Japanese Frog Research Articles

Conserved Evolution of MHC Supertypes among Japanese Frogs Suggests Selection for Bd Resistance.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) is a major threat to amphibians, yet there are no reports of major disease impacts in East Asian frogs. Genetic variation of the major histocompatibility complex (MHC) has been associated with resistance to Bd in frogs from East Asia and worldwide. Using transcriptomic data collated from 11 Japanese frog species (one individual per species), we isolated MHC class I and IIb sequences and validated using molecular cloning. We then compared MHC from Japanese frogs and other species worldwide, with varying Bd susceptibility. Supertyping analysis, which groups MHC alleles based on physicochemical properties of peptide binding sites, identified that all examined East Asian frogs contained at least one MHC-IIb allele belonging to supertype ST-1. This indicates that, despite the large divergence times between some Japanese frogs (up to 145 million years), particular functional properties in the peptide binding sites of MHC-II are conserved among East Asian frogs. Furthermore, preliminary analysis using NetMHCIIpan-4.0, which predicts potential Bd-peptide binding ability, suggests that MHC-IIb ST-1 and ST-2 have higher overall peptide binding ability than other supertypes, irrespective of whether the peptides are derived from Bd, other fungi, or bacteria. Our findings suggest that MHC-IIb among East Asian frogs may have co-evolved under the same selective pressure. Given that Bd originated in this region, it may be a major driver of MHC evolution in East Asian frogs.

Identification of ancestral sex chromosomes in the frog Glandirana rugosa bearing XX-XY and ZZ-ZW sex-determining systems.

Sex chromosomes constantly exist in a dynamic state of evolution: rapid turnover and change of heterogametic sex during homomorphic state, and often stepping out to a heteromorphic state followed by chromosomal decaying. However, the forces driving these different trajectories of sex chromosome evolution are still unclear. The Japanese frog Glandirana rugosa is one taxon well suited to the study on these driving forces. The species has two different heteromorphic sex chromosome systems, XX-XY and ZZ-ZW, which are separated in different geographic populations. Both XX-XY and ZZ-ZW sex chromosomes are represented by chromosome 7 (2n=26). Phylogenetically, these two systems arose via hybridization between two ancestral lineages of West Japan and East Japan populations, of which sex chromosomes are homomorphic in both sexes and to date have not yet been identified. Identification of the sex chromosomes will give us important insight into the mechanisms of sex chromosome evolution in this species. Here, we used a high-throughput genomic approach to identify the homomorphic XX-XY sex chromosomes in both ancestral populations. Sex-linked DNA markers of West Japan were aligned to chromosome 1, whereas those of East Japan were aligned to chromosome 3. These results reveal that at least two turnovers across three different sex chromosomes 1, 3 and 7 occurred during evolution of this species. This finding raises the possibility that cohabitation of the two different sex chromosomes from ancestral lineages induced turnover to another new one in their hybrids, involving transition of heterogametic sex and evolution from homomorphy to heteromorphy.

Taxon-specific zeta-crystallin of camel eye lens: A comparative in silico studies

The eye lens is a specialized organ of the visual system, which is transparent due to its complex geometry and unique protein composition. Nature has designed it to focus light on the retina throughout the life span. Several factors (post-translational modifications, thermal and solar radiations) causes aggregation of the lens proteins and result in early-onset of cataract. A cataract is one of the major causes of blindness worldwide. It's interesting how camel eyes maintain lens transparency in a harsh desert climate? The camel eye lenses contain a novel protein zeta (ζ)-crystallin in a bulk amount that is also present in two other animals (guinea pig and Japanese frog) but is adopted for milder habitats. The camel lens ζ-crystallin is a poorly characterized protein. This study has investigated the physicochemical and structural properties of camel, guinea pig, and Japanese frog’s ζ-crystallin. The docking results showed a strong affinity between NADPH and ζ-crystallin. RMSD, RMSF, radius of gyration, and SASA analysis in the ligand-free and bound states showed distinct prope6rties in these ζ-crystallins. Moreover, the hydrophobicity of camel and Japanese frog ζ-crystallin is lower than ζ-crystallin of other mammalian sources. The unique physicochemical and structural properties of taxon-specific ζ-crystallin are likely to maintain lens transparency.

Relative Differences in Na+/K+-ATPase Activity between Mountain and Coastal Populations of the Japanese Frog, Buergeria japonica

Buergeria japonica (Rhacophoridae) thrives in diverse habitats, from montane forests to the mouths of small rivers opening onto sandy beaches. The osmoregulatory system of B. japonica may differ between populations living in these contrasting environments. In this study, we compared the activity of cutaneous Na+/K+-ATPase in two populations of B. japonica from different habitats (montane vs. coastal). Our results reveal a significant difference between the Na+/K+-ATPase activity of the two populations of B. japonica, with the coastal population having higher Na+/K+-ATPase activity. Because the Na+/K+-ATPase plays a crucial role in osmoregulation in high salinity, these results are consistent with the higher salinity tolerance of the coastal population. It is not known whether the difference in Na+/K+-ATPase activity between populations is a result of local adaptation or phenotypic plasticity. More detailed work will be needed to disentangle the patterns of differentiation in physiological responses to salt in these amphibians.

Identification of Novel Proteins in Foam Nests of the Japanese Forest Green Tree Frog, Rhacophorus arboreus.

Foam nests of frogs are natural biosurfactants that contain potential compounds for biocompatible materials, Drug Delivery System (DDS), emulsifiers, and bioremediation. To elucidate the protein components in the foam nests of Rhacophorus arboreus, which is an endemic Japanese frog species commonly seen during the rainy season, we performed amino acid analysis, SDS-PAGE electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry using intact foam nests. Many proteins were detected in these foam nests, ranging from a few to several hundred kDa, with both essential and non-essential amino acids. Next, we performed transcriptome analysis using a next-generation sequencer on total RNAs extracted from oviducts before egg-laying. The soluble foam nests were purified by LC-MS and analyzed using Edman degradation, and the identified N-terminal sequences were matched to the transcriptome data. Four proteins that shared significant sequence homologies with extracellular superoxide dismutase of Nanorana parkeri, vitelline membrane outer layer protein 1 homolog of Xenopus tropicalis, ranasmurfin of Polypedates leucomystax, and alpha-1-antichymotrypsin of Sorex araneus were identified. Prior to purification of the foam nests, they were treated with both a reducing reagent and an alkylating agent, and LC-MS/ MS analyses were performed. We identified 22 proteins in the foam nests that were homologous with proteinase inhibitors, ribonuclease, glycoproteins, antimicrobial protein and barrier, immunoglobulin-binding proteins, glycoprotein binding protein, colored protein, and keratin-associated protein. The presence of these proteins in foam nests, along with small molecules, such as carbohydrates and sugars, would protect them against microbial and parasitic attack, oxidative stress, and a shortage of moisture.

Antifungal activity of bacteria isolated from Japanese frog skin against plant pathogenic fungi

Frogs carry bacterial communities on their skin (Harris et al., 2009) that benefit the host frogs by preventing harmful pathogen infections. Our study aimed to collect culturable bacteria from the skin of wild frogs sampled in Japan and evaluate the antagonistic activity of the bacteria toward plant pathogenic fungi. A collection of 106 bacterial isolates was obtained from three species of frogs, namely Hyla japonica, Pelophylax porosus porosus and Buergeria burgeri. Using a dual-culture method, three isolates, HjD52, HjD92 and B341, were selected based on their ability to significantly inhibit the growth of Colletotrichum orbiculare, the causal fungus of cucumber anthracnose disease. These three bacterial isolates also showed a broad-spectrum of antagonistic activity against plant pathogenic fungi. Furthermore, spray treatment with the bacterial suspensions (109 cfu/ml) effectively reduced the number of anthracnose lesions in greenhouse-grown, potted cucumber plants. Based on the 16S rDNA sequence analysis and similarity search, isolates HjD57, HjD92 and B341 were identified as Paenibacillus sp., Raoultella sp. and Citrobacter sp., respectively. This is the first report showing the potential of Paenibacillus sp., Raoultella sp. and Citrobacter sp. from frog skin to serve as potent biocontrol agents against plant diseases.

Feeding Habits of the Japanese Rice Frog, Fejervarya kawamurai, as an Invasive Species

Fejervarya kawamurai (Dicroglossidae) is an invasive species occurring in several populations in the Kanto region, including Fujisawa City, Kanagawa Prefecture, Japan. To estimate the ecological impacts of this frog, we investigated the food habits of invasive populations. We collected 119 individuals of F. kawamurai. Of these, 108 (90.8%) had stomach contents that included various small animals. Therefore, this invasive frog might influence the ecosystem via predation pressure on small native animals.

Participation of androgen and its receptor in sex determination of an amphibian species.

IntroductionIn the Japanese frog Rana (R.) rugosa the androgen receptor (AR) gene on the W chromosome (W-AR) is barely expressed. Previously we showed that incomplete female-to-male sex-reversal occurred in Z-AR transgenic female frogs. To date, however, there is no report showing that AR with androgens can determine genetically programed male sex fate in any vertebrate species. Here, we examined whether AR together with androgens functions as a sex determinant in an amphibian species.MethodsTo examine whether complete female-to-male sex-reversal occurs in R. rugosa frogs, we produced AR-transgenic (Tg) and -knockdown (KD) female R. rugosa frogs by the I-SceI meganuclease-mediated gene trap and CRISPR/Cas9 system, respectively. AR-Tg and -KD tadpoles were reared in water containing testosterone (T) at 0 to 7.1 ng/ml. Frozen sections were prepared from the gonads of metamorphosed frogs and immunostained for laminin, Vasa, Pat1a, CYP17 and AR. We also employed PCR analysis to examine Dmrt1, Pat1a and CYP17 expression in the gonads of KD and placebo-KD female frogs.ResultsComplete female-to-male sex-reversal occurred in the AR-Tg ZW female frogs when a low dosage of T was supplied in the rearing water of tadpoles. However, no sex-reversal was observed in AR-KD ZW female frogs when the gonads were treated with dosages of T high enough to induce complete female-to-male sex-reversal even in wild type frogs.DiscussionThese results suggest that AR with its androgen ligand functions as a male sex-determinant in the ZW type R. rugosa frogs.

A Threshold Dosage of Testosterone for Female-to-Male Sex Reversal in Rana rugosa Frogs.

Androgens play a critical role in testicular differentiation in many species of vertebrates. While female-to-male sex reversal can be induced by testosterone (T) in some species of amphibians, the mechanism still remains largely unknown even at the histological level. In this study, we determined a threshold dosage of T to induce female-to-male sex reversal in the Japanese frog Rana (R.) rugosa. Tadpoles were allowed to metamorphose into frogs with T present in the rearing water. At 0.2 ng/mL T, female frogs formed tissue comprising a mixture of ovary and testis, the so-called ovotestis, the size of which was significantly smaller than the wild-type ovary. Histological changes occurring in the oocytes of T-treated ovaries induced oocyte degeneration in the masculinizing ovaries leading to their final disappearance. In parallel, many germ cells emerged in the cortex of the ovotestis and, later, in the medulla as well. RT-PCR analysis revealed upregulated expression of CYP17 and Dmrt1 but not 17βHSD in the ovotestis, and downregulation of Pat1a expression. Furthermore, immunohistology revealed CYP17-positive signals in the cortex of the masculinizing ovary, spreading throughout the whole area as the testis developed. These results indicate that oocytes are sensitive to T in the ovary of R. rugosa and that male-type germ cells expand in the masculinizing gonad (testis) contemporaneous with oocyte disappearance.

A Case of Scavenging Behavior by the Japanese Rice Frog,Fejervarya kawamurai(Amphibia: Anura: Dicroglossidae)

Abstract: Most of metamorphosed terrestrial frogs feed only on moving organisms and rarely exhibit scavenging behavior in nature. We here report a case of scavenging by the Japanese Rice Frog, Fejervarya kawamurai, which fed on a dead Black-spotted Frog, Pelophylax nigromaculatus at the edge of paddy field in central Japan. This observation is the first case of Fejervarya, feeding on a dead animal in nature and is an important addition to the cases of this unique behavior, which has been reported only for limited number of anuran species so far. In contrast, the behavior is more often reported for frogs in captivity, and F. kawamurai may be a useful species for examining scavenging behavior in post metamorphic anurans.

Expression, Purification and Properties of Redox-Sensitive Eye Lens Zeta-Crystallin of Arabian Camel.

The high protein concentration, unique composition and complex geometry of the lens makes it transparent. α-, β-, and γ-crystallins are present in all the lenses. In addition, taxon-specific crystallins are present in lenses in bulk quantity. Zeta (ζ)-crystallin is an NADPH-dependent quinone oxidoreductase, which constitutes nearly 10 % of the total eye lens protein in the evolutionary divergent animals (Camel, guinea pig and Japanese frog eye lenses) living in different ecological conditions. ζ -Crystallin is also present in human and other animal lenses but at catalytic amount. The physiological role of γ-crystallin in the eye lens is not well understood, however, truncated ζ-crystallin causes congenital cataract in guinea pig. In earlier study, redox regulated reversible activity of ζ-crystallin was reported. In this study, recombinant camel ζ-crystallin was overexpressed in E.coli and purified to homogeneity. Effect of different concentrations of reducing agent, dithiothretol (DTT) on the quinone oxidoreductase activity of recombinant ζ-crystallin was studied by enzymatic assay. To evaluate the effect of the reducing agent on the ζ-crystallin conformation, we have used far-UV and near-UV CD, intrinsic fluorescence, ANS binding assay and size exclusion chromatography. Our results showed that nearly 50% of the of ζ-crystallin activity was lost at 50 µM DTT. However, no detectable changes in secondary structure were observed. No changes in the tertiary structure and surface hydrophobicity of ζ-crystallin were detected; however, marginal changes were seen at saturating concentration of DTT (1 mM).

Evolutionary Changes in Sensitivity to Hormonally Induced Gonadal Sex Reversal in a Frog Species

The Japanese frog Glandirana rugosa is unique in that it shows geographic variation in sex chromosome differentiation and heterogametic sex determination. To elucidate the cause of interpopulation differences in gonadal sex differentiation, we investigated hormonally induced sex reversal and the expression patterns of genes associated with sex determination during early tadpole development. We found that sex reversal was easily induced in XX females and XY males of 2 forms (West-Japan and East-Japan) of G. rugosa with the ancestral homomorphic sex chromosomes under male heterogametic sex determination. During sex reversal, expression of CYP19 and/or FOXL2 was dependent on the phenotypic sex of the gonad. In contrast, sex reversal was not induced in ZW females of a population with a heteromorphic ZW sex chromosome system or in XX females or XY males in a population with a heteromorphic XY sex chromosome system. The latter 2 populations are evolutionarily derived forms. These results indicate an evolutionary direction for the gonadal sex differentiation mechanism. The original system was highly sensitive to sex hormones and allowed almost complete sex reversal. From this ancestral form, a new system evolved that was resistant to hormones and showed a change in the heterogametic sex and the sex chromosome differentiation mechanism.

Involvement of androgen receptor in sex determination in an amphibian species.

In mice and humans, the androgen receptor (AR) gene, located on the X chromosome, is not known to be involved in sex determination. In the Japanese frog Rana rugosa the AR is located on the sex chromosomes (X, Y, Z and W). Phylogenetic analysis shows that the AR on the X chromosome (X-AR) of the Korean R. rugosa is basal and segregates into two clusters: one containing W-AR of Japanese R. rugosa, the other containing Y-AR. AR expression is twice as high in ZZ (male) compared to ZW (female) embryos in which the W-AR is barely expressed. Higher AR-expression may be associated with male sex determination in this species. To examine whether the Z-AR is involved in sex determination in R. rugosa, we produced transgenic (Tg) frogs carrying an exogenous Z-AR. Analysis of ZW Tg frogs revealed development of masculinized gonads or ‘ovotestes’. Expression of CYP17 and Dmrt1, genes known to be activated during normal male gonadal development, were up-regulated in the ZW ovotestis. Testosterone, supplied to the rearing water, completed the female-to-male sex-reversal in the AR-Tg ZW frogs. Here we report that Z-AR is involved in male sex-determination in an amphibian species.

Highly Complex Mitochondrial Dna Genealogy in an Endemic Japanese Subterranean Breeding Brown FrogRana Tagoi(Amphibia, Anura, Ranidae)

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.

Is a Sex-Determining Gene(s) Necessary for Sex-Determination in Amphibians? Steroid Hormones May Be the Key Factor

Amphibians have 2 genetic sex-determining systems, one with male (XX/XY) and one with female (ZZ/ZW) heterogamety. While the ancestral state of sex-determination is thought to be female heterogamety, male and female heterogametic types were probably once interchangeable. The Japanese frog Rana rugosa has both XX/XY and ZZ/ZW systems within a single species in certain local populations. However, steroid hormones can alter the phenotypic sex epigenetically. In R. rugosa, steroidogenic enzyme expression starts before sex-determination in the indifferent gonad, and these enzymes become active in both male and female tadpoles. Androgens are produced in the indifferent gonad of male tadpoles at high levels, whereas estrogens are synthesized in females. In this regard, the observed enhanced expression of the hormone-metabolizing genes, CYP19 in the female gonad and CYP17 in males, may be crucial for sex-determination. Moreover, with FSH known to increase estrogen synthesis in the vertebrate ovary, observed upregulation of FSH receptor (FSHR) expression in the indifferent gonad of female tadpoles is intriguing. These data suggest that steroid hormones could be crucial for sex-determination in R. rugosa, with the consequence that upregulation of CYP19 and FSHR expression is necessary for female and CYP17 for male sex-determination.

The nomenclature of the recent Pentastomida (Crustacea), with a list of species and available names.

The taxonomy of the Recent members of the crustacean subclass Pentastomida is based on nine accepted family names derived from 12 available names, 24 generic names derived from 37 available names (plus two incorrect subsequent spellings and one nomen nudum) and 124 accepted species names derived from 183 available names of which six remain incertae sedis as to their generic assignment. Compilation of this list has revealed that existing catalogues have included misspellings, wrong attributions and dates of the authors of taxa, and incorrectly nominated type-species. These are corrected here with reference to the original descriptions and diagnoses. Notably, all families except one were erected much earlier and by authors other than Fain (1961), who was credited by Martin & Davis (2001) and other authors before and afterwards with seven of the nine families they recognised. Other significant taxonomic anomalies are revealed. Raillietiellidae Sambon, 1922 is a senior synonym of Cephalobaenidae Heymons, 1922, the name in popular usage for the family including Cephalobaena Heymons, 1922 and Raillietiella Sambon, in Vaney & Sambon, 1910; here the two genera are placed in separate families following Almeida & Christoffersen (1999). Heymonsia Hett, 1934, considered a junior synonym of Raillietiella, is a nomen nudum. Raillietiella geckonis (Diesing, 1850) is a potential senior synonym of several SE Asian species of this genus. Raillietiella frenata Ali, Riley & Self, 1981 is a widely used species name but is a subjective junior synonym of R. hebitihamata Self & Kuntz, 1960 according to its own authors. Morphological and molecular evidence suggest that R. indica Gedoelst, 1921 is a subjective senior synonym of both species. The priority of Linguatulidae Haldeman, 1851 over Linguatulida Vogt, 1851, erected as a family in the same year, is established by applying the First Reviser rule. Linguatula serrata Frölich, 1789 is herein selected as the type-species of Prionoderma, making it an objective synonym of Linguatula Frölich, 1789. The priority of L. serrata over Taenia rhinaris Meyer, 1789 and T.capraea Abildgaard, 1789, all published in the same year, is established by applying the First Reviser rule. The purported synonymy of Netrorhynchus Zenker, 1827, also misspelled Nettorhynchus, with Armillifer Sambon, 1922 would seem to be ill-founded and without popular support. Armillifer australis Heymons, 1935, published as a subspecies of A. moniliformis (Diesing, 1836), is both a senior synonym and a homonym of A. australis Riley & Self, 1981. Humboldt (1812) is confirmed as the author of Porocephalus Humboldt, 1812 and P. crotali Humboldt, 1812. Pentastomidae Shipley, 1909 is an older family name than its subjective synonym Porocephalinae Sambon, 1922, but prevailing usage allows the latter to be retained as a family name. Cayerina mirabilis Kishida, 1927 is a genus and species from a Japanese frog that has not appeared in the more recent pentastome literature. Sebekia minor (Wedl, 1861) is an objective senior synonym of the more widely used S. wedli Gigioli, in Sambon, 1922. The importance of the many junior synonyms will become evident should refined morphological and molecular evidence reveal cryptic species or greater host-specificity than presently recognised.

Complete mitochondrial DNA sequence of the endangered frog Odorrana ishikawae (family Ranidae) and unexpected diversity of mt gene arrangements in ranids

We determined the complete nucleotide sequence of the mitochondrial (mt) genome of an endangered Japanese frog, Odorrana ishikawae (family Ranidae). We also sequenced partial mt genomes of three other Odorrana and six ranid species to survey the diversity of genomic organizations and elucidate the phylogenetic problems remaining in this frog family. The O. ishikawae mt genome contained the 37 mt genes and single control region (CR) typically found in vertebrate mtDNAs, but the region of Light-strand replication origin (OL) was triplicated in this species. Four protein-encoding genes ( atp6, nd2, nd3, and nd5) were found to have high sequence divergence and to be usable for population genetics studies on this endangered species. Among the surveyed ranids, only two species ( Rana and Lithobates) manifested the typical neobatrachian-type mt gene arrangement. In contrast, relatively large gene rearrangements were found in Amolops, Babina, and Staurois species; and translocations of single tRNA genes ( trns) were observed in Glandirana and Odorrana species. Though the inter-generic and interspecific relationships of ranid taxa remain to be elucidated based on 12S and 16S rrn sequence data, some of the derived mt gene orders were found to have synapomorphic features useful for solving problematic ranid phylogenies. The tandem duplication and random loss (TDRL) model, the traditional model for mt gene rearrangement, failed to easily explain several of the mt gene rearrangements observed here. Indeed, the recent recombination-based gene rearrangement models seemed to be more suitable for this purpose. The high frequency of gene translocations involving a specific trn block ( trnH-trnS1) and several single tRNA genes suggest that there may be a retrotranslocation in ranid mt genomes.

Identical Skin Toxins by Convergent Molecular Adaptation in Frogs

The Tree of Life is rife with adaptive convergences at all scales and biological levels of complexity. However, natural selection is not likely to result in the independent evolution of identical gene products. Here we report such a striking example of evolutionary convergence in the toxic skin secretions of two distantly related frog lineages. Caeruleins are important decapeptides in pharmacological and clinical research [1] and are commonly believed to represent a single evolutionary class of peptides [2-4]. Instead, our phylogenetic analyses combining transcriptome and genome data reveal that independently evolved precursor genes encode identical caeruleins in Xenopus and Litoria frogs. The former arose by duplication from the cholecystokinin (cck) gene, whereas the latter was derived from the gastrin gene. These hormone genes that are involved in many physiological processes diverged early in vertebrate evolution, after a segmental duplication during the Cambrian period. Besides implicating convergent mutations of the peptide-encoding sequence, recurrent caerulein origins entail parallel shifts of expression from the gut-brain axis to skin secretory glands. These results highlight extreme structural convergence in anciently diverged genes as an evolutionary mechanism through which recurrent adaptation is attained across large phylogenetic distances.

Amolopkinins W1 and W2—Novel bradykinin-related peptides (BRPs) from the skin of the Chinese torrent frog, Amolops wuyiensis: Antagonists of bradykinin-induced smooth muscle contraction of the rat ileum

Bradykinin-related peptides (BRPs) represent one of the most widespread and closely studied families of amphibian defensive skin secretion peptides. Apart from canonical bradykinin (RPPGFSPFR) that was first reported in skin extracts of the European brown frog, Rana temporaria, many additional site-substituted, N- and/or C-terminally extended peptides have been isolated from skin extracts and secretions from representative species of the families Ranidae, Hylidae, Bombinatoridae and Leiopelmatidae. The most diverse range of BRPs has been found in ranid frog skin secretions and this probably reflects the diversity and number of species studied and their associated life histories within this taxon. Amolops (torrent or cascade frogs) is a genus within the Ranidae that has been poorly studied. Here we report the presence of two novel BRPs in the skin secretions of the Chinese Wuyi Mountain torrent frog ( Amolops wuyiensis). Amolopkinins W1 and W2 are dodecapeptides differing in only one amino acid residue at position 2 (Val/Ala) that are essentially (Leu 1, Thr 6)-bradykinins extended at the N-terminus by either RVAL (W1) or RAAL (W2). Amolopkinins W1 and W2 are structurally similar to amolopkinin L1 from Amolops loloensis and the major BRP (Leu 1, Thr 6, Trp 8)-bradykinin from the skin of the Japanese frog, Rana sakuraii. A. wuyiensis amolopkinins were separately encoded as single copies within discrete precursors of 61 amino acid residues as deduced from cloned skin cDNA. Synthetic replicates of both peptides were found to potently antagonize the contractile effects of canonical bradykinin on isolated rat ileum smooth muscle preparations. Amolopkinins thus appear to represent a novel sub-family of ranid frog skin secretion BRPs.

A New Species of Proteocephalus (Cestoda: Proteocephalidae), Description of the Male of Desmogathiema papuensis (Nematoda: Quimperiidae), and Other Endoparasites in Sylvirana supragrisea (Anura: Ranidae) from Papua New Guinea

Sixty-two of 66 (94%) Sylvirana supragrisea collected from 4 localities in Papua New Guinea harbored 1,137 endoparasies representing 15 known species and 1 new species. Proteocephalus papuensis n. sp. and the male of Desmognathiema papuensis Moravec and Sey, 1990, from the small intestine of the ranid frog Sylvirana supragrisea are described and illustrated. Proteocephalus papuensis is the second proteocephalid cestode to be described from New Guinea; it differs from P. niuginii in that it possesses a post-equatorial cirrus sac and 72–84 uterine pouches compared to an equatorial cirrus sac and 28–36 uterine pouches in P. niuginii. Desmognathiema papuensis is the third species assigned to the genus. It parasitizes ranid frogs of New Guinea, D. toyamai is known from an endemic Japanese frog, and D. nantahalaensis is a parasite of North American salamanders. In addition to these 2 species, gravid individuals of the nematodes Aplectana macintoshii, Cosmocerca novaaeguineae, Icosiella papuensis, Oswaldocruzia bakeri, Paracapillaria spratti, Pseudorictularia dipsarilis, Seuratascaris numidica and the acanthocephalan Acanthocephalus bufonis were found. Juvenile individuals of the nematode Abbreviata sp. (in cysts), unidentified cestode cysticerci, acanathocephalan cystacanths, and nymphs of a pentostome, Kirkicephalus sp., were also found. Sylvirana supragrisea represents a new host record for each of these parasites. Of the 1,137 endoparasite individuals, 638 (56%) were juvenile forms not capable of completing their life cycles in frogs. Of the 16 endoparasite species, no 2 collection sites had more than 7 species in common.