Odorrana Schmackeri Research Articles

An Effective Modification Strategy to Build Multifunctional Peptides Based on a Trypsin Inhibitory Peptide of the Kunitz Family.

Peptides with antimicrobial activity or protease inhibitory activity are potential candidates to supplement traditional antibiotics or cancer chemotherapies. However, the potential of many peptides are limited by drawbacks such as cytotoxicity or susceptibility to hydrolysis. Therefore, strategies to modify the structure of promising peptides may represent an effective approach for developing more promising clinical candidates. In this study, the mature peptide OSTI-1949, a Kunitz-type inhibitor from Odorrana schmackeri, and four designed analogues were successfully synthesised. In contrast to the parent peptide, the analogues showed impressive multi-functionality including antimicrobial, anticancer, and trypsin inhibitory activities. In terms of safety, there were no obvious changes observed in the haemolytic activity at the highest tested concentration, and the analogue OSTI-2461 showed an increase in activity against cancer cell lines without cytotoxicity to normal cells (HaCaT). In summary, through structural modification of a natural Kunitz-type peptide, the biological activity of analogues was improved whilst retaining low cytotoxicity. The strategy of helicity enhancement by forming an artificial α-helix and ß-sheet structure provides a promising way to develop original bioactive peptides for clinical therapeutics.

A new peptide, VD11, promotes structural and functional recovery after spinal cord injury.

The regenerative capacity of the central nervous system is very limited and few effective treatments are currently available for spinal cord injury. It is therefore a priority to develop new drugs that can promote structural and functional recovery after spinal cord injury. Previous studies have shown that peptides can promote substantial repair and regeneration of injured tissue. While amphibians have a pronounced ability to regenerate the spinal cord, few studies have investigated the effect of amphibian spinal cord-derived peptides on spinal cord injury. Here we report for the first time the successful identification and isolation of a new polypeptide, VD11 (amino acid sequence: VDELWPPWLPC), from the spinal cord of an endemic Chinese amphibian (Odorrana schmackeri). In vitro experiments showed that VD11 promoted the secretion of nerve growth factor and brain-derived neurotrophic factor in BV2 cells stimulated with lipopolysaccharide, as well as the proliferation and synaptic elongation of PC12 cells subjected to hypoxia. In vivo experiments showed that intravertebral injection of VD11 markedly promoted recovery of motor function in rats with spinal cord injury, alleviated pathological damage, and promoted axonal regeneration. Furthermore, RNA sequencing and western blotting showed that VD11 may affect spinal cord injury through activation of the AMPK and AKT signaling pathways. In summary, we discovered a novel amphibian-derived peptide that promotes structural and functional recovery after spinal cord injury.

Sex‐specific shifts in morphology and diet in a frog after 50 years of anthropogenic habitat fragmentation

AbstractAimPhenotypic shifts are commonly observed when animals face insular habitat change and may reflect ongoing stresses on individuals. However, the generality and the driving processes of this ‘island rule’ remain equivocal, notably in amphibians. Here, we investigate both morphological and dietary shifts in a frog using a mosaic of human‐created islands to assess the potential operating mechanisms underlying these phenotypic responses.LocationThousand Island Lake, China.TaxonThe Chinese piebald odorous frog,Odorrana schmackeri.MethodsWe compared body size between insular and mainland populations and between sexes. We examined the potential underlying mechanisms regarding body size shifts using structural equation modelling (SEM). Finally, we analysed changes in diet composition and compared intersexual diet overlap between islands and mainland sites.ResultsWe found insular dwarfism in female but not male frogs. Meanwhile, insular females also had smaller gape widths than mainland females after accounting for snout‐vent lengths (SVLs). According to SEMs, resource availability had a direct positive effect on body size. Finally, diet composition differed between the island and mainland populations but only in females. Males and females on islands exhibited greater overlaps in the diet.Main conclusionsIn contrast with most studies in amphibians, we found insular dwarfism rather than gigantism in females. The smaller gape width after accounting for SVL in insular females suggests potential changes in prey utilization or food availability on these human‐created islands. This notion is further supported by the differentiation of diet composition between island and mainland females. The higher diet overlap between sexes implies stronger intersexual competition for food resources after habitat fragmentation. Overall, we found rapid shifts in morphology and diet in frogs, which might result from habitat fragmentation in only 50 years and underscore the need to consider intersexual differences when assessing responses of species to anthropogenic disturbances.

Large mountains make small barriers: Species composition and spatial dynamics history of the Odorrana schmackeri complex in the karst area of Guizhou, China

AbstractAimSpecies delimitation is the basis for identifying diversity, and the genetic structure and demographic history of species can shed light on the effects of geological and historical climate change, thus allowing us to develop appropriate conservation and management strategies. In this study, we aimed to clarify the species composition and geographic distribution of the Odorrana schmackeri complex in the karst areas of Guizhou, explore the influence of geological and climatic events on its evolutionary and demographic history, predict the impact of future climate on the distribution area, and investigate the ecological niche differentiation of the complex.LocationKarst areas of Guizhou Province, China.MethodsUsing three mitochondrial (12S, 16S, and ND2) and four nuclear gene (BDNF, RAG1, RAG2, and Tyr) sequences as genetic markers, Bayesian inference (BI) was used to infer phylogenetic relationships and BEAST was used to assess divergence times. We assessed and estimated demographic histories using neutrality tests, mismatch distributions, and Bayesian skyline plots (BSPs). Using a species distribution prediction model, past and future suitable ranges were assessed. Niche equivalency and similarity of the three species of the complex were examined using the R package “ecospat.”ResultsThe O. schmackeri complex in Guizhou Province consists of three species: O. kweichowensis, O. huanggangensis, and O. hejiangensis. Demographic analyses indicated that population expansion of O. kweichowensis and O. huanggangensis occurred during the late Pleistocene, coinciding with the expansion of suitable habitat during the last ice age and the mid‐Holocene. Our projections suggest that potentially suitable habitat for all three species will contract under all future climate scenarios. The ecological niche similarity test showed significant climatic ecological niche differentiation between O. huanggangensis and O. kweichowensis, and between O. kweichowensis and O. hejiangensis.Main conclusionsThe Guizhou distribution of the O. schmackeri complex comprises three species, most of whose recent common ancestors diverged at the early Miocene (ca. 14.30 Ma) and split at the late Miocene (ca. 8–9 Ma). The Miaoling Mountains were not a geographical barrier to the dispersal of O. kweichowensis and O. huanggangensis, and the Dalou Mountains, Foding‐Wuling‐Leigong‐Yueliang Mountains were geographical distribution boundaries in the formation of the current distribution of the three species in Guizhou Province. No population expansion was detected within O. hejiangensis. Population expansion in the late Pleistocene for O. kweichowensis and O. huanggangensis was less affected by the glacial period, and was related to the presence of refugia. Considering future climate change, the focus should be on suitable habitats, mainly in nature reserves. In conclusion, this study highlights the importance of the use of genetic markers for the identification of species in the complex, the small geographical barrier of the Miaoling Mountains, and the conservation of amphibians in karst areas.

Novel amphibian-derived antioxidant peptide protects skin against ultraviolet irradiation damage.

Given the adverse impact of ultraviolet irradiation on human skin, as well as currently limited interventions, the discovery of new molecules with anti-photodamage potency remains critical. In this research, we obtained a new bioactive peptide (named OS-LL11, amino acid sequence 'LLPPWLCPRNK') from Odorrana schmackeri. Results showed that OS-LL11 could directly scavenge free radicals and sustain the viability of mouse keratinocytes challenged by ultraviolet B (UVB) irradiation or hydrogen peroxide (H2O2) by decreasing the levels of lipid peroxidation, malondialdehyde, and reactive oxygen species while increasing the level of catalase, Keap-1, HO-1, GCLM, and NQO1. Interestingly, topical application of OS-LL11 protected mouse skin against UVB irradiation damage by up-regulating the levels of superoxide dismutase, glutathione, and nitric oxide, but down-regulating the levels of H2O2, IL-1α, IL-1β, IL-6, TNF-α, 8-OHdG, Bcl-2, and Bax, as well as the number of apoptotic bodies. Our research demonstrated the anti-photodamage activity of a novel amphibian-derived peptide and the potential underlying mechanisms related to its free radical scavenging ability and antioxidant, anti-inflammatory, and anti-apoptotic activities. This study provides a new molecule for the development of anti-skin photodamage drugs or cosmetics and highlights the prospects of amphibian-derived peptides in photodamaged skin intervention.

Modification Strategy of D-leucine Residue Addition on a Novel Peptide from Odorrana schmackeri, with Enhanced Bioactivity and In Vivo Efficacy.

Brevinins are a well-characterised, frog-skin-derived, antimicrobial peptide (AMP) family, but their applications are limited by high cytotoxicity. In this study, a wild-type des-Leu2 brevinin peptide, named brevinin-1OS (B1OS), was identified from Odorrana schmackeri. To explore the significant role of the leucine residue at the second position, two variants, B1OS-L and B1OS-D-L, were designed by adding L-leucine and D-leucine residues at this site, respectively. The antibacterial and anticancer activities of B1OS-L and B1OS-D-L were around ten times stronger than the parent peptide. The activity of B1OS against the growth of Gram-positive bacteria was markedly enhanced after modification. Moreover, the leucine-modified products exerted in vivo therapeutic potential in an methicillin-resistant Staphylococcus aureus (MRSA)-infected waxworm model. Notably, the single substitution of D-leucine significantly increased the killing speed on lung cancer cells, where no viable H838 cells survived after 2 h of treatment with B1OS-D-L at 10 μM with low cytotoxicity on normal cells. Overall, our study suggested that the conserved leucine residue at the second position from the N-terminus is vital for optimising the dual antibacterial and anticancer activities of B1OS and proposed B1OS-D-L as an appealing therapeutic candidate for development.

A Hylarana latouchii Skin Secretion-Derived Novel Bombesin-Related Pentadecapeptide (Ranatensin-HLa) Evoke Myotropic Effects on the in vitro Rat Smooth Muscles.

Amphibians have developed successful defensive strategies for combating predators and invasive microorganisms encountered in their broad range of environments, which involve secretion of complex cocktails of noxious, toxic and diverse bioactive molecules from the skins. In recent years, amphibian skin secretions have been considered as an extraordinary warehouse for the discovery of therapeutic medicines. In this study, through bioactivity screening of the Hylarana latouchii skin secretion-derived fractions, a novel peptide belonging to ranatensin subfamily (ranatensin-HLa) was discovered, and structurally and pharmacologically-characterised. It consists of 15 amino acid residues, pGlu-NGDRAPQWAVGHFM-NH2, and its synthetic replicate was found to exhibit pharmacological activities on increasing the contraction of the in vitro rat bladder and uterus smooth muscles. Corresponding characteristic sigmoidal dose-response curves with EC50 values of 7.1 nM and 5.5 nM were produced, respectively, in bladder and uterus. Moreover, the precursor of ranatensin-HLa showed a high degree of similarity to those of bombesin-like peptides from Odorrana grahami and Odorrana schmackeri. Hylarana latouchii skin continues to serve as a storehouse with diverse lead compounds for the development of therapeutically effective medicines.

Pharmacological Effects of Two Novel Bombesin-Like Peptides from the Skin Secretions of Chinese Piebald Odorous Frog (Odorrana schmackeri) and European Edible Frog (Pelophylax kl. esculentus) on Smooth Muscle.

Bombesin-like peptides, which were identified from a diversity of amphibian skin secretions, have been demonstrated to possess several biological functions such as stimulation of smooth muscle contraction and regulation of food intake. Here, we report two novel bombesin-like peptides, bombesin-OS and bombesin-PE, which were isolated from Odorrana schmackeri and Pelophylax kl. esculentus, respectively. The mature peptides were identified and structurally confirmed by high performance Scliquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). Subsequently, the effects of these purified chemically-synthetic peptides on smooth muscle were determined in bladder, uterus, and ileum. The synthetic replications were revealed to have significant pharmacological effects on these tissues. The EC50 values of bombesin-OS for bladder, uterus and ileum, were 10.8 nM, 33.64 nM, and 12.29 nM, respectively. Furthermore, compared with bombesin-OS, bombesin-PE showed similar contractile activity on ileum smooth muscle and uterus smooth muscle, but had a higher potency on bladder smooth muscle. The EC50 value of bombesin-OS for bladder was around 1000-fold less than that of bombesin-PE. This suggests that bombesin-OS and bombesin-PE have unique binding properties to their receptors. The precursor of bombesin-OS was homologous with that of a bombesin-like peptide, odorranain-BLP-5, and bombesin-PE belongs to the ranatensin subfamily. We identified the structure of bombesin-OS and bombesin-PE, two homologues peptides whose actions may provide a further clue in the classification of ranid frogs, also in the provision of new drugs for human health.

A structural and functional analogue of a Bowman–Birk-type protease inhibitor from Odorrana schmackeri

Frog skin secretions contain complex peptidomes and peptidic protease inhibitors that are one of the biologically and structurally described groups of components. In the present study, by use of molecular ‘shotgun’ cloning and LC MS/MS fractionation sequencing, a novel Bowman–Birk-type heptadecapeptide (AALKGCWTKSIPPKPCF-amide), named Odorrana schmackeri Trypsin Inhibitor (OSTI), with a canonical Cys6–Cys16 disulfide bridge, was isolated and identified in piebald odorous frog (O. schmackeri) skin secretion. A synthetic replicate of OSTI-exhibited trypsin inhibitory activity with a Ki value of 0.3 ± 0.04 nM and also a tryptase inhibitory effect with a Ki of 2.5 ± 0.6 μM. This is the first time that this property has been reported for a peptide originating from amphibian sources. In addition, substituting lysine (K) with phenylalanine (F) at the presumed P1 position, completely abrogated the trypsin and tryptase inhibition, but produced a strong chymotrypsin inhibition with a Ki of 1.0 ± 0.1 μM. Thus, the specificity of this peptidic protease inhibitor could be optimized through modifying the amino acid residue at the presumed P1 position and this novel native OSTI, along with its analogue, [Phe9]-OSTI, have expanded the potential drug discovery and development pipeline directed towards alleviation of serine protease-mediated pathologies.

Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs

Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to “shotgun” clone the cDNAs encoding two novel but structurally-related peptides from the lyophilised skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin.

Screening Method for the Discovery of Potential Bioactive Cysteine-Containing Peptides Using 3D Mass Mapping.

Animal venoms and toxins are a valuable source of bioactive peptides with pharmacologic relevance as potential drug leads. A large subset of biologically active peptides discovered up till now contain disulfide bridges that enhance stability and activity. To discover new members of this class of peptides, we developed a workflow screening specifically for those peptides that contain inter- and intra-molecular disulfide bonds by means of three-dimensional (3D) mass mapping. Two intrinsic properties of the sulfur atom, (1) its relatively large negative mass defect, and (2) its isotopic composition, allow for differentiation between cysteine-containing peptides and peptides lacking sulfur. High sulfur content in a peptide decreases the normalized nominal mass defect (NMD) and increases the normalized isotopic shift (NIS). Hence in a 3D plot of mass, NIS, and NMD, peptides with sulfur appear in this plot with a distinct spatial localization compared with peptides that lack sulfur. In this study we investigated the skin secretion of two frog species; Odorrana schmackeri and Bombina variegata. Peptides from the crude skin secretions were separated by nanoflow LC, and of all eluting peptides high resolution zoom scans were acquired in order to accurately determine both monoisotopic mass and average mass. Both the NMD and the NIS were calculated from the experimental data using an in-house developed MATLAB script. Candidate peptides exhibiting a low NMD and high NIS values were selected for targeted de novo sequencing, and this resulted in the identification of several novel inter- and intra-molecular disulfide bond containing peptides.Graphical ᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s13361-015-1282-z) contains supplementary material, which is available to authorized users.

Vicariance and Its Impact on the Molecular Ecology of a Chinese Ranid Frog Species-Complex (Odorrana schmackeri, Ranidae).

Paleogeological events and Pleistocene climatic fluctuations have had profound influences on the genetic patterns and phylogeographic structure of species in southern China. In this study, we investigated the population genetic structure and Phylogeography of the Odorrana schmackeri species complex, mountain stream-dwelling odorous frogs, endemic to southern China. We obtained mitochondrial sequences (1,151bp) of the complete ND2 gene and two flanking tRNAs of 511 individuals from 25 sites for phylogeographic analyses. Phylogenetic reconstruction revealed seven divergent evolutionary lineages, with mean pairwise (K2P) sequence distances from 7.8% to 21.1%, except for a closer ND2 distance (3.4%). The complex geological history of southern China drove matrilineal divergence in the O. schmackeri species complex into highly structured geographical units. The first divergence between lineage A+B and other lineages (C-G) had likely been influenced by the uplift of coastal mountains of Southeast China during the Mio-Pliocene period. The subsequent divergences between the lineages C-G may have followed the formation of the Three Gorges and the intensification of the East Asian summer monsoon during the late Pliocene and early Pleistocene. Demographic analyses indicated that major lineages A and C have been experienced recent population expansion (c. 0.045–0.245 Ma) from multiple refugia prior to the Last Glacial Maximum (LGM). Molecular analysis suggest that these seven lineages may represent seven different species, three described species and four cryptic species and should at least be separated into seven management units corresponding to these seven geographic lineages for conservation.

De novo sequencing of two novel peptides homologous to calcitonin-like peptides, from skin secretion of the Chinese Frog, Odorrana schmackeri

An MS/MS based analytical strategy was followed to solve the complete sequence of two new peptides from frog (Odorrana schmackeri) skin secretion. This involved reduction and alkylation with two different alkylating agents followed by high resolution tandem mass spectrometry. De novo sequencing was achieved by complementary CID and ETD fragmentations of full-length peptides and of selected tryptic fragments. Heavy and light isotope dimethyl labeling assisted with annotation of sequence ion series. The identified primary structures are GCD[I/L]STCATHN[I/L]VNE[I/L]NKFDKSKPSSGGVGPESP-NH2 and SCNLSTCATHNLVNELNKFDKSKPSSGGVGPESF-NH2, i.e. two carboxyamidated 34 residue peptides with an aminoterminal intramolecular ring structure formed by a disulfide bridge between Cys2 and Cys7. Edman degradation analysis of the second peptide positively confirmed the exact sequence, resolving I/L discriminations. Both peptide sequences are novel and share homology with calcitonin, calcitonin gene related peptide (CGRP) and adrenomedullin from other vertebrates. Detailed sequence analysis as well as the 34 residue length of both O. schmackeri peptides, suggest they do not fully qualify as either calcitonins (32 residues) or CGRPs (37 amino acids) and may justify their classification in a novel peptide family within the calcitonin gene related peptide superfamily. Smooth muscle contractility assays with synthetic replicas of the S–S linked peptides on rat tail artery, uterus, bladder and ileum did not reveal myotropic activity.

Sexual Dimorphism in Mass of the Hindlimb Muscles of the Piebald Odorous Frog ( Odorrana schmackeri)

Male-biased sexual dimorphism in hind limb muscles is widespread in anuran species where scramble competition is common among males. Such sexual difference is thought to result from sexual selection. In this view, we tested the differences in muscle mass between the sexes and between amplectant and non-amplectant males by quantifying the mass of four hindlimb muscles(triceps femoris, sartorius, gracilis and plantaris longus) of females and males of Odorrana schmackeri. The results showed that females significantly exceeded males for muscle triceps femoris, gracilis, plantaris longus and total mass when controlled for body size. There are no significant differences between amplectant and non-amplectant males. It is probable that the maintenance of the amplectant position in O. schmackeri may depend on the strength of hindlimb muscles in females to support the pair.

A novel mitogenomic rearrangement for Odorrana schmackeri (Anura: Ranidae) and phylogeny of Ranidae inferred from thirteen mitochondrial protein-coding genes

We determined the complete nucleotide sequence of the mitochondrial genome of Odorrana schmackeri (family Ranidae). The O. schmackeri mitogenome (18 302 bp) contained 13 protein-coding genes, 2 rRNA genes, 21 tRNA genes and a single control region (CR). In the new mitogenome, the distinctive feature is the loss of tRNA-His, which could be explained by a hypothesis of gene substitution. The new sequence data was used to assess the phylogenetic relationships among 23 ranid species mostly from China using maximum likelihood (ML) and Bayesian inference (BI). The phylogenetic analyses support two families (Ranidae, Dicroglossidae) for Chinese ranids. In Ranidae, we support the genus Amolops should be retained in the subfamily Raninae rather than in a distinct subfamily Amolopinae of its own. Meanwhile, the monophyly of the genus Odorrana was supported. Within Dicroglossidae, four tribes were well supported including Occidozygini, Dicroglossini, Limnonectini and Paini. More mitochondrial genomes and nuclear genes are required to decisively evaluate phylogenetic relationships of ranids.

Vasorelaxin: A Novel Arterial Smooth Muscle-Relaxing Eicosapeptide from the Skin Secretion of the Chinese Piebald Odorous Frog (Odorrana schmackeri)

The defensive skin secretions of amphibians are a rich resource for the discovery of novel, bioactive peptides. Here we report the identification of a novel vascular smooth muscle-relaxing peptide, named vasorelaxin, from the skin secretion of the Chinese piebald odorous frog, Odorrana schmackeri. Vasorelaxin consists of 20 amino acid residues, SRVVKCSGFRPGSPDSREFC, with a disulfide-bridge between Cys-6 and Cys-20. The structure of its biosynthetic precursor was deduced from cloned skin cDNA and consists of 67 amino acid residues encoding a single copy of vasorelaxin (vasorelaxin, accession number: HE860494). Synthetic vasorelaxin caused a profound relaxation of rat arterial smooth muscle with an EC50 of 6.76 nM.

Sex-Biased Dispersal of a Frog (Odorrana schmackeri) Is Affected by Patch Isolation and Resource Limitation in a Fragmented Landscape

Sex-biased dispersal is widespread in the animal kingdom and is affected by numerous factors including mating system, social factors and environmental conditions. Unlike birds and mammals, there is no common trend in amphibians and explaining the direction and degree of sex-biased dispersal in species-specific cases is difficult. We conducted a study on dispersal of the Chinese piebald odorous frog (Odorrana schmackeri) in a fragmented landscape associated with dam construction. Ten microsatellite loci were used to analyze 382 samples sourced from 14 fragmented ‘islands’. Assignment tests indicated a significant pattern of female-biased dispersal on one island with inconsistencies in the strength and direction of this pattern between nearby islands. The effects of four island attributes and two potential impact factors on the pattern of sex-biased dispersal were examined. We found that the extent of isolation from the mainland and the number of breeding sites both showed a negative correlation with female biased dispersal, such that the closer an island is to the mainland the more likely it is to display female biased dispersal, and the more breeding sites on an island the more male immigrants. Based on these results, we conclude that geographic isolation and limited breeding resources are the most likely explanation for the patterns of dispersal observed in this fragmented population of amphibians.

Amphibians and Reptiles of Dinghushan in Guangdong Province, China’s Oldest Nature Reserve

Dinghushan is the only nature reserve at the Tropic of Cancer in mainland China with extensive old growth monsoonal hardwood forest. We here assemble a comprehensive list, with life history notes, of amphibians and reptiles of Dinghushan. Relevant records were scattered over three centuries since the first in 1886. A total of 71 species are known to occur in the reserve, and this number of species per area far exceeds that predicted by classic island biogeographic theory. Among these 71 species, nine are new records: Five frogs (Amolops ricketti, Hylarana macrodactyla, Hylarana taipehensis, Odorrana schmackeri, and Occidozyga lima) and four snakes (Oligodon cinereus, Sinonatrix percarinata, Sinomicrurus macclellandi, and Trimeresurus stejnegeri). One undetermined skink may confound the generic definitions for the genera of Scincella and Sphenomorphus. Among these 71 species, 21 that are not closely associated with old growth forest have not been seen recently. Forest recovery and reforestation of farmland over five decades since the nature reserve was established in 1956 may have contributed to the decline of these species. Dinghushan has 97% of its species occurring strictly in the Oriental zone; among them, about 78% (55) occur both in the South China and Central China regions, 21% (15) occur only in the South China region, and one species was previously known only from the Central China region. The unique location of Dinghushan makes long-term monitoring of its herpetofaunal diversity important for future collaborative studies on a global scale.

Isolation and sequencing of the HMG domain of ten Sox genes from Odorrana schmackeri (Amphibia: Anura)

Sox (SRY-related HMG-box) genes encode a family of transcriptional regulators, which are characterized by a conserved 79-amino acid domain known as HMG-box. They play essential roles in a diverse range of processes including sex determination and the development of the central nervous system (CNS), neural crest and endoderm. In this paper, the HMG domain of ten distinct Sox gene family members (os-Sox2, os-Sox3a, os-Sox3b, os-Sox4, os-Sox11a, os-Sox11b, os-Sox14a, os-Sox14b, os-Sox21a, os-Sox21b) were isolated from both male and female Odorrana schmackeri (Boettger, 1892) using PCR, and no sexual differences were found. Molecular phylogenetic analysis of the HMG domain suggested that these ten Sox genes are members of the SoxB and SoxC groups. In addition, sequence analysis suggested that four Sox genes (os-Sox3, os-Sox11, os-Sox14, os-Sox21) were duplicated. The duplication-degeneration-complementation model should be implied to explain the evolution and diversity of the Sox gene family in O. schmackeri.

Novel brevinins from Chinese piebald odorous frog (Huia schmackeri) skin deduced from cloned biosynthetic precursors

Antimicrobial peptides represent the most characterized and diverse class of peptides within the defensive skin secretions of anuran amphibians. With an ever expanding database of primary structures, the current accepted rules for nomenclature have become increasingly difficult to apply to peptides whose primary structural attributes are either unique or that fall between those that define existing groups. An additional factor that adds to the confusion is the regular re-classification or revision of existing taxa. In the present study, we have identified five new antimicrobial peptide homologs in the defensive skin secretion of the Chinese piebald odorous frog, Huia schmackeri (formerly Rana (Odorrana) schmackeri), by cloning of their respective biosynthetic precursors. As these peptides are obvious homologs of the brevinin-1 and brevinin-2 families we have named these in accordance: (1) brevinin-1HS1, (2) brevinin-2HS1, (3) brevinin-2HS2, (4) brevinin-2HS3 and (5) brevinin-1HS2. The reasons for adopting these names are discussed. It is clear that with an ever-increasing number of amphibian skin antimicrobial peptides appearing in the literature that a consistent nomenclature scheme needs to be established.