Rana Nigromaculata Research Articles

Physiological Response of Pelophylax nigromaculatus Adults to Salinity Exposure.

Simple SummaryThis study explored physiological resilience to and recovery from saline exposure in Pelophylax nigromaculatus, a semi-aquatic frog that is widely distributed in East Asia. Analysis of 11 serum components revealed the physiological response of frogs to either severe saline exposure for six days or moderate saline exposure for forty days, followed by a twenty day recovery period. During exposure to both severe and moderate saline conditions, serum electrolytes increased, protein concentrations in serum decreased, and creatinine, an indicator of renal function, sharply increased. However, renal tissue sampled after the study did not show renal dysfunction. Moreover, serum components that changed during exposure to salinity returned to their initial values during the recovery period. Adult anurans seem capable of resilience, to some extent, to saline conditions.Many freshwater ecosystems are becoming more saline, and amphibians, which have permeable skin, are sensitive to this change. We studied the physiological responses to high salinity and recovery from saline exposure in adult frogs (Pelophylax nigromaculatus). Frogs that experienced severe salinity were exposed to saline conditions for 6 days, while those in the moderate group were exposed to saline conditions for 40 days, followed by a recovery period in freshwater for 20 days. Our data showed that during exposure to saline conditions of severe and moderate groups, serum electrolytes increased, protein concentrations decreased, and creatinine, an indicator of renal function, sharply increased. However, renal tissue sampled after exposure did not show renal dysfunction. In addition, serum components that changed during exposure to salinity returned to their initial values during the recovery period. Thus, adult anurans can be resilient, to some extent, to saline conditions in habitats that experience either rapid or slow salinity changes.

The Influence of Atrazine on the Growth, Development and Oxygen Consumption of Pelophylax nigromaculatus Tadpoles

The Influence of Atrazine on the Growth, Development and Oxygen Consumption of Pelophylax nigromaculatus Tadpoles

Microbiomic and transcriptomic insight into the pathogenesis of meningitis-like disease in cultured Pelophylax nigromaculatus

In recent years, a deadly infectious disease named frog meningitis (or cataract and torticollis) raged the frog farms in China. Diseased frogs manifested torticollis, cataract, edema, and finally died. Yet, the molecular pathogenesis of this disease has not been studied. In this study, the etiologic factor of frog meningitis broken in a black-spotted frog (Pelophylax nigromaculatus) farm was investigated by comparative microbiomics (brain, eyeball, and intestine). And the pathogenesis of two major types of symptomatic frogs (individuals with both torticollis and cataract and individuals with only torticollis) were investigated by histopathological section and comparative transcriptomics of brain, liver, and muscle. Our results indicated that Elizabethkingia bacteria had increased proportions in the brain and eyeball microbiomes (but not intestine microbiomes) of both types of symptomatic frogs, and E. miricola was dominant in the eyeball and brain microbiomes of most torticollis-cataract frogs. In the torticollis-only individuals, vascular congestion and reduced hepatic fat can be detected in their leptomeninges vessels and liver, respectively. While in the torticollis-cataract ones, we observed additional leukocyte extravasation in their leptomeninges vessel, and neurologic damage in their brain and spiral cord. Transcriptional analyses suggested that frogs could be well divided into asymptomatic, torticollis-only, and torticollis-cataract groups by the variation of their brain transcriptomes. The upregulated brain genes in symptomatic frogs were mainly enriched in immune-related pathways. And the Toll-like receptor and NOD-like receptor signaling pathways, inflammation, and adaptive humoral immunity were activated in response to E. miricola infection. Torticollis-only and torticollis-cataract individuals differed in the level of immune gene expression remarkably, and the emerge of cataract symptom indicated the onset of strong innate immune and inflammatory responses. Overall, our results suggested that E. miricola was likely the etiologic factor of meningitis emerged in this farm. In regards to pathological stages, the infection progressed to leptomeninges invasion in torticollis-only individuals, while it had caused meningitis in torticollis-cataract ones. Correspondingly, the torticollis symptom was likely due to leptomeninges infection, and the cataract was likely a concomitant symptom of meningitis due to disruption of vascular permeability during excessive inflammation. Our results provided a first molecular insight into the pathogenesis of frog meningitis.

Intraspecific competition reduces the quantity of excreted nutrients in tadpoles

Anuran larvae can form the largest animal biomass seasonally in freshwater environments, yet, they are still one of the least-studied taxa in terms of nutrient regeneration. The present study tested whether sympatric tadpoles regenerate nutrients at similar rates and ratios, and whether increased intraspecific competition (hereafter ‘competition’) alters patterns of excretion. We quantified rates and ratios of excretion (dissolved nitrogen (N) from ammonia, phosphorus (P) from total dissolved P, and N:P ratio) in three pond-dwelling Japanese tadpoles (Pelophylax nigromaculatus, Rhacophorus schlegelii, Hyla japonica), and tested the effect of competition on excretion in Pelophylax nigromaculatus and Rhacophorus arboreus. The three co-occurring species regenerated nutrients at different rates and ratios; H. japonica excreted nutrients and produced faecal pellets at the lowest rates. Inside field enclosures, increasing tadpole density reduced the quantity but not the quality of excretion by the tadpoles, suggesting higher nutrient sequestration, likely to maintain a stoichiometrically balanced growth under limited resources. Differences in rates and ratios of excretion have previously been shown to have various effects on community structure by affecting primary productivity, highlighting the importance of species identity and interactions on ecosystem function.

Low prevalence of spargana infection in farmed frogs in the Yangtze River Delta of China

Frogs are the main source of infection for human sparganosis. In this study, the prevalence and pathogenicity of plerocercoid larvae (sparganum) in frogs collected from the Yangtze River Delta in East China were investigated. A total of 386 frogs belonging to five species were purchased from farmers' markets across all three provincial level areas in the Yangtze River Delta region. The overall prevalence was 4.9% (19/386), and 39 spargana were detected visually, with the intensity ranging from 1 to 11. The spargana infection rate was 7.7% (11/143) in Jiangsu Province and 4.4% (8/181) in Shanghai City, while no spargana infection was detected in Zhejiang Province. In five tested frog species, only Rana nigromaculata and R. limnocharis were found to harbor spargana infection, with a prevalence of 7.7% (13/168) and 6.3% (6/95), respectively. There was no significant difference among the months of the experimental period, July to September. The spargana mostly parasitized the muscle tissues of frogs, especially in the hind legs. All the spargana were identified by molecular analysis based on cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes, and all plerocercoids were Spirometra erinaceieuropaei. Nine mice were infected orally with 1 to 3 scoleces, and 77.8% (14/18) of plerocercoids were found in mice at the 30th day post infection. No obvious clinical symptoms were observed in the mice; however, histopathological analysis showed an inflammatory cellular response in all tissues except intestinal tissue. Hematologic analysis showed an increased number of white blood cells (WBCs) at the 18th day post infection. These results indicated that R. nigromaculata and R. limnocharis are a potential source of zoonotic sparganosis in the Yangtze River Delta of China, and farmed frogs may substantially reduce zoonotic risk as compared to eating wild frogs. Our findings will provide data for frog food safety and prevention and control of sparganosis in the region.

Design of engineered modifications to allow frogs to escape from irrigation channels

Amphibian populations are under threat worldwide, one such threat is frogs getting trapped in concrete-lined irrigation channels. Earlier studies have found that concrete ditches are acting as barriers to animal movement, fragmenting habitat, and trapping amphibians. China has over 3,000,000 kms of irrigation channels and they are a major threat to frog populations. In this study, we assessed the capacity of a common Chinese frog, Rana nigromaculata, to escape from a typical concrete-lined irrigation channel via ‘frog-ways’ cut into the walls of the channel. The twelve different designs varied gradients, substrates and widths of the frog-ways. The results showed that these simple and inexpensive retrofits of irrigation channels are very effective at allowing the great majority of frogs to escape. Male frogs were more likely to be trapped in concrete ditches, probably due to their smaller body size. The best design was N11 (55 degree slopes, a crushed stone surface, and 100 cm width) that can help the frogs to successfully escape in just 1 min. Taking water conveyance efficiency of irrigation water delivery systems into consideration, we recommend a longitudinal design (the frog-way is parallel to the water-flow direction) of simple concrete with slopes less than 55° and crushed stone. The findings can serve as a reference for technicians involved in future ecological engineering designs of irrigation channels throughout the world.

Comparative Morphology of the Lungs and Skin of two Anura, Pelophylax nigromaculatus and Bufo gargarizans

The lungs and skin are important respiratory organs in Anura, but the pulmonary structure of amphibians remains unclear due to the lack of a suitable procedure. This study improved the procedure used for fixing lungs tissues and used light microscopy, transmission electron microscopy and scanning electron microscopy to reveal the differences in the lung and skin morphologies between Pelophylax nigromaculatus (P. nigromaculatus) and Bufo gargarizans (B. gargarizans). In P. nigromaculatus and B. gargarizans, the cystic lungs comprise a continuous outer pulmonary wall on which primary, secondary, and tertiary septa attach, and a number of regular lattices form from raised capillaries and the pulmonary epithelium on the surfaces of the pulmonary wall and septa. Each lattice in P. nigromaculatus consists of several elliptical sheets and flat bottom, and the septa are distributed with denser sheets and have a larger stretching range than the pulmonary wall. The lattice in B. gargarizans consists of thick folds and an uneven bottom with several thin folds, and the septa have more developed thick and thin folds than the pulmonary wall. However, the density of the pulmonary microvilli, the area of a single capillary, the thicknesses of the blood-air barrier, pulmonary wall and septum, and the lung/body weight percentage obtained for B. gargarizans were higher than those found for P. nigromaculatus. In P. nigromaculatus, the dorsal skin has dense capillaries and a ring surface structure with mucus layer on the stratum corneum, and the ventral skin is slightly keratinized. In B. gargarizans, the stratum corneum in both the dorsal and ventral skins is completely keratinized. A fine ultrastructure analysis of P. nigromaculatus and B. gargarizans revealed that the pulmonary septa are more developed than the pulmonary walls, which means that the septa have a stronger respiratory function. The more developed lungs are helpful for the adaptation of B. gargarizans to drought environments, whereas P. nigromaculatus has to rely on more vigorous skin respiration to adapt to a humid environment.

Habitat partitioning of two closely related pond frogs, Pelophylax nigromaculatus and Pelophylax porosus brevipodus, during their breeding season

Parapatric distributions of closely related species are common in many taxonomic groups. However, habitat overlap in sympatric areas within both species’ ranges is complex and driven by the nature of the interactions between species as well as each species’ ecological requirements. We therefore investigated the distribution of breeding habitats for two congeneric frogs, Pelophylax nigromaculatus and Pelophylax porosus brevipodus, in a region of Japan where these species co-occur, based on observation of mating calls as an index of the abundance of males engaging in reproductive activities. Among a total of 381 surveyed sites, P. nigromaculatus and P. porosus brevipodus were found at 171 and 229 sites, respectively. Pelophylax porosus brevipodus was widely distributed throughout the study area and was more abundant than P. nigromaculatus in sympatric sites. Analysis using cumulative link mixed models revealed that the abundance of calling males was negatively affected by the presence of calling males of the other species and by landscape factors. Interspecific differences in this negative effect appear to favor P. porosus brevipodus over P. nigromaculatus in our study area. Due to differences in the primary habitat preferences of the two species during the non-breeding season, habitat partitioning during the breeding season is likely driven by negative interspecific interactions during the reproductive process (i.e., reproductive interference).

Ranacyclin-NF, a Novel Bowman-Birk Type Protease Inhibitor from the Skin Secretion of the East Asian Frog, Pelophylax nigromaculatus.

Serine protease inhibitors are found in plants, animals and microorganisms, where they play important roles in many physiological and pathological processes. Inhibitor scaffolds based on natural proteins and peptides have gradually become the focus of current research as they tend to bind to their targets with greater specificity than small molecules. In this report, a novel Bowman–Birk type inhibitor, named ranacyclin-NF (RNF), is described and was identified in the skin secretion of the East Asian frog, Pelophylax nigromaculatus. A synthetic replicate of the peptide was subjected to a series of functional assays. It displayed trypsin inhibitory activity with an inhibitory constant, Ki, of 447 nM and had negligible direct cytotoxicity. No observable direct antimicrobial activity was found but RNF improved the therapeutic potency of Gentamicin against Methicillin-resistant Staphylococcus aureus (MRSA). RNF shared significant sequence similarity to previously reported and related inhibitors from Odorrana grahami (ORB) and Rana esculenta (ranacyclin-T), both of which were found to be multi-functional. Two analogues of RNF, named ranacyclin-NF1 (RNF1) and ranacyclin-NF3L (RNF3L), were designed based on some features of ORB and ranacyclin-T to study structure–activity relationships. Structure–activity studies demonstrated that residues outside of the trypsin inhibitory loop (TIL) may be related to the efficacy of trypsin inhibitory activity.

A game of patience between predator and prey: waiting for opponent’s action determines successful capture or escape

When predator and prey animals face each other, preemptive actions by both sides are considered to mediate successful capture or escape. However, in spite of the general presumption, some animals, such as predatory snakes and their frog prey, occasionally remain motionless or move slowly for a while before striking or escaping, respectively. To clarify the possible advantages of this behaviour, we examined interactions between Japanese Four-lined Ratsnakes (Elaphe quadrivirgata (H. Boie, 1826)) and Black-spotted Pond Frogs (Pelophylax nigromaculatus (Hallowell, 1861)), focusing especially on kinematic features of strike behaviour of snakes and flight behaviour of frogs in close quarters. Staged encounter experiments and field observations revealed that counteractions against an opponent’s preemptive actions are effective for both snakes and frogs until a certain distance because they are hardly able to change their trajectories once they initiate strike or escape behaviours. Snakes and frogs also appropriately switched their behaviour from waiting for the opponent’s action to taking preemptive action at this threshold distance. These results suggested the occurrence of a game of patience between snakes and frogs in which they wait for the opponent’s action to achieve effective countermeasures. Our study provides new insights for predicting optimal decision-making by predators and prey and will contribute to a better understanding of their strategies.

A full evaluation of chiral phenylpyrazole pesticide flufiprole and the metabolites to non-target organism in paddy field

Pesticides applied to paddy fields may pose considerable danger to non-target aquatic organisms and further threaten human health. Flufiprole is a pesticide used in rice fields; considering the widespread existence of rice-fish-farming ecosystems, the acute toxicities of flufiprole enantiomers and its six metabolites (fipronil, flufiprole sulfide, flufiprole sulfone, detrifluoromethylsulfinyl flufiprole, desulfinyl flufiprole, and flufiprole amide) to four common aquatic organisms in rice fields including Misgurnus anguillicaudatus (pond loach), Carassius gibelio (Prussian carp), Pelophylax nigromaculatus (black-spotted frog), and Daphnia magna (water flea) were investigated. Genotoxicity, pathological changes and the effects on the antioxidant system of M. anguillicaudatus were also evaluated after exposure. The LC50 (EC50) values showed that fipronil and desulfinyl flufiprole were the most toxic compounds and were approximately about six times as toxic as flufiprole. No enantioselective toxicity was observed between the two enantiomers. The activity of antioxidant defense enzymes and the content of malondialdehyde (MDA) in the liver and gills of M. anguillicaudatus were significantly increased by the chemicals in most cases. In addition, fipronil and desulfinyl flufiprole were found to induce an increase in the micronucleus rate in M. anguillicaudatus. Histopathological analysis showed that the liver of M. anguillicaudatus was not significantly affected by flufiprole. Our study demonstrated a potential negative effect on flufiprole-treated aquatic organisms. As an alternative to fipronil, the environmental risk of flufiprole and its metabolites to non-target organisms in rice fields cannot be ignored.

Characterization and genomic analysis of a ranavirus associated with cultured black-spotted pond frogs (Rana nigromaculata) tadpoles mortalities in China.

Ranaviruses are globally emerging pathogens causing mass mortality in both farmed and wild populations. In this study, we confirmed that a novel ranavirus was related to mass die-offs of black-spotted pond frogs (Rana nigromaculata) in Sichuan Province, China. Histological examination revealed marked degeneration or necrosis in parenchymatous organs, and intracytoplasmic inclusions can be observed in the liver and intestine cells. The virus was isolated from diseased tadpoles (tentatively named the virus as RNRV), and the ranavirus-like particles could be observed in infected EPC cells by the electron microscope. The tadpoles challenged with the isolated virus displayed similar clinical symptoms and pathogenicity as those naturally infected. Physicochemical characteristics showed that it was sensitive to chloroform treatment, trypsin treatment, heating treatment, acidity and alkalinity. Biological characteristics showed that RNRV can induce cytopathic effect (CPE) in various fish cell lines and the optimum growth temperature is 25~28°C. In addition, the complete genome sequence of RNRV was determined (GenBank accession number MG791866) and analysed. The results showed that the genome of RNRV consists of 104,286bp containing 55.2% GC content and 104 predicted ORFs were identified in the genome. Phylogenetic analysis of the complete genome revealed that RNRV should belong to the FV3-like group in genus Ranavirus, and is more closely related to RGV and STIV. These studies confirmed that the RNRV was the causative agent of this natural epizootic event and genome analysis indicated that it belongs to the FV3-like group. In addition, viral physicochemical and biological characteristics will provide a scientific basis for prevention and control.

Large-scale survey of a neglected agent of sparganosis Spirometra erinaceieuropaei(Cestoda: Diphyllobothriidae) in wild frogs in China.

BackgroundIn China, frogs play an understudied role in the spread of human sparganosis (caused by the larval form of Spirometra). However, our knowledge about the prevalence of sparganum infection in frogs remains fragmented, and the taxonomic identification of the parasite is still controversial.Methodology/Principal findingsThe prevalence of sparganum infection in wild frogs was surveyed at 145 geographical locations from 28 of the 34 provinces/autonomous regions/municipalities in China for six years. The collected sparganum isolates from the different locations were subjected to molecular identification by a multiplex PCR assay and then were analysed with clustering analysis. In the survey, sparganum infection was found in 8 out of 13 of the collected frog species, and the most frequently infected species was Pelophylax nigromaculatus (the infection rate was up to 14.07%). Infected frogs were found in 80 of the 145 surveyed locations. The sparganum infection rates in the wild frogs in several regions of China were still high (above 10%), especially in South and Southwest China. A total of 72 spargana were selected for molecular identification, and the clustering analysis showed that sequences from the Chinese isolates were very similar to those identified as from Spirometra erinaceieuropaei. However, the taxonomy of the genus remains confused and further analysis is required.ConclusionsEating wild frogs is associated with considerable health risks in China. Several traditional Chinese folk remedies may increase the risk of infection. The sparganum isolates in China are most likely from S. erinaceieuropaei, but new studies, especially comprehensive morphological analyses, are needed in the future.

Diversity of Antimicrobial Peptides in Three Partially Sympatric Frog Species in Northeast Asia and Implications for Evolution.

Antimicrobial peptides (AMPs) are evolutionarily ancient molecules that play an essential role in innate immunity across taxa from invertebrates to vertebrates. The evolution system of AMP system has not been well explained in the literature. In this study, we cloned and sequenced AMP transcriptomes of three frog species, namely Rana dybowskii, Rana amurensis, and Pelophylax nigromaculatus, which are partially sympatric in northeast Asia, but show different habitat preferences. We found that each species contained 7 to 14 families of AMPs and the diversity was higher in species with a large geographic range and greater habitat variation. All AMPs are phylogenetically related but not associated with the speciation process. Most AMP genes were under negative selection. We propose that the diversification and addition of novel functions and improvement of antimicrobial efficiency are facilitated by the expansion of family members and numbers. We also documented significant negative correlation of net charges and numbers of amino acid residues between the propiece and mature peptide segments. This supports the Net Charge Balance Hypothesis. We propose the Cut Point Sliding Hypothesis as a novel diversification mechanism to explain the correlation in lengths of the two segments.

Predation of frog eggs by the water strider Gerris latiabdominis Miyamoto (Hemiptera: Gerridae)

AbstractWater striders (Hemiptera: Gerridae) are predators found on the water surface that prey mainly on arthropods. The feeding on other organisms (dead vertebrates, fishes and tadpoles) is a rare event. To our knowledge, predation of frog eggs by water striders has not yet been reported. We observed that adult water striders, Gerris latiabdominis Miyamoto, 1958 (Hemiptera: Gerridae), preyed on the eggs of three frog species, Pelophylax nigromaculata Hallowell, 1861 (Anura: Ranidae), Rana japonica Boulenger, 1879 (Anura: Ranidae), and Rhacophorus schlegelii Günther, 1858 (Anura: Rhacophoridae). We found predation by the water striders affects the survival rate of frog eggs floating on the water surface. We suggest that this hunting event would occur in water bodies in which water striders and frogs coexist, especially the region where their phenology overlaps.

黑斑侧褶蛙蝌蚪断尾后的补偿生长和发育研究

黑斑侧褶蛙蝌蚪断尾后的补偿生长和发育研究

Immunohistochemical distribution of glucagon-like peptide 1 in the digestive system of different aquatic vertebrates.

The distribution of glucagon-like peptide 1 (GLP-1)-positive cells in digestive tracts and pancreases of aquatic vertebrates was investigated by immunohistochemical staining method. The results suggested that GLP-1-positive cells were distributed in the columnar mucous epithelium and tubular glands of lamina propria in the digestive system. However, GLP-1-positive cells were also found in subepithelial lamina propria of the mucosae and muscularis in each segment of the digestive tract of Rana nigromaculata. The distribution densities of these cells reached peaks in the stomachs, and the middle or end segments of small intestines of Chinese softshell turtle, Bufo gargarizans, R.nigromaculata and catfish, and there was the third distribution density peak in the rectum of catfish. The total amount or overall density of GLP-1-positive cells varied a lot in the digestive tracts of different animal species. The distribution density was relatively low in the digestive tract of chub and reached the maximum in the digestive tracts of snakehead and catfish, but no GLP-1-positive cells were found in the digestive tract of bighead carp. GLP-1-positive cells were densely distributed in the pancreases of Chinese softshell turtle, B.gargarizans and R.nigromaculata. These cells spread over the superficial layers of islets or scattered in exocrine pancreas in the pancreas of B.gargarizans, spread in the endocrine cells or scattered in the pancreas of Chinese softshell turtle, scattered in the pancreas of R.nigromaculata and distributed in the superficial layers of islets in the pancreas of catfish.

Candidate Animal Disease Model of Elizabethkingia Spp. Infection in Humans, Based on the Systematic Pathology and Oxidative Damage Caused by E. miricola in Pelophylax nigromaculatus.

Most species of the genus Elizabethkingia are pathogenic to humans and animals, most commonly causing meningitis. However, our understanding of the pathogenic mechanisms involved is poor and there have been few pathological studies of Elizabethkingia spp. in animals. To understand the host injury induced by Elizabethkingia spp., we established a model of E. miricola infection in the black-spotted frog (Pelophylax nigromaculatus). The systematic pathology in and oxidative damage in the infection model were investigated. Our results show that recently isolated E. miricola is a bacterium that mainly parasitizes the host brain and that neurogenic organs are the predominant sites of damage. Infection mainly manifested as severe brain abscesses, meningoencephalitis, necrotic spondylitis, and necrotic retinitis. The liver, spleen, kidney, gastrointestinal tract, and lung were also affected to varying degrees, with bacterial necrotic inflammation. P. nigromaculatus also suffered enormous damage to its oxidative system during E. miricola infection, which may have further aggravated its disease state. Our results provide a preliminary reference for the study and treatment of Elizabethkingia spp.-induced neurological diseases in animals.

Herbalogical studies of Ranae oviductus (Hamayou) and its species identification using DNA barcoding method

<p indent=0mm>Ranae oviductus is a rare and precious Chinese medicine; however, there are many challenges connected with identifying this medicine, such as presence of many adulterants, the controversy over its origin, and the complexity of its classification history. First, we performed textual criticism of herbal literature and modern research data, constructed a phylogenetic relationship using whole mitochondrial genomes, and created a spatial overlay for the traditional production areas of Ranae oviductus and animal geographical distribution. The results obtained confirm that <italic>Rana chensinensis</italic>, <italic>R. dybowskii</italic>, and <italic>R. amurensis</italic> can be considered as a valid species for Ranae oviductus and that the <italic>Chinese Pharmacopoeia’s </italic>use of <italic>Rana temporaria chensinensis</italic> David. as the scientific name for Chinese brown frog is inappropriate. We further believe that the original species for Ranae oviductus could be <italic>R. dybowskii</italic>. Second, a debonding solution was prepared and used to pretreat medicinal materials to overcome the harmful effects of high expansion properties of Ranae oviductus during its DNA extraction. A new polymerase chain reaction primer pair was designed to resolve nonspecific amplification of cytochrome c oxidase subunit I (<italic>COI</italic>) sequences of Ranae oviductus using universal primers. A DNA barcoding reference library was established that contained 259 <italic>COI</italic> barcodes for 43 species covering Ranae oviductus and most of its adulterants. Finally, the DNA barcoding procedure for the identification of Ranae oviductus was verified based on simulation of real identification practices using commercially available medicinal materials. The results indicate that there are several adulterants, such as frog oil (<italic>Pelophylax nigromaculatus</italic>) and bullfrog oil (<italic>R. catesbeiana</italic>), in the herbal market. This study shows that DNA barcoding can successfully distinguish Ranae oviductus and its adulterants. In addition, the procedure established in this study can provide strong support for the safety of Ranae oviductus and protect the interests of consumers.

Temporal variation in behavioral responses to dietary cues from a gape‐limited predator in tadpole prey: A test of the phylogenetic relatedness hypothesis

AbstractThe ability of prey to recognize and adequately respond to predators determines their survival. Predator‐borne, post‐digestion dietary cues represent essential information for prey about the identity and the level of risk posed by predators. The phylogenetic relatedness hypothesis posits that prey should respond strongly to dietary cues from closely related heterospecifics but respond weakly to such cues from distantly related prey, following a hierarchical pattern. While such responses have mostly been observed in prey at their first encounter with predators, whether prey maintain such hierarchical levels of investment through time remains unclear. We investigated this question by exposing Rhacophorus arboreus tadpoles to the non‐consumptive effect of gape‐limited newt predators Cynops pyrrhogaster that were fed one of five prey diets across a gradient of phylogenetic relatedness: frog tadpoles (Rhacophorus arboreus, Rhacophorus schlegelii, Pelophylax nigromaculatus, and Hyla japonica) and medaka fish (Oryzias latipes). Predators’ diet, time, and their interaction significantly influenced tadpole activity level. We found support for the phylogenetic relatedness hypothesis: Investments in defense were stronger to cues from tadpole diets than to cues from fish diet. However, such a hierarchical response was recorded only in the first four days following predator exposure, then gradually disappear by day 8 on which the tadpoles exhibited similar activity level across all predator treatments. The findings suggest that, at least under the threat of gape‐limited predators, prey use phylogenetic information to evaluate risk and appropriately invest in defense during early encounters with predators; however, energy requirements may prevent prey from maintaining a high level of defense over long exposure to predation risk.