Bufo Japonicus Research Articles

国内外来種として北海道に定着した アズマヒキガエル<i>Bufo japonicus formosus </i>の寄生蠕虫類

国内外来種化による寄生虫相の変遷を分析するため，2010年と 2011年に旭川市内で捕獲されたアズマヒキガエル Bufo japonicus formosusの蠕虫調査をした。その結果，このカエルで既報告の 3線虫種と 1鉤頭虫種が検出された。北海道における当該カエル種の調査はなく，新記録となったが，寄生蠕虫相は本州に生息していた時の状態をほぼ保持したまま定着していたことが判明した。

Cloning and sequence analysis of prothymosin-α cDNA of Bufo japonicus formosus

To study the polypeptide effective components included in the skin of Bufo japonicus formosus （Japanese toad） and their secretions，the full length cDNAs were screened from the plasmid cDNA library of adult Japanese toad skin using colony polymerase chain reaction with a pair of primers，SP6 （the upstream primer of the vector） and poly（T） （a self-designed primer recognizing the area connecting polyA tail of cDNA and the vector）. Results showed that a prothymosin- （ProT） cDNA was obtained from 1 344 colonies checked，whose transcript was 1 480 bp in length consisting of 125 bp in the 5 prime untranslated region （5 UTR） and 1 016 bp 3 UTR with a complete open reading frame （ORF） of 339 bp encoding a polypeptide of 112 amino acid residues. The homologous analysis indicated a similarity between B. japonicus formosus and Rana esculenta of up to 82%，but for other species it was 54%-73%. Previous studies have shown significant anti-tumor effects of ProT; therefore，this study could contribute to further studies on ProT.［Ch，4 fig. 17 ref.］

Population admixture and high larval viability among urban toads

In terms of evolutionary biology, a population admixture of more than two distinct lineages may lead to strengthened genetic variation through hybridization. However, a population admixture arising from artificial secondary contact poses significant problems in conservation biology. In urban Tokyo, a population admixture has emerged from two lineages of Japanese common toad: native Bufo japonicus formosus and nonnative B. japonicus japonicus, of which the latter was introduced in the early 20th century. To evaluate the degree of genetic disturbance in the admixed population of these two subspecies, we analyzed genotypes of toads distributed within and outside Tokyo by assessing mtDNA and seven microsatellite loci. We found that the introduced B. japonicus japonicus genotype dominates six local populations in the Tokyo admixture zone and was clearly derived from past introgressive hybridization between the two subspecies. These observations were supported by morphological assessments. Furthermore, the average larval survival rate in Tokyo was significantly higher than that outside Tokyo, suggesting that the temporary contribution of introduced toads occurred through introgression. The fitness of toads in urban Tokyo may thus be increasing with the assistance of nonnative individuals.

Chemical defense of an Asian snake reflects local availability of toxic prey and hatchling diet

Species that sequester toxins from prey for their own defense against predators may exhibit population-level variation in their chemical arsenal that reflects the availability of chemically defended prey in their habitat. Rhabdophis tigrinus is an Asian snake that possesses defensive glands in the skin of its neck (‘nuchal glands’), which typically contain toxic bufadienolide steroids that the snakes sequester from consumed toads. In this study, we compared the chemistry of the nuchal gland fluid of R. tigrinus from toad-rich and toad-free islands in Japan and determined the effect of diet on the nuchal gland constituents. Our findings demonstrate that captive-hatched juveniles from toad-rich Ishima Island that had not been fed toads possess defensive bufadienolides in their nuchal glands, presumably due to maternal provisioning of these sequestered compounds. Wild-caught juveniles from Ishima possess large quantities of bufadienolides, which could result from a combination of maternal provisioning and sequestration of these defensive compounds from consumed toads. Interestingly, juvenile females from Ishima possess larger quantities of bufadienolides than do juvenile males, whereas a small sample of field-collected snakes suggests that adult males contain larger quantities of bufadienolides than do adult females. Captive-born hatchlings from Kinkasan Island lack bufadienolides in their nuchal glands, reflecting the absence of toads on that island, but they can sequester bufadienolides by feeding on toads (Bufo japonicus) in captivity. The presence of large quantities of bufadienolides in the nuchal glands of R. tigrinus from Ishima may reduce the risk of predation by providing an effective chemical defense, whereas snakes on Kinkasan may experience increased predation due to the lack of defensive compounds in their nuchal glands.

High Degree of Mitochondrial Haplotype Diversity in the Japanease Common Toad Bufo japonicus in Urban Tokyo

The Japanese common toad Bufo japonicus is widely distributed across mainland Japan and is classified into two subspecies, B. japonicus japonicus and B. japonicus formosus, in the western and eastern regions, respectively. To investigate the genetic diversity of B. japonicus at the breeding pond (local population) level, we sequenced 831 base pairs of mitochondrial DNA (mtDNA) cytochrome b (Cyt b) from 75 individuals collected from nine ponds in urban Tokyo and the surrounding area. Phylogenetic and population genetic analyses revealed high mtDNA haplotype diversity (Hd, 0.716 (mean) ± 0.230 (SD)) within local populations (breeding ponds). Most local populations had multiple haplotypes of the mitochondrial Cyt b gene, and seven of the 18 haplotypes were identified in two or more local populations. These results indicate that mitochondrial gene flow had occurred across different breeding sites. We also identified five haplotypes that belonged to the western clade and correspond to B. japonicus japonicus. Our results provide genetic evidence that B. japonicus japonicus was introduced artificially from Western Japan to Tokyo, where it occupied the natural habitat of B. japonicus formosus. The urban Tokyo area was found to represent an admixed population consisting of both native and non-native B. japonicus subspecies.

Involvement of Gαolf‐expressing neurons in the vomeronasal system of Bufo japonicus

Most terrestrial vertebrates possess anatomically distinct olfactory organs: the olfactory epithelium (OE) and the vomeronasal organ (VNO). In rodents, olfactory receptors coupled to Gα(olf) are expressed in the OE, whereas vomeronasal receptors type 1 (V1R) and vomeronasal receptors type 2 (V2R), coupled to Gα(i2) and Gα(o) , respectively, are expressed in the VNO. These receptors and G proteins are thought to play important roles in olfactory perception. However, we previously reported that only V2R and Gα(o) expression is detected in the Xenopus laevis VNO. As X. laevis spends its entire life in water, we considered that expression of limited types of chemosensory machinery in the VNO might be due to adaptation of the VNO to aquatic life. Thus, we analyzed the expression of G proteins in the VNO and the accessory olfactory bulb (AOB) of the adult Japanese toad, Bufo japonicus, because this species is well adapted to a terrestrial life. By using immunohistochemical analysis in combination with in situ hybridization and DiI labeling, we found that B. japonicus Gα(olf) and Gα(o) were expressed in the apical and middle-to-basal layer of the vomeronasal neuroepithelium, and that the axons of these Gα(olf) - and Gα(o) -expressing vomeronasal neurons projected to the rostral and caudal accessory olfactory bulb, respectively. These results strongly suggest that both the Gα(olf) - and Gα(o) -mediated signal transduction pathways function in the B. japonicus VNO. The expression of Gα(olf) in the B. japonicus VNO may correlate with the detection of airborne chemical cues and with a terrestrial life.

Body Size and Age Structure of a Breeding Population of the Japanese Common Toad, Bufo Japonicus Formosus (Amphibia: Bufonidae)

Abstract: The body size and age structure of a breeding population of the Japanese common toad, Bufo japonicus formosus, was studied at Yamakitamachi in Kanagawa Prefecture for three breeding seasons from 1992 to 1994. Yearly variation in snout-vent length (SVL) was not significant, but the difference in SVL between the sexes was highly significant; the mean SVL of males and females was 125.8 and 134.2 mm, respectively. The breeding adults were aged successfully by skeletochronology using phalanges. The breeding adults were 1-8 yr old. The mean and median ages were 4.5 and 4 yr in males, and 5.2 and 5 yr in females, respectively. The males were estimated to begin to breed when one to four yr old, with a mean of 2.4 yr, while the females were estimated to breed when two to four yr old, with a mean of 2.7 yr. Age at first reproduction was significantly younger for males than females. We discuss the sexual size dimorphism of B. j. formosus in relation to the difference in age structure of the breeding popula...

Seasonal Increase in Olfactory Receptor Neurons of the Japanese Toad, Bufo japonicus, is Paralleled by an Increase in Olfactory Sensitivity to Isoamyl Acetate

Japanese toads (Bufo japonicus) migrate to and from breeding sites in the early spring, possibly guided by olfactory cues. We previously showed that the electrical activity of olfactory receptor neurons (ORNs) in the toads was enhanced in the breeding period. We undertook morphological and physiological studies of the olfactory epithelium to determine whether any cellular substrate of the epithelium underlies the enhanced electrical activity of ORNs. The ORNs of the toads were labeled by antiserum to olfactory marker protein (OMP), and the morphology of the labeled cells and their distribution in the epithelium were examined throughout the year. The OMP-positive cells, distributed mainly in the basal and intermediate layers of the epithelium, were most numerous in the early breeding period. Cell proliferation in the epithelium detected by 5-bromo-2'-deoxyuridine labeling was most elevated in this period. The electrical activity of ORNs was examined by recording the electroolfactogram (EOG) in the toads throughout the year. Statistical analysis showed a positive correlation between the density of OMP-positive cells in the epithelium and the amplitude of the EOG responses. A greater number of ORNs in the breeding period possibly aids the toads in migrating to their breeding sites.

The cerebral hemisphere patterns in hybrids produced by artificial crosses of a new species, Bufo torrenticola with Bufo bufo japonicus

Summary In Japanese toads, B. torrenticola, B. japonicus and their hybrids the cerebral hemisphere patterns were quantitatively studied. The posterior portion of pallium is less developed in B. torrenticola as a relict-like species than in B. japonicus as a advanced toad. The developmental degrees of the posterior portion of pallium in hybrids are intermediate between that of parental species, and furthermore, tend towards maternal side. Zusammenfassung Muster der Groshirnhemispharen von Hybriden, die durch kunstliche Kreuzungen zwischen Bufo torrenticola, einer neuen Art, und Bufo bufo japonicus erzeugt wurden Bei japanischen Kroten, B. torrenticola, B. japonicus und ihren Hybriden wurden die Muster der Groshirnhemispharen quantitativ studiert. Der hintere Abschnitt des Palliums von B. torrenticola, als reliktahnlicher Art, ist weniger entwickelt, im Vergleich zu B. japonicus, einer hoher evoluierten Art. Die Enrwicklungsgrade des hinteren Abschnitts des Palliums sind bei den Hybriden intermediar zwischen den elterlichen Arten, sie neigen zur mutterlichen Seite.

Molecular Cloning of Novel Cytochrome P450 1A Genes from Nine Japanese Amphibian Species

Novel cytochrome P450 1A (CYP1A) cDNA fragments were isolated from the liver of nine Japanese amphibian species using reverse transcription polymerase chain reaction (RT-PCR). Degenerate PCR primers were used to amplify 122-bp fragments of CYP1A cDNAs. Construction of a phylogenetic tree revealed that urodele and anuran amphibians formed two branches. Within the anuran species, three branches were formed: 1) Ranidae and Rhacophoridae, 2) Bufo japonicus formosus and Hyla japonica, and 3) Xenopus laevis. The cDNA nucleotide sequence of these CYP1A fragments showed identities ranging 72-98% (all), 72-78% (Anura vs. Urodela), 75 to 98% (Anura), 81% (Urodela), 74-80% (Xenopus laevis vs. nine Japanese amphibians).

Natural Hybridization and Directional Introgression of Two Species of Japanese Toads Bufo japonicus formosus and Bufo torrenticola (Anura: Bufonidae) Resulting from Changes in Their Spawning Habitat

Two species of Japanese toads, Bufo japonicus formosus and Bufo torrenticola, were identified in a mountain stream using random amplified polymorphic DNA (RAPD) and mitochondrial (mt) DNA sequencing techniques. We detected RAPD fragments and mtDNA sequences specific for each species collected from allopatric areas. These molecular data indicated that most of the toads collected from their sympatric areas in a mountain stream in central Japan were backcross hybrids to B. torrenticola, even though they possessed the mtDNA of B. j. formosus. Directional introgression probably occurred between the males of B. torrenticola and females possessing the mtDNA of B. j. formosus, resulting from B. j. formosus being compelled to spawn in stream side pools, especially during the initial period of contact between the two species. Furthermore, most of the hybrid individuals probably backcrossed to the numerically predominant B. torrenticola. Such conditions might have resulted from recent and continuous flooding which created changes in the topography of the Bufo spawning habitat.

Phylogenic Aspects of the Amphibian Dual Olfactory System

The phylogenic significance of the subdivision of dual olfactory system is reviewed mainly on the basis of our findings by electron microscopy and lectin histochemistry in the three amphibian species. The dual olfactory system is present in common in these species and consists of the projection from the olfactory epithelium (OE) to the main olfactory bulb (MOB) and that from the vomeronasal epithelium (VNE) to the accessory olfactory bulb (AOB). The phylogenic significance of subdivisions in the dual olfactory system in the amphibian must differently be interpreted. The subdivision of the MOB into its dorsal region (D-MOB) and ventral region (V-MOB) in Xenopus laevis must be attributed to the primitive features in their olfactory receptors. The middle cavity epithelium lining the middle cavity of this frog possesses both ciliated sensory cells and microvillous sensory cells, reminding the OE in fish. The subdivision of the AOB into the rostral (R-AOB) and caudal part (C-AOB) in Bufo japonicus formosus must be regarded as an advanced characteristic. The lack of subdivisions in both MOB and AOB in Cynops pyrrhogaster may reflect their phylogenic primitiveness. Since our lectin histochemistry to detect glycoconjugates expressed in the olfactory pathway reveals the subdivisions in the dual olfactory system in the amphibian, the glycoconjugates may deeply participate in the organization and function of olfactory pathways in phylogeny.

Complete mitochondrial genomes of three neobatrachian anurans: A case study of divergence time estimation using different data and calibration settings

We sequenced the whole mitochondrial (mt) genomes of three neobatrachian species: Japanese tree frog Hyla japonica, Japanese common toad Bufo japonicus, and narrow-mouthed toad Microhyla okinavensis. The gene arrangements of these genomes diverged from that of basal anurans (suborder Archaeobatrachia), but are the same as that of the members of derived frogs (i.e., superfamily Hyloidae and Ranoidae in suborder Neobatrachia), suggesting the one-time occurrence of a gene rearrangement event in an ancestral lineage of derived anurans. Furthermore, several distinct repeat motifs including putative termination-associated sequences (TASs) and conserved sequence blocks (CSBs) were observed in the control regions (CRs) of B. japonicus and H. japonica, while no repeat motifs were found in that of M. okinavensis. Phylogenetic analyses using both nucleotide and amino acid data of mt genes support monophyly of neobatrachians. The estimated divergence time based on amino acid data with multiple reference points suggests that the three living amphibian orders may have originated in the Carboniferous period, and that the divergences of anurans had occurred between the Permian and Tertiary periods. We also checked the influence of the data types and the settings of reference times on divergence time estimation. The resultant divergence times estimated from several datasets and reference time settings suggest that the substitution saturation of nucleotide data may lead to overestimated (i.e., older) branching times, especially for early divergent taxa. We also found a highly accelerated substitution rate in neobatrachian mt genes, and fast substitution possibly resulted in overestimation. To correct this erroneous estimation, it is efficient to apply several reference points among neobatrachians.

Somaclonal variation in Tricyrtis hirta plants regenerated from 1-year-old embryogenic callus cultures

Abstract The Liliaceous perennial Tricyrtis hirta, sometimes called ‘Japanese toad lily’, has recently become popular as an ornamental for pot and garden uses. Highly embryogenic callus cultures of this plant predominately consisted of diploid cells but also contained tetraploid cells after 1 year of establishment. In the present study, plans regenerated from the 1-year-old embryogenic callus cultures were subjected to ploidy level analysis and morphological characterization following 3 years of cultivation. Among 37 plants examined, 28 kept the diploid level (2n = 2x = 26) but nine were tetraploid (2n = 4x = 52) as indicated by FCM analysis and chromosome observation. Although no morphological alterations were detected in 26 out of 28 diploid regenerants, the remaining two showed noticeable variations: both were severely dwarf and had crimped leaves and many malformed flowers. The tetraploid regenerants had several horticulturally attractive characteristics compared with the diploid controls, such as longer shoots, thicker stems, and larger flowers. Thus regeneration of tetraploid plants from 1-year-old embryogenic callus cultures offers a possibility to improve the horticultural value of T. hirta, although regeneration of trueness-to-type plants is essential for utilizing the cultures for micropropagation and genetic transformation.

Subdivision of the accessory olfactory bulb in the Japanese common toad, Bufo japonicus, revealed by lectin histochemical analysis

Lectin binding patterns in the olfactory bulb of the Japanese common toad, Bufo japonicus, were examined using 21 types of lectin. Ten out of 21 lectins, WGA, s-WGA, LEL, STL, DBA, VVA, SJA, RCA-I, PNA, and PHA-L, stained the olfactory nerve, the glomeruli in the main olfactory bulb (MOB), the vomeronasal nerve, and the glomeruli in the accessory olfactory bulb (AOB). The binding patterns of LEL, STL, DBA, and PHA-L subdivided AOB glomeruli into rostral and caudal regions, where LEL, STL, and DBA stained the rostral region more intensely than the caudal region, and PHA-L had the opposite effect. Another lectin, BSL-I, stained both AOB glomeruli and the vomeronasal nerve, but not MOB glomeruli or the olfactory nerve. This is the first report of histological subdivision in the AOB of an amphibian, which suggests that the AOB development in Bufo may be unique.

Exfoliation of the Epidermal cells and Defecation by Amphibian Larvae in Response to Coelomic Fluid and Lysenin from the Earthworm Eisenia foetida

Coelomic fluid (CF) and lysenin from the earthworm Eisenia foetida induced heavy epidermal exfoliation in the larvae of Bufo japonicus formosus at developmental stages from hatching (stage 22) to operculum completion (stage 34). In experiments with Xenopus laevis, we observed that exfoliated cells were not stained by trypan blue. Thus, it appeared that these cells were still alive. It is likely, therefore, that both CF and lysenin might disrupt the adhesion between epidermal cells of larvae prior to stage 34. Since it is known that lysenin exerts its toxic effects through its specific binding to sphingomyelin (SM), SM might be involved in such adhesion. This hypothesis was supported by the observations that CF and lysenin which had been incubated with SM-liposomes lost their exfoliative activity. In larvae after stage 34, the mechanism of adhesion between epidermal cells seemed to change and the adhesion was no longer disrupted by CF and lysenin. In larvae at around stage 34, a collagen layer started to form beneath the basement membrane of the epidermis. Furthermore, larvae at around this stage started to eat solid food. The developing collagen layer and food intake might be related indirectly to the chemical change in epidermal adhesion. The induction of exfoliation by CF and lysenin was also observed in other amphibian species. In Bufo larvae, defecation was induced both by CF and by lysenin but this effect was independent of exfoliation.

Growth of Japanese Toad (Bufo japonicus formosus) Tadpoles Fed Different Food Items

Suitability of different food items for tadpoles of the Japanese toad (Bufo japonicus formosus) was studied by rearing experiments. Tadpoles were fed single food items (leaf litter, algae, or sludgeworms) individually in the laboratory, and their survival, growth, and development were compared. All individuals metamorphosed successfully. Litter-fed tadpoles metamorphosed at a significantly smaller size than algae- or worm-fed tadpoles, but the latter two metamorphosed at a similar size. The larval period showed little difference among the three food treatments.

Seasonal changes of odor sensitivity in the Japanese toad (Bufo japonicus) in relation to the maturation of olfactory receptor neurons

Seasonal changes of odor sensitivity in the Japanese toad (Bufo japonicus) in relation to the maturation of olfactory receptor neurons

大分川中流部の農村景観における両生類の分布パターン

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.

A survey of Blastocystis infection in anuran and urodele amphibians

Blastocystis infection in amphibians was surveyed in three species of anuran and one species of urodele amphibians captured at two distinct locations in Japan. All three species of frogs were highly infected with Blastocystis, while 69 individual urodele newts, Cynopus pyrrhogaster, were negative for infection. Eleven Blastocystis isolates (47.8%) were recovered from 23 Rana nigromaculata leopard frogs. Twenty-three (92%) of 25 Rana catesbeiana bullfrogs and all (100%) of 24 Bufo japonicus japonicus toads were positive for Blastocystis. Two distinct populations of the toad and bullfrog showed a high prevalence (100 or 84.6%) of Blastocystis infection, while in two populations of the leopard frog only one population was positive for Blastocystis (84.6%). Three Blastocystis isolates from different species of the frogs were established. Since none of the three isolates could survive at 37 °C, a temperature tolerance assay was performed to assess the optimal growth temperature and to determine the range of non-lethal temperatures. During the exponential growth phase of 3- or 4-day cultures at 25 °C, three isolates were exposed to 4, 28, 31, or 34 °C for 3 days and then returned to 25 °C to monitor the cell growth. Based on the optimal growth temperatures and different ranges of temperature tolerance among the three new isolates from frogs and two known species, Blastocystis hominis and Blastocystis lapemi, it was established that the three isolates recovered from different species of frogs had different physiological features from B. hominis and B. lapemi.