Japanese Brown Frog Rana Japonica Research Articles

Fine-scale Genetic Structure and Estimation of Gene Flow of the Japanese Brown FrogRana japonicain a Satoyama Landscape on the Western Side of Inba Lake, Eastern Japan

Habitat fragmentation is one of the major threats to amphibian species. In a previous study, population genetic analyses of the Japanese brown frog Rana japonica were conducted using a mitochondrial DNA (mtDNA) marker in a typical Japanese agricultural landscape (known as satoyama) in Chiba, Japan. This previous study revealed that gene flow was restricted by the roads and cement-walled urban river that divide this site. In the present study, we reanalyzed the genetic structure of the same meta-population using microsatellite markers in comparison with the mtDNA results and elucidated fine-scale gene flow. The genetic structure derived from the microsatellite clustering analysis was almost identical to that of the mtDNA results, although some important details differed. We recognized boundaries of genetic structure are consistent with the major roads and cement-walled river, however, we also detected gene flow across those artificial barriers. We concluded that the current genetic structure was formed in the past when gene flow was strongly restricted. Gene flow among breeding populations is now being restored by the maintenance of breeding sites, although it is not sufficient to erase the signature of historical isolation.

Transcriptome analyses of immune tissues from three Japanese frogs (genus Rana) reveals their utility in characterizing major histocompatibility complex class II

BackgroundIn Japan and East Asia, endemic frogs appear to be tolerant or not susceptible to chytridiomycosis, a deadly amphibian disease caused by the chytrid fungus Batrachochytridium dendrobatidis (Bd). Japanese frogs may have evolved mechanisms of immune resistance to pathogens such as Bd. This study characterizes immune genes expressed in various tissues of healthy Japanese Rana frogs.ResultsWe generated transcriptome data sets of skin, spleen and blood from three adult Japanese Ranidae frogs (Japanese brown frog Rana japonica, the montane brown frog Rana ornativentris, and Tago’s brown frog Rana tagoi tagoi) as well as whole body of R. japonica and R. ornativentris tadpoles. From this, we identified tissue- and stage-specific differentially expressed genes; in particular, the spleen was most enriched for immune-related genes. A specific immune gene, major histocompatibility complex class IIB (MHC-IIB), was further characterized due to its role in pathogen recognition. We identified a total of 33 MHC-IIB variants from the three focal species (n = 7 individuals each), which displayed evolutionary signatures related to increased MHC variation, including balancing selection. Our supertyping analyses of MHC-IIB variants from Japanese frogs and previously studied frog species identified potential physiochemical properties of MHC-II that may be important for recognizing and binding chytrid-related antigens.ConclusionsThis is one of the first studies to generate transcriptomic resources for Japanese frogs, and contributes to further understanding the immunogenetic factors associated with resistance to infectious diseases in amphibians such as chytridiomycosis. Notably, MHC-IIB supertyping analyses identified unique functional properties of specific MHC-IIB alleles that may partially contribute to Bd resistance, and such properties provide a springboard for future experimental validation.

The first see-through frog created by breeding: description, inheritance patterns, and dermal chromatophore structure.

We have succeeded in creating see-through frogs from natural color mutants of the Japanese brown frog Rana japonica, which usually possesses an ochre or brown back; this coloration enables the organs, blood vessels, and eggs to be observed through the skin without performing dissection. We crossed two kinds of recessive color mutant (black-eyed and gray-eyed) frogs through artificial insemination, and F2 offspring produced frogs whose skin is translucent throughout the life cycle. Three kinds of dermal chromatophores—xanthophores, iridophores, and melanophores—are observed in a layered arrangement in the skin of wild-type frogs, but few chromatophores were present in the skin of the see-through frogs. The translucent skin enables observation of organ growth and cancer formation and progression in the animal, which can be monitored over its entire life without the need for dissection. See-through frogs thus provide a useful animal model for environmental, medical, and biological research.

Molecular Cloning of a cDNA Encoding Atypical Antimicrobial and Cytotoxic Brevinin-1Ja from the Skin of the Japanese Brown Frog, Rana japonica

Antimicrobial peptides (AMPs) are important components of the host innate defense system against pathogenic microbial invasion in many organisms. In the present study, we cloned by RT-PCR a cDNA from total RNA prepared from the skin of the Japanese brown frog Rana japonica. The cDNA directs the synthesis of a novel, non-C-terminally alpha-amidated peptide composed of 21 amino acid residues (FLGSLIGAAIPAIKQLLGLKK). The putative peptide showed limited sequence similarity to atypical acyclic brevinin-1OK family AMPs originally isolated from the skin of the Ryukyu brown frog (R. okinavana), which lacks the COOH-terminal cyclic domain commonly observed in typical brevinin-1 groups, but that contains a C-terminally alpha-amidated residue. Although it is unclear whether the peptide, designated brevinin-1Ja, is produced in R. japonica skin, a synthetic replicate of the peptide showed differential growth-inhibiting activity against the Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli (minimal inhibitory concentrations: 15 microM and 119 microM, respectively), and produced cell death of mammalian COS7 cells (LD50=28 microM).

Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica

Japonicin-1 (FFPIGVFCKIFKTC) and japonicin-2 (FGLPMLSILPKALCILLKRKC), two peptides with differential growth-inhibitory activity against the Gram-negative bacterium, Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, were isolated from an extract of the skin of the Japanese brown frog Rana japonica. Both peptides show little amino acid sequence similarity to previously characterized antimicrobial peptides isolated from the skins of Ranid frogs. Circular dichroism studies, however, demonstrate that japonicin-2 adopts an α-helical conformation in 50% trifluoroethanol in common with many other cationic antimicrobial peptides synthesized in amphibian skin. Peptides belonging to the brevinin-1, brevinin-2, and tigerinin families, previously identified in the skins of Asian Ranid frogs, were not detected but a temporin-related peptide (ILPLVGNLLNDLL.NH2; temporin-1Ja), that atypically bears no net positive charge, was isolated from the extract. The minimum inhibitory concentrations (MICs) of the peptides against E. coli were japonicin-1, 30 μM; japonicin-2, 12 μM; and temporin-1Ja > 100 μM. The MICs against S. aureus were japonicin-1, > 100 μM; japonicin-2, 20 μM; and temporin-1Ja, > 100 μM.

Function of the Mos/MAPK pathway during oocyte maturation in the Japanese brown frog Rana japonica.

Fully grown immature oocytes acquire the ability to be fertilized with sperm after meiotic maturation, which is finally accomplished by the formation and activation of the maturation-promoting factor (MPF). MPF is the complex of Cdc2 and cyclin B, and its function in promoting metaphase is common among species. The Mos/mitogen-activated protein kinase (MAPK) pathway is also commonly activated during vertebrate oocyte maturation, but its function seems to be different among species. We investigated the function of the Mos/MAPK pathway during oocyte maturation of the frog Rana japonica. Although MAPK was activated in accordance with MPF activation during oocyte maturation, MPF activation and germinal vesicle breakdown (GVBD) was not initiated when the Mos/MAPK pathway was activated in immature oocytes by the injection of c-mos mRNA. Inhibition of Mos synthesis by c-mos antisense RNA and inactivation of MAPK by CL100 phosphatase did not prevent progesterone-induced MPF activation and GVBD. However, continuous MAPK activation and MAPK inhibition through oocyte maturation accelerated and delayed MPF activation, respectively. Furthermore, Mos induced a low level of cyclin B protein synthesis in immature oocytes without the aid of MAPK. These results suggest that the general function of the Mos/MAPK pathway, which is not essential for MPF activation and GVBD in Rana oocytes, is to enhance cyclin B translation by Mos itself and to stabilize cyclin B protein by MAPK during oocyte maturation.

Intraspecific Differentiation in the Japanese Brown Frog Rana japonica Inferred from Mitochondrial DNA Sequences of the CytochromebGene

Intraspecific differentiation of the Japanese brown frog Rana japonica was investigated by analyzing nucleotide sequences of the mitochondrial cytochrome b (cyt b) gene in order to clarify phylogenetic relationships among three population groups known to exist in this species. The nucleotide sequences of 447 base pair (bp) segments were determined by the PCR-direct sequencing method on 31 individuals from 14 populations of R. japonica from Honshu, and phylogenetic trees were constructed by the UPGMA and NJ methods using Rana catesbeiana as an outgroup. A sequence alignment provided 92 variable sites (15 corresponded to the first codon position, three to the second, and 74 to the third), and 19 haplotypes were identified from 31 frogs. The sequence divergences were 0.22∼2.50% (x̄ = 0.65%) within populations, 0.22∼12.02% (x̄ = 7.34%) between populations, and 23.59∼27.89% (x̄ = 26.19%) between R. japonica and R. catesbeiana. Although many nucleotide substitutions were silent mutations, 12 amino acid replacements were found to occur within R. japonica. A high frequency of transitions relative to transversions was observed within R. japonica. The present nucleotide sequence data showed that the eastern and western groups of R. japonica was considerably differentiated to each other, and that the Akita population of the northwestern group was evidently derived from the eastern group, but the Nakajo and Izumozaki populations of the northwestern group diverged considerably from each of the eastern and the western groups.

Inheritance and linkage analysis of ten enzyme and blood protein loci in the Japanese brown frog Rana japonica.

In order to clarify the genetic linkage groups of the Japanese brown frog Rana japonica, and compare them with those of other vertebrates, the inheritance of 10 enzyme and blood protein loci was examined in 267 offspring derived from 18 crosses using 10 males heterozygous at these loci. Most of the segregation tests exhibited no significant deviations from the expected normal Mendelian ratios. Of 32 pairs of loci tested for linkage, 29 pairs showed independent assortment in all crosses examined. In the three other locus pairs, between ADH-1 and Alb, IDH-1 and Hb-1, and LDH-B and MPI, all offspring analyzed were parental, and there were no recombinants. These three linkage groups comprising six loci were thus established in R. japonica, whereas no linkage between the other four loci, AAT-1, ADA, GPI, and PEP-A, was observed.

Incipient Intraspecific Isolating Mechanisms in the Japanese Brown Frog Rana japonica

In order to elucidate the extent to which the reproductive isolating mechanism is established during the intraspecific differentiation of Rana japonica, hybridization experiments were carried out in 101 combinations using 161 frogs collected from 20 populations throughout the Honshu and Kyushu Islands of Japan. Although neither gametic isolation nor hybrid inviability existed among these populations, a remarkable preponderance of males occurred in the reciprocal hybrids among the eastern, western, and northwestern groups. In crosses within each group, 52.7-55.7% of frogs were males, whereas 93.0 and 88.0% of frogs were males in the hybrid crosses between the eastern and western groups and between the northwestern and western groups, respectively. Spermatogenesis in male progeny from crosses within each of the three groups was almost always normal, whereas most of the male reciprocal hybrids among the three groups were more or less abnormal in spermatogenesis. The abnormality in spermatogenesis was largest in the hybrids between the western and eastern groups and smallest in the hybrids between the northwestern and eastern groups. These results suggest that the eastern and western groups had come into contact in the northwestern region before they were reproductively isolated from each other through hybrid sterility. It is thus not unreasonable to label the western and eastern groups as the Hiroshima and Ichinoseki races, respectively, and the northwestern region as the hybrid zone. The Japanese brown frog Rana japonica is widely distributed through Japan, from Honshu (except for the northern end), to Kyushu, and into China (except for the northeastern regions) (Okada, 1931, 1966; Maeda and Matsui, 1989). In Japan, the Ichinoseki population (eastern Honshu) of Rana japonica is reproductively isolated from the Hiroshima population (western Honshu) by the sterility of the male hybrids (Sumida, 1981). Although the two populations are not morphologically distinct, they are distinguishable both karyologically and biochemically. The karyotypes of two populations differ slightly in the centromere positions of chromosomes Nos. 6 and 9, and these two populations are further distinguished by diagnostic alleles at the Pep-A and Hb-II loci. In reciprocal crossings between these two populations, the sex ratio is skewed in favor of males in the reciprocal hybrids. These male hybrids show e Japan se brown frog Rana japonica is idely distributed through Japan, fr m Hon(except for the north rn end), to Kyushu, into China (except for the nor heastern ions) (Ok da, 193 , 1966; M e a and Matsui, 89). In Japan, the Ichinoseki population (eastHonshu) of Rana japonica is reproductively lated from the Hiroshima population (westHonshu) by the sterility of th male hybrids ida, 1981). Althoug he two populations e not morphologically dis inct, th y are disguishable both karyologic lly and biochema ly. The karyotypes of two populations differ i htly in the centromere p sitions of chrosomes Nos. 6 and 9, and these two populans are further distinguished by diagnostic leles at the Pep-A and Hb-II loci. In reciprocal o sings between these two populations, the varying degrees of testicular abnormality, and in the first spermatogenetic meiosis, homologo s chromosomes fail to pair, resulting in formation of univalents and varying degrees of degeneration of the spermatids (Sumida, 1994). Starch gel electrophoresis of enzymes and blood proteins also distinguishes the eastern and western groups of Rana japonica (Sumida and Nishioka, 1994a). The locus linked with the sexdetermining gene differs with the local popul tions (Sumida and Nishioka, 1994b). It is assumed that the eastern and western groups of R. japonica are at the stage of incipient speciation. It would be very interesting to measure the extent to which the reproductive isolating mechanisms are established during the intraspecific differentiation of R. japonica. The present study reports extensive intraspecific crossing experiments among many populations from Honshu to Kyushu to elucidate the arying degrees of testicular abno mality, and the first permatogenetic meiosis, homoloous c romosomes fail to pair, resulting in fortion of univalents and v rying degrees of egeneration of the spermatids (Sumida, 1994). tarch gel e ectrophoresis of enzymes and blood roteins also distinguishes the eastern and est rn groups of Rana japonic (Sumida and i hioka, 1994a). The locus linked with the sexe ermin g gene differs with the local popu333 This content downloaded from 207.46.13.76 on Fri, 09 Sep 2016 04:37:35 UTC All use subject to http://about.jstor.org/terms

A pronounced sex difference when two linked loci of the Japanese brown frogRana japonica are recombined

A pronounced sex difference when two linked loci of the Japanese brown frogRana japonica are recombined

A pronounced sex difference when two linked loci of the Japanese brown frogRana japonica are recombined

To investigate the sex differences of crossing-over in Rana japonica, the recombination frequencies between two linked loci, LDH-B and MPI, were examined in offspring obtained from 29 matings involving either females or males heterozygous at both loci. No recombinants were found among 303 offspring bred from 6 heterozygous males, whereas 160 were parental and 138 were recombinants among 298 offspring bred from 9 heterozygous females. The recombination frequency was 46.3%. These results show that the sex difference when two loci were recombined was marked and significant in Rana japonica.