Rana Rugosa Research Articles

Territory Defense Strategy of the Wrinkled Frog, Rana rugosa

The advertisement call of anurans functions to attract potential mates. The dominant frequency of an advertisement call is generally getting lower with increased snout-vent length (SVL) of the caller Rana rugosa has an advertisement call tilth a particularly high frequency modulation. We conducted a playback experiment to verify the function of frequency modulation, and investigated the territorial behavior of the frog. The frog has five types of territory defense strategy. Strategy choice depended on the caller`s SVL. Small males became satellites or lowered the dominant frequency of their advertisement call, whereas large males actively defended their territory with encounter calls. In response to high frequency (1107 Hz) playback, the frogs lowered their advertisement call frequency, and towered them further in response to the low frequency (1028 Hz) playback. In addition, the number of pulses in a call was increased in response to the playback. These results indicate that the frog avoids physical conflict with competitors by selecting a territory defense strategy suitable for the caller`s size, and by lowering its call frequency to disguise its SVL.

Expression of Dax-1 during gonadal development of the frog

Dax-1, a member of the nuclear hormone receptor superfamily of transcription factors, is known to be involved in gonadal development in mammals. To date, Dax-1 has only been isolated in reptiles, birds and mammals. The expression of Dax-1 is down-regulated in the developing testis, but persists in the ovary of mice (Swain et al., Nat. Genet. 12 (1996) 404) and chicken (Smith et al., J. Mol. Endocrinol. 24 (2000) 23). Curiously, there is no sex difference in the expression patterns of Dax-1 in the American alligator (Western et al., Gene 241 (2000) 223). To understand its role(s) in gonadal development in vertebrates, molecular cloning of Dax-1 in amphibians is required. In this study, we cloned an amphibian Dax-1 homologue of the frog Rana rugosa and examined its expression profile during gonadal development. Cloned Dax-1 cDNA encoded a protein of 287 amino acids. Unlike mammalians that possess the three and one half repeat elements representing the putative DNA binding domain in the predicted sequence of Dax-1 protein, the frog had a single poorly conserved copy of the repeat unit. By RT-PCR analysis, the Dax-1 mRNA was detected in the liver and pancreas, but not in the testis and ovary of adult frogs. However, Dax-1 expression was seen first in the embryo at stage 12 and became stronger in tadpoles until stage X. The Dax-1 was transcribed in the testis stronger than in the ovary of frogs at stage XXV (just after completion of metamorphosis). In the gonad of frogs 2 months after metamorphosis (at this stage postmeiotic cells can be seen in the seminiferous tubules), the Dax-1 was expressed only in males. In addition, the Dax-1 transcription declined gradually as ovarian development proceeded, but its expression was down-regulated and then up-regulated rapidly when female-to-male sex reversal was caused by administration of testosterone into female tadpoles. Taken together, the results suggest that the Dax-1 may be closely involved in testicular development of amphibians.

Effects of Styrene Monomer and Trimer on Gonadal Sex Differentiation of Genetic Males of the Frog Rana rugosa

To ascertain whether styrene monomer and trimer induce an estrogen-like effect, all-male tadpoles of Rana rugosa were exposed to dilute solutions of styrene monomer and trimer (2,4,6-tripfenyl-1-hexene) at concentrations of 0.1, 1, or 10 μM during days 19–23 after fertilization, which is the critical period of gonadal sex differentiation. As positive and vehicle controls, tadpoles were exposed to dilute solutions of estradiol-17β at concentrations of 0.01, 0.1, or 1 μM and 0.01% ethanol, respectively, during the same period. The influence was estimated by examining the inner structure of the gonads of 40-day-old tadpoles. All the gonads of tadpoles in the vehicle and untreated controls showed histological characteristics of testes, whereas all those of tadpoles treated with 1 μM estradiol-17β showed a structure typical for ovaries, having an ovarian sac and many meiotic germ cells. Of the genetically male tadpoles treated with styrene monomer and trimer, 97 and 96%, respectively, showed normal testicular structure of the gonads. In contrast, the remaining tadpoles had gonads showing coexistence of testicular and ovarian structure, irrespective of the strength of styrene concentration. These findings suggest that the styrene monomer and trimer induced a weak estrogen-like effect on the pathways of testicular differentiation in genetically male tadpoles.

The yellow mutation in the frog Rana rugosa: pigment organelle deformities in the three types of chromatophore.

Crossing experiments revealed that a single recessive gene mutation (yellow) gives rise to the yellow phenotype of Rana rugosa in Japan. Ultrastructural observation of dermal chromatophores showed that the pigment organelles; melanosomes, pterinosomes, and reflecting platelets, all had structural deformities. This suggests that the yellow gene acts at the level of a primordial pigment organelle common to the three types of chromatophore.

Expression of Steroidogenic Factor-1 in Frog Embryo and Developing Gonad

Steroidogenic factor-1 (SF-1), originally identified as an orphan nuclear receptor that regulates expression of genes encoding cytochrome P-450 steroid hydroxylases, is an essential transcriptional factor for adrenal and gonadal development in mammals. Since sex steroid hormones have been shown to play important roles in the sex determination of frogs, it is of interest to know how SF-1 gene expression is regulated during the sexual development of this organism. A previous study isolated the cDNA of the frog Rana rugosa SF-1 (rrSF-1) and found sexual differences in its gene expression in adult frogs; positive in testis and negative in ovary (Kawano et al., 1998). This study examined rrSF-1 gene expression in frog embryos and developing and mature gonads by immunohistochemical staining using anti-rrSF-1 protein antibody for protein localization and by in situ hybridization analysis for mRNA transcription. The results obtained in this study indicated that cells expressing SF-1 that originate in the mesoderm and endoderm probably migrate into the developing gonad via the dorsal mesentery, genital ridge, and mesorchium or mesovarium. Thus, SF-1 may play an important role in gonadal development in amphibians.

Expression of FTZ-F1alpha in frog testicular cells.

Fushi tarazu transcription factor-1 (FTZ-F1), a member of a nuclear hormone receptor superfamily, is a transcriptional regulator for fushi tarazu gene expression in Drosophila (Ueda et al., '90). We have cloned a homologue (rrFTZ-F1alpha) of the FTZ-F1 gene of the frog Rana rugosa. The gene, in frogs, has been shown to have high expression level in the testis (Nakajima et al., 2000). In this study, the RT-PCR analysis showed that the FTZ-F1alpha mRNA level in adult frogs did not change throughout the year, even during hibernation. However, when immunohistological studies using the anti-rrFTZ-F1alpha antibody were employed to examine which testicular cells expressed this gene, Sertoli cells were found to produce rrFTZ-F1alpha in the two seasons: the breeding season (from March through May) and the pre-hibernating season (from October through November). Interstitial cells, however, did it in only the breeding season (from April through May). Taken together, the results suggest that the rrFTZ-F1alpha expression is regulated at the post-transcriptional step, and that the rrFTZ-F1alpha may play an important role(s) in the seasonal activities of Sertoli and interstitial cells in the frog testis. J. Exp. Zool. 290:182-189, 2001.

Age Determination of Wrinkled Frog, Rana rugosa with Special Reference to High Variation in Postmetamorphic Body Size (Amphibia: Ranidae)

Individual ages of postmetamorphic R. rugosa from three populations of Kyoto, Japan (Gondoike, Iwakura, and Kiyotaki), were determined skeletochronologically. From recaptured individuals, one LAG was confirmed to be formed per year. In all three populations, age at sexual maturity was 1–2 yrs in males and 2–3 yrs in females, though some females matured at the age of 1 yr. Longevity was at least 4 yrs in males and 5 yrs in females. Sexual size dimorphism was conspicuous in adults, and females were invariably larger than males. Females had a larger asymptotic size than males but growth rate differed between some localities only in males. In all populations, both sexes showed high variations in SVL and the variation was even higher in juveniles than in adults. This high variation is explained by a prolonged breeding season and resultant mixture of metamorphs with and without overwintering larval stage within a population of this species.

Effects of Dibutyl Phthalate as an Environmental Endocrine Disruptor on Gonadal Sex Differentiation of Genetic Males of the Frog Rana rugosa

To examine the effects of dibutyl phthalate (DBP) on gonadal sex differentiation, genetically male tadpoles of Rana rugosa were exposed to dilute solutions of DBP at concentrations of 0.1, 1, or 10 microM during days 19-23 after fertilization, which is the critical period of gonadal sex differentiation in R. rugosa. Tadpoles were necropsied on day 40. The genetically male tadpoles were produced from crossings between males (ZZ) of one local population, in which females are the heterogametic sex, and females (XX) of another local population, in which males are the heterogametic sex. As positive control groups, tadpoles were exposed to dilute solutions of 17beta-estradiol (E(2)) at concentrations of 0. 01, 0.1, or 1 microM during the same period. The internal structure of the gonads was histologically examined in a total of 30 control tadpoles, 86 E(2)-treated tadpoles, and 90 DBP-treated tadpoles. The gonads of the control tadpoles all showed the typical structure of testes. In contrast, 0.01, 0.1, and 1 microM E(2) treatments caused the undifferentiated gonads of 18, 63, and 100% of the tadpoles, respectively, to develop into gonads of complete or partial ovarian structure. After 0.1, 1, and 10 microM DBP treatment, 0, 7, and 17% of tadpoles, respectively, were similarly affected. These findings suggest that DBP was about 1,000-fold less potent than E(2). Nevertheless, DBP is an environmentally dangerous hormone that disrupts the pathways of testicular differentiation in genetically male animals.

Effects of dibutyl phthalate as an environmental endocrine disruptor on gonadal sex differentiation of genetic males of the frog Rana rugosa.

To examine the effects of dibutyl phthalate (DBP) on gonadal sex differentiation, genetically male tadpoles of Rana rugosa were exposed to dilute solutions of DBP at concentrations of 0.1, 1, or 10 microM during days 19-23 after fertilization, which is the critical period of gonadal sex differentiation in R. rugosa. Tadpoles were necropsied on day 40. The genetically male tadpoles were produced from crossings between males (ZZ) of one local population, in which females are the heterogametic sex, and females (XX) of another local population, in which males are the heterogametic sex. As positive control groups, tadpoles were exposed to dilute solutions of 17beta-estradiol (E(2)) at concentrations of 0. 01, 0.1, or 1 microM during the same period. The internal structure of the gonads was histologically examined in a total of 30 control tadpoles, 86 E(2)-treated tadpoles, and 90 DBP-treated tadpoles. The gonads of the control tadpoles all showed the typical structure of testes. In contrast, 0.01, 0.1, and 1 microM E(2) treatments caused the undifferentiated gonads of 18, 63, and 100% of the tadpoles, respectively, to develop into gonads of complete or partial ovarian structure. After 0.1, 1, and 10 microM DBP treatment, 0, 7, and 17% of tadpoles, respectively, were similarly affected. These findings suggest that DBP was about 1,000-fold less potent than E(2). Nevertheless, DBP is an environmentally dangerous hormone that disrupts the pathways of testicular differentiation in genetically male animals.

Maternal control of sex ratio in Rana rugosa: evidence from DNA sexing.

Parental control of primary sex ratio has been reported in a mammal (red deer), some birds, and a snake. However, it remains uncertain whether other vertebrates including Amphibia can control sex ratio. In this paper, we examined the possibility in a wild population of the Japanese frog Rana rugosa which has female heterogamety. Sex ratios of their eggs were determined using DNA markers. The eggs were sampled in the field from May to August in 1998. Each egg was then sexed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using a sex-specific DNA marker. The result showed a male bias early in the season which changed to a female bias later, suggesting that females of R. rugosa can control the primary sex ratio.

FTZ-F1α is expressed in the developing gonad of frogs

Fushi tarazu transcription factor-1 (FTZ-F1), a member of the nuclear hormone receptor superfamily, is a regulator for fushi tarazu gene expression in Drosophila. Its expression pattern during organogenesis in vertebrates, however, is not known yet. In this study, we cloned a frog FTZ-F1 homologue ( rrFTZ-F1α) and analyzed its expression and localization during gonadal development of the frog Rana rugosa. Cloned rrFTZ-F1α cDNA encoded a protein of 501 amino acids including the regions I–III and FTZ-F1 box that are evolutionally conserved in the FTZ-F1 superfamily. rrFTZ-F1α shared high similarity at the amino acid level with mouse LRH-1 (76%), human FTF (92%), chicken OR2.0 (92%), Xenopus laevis FF1rA (94%) and zebrafish FF1A (82%). Northern blot analysis showed that the rrFTZ-F1α mRNA at a size of 7.4 kb was the most prominent in the testis among various tissues of adult frogs examined. The RT-PCR analysis revealed that the expression of rrFTZ-F1α was weak in the gonad of tadpoles before stage XVI, but it became stronger in the testis of froglets at stage XXV and much higher in the testis of frogs 2 months after metamorphosis. In addition, in situ hybridization analysis revealed that the rrFTZ-F1α gene was transcribed in germ cells except for sperm in the testis, and in oocytes at stage A in the ovary of frogs 2 months after metamorphosis. Together, these results suggest that FTZ-F1α probably plays an important role in differentiation of germ cells in the gonad of frogs in both sexes.

Structural organization and expression of the gaegurin 4 gene of Rana rugosa

Gaegurin 4 (GGN4) is a member of the antimicrobial peptide subfamily isolated from the skin of Rana rugosa. We cloned gDNA encoding GGN4 to study its gene organization and regulation of expression. The GGN4 gene occurs in single copy in the R. rugosa genome and contains a single intron of about 3.4 kb. The transcription start site is located 68 bases upstream of the translation initiation codon. The GGN4 gene was expressed both in Xenopus kidney epithelial cells (A6) and in Xenopus oocytes using the chloramphenicol acetyltransferase reporter gene system. The 5′ flanking region of the GGN4 gene contains a dl binding site that is known to regulate acute phase immune response related gene expression in mammals and insects. The dl protein bound specifically to the GGN4 gene promoter region. Mutants that serially delete the 5′ flanking region show that removal of the dl binding site inhibited GGN4 gene expression in both A6 cells and Xenopus oocytes. From these results, we propose that expression of the GGN4 gene may be regulated by the region containing the dl element which plays a key role in the regulation of antimicrobial peptide genes in Drosophila and mammals.

Myrmecophagy in a Ranid Frog Rana rugosa: Specialization or Weak Avoidance to Ant Eating

Abstract We studied diets of an Asian ranid, Rana rugosa, inhabiting three different environments (reservoir, river shoreline, and paddy fields) in Kyoto, Japan. In all the three habitats, ants were the most frequently consumed prey, representing surprisingly similar proportions in both the frequency of occurrence (81.9–85.7%) and the number of total prey items (56.8–59.4%). These values are exceptionally large for Rana, and equivalent to those reported for ant specialists in other families of frogs such as dendrobatids or bufonids. However, R. rugosa consumed ants lower in proportions than those found in the environment, and could not be regarded as purely ant specialists. Instead, we conclude that this species tends to avoid ants more weakly than other species of Rana. Other than ants, larger prey were more and smaller ones less frequently taken in proportion to frog body size, indicating that the frog consumes ants because of its weak avoidance of these abundant potential prey.

Solution structure of the antimicrobial peptide gaegurin 4 by H and 15N nuclear magnetic resonance spectroscopy.

Gaegurin 4 (GGN4) is a 37-residue antimicrobial peptide isolated from the skin of a Korean frog, Rana rugosa. This peptide shows a broad range of activity against prokaryotic cells but shows very little hemolytic activity against human red blood cells. The solution structure of GGN4 was studied by using circular dichroism (CD) and NMR spectroscopy. CD investigations revealed that GGN4 adopts mainly an alpha-helical conformation in trifluoroethanol/water solution, in dodecylphosphocholine and in SDS micelles, but adopts random structure in aqueous solution. By using both homonuclear and heteronuclear NMR experiments, complete 1H and 15N resonance assignments were obtained for GGN4 in 50% trifluoroethanol/water solution. The calculated structures of GGN4 consist of two amphipathic alpha-helices extending from residues 2-10 and from residues 16-32. These two helices are connected by a flexible loop spanning between the residues 11 and 15. By using enzyme digestion and matrix-assisted laser desorption/ionization mass spectroscopy, we confirmed that GGN4 contains a disulfide bridge formed between the residues Cys31 and Cys37 in its C-terminus. The effect of disulfide bridge on the structure and the activity of GGN4 was investigated. The reduced form of GGN4 revealed a similar activity and conformation to native GGN4, suggesting that the disulfide bridge does not strongly affect the conformation and the antimicrobial activity of GGN4.

Ligand binding inhibitors of A1 adenosine receptor from Rana rugosa are phospholipase A2s.

Inhibitors of the A1 adenosine receptor were isolated from the skin extract of Korean frog, Rana rugosa. The frog-skin extract was prepared by an electrical shock and fractionated with C4 followed by C18 reverse-phase HPLC. Two A1 receptor inhibitors were isolated using a filter binding assay and the molecular masses of the proteins were estimated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to be 15 347 and 15 404 Da, respectively. The inhibitory activity was also measured against other membrane receptors, such as the A2 adenosine receptor, muscarinic acetylcholine receptor and capsaicin receptor. Ligand binding to the A2 and muscarinic receptors was also severely inhibited by these proteins. However, they did not inhibit the functional activation of the capsaicin receptor by its ligand, capsaicin, suggesting that inhibition of ligand-receptor binding occurs specifically. Their N-terminal sequences were determined by Edman degradation. Surprisingly, they showed sequence similarity to the secretory protein, phospholipase A2 from various organisms. The phospholipase A2 activity of both proteins was tested using Dole's assay technique. Both proteins showed phospholipase A2 activity, and therefore, they were designated as PLA2-R1 and PLA2-R2, respectively. In addition, their ligand-binding inhibitory activity depended on their phospholipase A2 activity. This is the first finding that the frog secretes a phospholipase A2 similar to that of snake venoms, which posess inhibitory activity against the adenosine A1, adenosine A2 and muscarinic receptors.

Alteration of the sex determining system resulting from structural change of the sex chromosomes in the frog Rana rugosa

Alteration of the sex determining system resulting from structural change of the sex chromosomes in the frog Rana rugosa

Alteration of the sex determining system resulting from structural change of the sex chromosomes in the frogRana rugosa

Rana rugosa in Japan is divided into four geographical races on the basis of the karyotype of the sex chromosomes: one in which heteromorphic sex chromosomes occur in the female sex (ZW/ZZ-system), another in which they are present in males (XX/XY-system), and the remaining two in which no heteromorphism is seen in either sex. The last two inherit the XX/XY sex determining system. Y and Z chromosomes in the former two are of the same karyotype as the no. 7 chromosomes seen in one of the latter two, whereas X and W are caused by two inversions that occurred in the original Xs (no. 7). In this study, we first attempted to detect the structural difference between the resulting X and W by examining their chiasma formation. The chiasma distribution between X and W was closely similar to that between two Xs, suggesting that the W and X are identical in structure. Regarding the change from XX/XY- to ZW/ZZ-system, the simplest explanation is that the putative female-determining gene(s) on the W grew functionally stronger by inversions. Next, we examined the sex of triploids having two Xs and one Z. The data showed that the triploids with two original Xs and a Z were all male, whereas most of those with two resulting Xs and a Z developed into females as expected. We speculated that the female-determining gene(s) on the resulting X grew mildly stronger functionally by position effect, whereas those on the W grew much stronger for some other reason (e.g., duplication). J. Exp. Zool. 286:313-319, 2000.

Two Sox9 messenger RNA isoforms: isolation of cDNAs and their expression during gonadal development in the frog Rana rugosa

Sox is a family of SRY-related testis-determining genes. We have isolated two different mRNA isoforms of the frog Sox9 gene from adult frog testis cDNAs. One form ( Sox9 α) encodes a 482 amino acid protein containing the HMG box, whereas the other form ( Sox9 β), which completely lacks the HMG box, is a truncated 265 amino acid protein of Sox9 α. Sox9 α is 82% similar to mouse, 86% to chicken, and 77% to trout Sox9 at the amino acid level. Sox9 expression was up-regulated in embryos after stage 16, and was seen in both developing testes and ovaries. The size of Sox9 transcripts was determined to be 7.8 knt by Northern blot analysis. In addition, Sox9 α expression was found prominently in the testis and brain among various tissues of adult frogs examined, and was considerably higher than Sox9 β. The fact that Sox9 is expressed in both sexes suggests that this gene is involved in gonadal development of male and female frogs. This is dissimilar to the pattern in birds and mammals, in which Sox9 expression is male-specific.

Genetic diversity and speciation ofrana rugosa(amphibia; ranidae)

Horizontal starch gel electrophoresis for 29 populations (n=543) of the wrinkled frog, Rana rugosa, from Korea and Japan was performed to assess the degree of genic variation and genetic diversity, and to understand the biogeographic pattern of distribution and speciation. A sum of 22 presumptive loci was screened from 17 enzymes and general proteins. Four loci, Aco, Est‐3, Me‐2, and Pgm, demonstrated high levels of polymorphism. The degree of average genetic variation of R. rugosa was P=22.7% (9.1–40.9%), Ho=0.086 (0.048–0.165) and He=0.090 (0.042–0.168). In the south‐eastern region of the Korean peninsula (Chongsong, Yongchon, Ulsan, Kyongju, Pohang, Yongdok and Ulchin), a few unique alleles in the Mpi locus were detected and their biogeographic implications were considered. The degree of genetic differentiation among the Korean populations was moderate (S=0.900), whereas the degree of genetic diversity between Korean and Japanese populations was notably high (S=0.687, D=0.293). This result corresponds with the data obtained by the mitochondrial cytochrome b gene sequence (Lee et al., 1999) suggesting that the Korean and Japanese R. rugosa might have evolved a specific level of genetic differentiation since their geographic isolation.

Myrmecophagy in a Ranid Frog Rana rugosa. Specialization or Weak Avoidance to Ant Eating?

Myrmecophagy in a Ranid Frog Rana rugosa. Specialization or Weak Avoidance to Ant Eating?