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The amphibian acanthocephalan, Pseudoacanthocephalus toshimai, was considered to be an island-endemic species in Hokkaido, Japan. However, the parasite was found from Rana ornativentris, Rana tagoi, Zhangixalus arboreus, and Bufo japonicus formosus in northern Honshu (Aomori and Iwate Prefectures), which is separated from Hokkaido by the Tsugaru Strait. The mitochondrial DNA-based phylogenetic and population genetic analyses of P. toshimai showed that the northern Honshu isolates are far distantly related to the Hokkaido isolates, and that a demographic population expansion occurred in Hokkaido during the recent geological past. The rich genetic diversity of P. toshimai in northern Honshu suggests a scenario that anuran hosts invaded Hokkaido together with P. toshimai via the land bridge of the Tsugaru Strait. However, the evolutionary history of Rana pirica, a main definitive host for P. toshimai in Hokkaido, is contradictory to the introduction scenario inferred from the parasite. The finding of several geographically mismatched isolates of P. toshimai from both northern Honshu and Hokkaido suggests a possibility that the migration of the parasite infrequently occurred between the two areas even after the land bridge disappeared. More detailed information on the evolutionary history of anurans is needed to resolve the biogeographical enigma of P. toshimai.

Recent advancement of environmental DNA (eDNA) methods for surveying species in aquatic ecosystems has been used for various organisms and contributed to monitoring and conservation of species and environments. Amphibians are one of the promising taxa which could be monitored efficiently by applying quantitative PCR (qPCR) or next generation sequencing to eDNA. However, the cost of eDNA detection using these approaches can be quite high and requires instruments that are not usually installed in ecology laboratories. For aiding researchers in starting eDNA studies of amphibians, especially those not specialized in molecular biology, we developed a cost efficient protocol using PCR-RFLP method. We attempted to detect eDNA of three Japanese Rana species (Rana japonica, Rana ornativentris, and Rana tagoi tagoi) in various spatial scales including an area close to the Fukushima nuclear power plant where the environment is recovering after the disaster in 2011. Our PCR-RFLP protocol was successful in detecting Rana species in static water in both laboratory and field; however, it could not detect Rana species in non-static water samples from the field. Even a more sensitive detection method (standard qPCR) was unable to detect frogs in all non-static water samples. We speculate that our new protocol is effective for frogs living in lentic habitats, but not for lotic habitats which may still require the gold standard of field observation for detection approach.

Feeding habits of snakes on Kinkasan Island, where potential food resources for snakes is limited, was investigated based on field surveys of six years. In total 280 snakes, which consisted of five species (Elaphe quadrivirgata, E. climacophora, Euprepiophis conspicillatus, Rhabdophis tigrinus, and Gloydius blomhoffii), were collected. A total of 168 prey items were recovered from the stomach contents of these snakes. The diet of R. tigrinus and G. blomhoffii exclusively consisted of a frog, Rana tagoi, which is the only amphibian species inhabiting Kinkasan Island. Diet of El. quadrivirgata was also predominantly comprised of Ra. tagoi ( 97.7%). O nly e ndothermic animals were found in the stomach contents of El. climacophora and Eu. conspicillatus. Direct observations of feeding behavior and a broad range of body temperature of El. quadrivirgata indicated that during the breeding season of Ra. tagoi, this snake forages for frogs hiding in crevices of rocks immersed in cold running water of mountain streams. Our study demonstrated that, given the limited prey fauna on Kinkasan Island, two dietary generalists, El. quadrivirgata and G. blomhoffii, and one amphibian specialist, R. tigrinus, largely rely on the single frog species as major diet. High exploitation of Ra. tagoi by El. quadrivirgata would be attained by its unique foraging tactics that are characterized by a shuttling movement between hot basking rocks and cold streams for foraging.

Amphibians are key components in forest food webs. When examining radioactive contamination in anurans, it is important to understand how radiocesium transfer occurs from lower to higher trophic levels in forest ecosystems. We investigated the activity concentration of radiocesium ( 134 Cs and 137 Cs) in Tago's brown frog ( Rana tagoi tagoi ) captured on the forest floor approximately 2.5 years after the Fukushima Nuclear Power Plant (FNPP) accident. We collected 66 R. tagoi tagoi at different distances from the FNPP. Radiocesium accumulation showed positive correlations with the air radiation dose rate and litter contamination but not with distance from the FNPP. Whole-body radioactivity showed no correlation with body mass or length. Our results suggest that differences in the available food items result in large variability in individual contamination. Contamination level monitoring in terrestrial and aquatic amphibian is necessary for clarifying the processes and mechanisms of radiocesium transfer through forest food webs.

Prior studies of mitochondrial genomic variation reveal that the Japanese brown frog Rana tagoi comprises a complex of cryptic species lineages, and that R. sakuraii arose from within this complex. Neither species forms a monophyletic group on the mitochondrial haplotype tree, precluding a simple explanation for the evolutionary origins of R. sakuraii. We present a more complete sampling of mitochondrial haplotypic variation (from the ND1 and 16S genes) plus DNA sequence variation for five nuclear loci (from the genes encoding NCX1, NFIA, POMC, SLC8A3, and TYR) to resolve the evolutionary histories of these species. We test hypotheses of population assignment (STRUCTURE) and isolation-with-migration (IM) using the more slowly evolving nuclear markers. These demographic analyses of nuclear genetic variation confirm species-level distinctness and integrity of R. sakuraii despite its apparent polyphyly on the mitochondrial haplotype tree. Divergence-time estimates from both the mitochondrial haplotypes and nuclear genomic markers suggest that R. sakuraii originated approximately one million years ago, and that incomplete sorting of mitochondrial haplotype lineages best explains non-monophyly of R. sakuraii mitochondrial haplotypes. Cytonuclear discordance elsewhere in R. tagoi reveals a case of mitochondrial introgression between two species lineages on Honshu. The earliest phylogenetic divergence within this species group occurred approximately four million years ago, followed by cladogenetic events in the Pliocene and early Pleistocene yielding 10-13 extant species lineages, including R. sakuraii as one of the youngest.

Two morphotypes, with a large and small body size, of a brown frog Rana t. tagoi occur sympatrically in the Kinki region, central Honshu of Japan. Previous mitochondrial (mt) DNA genealogical study recognized two main lineages (A and B) and several sublineages in R. tagoi, where the small type was placed in the group A-1b, and the large type in groups A-1a and B-2a. Using haplotype network and structure analysis of three nuclear genes, we examined the discrepancy between morphology and mitochondrial genealogy. The results showed that the small type is reproductively isolated from its co-occurring large type (A-1a or B-2a), and that unlimited gene flow occurred between parapatrically occurring two mtDNA lineages of large types (A-1a and B-2a). Discordant genetic relationships between mtDNA and nuclear DNA results may be caused by the past mitochondrial introgression, and possibly, the incomplete lineage sorting. These results also suggest a heterospecific relationship between the large (A-1a and B-2a) and small types (A-1b). The large type is identified as Rana t. tagoi as it is genetically very close to the topotypes of the nominal subspecies, while the small type remains unnamed.

Two morphotypes, with a large and small body size, of a brown frog Rana t. tagoi occur sympatrically in the Kinki region, central Honshu of Japan. Previous mitochondrial (mt) DNA genealogical study recognized two main lineages (A and B) and several sublineages in R. tagoi, where the small type was placed in the group A-1b, and the large type in groups A-1a and B-2a. Using haplotype network and structure analysis of three nuclear genes, we examined the discrepancy between morphology and mitochondrial genealogy. The results showed that the small type is reproductively isolated from its co-occurring large type (A-1a or B-2a), and that unlimited gene flow occurred between parapatrically occurring two mtDNA lineages of large types (A-1a and B-2a). Discordant genetic relationships between mtDNA and nuclear DNA results may be caused by the past mitochondrial introgression, and possibly, the incomplete lineage sorting. These results also suggest a heterospecific relationship between the large (A-1a and B-2a) and small types (A-1b). The large type is identified as Rana t. tagoi as it is genetically very close to the topotypes of the nominal subspecies, while the small type remains unnamed.

The endemic Japanese frog Rana tagoi is unique among Holarctic brown frogs in that it breeds in small subterranean streams. Using mitochondrial 16S ribosomal RNA and NADH dehydrogenase subunit 1 genes, we investigated genealogical relationships among geographic samples of this species together with its relative R. sakuraii, which is also a unique stream breeder. These two species together form a monophyletic group, within which both are reciprocally paraphyletic. Rana tagoi is divided into two major clades (Clade A and B) that are composed of 14 genetic groups. Rana sakuraii is included in Clade A and split into two genetic groups, one of which forms a clade (Subclade A-2) with sympatric R. tagoi. This species-level paraphyly appears to be caused by incomplete taxonomy, in addition to introgressive hybridization and/or incomplete lineage sorting. Rana tagoi strongly differs from other Japanese anurans in its geographic pattern of genetic differentiation, most probably in relation to its unique reproductive habits. Taxonomically, R. tagoi surely includes many cryptic species.

Peptidomic analysis of an extract of the skin of the stream brown frog Rana sakuraii Matsui and Matsui, 1990 led to the isolation of a C-terminally α-amidated peptide (VR-23; VIGSILGALASGLPTLISWIKNR·NH 2 ) with broad-spectrum antimicrobial activity that shows structural similarity to the bee venom peptide, melittin together with two peptides belonging to the temporin family (temporin-1SKa; FLPVILPVIGKLLNGIL·NH 2 and temporin-1SKb; FLPVILPVIGKLLSGIL·NH 2 ), and peptides whose primary structures identified them as belonging to the brevinin-2 (2 peptides) and ranatuerin-2 (1 peptide) families. Using a forward primer that was designed from a conserved region of the 5′-untranslated regions of Rana temporaria preprotemporins in a 3′-RACE procedure, a cDNA clone encoding preprotemporin-1SKa was prepared from R. sakuraii skin total RNA. Further preprotemporin cDNAs encoding temporin-1SKc (AVDLAKIANIAN KVLSSL F·NH 2 ) and temporin-1SKd (FLPMLAKLLSGFL·NH 2 ) were obtained by RT-PCR. Unexpectedly, the 3′-RACE procedure using the same primer led to amplification of a cDNA encoding a preprobradykinin whose signal peptide region was identical to that of preprotemporin-1SKa except for the substitution Ser 18 → Asn. R. sakuraii bradykinin ([Arg 0 ,Leu 1 ,Thr 6 ,Trp 8 ] BK) was 28-fold less potent than mammalian BK in effecting B2 receptor-mediated relaxation of mouse trachea and the des[Arg 0 ] derivative was only a weak partial agonist. The evolutionary history of the Japanese brown frogs is incompletely understood but a comparison of the primary structures of the R. sakuraii dermal peptides with those of Tago's brown frog Rana tagoi provides evidence for a close phylogenetic relationship between these species .

Amphibian fossils excavated from two Late Pleistocene-Holocene fissures in the southern part of Okinawajima Island in the Ryukyu Archipelago were identified through detailed comparisons with skeletal specimens of extant taxa. The identified species (eight frogs and two newts) were confined to extant elements on the island and were mostly endemic to either the island or the central part of the archipelago including it. Previous Late Pleistocene records of extralimital frogs (two mainland Japanese species [Glandirana rugosa and Rana tagoi] and one Southeast Asian frog currently naturalized in the Ryukyus [Polypedates leucomystax]) from one of the study sites (the Minatogawa Fissure) are therefore considered misidentifications. This may also be the case with Fejervarya kawamurai, a synanthropic frog currently widely distributed in East Asia, including the Central Ryukyus. Of the species obtained from the Late Pleistocene deposits at these sites, five frogs (Limnonectes namiyei, Babina holsti, Odorrana ishikawae, O. narina, and Rana ulma) are forest-dwellers currently confined to the northern forested area of Okinawajima. Additionally, none of these species were detected in the middle Holocene deposits from one of the sites, the Sashiki Fissure. Our findings indicate that the southern limestone area of Okinawajima in the Late Pleistocene harbored a dense and humid forest with diverse lotic habitats, similar to the northern forested area of the island at present, and that these environmental conditions were lost by the midpoint of the Holocene Period.

The temporins are a family of hydrophobic, C-terminally alpha-amidated antimicrobial peptides that are synthesized in the skins of a wide range of species of frogs belonging to the genus Rana. In the present study, we investigated using RT-PCR the expression of preprotemporin mRNAs in extradermal tissues of Tago's brown frog Rana tagoi. cDNAs encoding temporin-1TGa (FLPILGKLLS(10)GIL.NH2), previously isolated from an extract of the skin of R. tagoi skin, were amplified and cloned from the stomach, liver, kidney, skeletal muscle. However, a net insertion of 10 nucleotides resulted in the presence of a premature stop codon in the open reading frame that was not present in the corresponding region of preprotemporin-1TGa from skin. The preprotemporin cDNA obtained from small intestine contained an additional 12 nucleotide insertion in the region that encodes the temporin sequence so that a novel peptide (FLPVILPVIG(10)KLLSGIL.NH2), termed temporin-1TGc, is specified. This cDNA also contained a premature stop codon in the open reading frame. Although it is unclear whether temporin-1TGc is produced in R. tagoi tissues, a synthetic replicate of the peptide of was biologically active, inhibiting the growth of Staphylococcus aureus (minimal inhibitory concentration = 37.5 microM) and producing hemolysis of human erythrocytes (LD50 = 50 microM).

Using RT-PCR, two cDNAs encoding preprotemporins were cloned from a total RNA preparation of the skin of Tago's brown frog Rana tagoi. Preprotemporin-1TGa cDNA directs the synthesis of temporin-1TGa (FLPILGKLLSGIL.NH2) previously isolated from R. tagoi skin. Preprotemporin-1TGb cDNA directs the synthesis of a novel 16-amino-acid-residue peptide (AVDLAKIANKVLSSLF.NH2) that, atypically for members of the temporin family, inhibits the growth of Gram-negative bacteria more effectively than Gram-positive bacteria. Preprotemporin-1TGa mRNA and preprotemporin-1TGb mRNA were not detected in skin prior to the onset of metamorphosis (stage 35) but the levels of the transcripts increased markedly during metamorphosis reaching a maximum at stage 38. Exposure of adult animals to 10(-8) M triiodothyronine (T3) for 72 h enhanced expression of the preprotemporin-1TGb gene (approximately threefold) but did not significantly change the level of expression of the preprotemporin-1TGa gene. Exposure of the animals to 10(-8) M T3 and 10(-6) M bisphenol A, an endocrine disrupting chemical that potently inhibits the action of thyroid hormones (THs), reduced expression of the preprotemporin-1TGb gene by 10-fold and the preprotemporin-1TGa gene by threefold. We propose that T3-stimulated synthesis of antimicrobial peptides is important in protecting the animal against microorganisms, particularly at metamorphosis and during skin moulting, but environmental pollutants can inhibit peptide synthesis and render the animal susceptible to invasion by pathogens.

This study aimed at acquiring the knowledge about the distribution pattern of amphibians on the rural landscape in the middle reaches of the Oita River. Here were recognized two URODELA species and ten ANURA species, twelve species of amphibians in total. In the running water area, Hynobius naevius and Rana tagoi inhabited in streams of a headwater in wooded regions, and Buergeria buergeri inhabited only the main current of the Oita River. In standing water area, the subject of our investigation was classified into the extensive distribution species, Hyla japonica, R. nigromaculata and Rhacophorus schlegelii, the uneven distribution species, R. limnocharis, R. rugosa, the limited distribution species, Cynops pyrrhogaster, R. ornativentris, and the rare species, Bufo japonicus, R. japonica. Especially the limited distribution species were guessed to be under the influence of the distribution restrictions by the altitude and the ability to colonize upper paddy fields. The distribution of each species corresponded to landscape elements relevant to the wetlands from the hillside to the Oita River, much the same pattern applying to every part of the three target basins. We were able to clarify the relationship between the landscape structure and the amphibians in this target rural district which mainly consists of step-like paddy field.

Two hundred and sixteen anurans representing 10 species (Bufo japonicus, Rana japonica, Ra. limnocharis, Ra. nigromaculata, Ra. ornativentris, Ra. porosa, Ra. rugosa, Ra. tagoi, Buergeria buergeri, andRhacophorus schlegelii) and 3 families were collected on Honshu Island, Japan, between 1994 and 1999 and examined for helminths. Gravid individuals of 5 species of Trematoda, Diplodiscus japonicus, Glypthelmins rugocaudata, Haematoloechus sibiricus, Mesocoelium brevicaecum, and Polystoma ozakii, 2 species of Cestoda, Cylindrotaenia japonica, Ophiotaenia ranae, 10 species of Nematoda, Amphibiocapillaria bufonis, Cosmocerca japonica, Cosmocercoides pulcher, Meteterakis japonica, Oswaldocruzia insulae, Os. socialis, Pharyngodon polypedatis, Rhabdias incerta, Rhab. nipponica, and Spiroxys japonica, and 1 species of Acanthocephala, Acanthocephalus lucidus, were recovered. Six new host records are reported. Rhabdias montana and Rhab. polypedates are proposed as synonyms of Rhab. nipponica.

Karyotypes of the Tago brown frog Rana tagoi and stream Tago brown frog Rana sakuraii from a mountain region in the Nishitama district in Tokyo were examined by conventional Giemsa staining, C-banding and late replication (LR)-banding. Chromosome number was 2n = 26 in all cases. The 26 chromosomes consisted of five (1–5) pairs of large chromosomes and eight (6–13) pairs of small chromosomes. Chromosome 10 had a secondary constriction on the long arm. In all frogs, on chromosome pair 8, the XX/XY type sex chromosome was present. C-banding analysis indicated that, in R. sakuraii, neither the X nor Y chromosome possessed interstitial C-bands but each had centromere staining, while in R. tagoi, an interstitial C-band was present on the long arm of the X chromosome. The Y chromosome had no interstitial C-band. LR-banding analysis demonstrated the X and Y chromosomes to have a LR-band on the short arm and two LR-bands, each on the long arm, and the bands on both species to be essentially the same. Heteromorphic sex chromosomes in males of R. sakuraii and R. tagoi were identified for the first time in this study.

The present study examines amphibian metabolic activity - particularly conjugation - by analysis of pyrene (a four ring, polycyclic aromatic hydrocarbon) metabolites using high-performance liquid chromatography (HPLC) with fluorescence detector (FD), a mass spectrometry detector (MS) system and kinetic analysis of conjugation enzymes. Six amphibian species were exposed to pyrene (dissolved in water): African claw frog (Xenopus laevis); Tago's brown frog (Rana tagoi); Montane brown frog (Rana ornativentris); Wrinkled frog (Rana rugosa); Japanese newt (Cynops pyrrhogaster); and Clouded salamander (Hynobius nebulosus); plus one fish species, medaka (Oryzias latipes); and a fresh water snail (Clithon retropictus), and the resultant metabolites were collected. Identification of pyrene metabolites by HPLC and ion-trap MS system indicated that medaka mainly excreted pyrene-1-glucuronide (PYOG), while pyrene-1-sulfate (PYOS) was the main metabolite in all amphibian species. Pyrene metabolites in amphibians were different from those in invertebrate fresh water snails. Inter-species differences were also observed in pyrene metabolism among amphibians. Metabolite analysis showed that frogs relied more strongly on sulfate conjugation than did Japanese newts and clouded salamanders. Furthermore, urodelan amphibians, newts and salamanders, excreted glucose conjugates of pyrene that were not detected in the anuran amphibians. Kinetic analysis of conjugation by hepatic microsomes and cytosols indicated that differences in excreted metabolites reflected differences in enzymatic activities. Furthermore, pyrenediol (PYDOH) glucoside sulfate was detected in the Japanese newt sample. This novel metabolite has not been reported previously to this report, in which we have identified unique characteristics of amphibians in phase II pyrene metabolism.

Previous studies led to the isolation from skin extracts of Oki Tago's brown frog, Rana tagoi okiensis of five antimicrobial peptides belonging to the brevinin-1 (brevinin-1TOa), temporin (temporin-TOa and -TOb), and ranatuerin-2 (ranatuerin-2TOa and -2TOb) families, and bradykinin (BK) identical to mammalian BK. Using the reverse-transcription polymerase chain reaction (RT-PCR), we have now cloned from skin total RNA preparations cDNAs encoding biosynthetic precursors of brevinin-1TOa and brevinin-1TOb (containing the substitution Gly(1)-->Val), temporin-TOa and -TOb, and ranatuerin-2TOa and -2TOb. In addition, three cDNA clones encoding preprobradykinins were obtained that contained either one, two, or three tandem repeats of the sequence of BK followed by the sequence of [Thr(6)]-BK. In tissue expression analyses, preprobrevinin-1, preprotemporin, and preproranatuerin-2 gene transcripts were detected at higher levels in brain compared with peripheral tissues (heart, small intestine, kidney, liver lung, skeletal muscle, stomach, and testis). RT-PCR of brain RNA resulted in the amplification of cDNAs encoding ranatuerin-2TOc and ranatuerin-2TOd that contained the amino acid substitutions Lys(6)-->Arg and Ala(14)-->Thr, respectively compared with ranatuerin-2TOb. cDNAs encoding preprobrevinin-1TOa and preprotemporin-TOa were amplified from brain RNA as well as a second preprotemporin cDNA that contained a 10-nucleotide insertion that introduced a frame shift resulting in a premature stop codon. A cDNA encoding a novel peptide, DK25 (DVNDLKNLCAKTHNLLPMCAMFGKK) was amplified from brain RNA but neither DK25 nor its putative post-translationally modified form, DF22-amide (DVNDLKNLCAKTHNLLPMCAMF.NH(2)) displayed antimicrobial or hemolytic activities.

Morphological evidence and data from comparisons of nucleotide sequences of mitochondrial genes demonstrate considerable intraspecies variation among populations of the Japanese brown frog Rana tagoi Okada 1928 (Tago's brown frog). Five peptides with antimicrobial activity were isolated from an extract of the skins of specimens of Rana tagoi okiensis collected on the Oki Islands, Japan. Determination of their primary structures demonstrated that two peptides belong to the ranatuerin-2 family, two peptides to the temporin family, and one peptide to the brevinin-1 family. Ranatuerin-2 peptides were not previously identified in the skin of specimens of R. t. tagoi collected in Chiba Prefecture, Japan and the structures of the temporin peptides from R. t. okiensis (temporin-TOa: FLPILGKLLSGFL.NH 2 and temporin-TOb: FLPILGKLLSGLL.NH 2 ) are different from temporin-TGa (FLPILGKLLSGIL.NH 2 ) isolated from R. t. tagoi. Similarly, the acyclic C-terminally α-amidated brevinin-1 peptide from R. t. okiensis (Brevinin-1TOa, GIGSILGVIAKGLPTLISWIKNR.NH 2 ) shows three amino acid substitutions (Gly 1 → Ala, Val 8 → Ala, Ile 9 → Leu) compared to the ortholog from R. t. tagoi . In addition, bradykinin, identical to the mammalian peptide, is present in high concentration in the skin of R. t. okiensis but not R. t. tagoi . The data provide evidence to support the proposal that R. t. tagoi and R. t. okiensis should be regarded as separate species ( R. tagoi and R. okiensis ) rather than conspecific subspecies.

The antimicrobial melittin-related peptides (MRPs) isolated from skin extracts of the Japanese frogs, Rana sakuraii and Rana tagoi, show amino acid sequence similarity with melittin from the venom of honeybees but the evolutionary relationship between the amphibian and insect peptides is unknown. cDNA clones encoding the MRP precursor (preproMRP) were obtained from R. sakuraii and R. tagoi skin total RNA. The nucleotide and deduced amino acid sequences of the clones indicated that the preproMRPs are organized like typical amphibian antimicrobial peptide precursors, with a highly conserved signal peptide, a more variable intervening sequence, and a hypervariable mature peptide region. This organization is markedly different from that of prepromelittin, in which the melittin sequence is flanked by multiple Xaa-Pro and Xaa-Ala dipeptides. The data indicate, therefore, that the genes encoding frog skin MRPs are not orthologous to the genes encoding melittins from bee venom. In adult R. sakuraii specimens, preproMRP gene transcripts were detected in total RNA from skeletal muscle as well as skin but not from heart, stomach, small intestine, or liver. In R. tagoi, preproMRP mRNA was not detected in skin prior to the onset of metamorphosis, but its level increased markedly during metamorphosis reaching a maximum at the stages of metamorphic climax.

Karyotypes of Tago’s brown frog Rana tagoi from the Chausu mountains in Minamishinshu of Nagano Prefecture were examined by conventional Giemsa staining, C-banding and late replication (LR)-banding. Chromosome number was 2n = 28 in all cases. The 28 chromosomes consisted of four pairs (1–4) of large biarmed chromosomes, two pairs (5–6) of telocentric chromosomes and eight pairs (7–14) of small biarmed chromosomes. Chromosome pair 11 had a secondary constriction on the long arm. In females, the C-band on the long arm of chromosome pair 6 was detected in both homologs, but was absent from the arms of the homologs of chromosome pairs 5 and 9. In males, C-bands were found in the long arms of both homologs of chromosome pairs 5 and 6, were present only in one homolog of chromosome pair 5 for certain male specimens and found in only one homolog of chromosome pair 9. Specimens of R. tagoi (2n = 28) should thus have two pairs of telocentric chromosomes to provide the same number of chromosome arms, these originating quite likely from chromosome pair 1 in the 26-chromosome specimens by centric fission. Heteromorphic sex chromosomes of the XX-XY type in R. tagoi (2n = 28) in the Chausu mountains were identified. Karyotypes of tail-tip cells from a hybrid tadpole between female R. tagoi (2n = 26) from the Hinohara village in Tokyo and male R. tagoi (2n = 28) from the Chausu mountain population were examined by squash preparation. Chromosome number was 2n = 27 in all tadpoles. The 27 chromosomes consisted of one chromosome set of R. tagoi (2n = 28) and one of R. tagoi (2n = 26).

Two peptides with antimicrobial and cytolytic properties were purified from an extract of the skin of Tago's brown frog Rana tagoi. The primary structure of one peptide (FLPILGKLLS(10)GIL.NH(2)) identifies it as a member of the temporin family, whereas the second peptide (AIGSILGALA(10)KGLPTLISWI(20)KNR.NH(2)) displays 78% sequence identity to melittin from the venom of the honeybee Apis florea. Compared with melittin, the melittin-related peptide (MRP) was equipotent in inhibiting the growth of the Gram-positive bacterium Staphylococcus aureus, 5-fold less potent against the Gram-negative bacterium Escherichia coli and against the fungal pathogen, Candida albicans. MRP was 13-fold less hemolytic than melittin against human erythrocytes and 4- and 5-fold less cytolytic against mouse EL4 T-lymphoma-derived cells and L929 fibroblasts, respectively. However, at non-cytotoxic concentrations (or=8 microM), MRP did not protect HeLa cells from cell death produced by human rhinovirus type 2 infection.

The major histocompatibility complex (MHC) is a key component of adaptive immunity in all jawed vertebrates, and understanding the evolutionary mechanisms that have shaped these genes in amphibians, one of the earliest terrestrial tetrapods, is important. We characterised MHC class I variation in three common Japanese Rana species (Rana japonica, Rana ornativentris and Rana tagoi tagoi) and identified a total of 60 variants from 21 individuals. We also found evolutionary signatures of gene duplication, recombination and balancing selection (including trans-species polymorphism), all of which drive increased MHC diversity. A unique feature of MHC class I from these three Ranidae species includes low synonymous differences per site (d S) within species, which we attribute to a more recent diversification of these sequences or recent gene duplication. The resulting higher d N/d S ratio relative to other anurans studied could be related to stronger selection pressure at peptide binding sites. This is one of the first studies to investigate MHC in Japanese amphibians and permits further exploration of the polygenetic factors associated with resistance to infectious diseases. Electronic supplementary material The online version of this article (doi:10.1007/s00251-016-0934-x) contains supplementary material, which is available to authorized users.

Seventeen anurans occur on the four main islands of Japan and on the Islands of Tsushima situated between Japan and Korea. Two of these, Rana nigromaculata and R. brevipoda, are pond frogs allied to Rana esculenta.1 Six other species, Rana japonica, R. ornativentris, R. tagoi, R. chensinensis, R. tsushimensis and R. dyboswkii, are brown frogs allied to Rana temporaria, although Rana tagoi differs from the others in many respects. In addition to these species, two small, dark-colored frogs, Rana rugosa and R. limnocharis, and a large American frog, Rana catesbeiana, are widely distributed in Japan. In other genera Hyla arborea japonica, Bufo bufo japonicus, Bufo torrenticola and three species of Rhacophorus occur. Although it is generally believed that Bombina orientalis occurs on Tsushima, no one has recently found it there. During the last ten years, we have made a number of studies of reproductive biology using most of the Japanese anurans and some foreign ones.