu, which contains an allopatric population of E. andersoni isolated from its main range, the rDNA-RFLP data provide robust evidence of past mutual interspecific gene introgression, while the Cytb and Sry sequences were specific to this population. In central Honshu, where E. andersoni and E. smithii inhabit higher and lower altitudes, respectively, with a narrow sympatric zone, the rDNA-RFLP and Sry variation was specific for each species, while introgression of the mtDNA from E. smithii to E. andersoni was seen. These complex patterns in the gene markers are consistent with our previous notions derived from sex chromosome variation. Our previous and present data strongly suggest that the evolution of these vole species, which are morphologically and cytogenetically distinct, involves complex genetic interactions and the resultant combinations of genes are sometimes peculiar, mainly due to the Cytb haplotypes. However, phylogenetic analysis using a combination of maternal, paternal, and biparental markers has proven useful for understanding the evolutionary history given the complex phylogenetic background.

Evolutionary significance of chromosome changes in northeastern Asiatic red-backed voles inferred with the aid of intron 1 sequences of the G6pd gene.

To evaluate the evolutionary significance of karyotypic characters in red-backed voles (Rodentia, Arvicolinae, genera Clethrionomys and Eothenomys) from northeastern Asia, we constructed phylogenetic trees based on intron 1 sequences (ca. 580 bp) of the X-linked gene G6pd. Phylogenetic trees, constructed using neighbor-joining and maximum-likelihood methods, revealed two major clades in red-backed voles corresponding to the glareolus- and the rufocanus-cytotypes defined by a reciprocal translocation between chromosomes 1 and 9 based on G-band patterns. If confirmed, two types of X chromosome, acrocentric X and subtelocentric X, which have often been claimed to be traits of evolutionary and taxonomic significance, should be considered as examples of homoplasy because they occur sporadically in members of both rufocanus- and glareolus-cytotypes in red-backed voles.

A spatial aspect on mitochondrial DNA genealogy in Apodemus peninsulae from East Asia.

Apodemus peninsulae is a field mouse that inhabits the broad-leafed forests of temperate Eurasia. We examined the mitochondrial cytochrome b gene in 57 in dividuals of A. peninsulae from northeastern Asia, including Siberia, Primorye, Magadan region, Sakhalin, Hokkaido, and the Korean Peninsula. The genealogy of the mitochondrial DNA (mtDNA) in A. peninsulae was shown to have substantial geographic affinity, suggesting geographic architecture of northeastern Asia, including the islands of Sakhalin and Hokkaido, played important roles on the cladogenesis. Taking into account the presence of region-specific anciently divergent mtDNA types, three parts of the regions of Primorye, Siberia, and the Korean Peninsula can be denoted as refugia for A. peninsulae during the substantial period of the Quaternary glacial ages. Among the geographic regions examined, Primorye is likely to be the most influential one, from which the mtDNA is thought to have migrated to the neighboring regions of Sakhalin, Hokkaido, the Magadan region, and Siberia during the evolution of this species.