Black-spotted Pond Frog Research Articles

Life on both environment in semi-aquatic frogs: Impact of aquatic microplastic (MP) from MP enrichment to growth, immune function and physiological stress

The pervasive distribution of microplastics (MPs) in aquatic ecosystems presents a significant threat to wildlife, with amphibians being particularly vulnerable due to their complex life cycles and ecological roles. This study investigates physiological and ecological impacts of aquatic MP exposure on juvenile black-spotted pond frogs (Pelophylax nigromaculatus), focusing on juvenile frog stage, history of life after metamorphosis. MP examinations in the intestine and body revealed accumulation primarily in the gastrointestinal tracts without evidence of systemic distribution. Experimental exposure to different concentrations of MPs demonstrated adverse effects on growth, physiological stress, and immune function. Notably, higher MP concentrations led to significant reductions in growth and innate immunity, indicative of compromised health. High concentrations of MPs were associated with elevated levels of corticosterone and antioxidant enzymes, indicating physiological stress. However, there was no evidence of extreme hormonal surges or imbalances in antioxidant enzyme activity, suggesting that amphibians were able to effectively cope with the levels of MPs used in the study. Changes in gastrointestinal morphology and fecal microbiota composition were observed, reflecting response of metabolic adaptation to MP exposure. At low concentrations of MPs, adaptive changes in digestive tract morphology and the maintenance of gut microbiota balance were observed, indicating that the frogs were able to manage the exposure below a certain threshold. In contrast, high concentrations of MPs had clear negative effects on amphibians, which could impact biodiversity and ecosystem stability. These findings also suggest that MPs may trigger adaptive responses at lower concentrations, while still posing significant environmental risks at higher levels.

Environmentally Relevant Levels of Antiepileptic Carbamazepine Altered Intestinal Microbial Composition and Metabolites in Amphibian Larvae.

There is growing concern about the potential ecological risks posed by pharmaceutical residues in the aquatic environment. However, our understanding of the toxic effects of antiepileptic pharmaceuticals, such as carbamazepine (CBZ), on aquatic animal larvae is still limited. In this study, the tadpoles of the black-spotted pond frog (Pelophylax nigromaculatus) were exposed to environmentally relevant concentrations of CBZ (0.3 and 3.0 μg/L) for 30 days, and their growth, intestinal microbial composition, and metabolites were investigated to assess the potential toxic effects of CBZ in non-targeted aquatic organisms. Some tadpoles died during exposure, but there was no significant among-group difference in the survival and growth rates. CBZ exposure significantly altered the composition of tadpole intestinal microbiota. Relative abundances of some bacterial genera (e.g., Blautia, Prevotella, Bacillus, Microbacterium, etc.) decreased, while others (e.g., Paucibacter, etc.) increased in CBZ-exposed tadpoles. Interestingly, CBZ-induced alterations in some bacteria might not necessarily lead to adverse outcomes for animals. Meanwhile, small molecular intestinal metabolites related to energy metabolism, and antioxidant and anti-inflammatory activities were also altered after exposure. Taken together, environmentally relevant levels of CBZ might alter the metabolic and immune performances of amphibian larvae by modifying the abundance of some specific bacteria and the level of metabolites in their intestines, thereby potentially causing a long-term effect on their fitness.

Microcystin-LR Induces Estrogenic Effects at Environmentally Relevant Concentration in Black-Spotted Pond Frogs (Pelophylax nigromaculatus): In Situ, In Vivo, In Vitro, and In Silico Investigations.

Harmful cyanobacterial blooms are frequent and intense worldwide, creating hazards for aquatic biodiversity. The potential estrogen-like effect of Microcystin-LR (MC-LR) is a growing concern. In this study, we assessed the estrogenic potency of MC-LR in black-spotted frogs through combined field and laboratory approaches. In 13 bloom areas of Zhejiang province, China, the MC-LR concentrations in water ranged from 0.87 to 8.77 μg/L and were correlated with sex hormone profiles in frogs, suggesting possible estrogenic activity of MC-LR. Tadpoles exposed to 1 μg/L, an environmentally relevant concentration, displayed a female-biased sex ratio relative to controls. Transcriptomic results revealed that MC-LR induces numerous and complex effects on gene expression across multiple endocrine axes. In addition, exposure of male adults significantly increased the estradiol (E2)/testosterone (T) ratio by 3.5-fold relative to controls. Downregulation of genes related to male reproductive endocrine function was also identified. We also showed how MC-LR enhances the expression of specific estrogen receptor (ER) proteins, which induce estrogenic effects by activating the ER pathway and hypothalamic-pituitary-gonadal (HPG) axis. In aggregate, our results reveal multiple lines of evidence demonstrating that, for amphibians, MC-LR is an estrogenic endocrine disruptor at environmentally relevant concentrations. The data presented here support the need for a shift in the MC-LR risk assessment. While hepatoxicity has historically been the focus of MC-LR risk assessments, our data clearly demonstrate that estrogenicity is a major mode of toxicity at environmental levels and that estrogenic effects should be considered for risk assessments on MC-LR going forward.

Metformin exposure altered intestinal microbiota composition and metabolites in amphibian larvae

The antidiabetic pharmaceutical metformin (MET) is largely unmetabolized by the human body. Its residues are readily detectable in various aquatic environments and may have adverse impacts on the growth and survival of aquatic species. To date, its toxicological effects have scarcely been explored in non-fish species. Here, we exposed the tadpoles of black-spotted pond frog (Pelophylax nigromaculatus) to different concentrations (0, 1, 10 and 100 μg/L) of MET for 30 days and measured the body size, intestinal microbiota and metabolites to evaluate potential effects of MET exposure in amphibian larvae. MET exposure did not affect the growth and intestinal microbial diversity of tadpoles. However, intestinal microbial composition changed significantly, with some pathogenic bacteria (e.g., bacterial genera Salmonella, Comamonas, Stenotrophomonas, Trichococcus) increasing and some beneficial bacteria (e.g., Blautia, Prevotella) decreasing in MET-exposed tadpoles. The levels of some intestinal metabolites associated with growth and immune performance also changed significantly following MET exposure. Overall, our results indicated that exposure to MET, even at environmentally relevant concentrations, would cause intestinal microbiota dysbiosis and metabolite alteration, thereby influencing the health status of non-target aquatic organisms, such as amphibians.

Phenotypic Plasticity in Juvenile Frogs That Experienced Predation Pressure as Tadpoles Does Not Alter Their Locomotory Performance

Anuran species can respond to environmental changes via phenotypic plasticity, which can also result in ecological impacts across the life history of such species. We investigated the effects of predation pressure (i.e., the non-consumption effect) from the dragonfly larva (Anax parthenope) on the phenotypical change of tadpoles into juvenile frogs (specifically the black-spotted pond frog, Pelophylax nigromaculatus), and also analyzed the impact of morphological changes on locomotory performance after metamorphosis. The experiments on predator impact were conducted in the laboratory. Body length, weight, development timing, and metamorphosis timing in the presence of dragonfly nymphs were measured in both tadpoles and juvenile frogs. The body and tail shapes of the tadpoles, as well as the skeletal shape of the juvenile frogs, were analyzed using landmark-based geometric morphometrics. Furthermore, the locomotory performance of the juvenile frogs was tested by measuring their jumping and swimming speeds. Tadpoles that had grown with predators possessed smaller bodies, deeper tail fins, and slower development rates, and they waited longer periods of time before commencing metamorphosis. Having said this, however, the effect of predator cues on the body length and weight of juvenile frogs was not found to be significant. These juvenile frogs possessed longer limbs and narrower skulls, with subtle morphological changes in the pelvis and ilium, but there was no subsequent difference in their swimming and jumping speeds. Our results showed that the changes in anatomical traits that can affect locomotor performance are so subtle that they do not affect the jumping or swimming speeds. Therefore, we support the view that these morphological changes are thus by-products of an altered tadpole period, rather than an adaptive response to predator-escape ability or to post-metamorphosis life history. On the other hand, delayed metamorphosis, without an increase in body size, may still be disadvantageous to the reproduction, growth, and survival of frogs in their life history following metamorphosis.

Population Structure and Morphological Pattern of the Black-Spotted Pond Frog (Pelophylax nigromaculatus) Inhabiting Watershed Areas of the Geum River in South Korea

Black-spotted pond frogs (Pelophylax nigromaculatus), widely distributed in East Asia, can be suitably used for the study of population genetic patterns and ecosystem monitoring. To systematically manage, conserve, and study this species, it is necessary to understand its habitat range. We analyzed the genetic and morphological range of black-spotted pond frog populations within a watershed of the Geum River, one of the main rivers in South Korea. We genotyped the frogs based on seven microsatellite loci and defined the skull shape based on landmark-based geometric morphometrics. One watershed area was divided into 14 sub-watershed areas, the smallest unit of the Geum River basin. The genetic structure of frogs among the 14 sub-watershed areas did not differ significantly, nor was correlated with geographic distance. Therefore, frogs within these watershed areas constitute a single population. Morphologically, they differed between some sub-watershed areas, but morphological distance did not correlate with genetic distance but rather with geographic distance. This morphological change differs depending on the environmental gradient rather than the genetic structure. As a single population, frogs in this watershed area need to be managed in an integrated way. We suggest that the identification of response and adaptation by population genetics must be compared across and beyond the watershed range.

Trophic niche differentiation between invasive and native frog species in urban wetlands of Kunming, China

Non-native species often proliferate and become invasive in urban ecosystems. Knowing the interactions between the overlapping trophic niches of invasive and native species is imperative, and answering this question of how they succeed in these ecosystems is an area of active research. Here, frogs were collected from eight sites in the suburbs of Kunming, including 74 non-native American bullfrogs (Lithobates catesbeianus), 115 native Yunnan pond frogs (Nidirana pleuraden, YPF) and 26 non-native black-spotted pond frogs (Pelophylax nigromaculatus, BPF). The diversity indices and niche widths of the sampled frogs’ stomach contents were determined, and δ13C and δ15N isotopes in their muscle tissue measured for a subset of samples. At sites where multiple species occurred, the prey Shannon-Wiener diversity index and niche width of the bullfrog were higher than those of BPF yet lower than those of YPF. The prey diversity and niche width of YPF decreased significantly at the level of individual populations. The overlap between the trophic niches of bullfrog and sympatric frogs ranged from 0.3 to 0.37 based on their stomach contents, while that between the YPF and BPF was 0.68. These results demonstrate that bullfrog and YPF eat some items in common, for which competition is likely, but this is not as pronounced as between YPF and BPF. Plotting values of δ13C (x-axis) against δ15N (y-axis) isotopes, the standard ellipse areas at the LaoY site, where all three species co-occurred, were 23.75, 12.9, and 11.95 for bullfrog, YPF, and BPF, respectively. The maximal standard ellipse area of YPF across the sites without bullfrog invasion was 4.74. These results showed that bullfrog’s prey diversity did not exceed that of YPF in the co-distributed environments, yet its isotopic niche was nonetheless larger. Stomach contents can only convey short-term dietary composition whereas carbon isotopes can reveal relatively long-term nutritional trends, so we conclude the trophic niche width is larger for Bullfrog than YPF. Their degree of overlap is large, which suggests both species have similar ecological functions and experience intense interspecific competition.

Taxonomic status of Rana nigromaculata mongolia and the validity of Pelophylax tenggerensis (Anura, Ranidae).

The Black-spotted Pond Frog, Pelophylax nigromaculatus, is widely distributed across mainland China, Korean Peninsula, and Japan. The taxonomic relationships among P. n. nigromaculatus, Rana nigromaculata mongolia (sensu P. n. mongolicus), and P. tenggerensis have long been ambiguous. Here we examine the topotype specimens of P. tenggerensis and R. n. mongolia, and provide phylogenic analyses based on four mitochondrial DNA sequences. The combined evidences from morphology and molecular phylogeny have shown the distinct specific-level of P. n. mongolicus that distant from P. nigromaculatus, while indicating the homogeneity between P. n. mongolicus and P. tenggerensis. Thus, we suggest elevating P. n. mongolicus as a full species Pelophylax mongolicus comb. nov., and place P. tenggerensis to be a secondary synonym of P. mongolicus comb. nov.

Amphibian-Friendly Water Drainages for Agricultural Landscapes, Based on Multiple Species Surveys and Behavioural Trials for Pelophylax nigromaculatus

Amphibians are the most threatened vertebrate group on earth, and one of the reasons for their decline is habitat loss. While some populations have persisted in agricultural wetlands such as rice paddies, the current anthropisation of landscapes is dealing a new blow to the survival of these species. In rice paddies, the new threats are especially visible through the increased channelization of water bodies with increasingly efficient drainage ditches, which become deadly traps. We first conducted surveys over three years to determine the use of ditches by frog species for natural versus concrete ditches, and thus relate to habitat adequacy as well as the probability of becoming trapped in concrete ditches. We then set up four types of experimental arena for escape trials. Experiments were replicated for the Black-spotted pond frog (Pelophylax nigromaculatus), as a proxy for other species abundant in rice paddies in the Republic of Korea. We determined that a slope of at least 70 degrees, with engraved patterns, was the only set-up from which frogs managed to escape. We recommend the implementation of this type of device in areas where a high concentration of animals is trapped, before phasing out the ancient design and relying on amphibian-friendly drainage ditches as they also support higher amphibian biodiversity.

Prevalence of Ranavirus Infection in Three Anuran Species across South Korea.

To cope with amphibian die-offs caused by ranavirus, it is important to know the underlying ranavirus prevalence in a region. We studied the ranavirus prevalence in tadpoles of two native and one introduced anuran species inhabiting agricultural and surrounding areas at 49 locations across eight provinces of South Korea by applying qPCR. The local ranavirus prevalence and the individual infection rates at infected locations were 32.6% and 16.1%, respectively, for Dryophytes japonicus (Japanese tree frog); 25.6% and 26.1% for Pelophylax nigromaculatus (Black-spotted pond frog); and 30.5% and 50.0% for Lithobates catesbeianus (American bullfrog). The individual infection rate of L. catesbeianus was significantly greater than that of D. japonicus. The individual infection rate of P. nigromaculatus was related to the site-specific precipitation and air temperature. The individual infection rate gradually increased from Gosner development stage 39, and intermittent infection was confirmed in the early and middle developmental stages. Our results show that ranavirus is widespread among wild amphibians living in agricultural areas of South Korea, and mass die-offs by ranavirus could occur at any time.

Examination of Physiological and Morphological Differences between Farm-Bred and Wild Black-Spotted Pond Frogs (Pelophylax nigromaculatus).

Simple SummaryWe compared the health status and ecological characteristics of farm-bred and wild frogs using veterinary clinical examinations. Wild frogs showed a better nutritional status and superior traits of locomotory performance than farm-bred frogs. However, wild animals are not necessarily in good health. This comparison can help determine the condition of animals and suggest improvements to the breeding environment.Due to the decline in the population and the difficulty of in situ conservation, several anuran species are being reared in captivity. In this study, we identified physiological and morphological differences between farm-bred and wild frogs. Nine different serum components were used as indicators of osmotic pressure, homeostatic state, organ function, and nutritional status of farm-bred frogs and wild frogs, while radiographic techniques were used to visualize differences in bone mineral density and body composition ratio. Additionally, X-ray skeletal images were used for morphological analysis to estimate differences in locomotory performance between the two groups. Wild frogs harbor traits that aid in better locomotory performance than farm-bred frogs. They also have a relatively lower fat content ratio and higher calcium and phosphorus serum levels than farm-bred frogs, suggesting a difference in nutritional status. However, hepatic stress was higher in wild frogs than in farm-bred frogs. Veterinary clinical examinations allow for the identification of differences in nutritional and morphological conditions between farm-bred and wild frogs. Determining the health of animals can help improve their living conditions, eliminate conditions that can negatively affect them, and effectively manage them on farms, in zoos, and at ex situ conservation institutes.

Reference Intervals in Combined Veterinary Clinical Examinations of Male Black-Spotted Pond Frogs (Pelophylax nigromaculatus).

Simple SummaryThe reference intervals (RIs) of immunity, serum components, bone mineral density (BMD), and body composition in 151 males of Pelophylax nigromaculatus were established. These analyses are easily replicable and can safely and accurately diagnose the physiological condition of animals. The use of combined examination allows for the establishment of a successful conservation strategy through the identification of conservation problems in many vertebrate groups.In conservation physiology, analyzing the physiological response of an organism to understand its ability to adapt to environmental changes is a key technique in establishing a successful conservation strategy. Veterinary clinical examinations determine the physiological condition of animals accurately and safely, and this examination is synergistic when combined. The accuracy and safety of a clinical examination makes it advantageous for use in amphibians with high species diversity and numerous endangered species. However, it is necessary to establish a reference interval (RI) for precise interpretations and identification of animals with abnormalities through individual unit testing. We have established RIs for the immunity, serum components, bone mineral density (BMD), and body composition of black-spotted pond frogs (Pelophylax nigromaculatus). Black-spotted pond frogs are a common species and are widely distributed in East Asia, with suitable characteristics for environmental monitoring. Serum was extracted from 151 male frogs to establish the RI for bacterial killing ability in order to represent immunity. We also used the serum to establish an RI of ten additional serum components to determine the nutritional status, organ function status, body osmotic pressure, and homeostasis conditions. The BMD and three body composition measures for diagnosing food intake status and nutritional condition were measured using dual energy X-ray absorptiometry. The RI was recorded as the mean ± standard deviation, median, first (25%) to third (75%) quantile range, 95% confidence interval of the mean and median, and the 95% percentile (2.5%–97.5% range) of all components. The use of combined clinical veterinary examinations aids our understanding of the physiological conditions of an individual according to biotic and abiotic factors on a complex spatiotemporal scale in an ecosystem.

Trophic Ecology of Endangered Gold-Spotted Pond Frog in Ecological Wetland Park and Rice Paddy Habitats.

Simple SummaryGaining information about the habitat environment and biological interactions is important for conserving gold-spotted pond frogs, which are faced with a threat of local population extinction in Korea due to artificial habitat changes. Based on stable isotope ratios, we estimated the ecological niche space (ENS) of gold-spotted pond frogs in an ecological wetland park and a rice paddy differing in habitat patch connectivity and analyzed the possibility of their ENS overlapping that of competitive and predatory frogs. Gold-spotted pond frogs showed a broader ENS in the ecological wetland park, wherein predation was relatively easy, than in the rice paddy. However, the ENS of the gold-spotted pond frogs was highly probably overlapped with that of other competing frog species that shared some of the food sources. Nevertheless, since the stable isotope analysis showed that gold-spotted pond frogs fed on more diverse prey than their competitors, it would remain relatively easy to procure alternative food sources, which are less affected by the competition in an environment with abundant food. Therefore, for stable settling of gold-spotted pond frogs into habitats and preserving their population, establishing habitat environments with highly diverse food sources is crucial, following consideration of their feeding behavior.The gold-spotted pond frog (Pelophylax chosenicus) is an endangered amphibian species in South Korea. In order to obtain ecological information regarding the gold-spotted pond frog’s habitat environment and biological interactions, we applied stable isotope analysis to quantify the ecological niche space (ENS) of frogs including black-spotted pond frogs (P. nigromaculatus) and bullfrogs (Lithobates catesbeianus) within the food web of two different habitats—an ecological wetland park and a rice paddy. The gold-spotted pond frog population exhibited a broader ENS in the ecological wetland park than in the rice paddy. According to the carbon stable isotope ratios, gold-spotted pond frogs mainly fed on insects, regardless of habitat type. However, the results comparing the range of both carbon and nitrogen stable isotopes showed that gold-spotted pond frogs living in the rice paddy showed limited feeding behavior, while those living in the ecological wetland park fed on various food sources located in more varied trophic positions. Although the ENS of the gold-spotted pond frog was generally less likely to be overlapped by that of other frog species, it was predicted to overlap with a high probability of 87.3% in the ecological wetland park. Nevertheless, gold-spotted pond frogs in the ecological wetland park were not significantly affected by the prey competition with competitive species by feeding on other prey for which other species’ preference was low. Since these results show that a habitats’ food diversity has an effect on securing the ENS of gold-spotted pond frogs and prey competition, we recommend that the establishment of a food environment that considers the feeding behavior of gold-spotted pond frogs is important for the sustainable preservation of gold-spotted pond frogs and their settlement in alternative habitats.

Motilin- and ghrelin-induced contractions in isolated gastrointestinal strips from three species of frogs

Ghrelin (GHRL) and motilin (MLN), gut peptides isolated from the mucosa of the stomach and duodenum, respectively, stimulate gastrointestinal (GI) motility in mammals and birds. However, the functions of MLN and GHRL in amphibian GI tracts have not been examined in detail. To clarify the regulation of GI motility by the two peptides, the effects of human MLN and rat GHRL on contractility of isolated GI strips from three species of frogs, the black-spotted pond frog (pond frog; Pelophylax nigromaculata), bullfrog (Lithobates catesbeiana) and Western clawed frog (Xenopus; Xenopus tropicalis), were examined in in vitro experiments. The GI tract of each frog was divided into the stomach, upper intestine, middle intestine and lower intestine. Human MLN caused contractions of the stomach in the pond frog and upper intestine in the bullfrog and Xenopus, but other GI regions were insensitive to human MLN. Erythromycin did not cause contraction of the upper intestine of the bullfrog and Xenopus. Rat GHRL did not cause contraction of the stomach and small intestines in the pond frog and bullfrog, but it caused a concentration-dependent contraction in the stomach and upper intestine of Xenopus, while des-acyl rat GHRL did not cause any contraction of them. In conclusion, human MLN caused the contraction of the stomach or upper intestine in the three species of frogs, but GHRL was effective only in the stomach and upper intestine of Xenopus. On the basis of these data, MLN but not GHRL causes the GI region-dependent contractions in the frogs.

The early frog gets eaten

![Graphic][1] If a giant grizzly bear was to pop out from behind a bush and start approaching you, your first reaction would probably be to run immediately for your life in the hopes of reaching safety as quickly as possible. However, when a black-spotted pond frog sees an approaching

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.

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.

FV3-like ranavirus infection outbreak in black-spotted pond frogs (Rana nigromaculata) in China

In April 2016, an outbreak emerged in a cultured population of black-spotted pond frog tadpoles in Shuangliu County, China, whereas tadpoles were suffering from substantial mortality (90%). Principal clinical signs of diseased tadpoles were comprised haemorrhage on their body surface, swollen abdomen with yellow ascites, congestion and swelling of the liver. The diseased tadpole's homogenates tissue were inoculated into epithelioma papulosum cyprini (EPC) cells at 25 °C for 4 days which caused typical cytopathic effect, and the viral titer TCID50 reached 107/0.1 mL. In pathogenicity tests, tadpoles were immersed in 2‰ virus fluid for 8 h, the clinical signs were observed similar to those recognized in naturally infected tadpoles and mortality rate were reached up to 80%, which affirms that the virus was the main cause for this disease. In addition, transmission electron microscopy of EPC cells infected with isolated virus reflected that the virus was in a regular hexagon way (shape) with capsule like structure. The diagonal diameter was recorded 135 ± 8 nm, wherever virus particles were arrayed in crystalline manner in the cytoplasm. The electrophoresis of MCP gene PCR-product showed that the samples of diseased tadpoles, aquaculture water source and isolated virus were all positive. The sequence of the isolate revealed more than 99% similarities to ranavirus based on homology and genetic evolution analysis of the whole MCP gene, and the isolate belongs to FV3-like virus group. This study confirmed that ranavirus was the causative agent of this outbreak, and named the virus as Rana nigromaculata ranavirus (RNRV).

Pathogen richness and abundance predict patterns of adaptive major histocompatibility complex variation in insular amphibians

The identification of the factors responsible for genetic variation and differentiation at adaptive loci can provide important insights into the evolutionary process and is crucial for the effective management of threatened species. We studied the impact of environmental viral richness and abundance on functional diversity and differentiation of the MHC class Ia locus in populations of the black-spotted pond frog (Pelophylax nigromaculatus), an IUCN-listed species, on 24 land-bridge islands of the Zhoushan Archipelago and three nearby mainland sites. We found a high proportion of private MHC alleles in mainland and insular populations, corresponding to 32 distinct functional supertypes, and strong positive selection on MHC antigen-binding sites in all populations. Viral pathogen diversity and abundance were reduced at island sites relative to the mainland, and islands housed distinctive viral communities. Standardized MHC diversity at island sites exceeded that found at neutral microsatellites, and the representation of key functional supertypes was positively correlated with the abundance of specific viruses in the environment (Frog virus 3 and Ambystoma tigrinum virus). Theseresults indicate that pathogen-driven diversifying selection can play an important role in maintaining functionally important MHC variation following island isolation, highlighting the importance of considering functionally important genetic variation and host-pathogen associations in conservation planning and management.

Modernization of drainage systems decreases gray-faced buzzard occurrence by reducing frog densities in paddy-dominated landscapes

Modernization of drainage systems is suspected of causing a deterioration in the biodiversity of paddy-dominated landscapes that are substitutes for natural wetlands. Here, we focused on the gray-faced buzzard (Butastur indicus), a top predator that inhabits agricultural landscapes in East Asia. We evaluated the impacts of drainage system modernization on buzzards by measuring reductions in paddy-dwelling frogs, a major prey for buzzards that are common in rice fields. Field surveys were conducted in yatsu valleys (“yatsu” refers to small, narrow valleys in which rice cultivation often dominates the valley bottom) in Shimane prefecture, western Japan. We found that the distribution of buzzards was positively correlated with the densities of black-spotted pond frogs (Pelophylax nigromaculatus) and forest green tree frogs (Rhacophorus arboreus), although the relationship between buzzards and pond frogs was stronger. In addition, pond frog density was negatively correlated with modern drainage systems, whereas tree frog density did not show such a pattern. Therefore, modern drainage systems may reduce the buzzard population indirectly by reducing pond frog densities.