Odorrana Research Articles

A new odorous frog species of Odorrana (Amphibia, Anura, Ranidae) from Guizhou Province, China.

The frog genus Odorrana is distributed across east and southeastern Asia. Based on morphological differences and molecular phylogenetics, a new species of the genus occurring from Leigong Mountain in Guizhou Province, China is described. Phylogenetic analyses based on DNA sequences of the mitochondrial 12S rRNA, 16S rRNA, and ND2 genes supported the new species as an independent lineage. The uncorrected genetic distances between the 12S rRNA, 16S rRNA, and ND2 genes between the new species and its closest congener were 5.0%, 4.9%, and 16.3%, respectively. The new species is distinguished from its congeners by a combination of the following characters: body size moderate (SVL 39.1-49.4 mm in males, 49.7 mm in female); head width larger than head length; tympanum distinctly visible; small rounded granules scattered all over dorsal body and limbs; dorsolateral folds absent; heels overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level between eye to nostril when leg stretched forward; vocal sacs absent in male and nuptial pads present on the base of finger I.

A new species of Odorrana Fei, Ye & Huang, 1990 (Amphibia, Anura, Ranidae) from central Guangxi, China with a discussion of the taxonomy of Odorrana (Bamburana).

A new species of odorous frog, Odorranadamingshanensissp. nov., was found at the Damingshan National Nature Reserve in Guangxi, China. This species can be distinguished from its congeners by a combination of the following characters: medium body size (SVL 52.3-54.8 mm in males and 74.8-81.2 mm in females), sawtooth spinules on the upper lip, obtusely rounded snout that extends beyond the lower margin, distinct dorsolateral folds, horny tubercles on the rear of the back, presence of outer metatarsal tubercles, dilated nuptial pad with velvety spinules, distinct maxillary gland with tiny spines, and external lateral vocal sac. Through analysis of the 16S mitochondria gene, the new species is closely related to O.nasica and O.yentuensis, but the genetic divergence between the new species and the latter exceeds 7% (uncorrected p-distance). Currently, the new species is only known from its original discovery site. Furthermore, a discussion on the taxonomy of Odorrana (Bamburana) was conducted, identifying seven species within the subgenus Odorrana (Bamburana).

First Evidence of Anti-Steatotic Action of Macrotympanain A1, an Amphibian Skin Peptide from Odorrana macrotympana.

Many different amphibian skin peptides have been characterized and proven to exert various biological actions, such as wound-healing, immunomodulatory, anti-oxidant, anti-inflammatory and anti-diabetic effects. In this work, the possible anti-steatotic effect of macrotympanain A1 (MA1) (FLPGLECVW), a skin peptide isolated from the Chinese odorous frog Odorrana macrotympana, was investigated. We used a well-established in vitro model of hepatic steatosis, consisting of lipid-loaded rat hepatoma FaO cells. In this model, a 24 h treatment with 10 µg/mL MA1 exerted a significant anti-steatotic action, being able to reduce intracellular triglyceride content. Accordingly, the number and diameter of cytosolic lipid droplets (LDs) were reduced by peptide treatment. The expression of key genes of hepatic lipid metabolism, such as PPARs and PLINs, was measured by real-time qPCR. MA1 counteracted the fatty acid-induced upregulation of PPARγ expression and increased PLIN3 expression, suggesting a role in promoting lipophagy. The present data demonstrate for the first time a direct anti-steatotic effect of a peptide from amphibian skin secretion and pave the way to further studies on the use of amphibian peptides for beneficial actions against metabolic diseases.

Discovery of a new limestone karst-restricted odorous frog from northern Guangdong, China (Anura, Ranidae, Odorrana).

Karstic landscapes play an important role in biodiversity formation and often contain high levels of endemism. However, site-endemic taxa in karstic landscapes are being threatened by exploitation and weak legal protection. In this study, we describe Odorranaconcelata Wang, Zeng, & Lin, sp. nov., a limestone karst-restricted odorous frog from northern Guangdong, China. This new species shows distinctive genetic divergence and morphological differences from its congeners. Phylogenetic results suggest that the new species represents an independent lineage that is grouped with O.lipuensis and O.liboensis based on the mitochondrial 16S and 12S ribosomal RNA genes. We recommend the new species be listed as Vulnerable (VU) in the IUCN categorization as it is only known from the type locality with limited microhabitats and is threatened by habitat degradation.

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.

Male antiphonal calls and phonotaxis evoked by female courtship calls in the large odorous frog (Odorrana graminea).

Acoustic communication plays a vital role in frog reproduction. In most anuran species, long-distance sound communication is one-way from males to females; during the reproductive season, males produce species-specific advertisement calls to attract gravid females, and females are generally silent but perform phonotactic movements that lead to amplexus. One exception is the concave-eared torrent frog (Odorrana tormota). In this species, females produce courtship calls that elicit antiphonal vocalizations by males, followed by precise phonotactic movements. The large odorous frog O. graminea (previously Odorrana livida) in southern China is subject to the same environmental constraints as O. tormota, with which it is sympatric; it is unclear whether their sound communication is one-way or bidirectional. Here, we provide the first data on female O. graminea vocalizations and their functions. Using playbacks of female calls, we conducted acoustic behavioral experiments in the laboratory in response to which males emitted single- or multi-note antiphonal calls with a varying fundamental frequency. Moreover, they were attracted to female call playbacks, exhibiting precise phonotaxis. The female courtship call-male response interaction thus forms a duet between partners of a receptive pair. These results demonstrate that this unique communication system likely reflects an adaptation to an environment in which short-distance communication is at a premium given the high levels of ambient noise.

OL-FS13 alleviates experimental cerebral ischemia-reperfusion injury

Cerebral ischemia-reperfusion (I/R) is the main cause of neurological injury after stroke. However, existing treatments for I/R injury are relatively poor, and relevant drugs need to be further explored. Amphibians have received increasing attention as a resource bank of bioactive peptides. However, reports on neuroprotective peptides from amphibians remain extremely rare. Here, we identified a new neuroprotective peptide (OL-FS13, amino acid sequence: FSLLLTWWRRRVC) from the odorous frog species Odorrana livida using a constructed cDNA library. OL-FS13 significantly improving infarct volume, behavioral and histological abnormalities in rats, and also showed neuroprotective activities in PC12 cell (by oxygen glucose deprivation/reoxygenation, OGD/R). Mechanistically, OL-FS13 increased the level of antioxidative enzymes to resist oxidative stress and alleviated endoplasmic reticulum (ER) stress induced by I/R and OGD/R. The use of ML385 (Nrf2 inhibitor) indicated that OL-FS13 relieved nerve damage caused by oxidative and ER stress by increasing the nuclear displacement of Nrf2. Collectively, this research provides a novel drug candidate for the clinical cerebral I/R curation.

A new peptide originated from amphibian skin alleviates the ultravioletB-induced skinphotodamage.

Although amphibian-derived bioactive peptides have attracted increasing attention for their potential use in the treatment of photodamage, research is still in its infancy. In this study, we obtained a new antioxidant peptide, named OA-GI13 (GIWAPWPPRAGLC), from the skin of the odorous frog Odorrana andersonii and determined its effects on ultraviolet B (UVB)-induced skin photodamage as well as its possible molecular mechanisms. Results showed that OA-GI13 directly scavenged free radicals, maintained the viability of hydrogen peroxide-challenged keratinocytes, promoted the release of superoxide dismutase, catalase, and glutathione, and reduced the level of lactate dehydrogenase. Furthermore, topical application of OA-GI13 in mice alleviated dorsal skin erythema and edema and protected the skin against UVB irradiation by increasing antioxidant levels and decreasing peroxide, malondialdehyde, and 8-hydroxydeoxyguanosine levels. OA-GI13 also alleviated oxidative stress injury in vivo and in vitro, possibly by inhibiting p38 protein phosphorylation. Our study confirmed the anti-photodamage effects of this novel amphibian-derived peptide, thus providing a new molecule for the development of drugs and topical agents for the treatment of skin photodamage.

Female large odorous frogs (Odorrana graminea) prefer males with higher nonlinear vocal components.

In anurans, the complexity of courtship calls may affect female mate choice. The current study suggests that nonlinear phenomena (NLP) components can contribute to increasing complexity in courtship calls and attracting female attention. The results of a recent study showed that calls of large odorous frog (Odorrana graminea) contained NLP components. However, whether the nonlinear components of courtship calls in O. graminea improve male attractiveness remains unknown. We hypothesized that female O. graminea would prefer males producing calls with a higher proportion of NLP components (P‐NLP‐C). To test this hypothesis, we recorded the advertisement calls of 28 males and confirmed that the P‐NLP‐C was significantly positively related to body size. We also measured the body size of natural amplectant males and non‐amplectant males in the field and found that amplectant males had larger body sizes than non‐amplectant males, and the results of two‐choice amplexus experiments similarly revealed a female preference for males with larger body sizes. Additionally, phonotaxis experiments also revealed that females preferred male calls with a high P‐NLP‐C. The results suggest that a higher P‐NLP‐C in calls can enhance male attractiveness, and the P‐NLP‐C may provide key information about male body conditions for female O. graminea. Our study provides a new insight for better understanding the role of NLP in anuran mate selection.

Novel Peptide from an Odorous Frog Provides Strategies for Intervention in Skin Wound Healing

Novel Peptide from an Odorous Frog Provides Strategies for Intervention in Skin Wound Healing

Amphibian-derived peptide homodimer promotes regeneration of skin wounds

Despite the increasing treatments in skin wound repair, existing therapeutic drugs cannot meet current needs. As such, skin wound repair remains a considerable clinical challenge, and thus the discovery of new pro-healing agents is crucial. Here, we identified the first naturally occurring peptide homodimer named as OA-GP11 dimer (OA-GP11d) from Odorrana andersonii (odorous frog) through the combinational methods of peptidomics and genomics. OA-GP11d was linked by the intramolecular disulfide formed by the 10th cysteine residues from the monomer of peptide with sequence of GPLSGINAECM, which effectively promoted the repair of full-thickness and burn wounds in mice. The underlying molecular mechanisms revealed that OA-GP11d not only accelerated the migration and cell-scratch healing of mouse keratinocytes, but also activated the mitogen-activated protein kinases (MAPKs) signaling pathway (phosphorylation of p38 and ERK subgroups) in immortalized human keratinocytes (HaCaT). Besides, OA-GP11d reduced the phosphorylation of nuclear factor-κB (NF-κB) and inhibitor of NF-κB (I-κB) induced by lipopolysaccharide stimulation in mouse macrophages, and inhibited the release of associated inflammatory factors tumor necrosis factor (TNF)-α and interleukin (IL)-6. OA-GP11d is the first identified naturally occurring peptide dimer with significant pro-healing potency. Our results highlight the importance of amphibians as a source of novel pro-healing agents and suggest OA-GP11d as a potential new pro-regenerative drug candidate.

The beneficial roles of poisonous skin secretions in survival strategies of the odorous frog Odorrana andersonii.

The evolution of predatory, anti-predatory, and defensive strategies regarding environmental adaptation in animals is of significant research interest. In particular, amphibians, who represent a transition between aquatic and terrestrial vertebrates, play an important role in animal evolution. The bioactive skin secretions of amphibians are of specific interest due to their involvement in the crucial physiological functions of amphibian skin. We previously isolated and identified several bioactive peptides, including those showing antioxidant, antimicrobial, and wound-healing properties, from the skin secretions of the odorous frog species Odorrana andersonii. Currently, however, the biological significance of skin secretions in O. andersonii survival remains unclear. Here, we studied the biological significance of skin glands and secretions in regard to environmental adaptations of O. andersonii. Our research found that O. andersonii may secrete and excrete bioactive secretions through many glands (peptides and proteins as the main components in glands) distributed in the skin. The skin secretions not only displayed toxicity but also showed antioxidant, antibacterial, and repair promoting activities, suggesting that they play a protective role in O. andersonii when facing environmental threats. These bioactive skin secretions appear to act as a chemical survival strategy in O. andersonii, allowing the species to gain advantages in survival behavior.

Isolation and reconnection: Demographic history and multiple contact zones of the green odorous frog (Odorrana margaretae) around the Sichuan Basin.

The green odorous frog (Odorrana margaretae) displays a circular distribution around the Sichuan Basin of western China and possesses multiple replicate hybrid zones between lineages with high levels of divergence. To gain an understanding of the speciation process, we obtained 1540 SNPs from 29 populations and 227 individuals using ddRAD sequencing. Population structure analysis revealed three groups within the species: the West, North & South, and East groups. Demographic inference showed that they were initially isolated at ~2million years ago, and subsequent post-glacial expansion produced the current circular distribution with four secondary contact zones. Hybridization in those zones involved lineages with various levels of divergence and produced greatly different patterns of introgression. Contact zones between the East and North & South groups (E-S and E-N) had contrast admixture levels but both showed a general lack of potential barrier loci. Meanwhile, the reconnection of the West and North & South groups produced two contact zones along the rim of the Basin. The S-W zone had extensive admixture while the N-W zone had limited admixture within a narrow geographic distance. Both showed substantial barrier effects, and a large number of potential barrier loci were shared. We also detected strong coupling among these loci. The N-W hybrid zone involved two highly-diverged lineages (FST = 0.704) and many loci have reached fixation around the hybrid zone. This study system offers a unique opportunity to understand the dynamics of introgression in contact zones and the architecture of reproductive isolation at different stages of speciation.

The complete mitochondrial genome of Odorrana grahami (Anura: Ranidae)

The mitochondrial genome of the Disckless-fingered Odorous Frog, Odorrana grahami (Anura: Ranidae), was sequenced using high-throughput sequencing technology. The genome length was 17864 bp, including 22 tRNA genes, 13 protein-coding genes, 2 rRNA genes and 1 control region (D-loop). The AT content of the mitochondrial genome was 55.9%. The composition of mitochondrial genome of O. grahami is similar to that of other species of the genus Odorrana. Phylogenetic analysis of the mitochondrial genomes of six congeners shows that O. grahami is sister to O. margaretae, but the analysis using 16S rRNA gene of additional congeners do not resolve their relationships.

Mitochondrial DNA phylogeography of the Guizhou odorous frog: limited population genetic structure and evidence for recent population size expansion

The Guizhou odorous frog Odorrana kweichowensis is endemic to Guizhou Province, China. In this study, a comparative analysis of the mitochondrial COI and ND2 gene sequences was performed to examine genetic diversity in 109 individuals from ten localities across the geographic range of the species. Haplotype diversity and nucleotide diversity were 0.576 and 0.00055, respectively. Phylogenetic analyses almost nested all haplotypes into one lineage. AMOVA indicated that total variation was mainly derived from variation within individual populations. Neutral tests indicated that a recent expansion occurred in the total population. Fst estimations indicated that genetic divergence was not correlated with geographic distance. Accordingly, the species probably experienced a recent population expansion, and there no obvious population genetic structure is apparent. The findings provide useful information for the conservation of this species.

A novel amphibian-derived peptide alleviated ultraviolet B-induced photodamage in mice

Although the application potential of amphibian skin-derived active peptides in alleviating ultraviolet B (UVB)-induced damage has attracted increasing attention, research remains in its infancy. In this study, a new peptide (OM-GL15, GLLSGHYGRASPVAC) was identified from the skin of the green odorous frog (Odorrana margaretae). Results showed that OM-GL15 scavenged free radicals (2,2′-diazo-bis-3-ethylbenzothiazoline-6-sulfonic acid and 1,1-diphenyl-2-trinitrophenylhydrazine) and reduced Fe3+ to Fe2+. Moreover, topical administration of OM-GL15 significantly alleviated UVB-induced skin photodamage in mice. Exploration of the underlying mechanisms further showed that OM-GL15 exerted antioxidant potency. Specifically, the peptide reduced the levels of lipid peroxidation and malondialdehyde and protected epidermal cells from UVB-induced apoptosis by inhibiting DNA damage via down-regulation of p53, caspase-3, caspase-9, and Bax and up-regulation of Bcl-2. Our results highlight the potential application of amphibian skin-derived peptides in protection against UVB-induced photodamage and provide a novel peptide candidate for the development of anti-photodamage agents.

Habitat Selection and Genetic Structure of the Endangered Frog Species Odorrana wuchuanensis (Anura: Ranidae)

Understanding the habitat selection and population genetic structure of an endangered species can play important roles in its protection. The Wuchuan odorous frog (Odorrana wuchuanensis) is endemic to the karst regions of southwest China. This frog is currently listed as "Critically Endangered" by the IUCN, but little is known about its habitat selection and population genetics. In this study, we conducted analyses of habitat selection with occurrence/absence sites and environmental data, and assessed the genetic structure between north and south populations in Guizhou provinces in China using three mitochondrial markers. The results revealed that the probability of this frog occupying cave habitats increased with higher average humidity in July and higher lowest temperature in January, but was negatively related to precipitation in January. Analyses of F statistics combined with analyses of median-joining haplotype networks and the phylogenetic tree showed low genetic differentiation between the two populations of O. wuchuanensis. Considering the small population size and geographic isolation because of the complex karst terrains, we suggest careful management practices are needed to protect this species.

A Novel Kunitzin-Like Trypsin Inhibitor Isolated from Defensive Skin Secretion of Odorrana versabilis.

Protease inhibitors that were identified from amphibian skin secretions with low molecular weights and potent inhibitory activity were thought to be potential candidates for novel peptide drugs. Here, a novel peptide with trypsin inhibitory activity was found in the skin secretion of the Chinese bamboo leaf odorous frog, Odorrana versabilis. Based on the sequence alignments of sequencing results, the novel peptide (ALKYPFRCKAAFC) was named as Kunitzin-OV. The synthetic replicate of Kunitzin-OV was subjected to a series of functional assays, and it exhibited a trypsin inhibitory activity with a Ki value of 3.042 µM, whereas, when Lys-9 at P1 position was substituted by Phe, trypsin inhibitory activity was undetected and the chymotrypsin inhibitory activity was optimized with a Ki value of 2.874 µM. However, its protease-binding loop was catabolized by trypsin during the trypsin cleavage test. In conclusion, Kunizin-OV is a novel peptide with trypsin inhibitory activity as a member of kunitzins, which is a non-typical Kunitz-like trypsin inhibitor with a highly conserved reactive site (K-A) and quite a short sequence.

Antioxidant Peptide AOP-P1 Derived from Odorous Frog Showed Protective Effects Against UVB-Induced Skin Damages

The protection of skin from photo-damage induced by ultraviolet B (UVB) irradiation has been a research hotspot in recent years and gene-encoded antioxidant peptides from amphibians have aroused great attention. In our previous research, a short peptide AOP-P1 identified from amphibian exerted direct scavenging capacity against free radicals, which implied its possible application in protecting skin against photo-injury. In this research, our results revealed that, on both UVB-irradiative and hydrogen peroxide-induced oxidative stress models established with human immortalized keratinocyte, AOP-P1 could protect cell viability, promote the release of catalase, and decrease levels of lactate dehydrogenase & malondialdehyde. Moreover, AOP-P1 could promote the production of superoxide dismutase and glutathione, alleviate the thickness of epidermis & dermal and the production of light spot & collagen fibers on skin photo-injury animal model induced by UVB irradiation. In summary, our results implied the potential application of AOP-P1 in skin protection against photo-damage and highlighted the amphibian-derived antioxidant peptides as novel development of natural antioxidants for the treatment against photo-injury induced by ultraviolet irradiation.

Accelerated Wound Healing Induced by a Novel Amphibian Peptide (OA-FF10).

Despite the continued development of modern medicine, chronic wounds are still a critical issue in clinical treatment, placing a great physiological, psychological, and financial burden on patients. Researchers have investigated many methods to solve this problem, with bioactive peptides gaining increasing attention due to their considerable advantages and diverse functions, as well as low cost, simple storage, and easy transportation. In this research, a novel peptide (named OA-FF10) was identified from the skin secretions of the odorous frog species Odorrana andersonii. The sequence of mature OA-FF10 was "FFTTSCRSGC", which was produced by the post-translational processing of a 61-residue prepropeptide. Similar to most frog peptides, OA-FF10 showed an intramolecular disulfide bridge at the C-terminus. OA-FF10 demonstrated no antibacterial, antioxidant, hemolytic, or acute toxic activity, but promoted wound healing and proliferation of human keratinocytes (HaCaT) both time- and dose-dependently. Furthermore, while OA-FF10 had no effect on wound healing of Human Skin Fibroblasts (HSF), it did accelerate healing in a full-thickness skin-wound mouse model. Our research revealed the strong wound-healing activity of OA-FF10 in vivo and in vitro, thus providing a new candidate for the development of novel wound-healing drugs.