Rana Nigromaculata Research Articles

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.

Study effect and mechanism of levofloxacin on the neurotoxicity of Rana nigromaculata tadpoles exposed to imidacloprid based on the microbe-gut-brain axis

Aquatic organisms may be simultaneously exposed to antibiotics and pesticides. After levofloxacin (LVFX), imidacloprid (IMI) exposure and co-exposure at environmental levels, we found LVFX and IMI had antagonistic effect on the neurotoxicity of tadpoles. IMI-induced neurotoxicity on tadpoles can be explained by oxidative stress and hormone levels in some degree. By regulating ornithine, l-asparagine, putrescine and tryptamine in the intestine, LVFX affected glutathione metabolism, arginine and proline metabolism, alanine, aspartate and glutamate metabolism, tyrosine metabolism and aminoacyl tRNA biosynthesis, so then eased the neurotoxicity caused by IMI. More interestingly, Fusobacteriota and Cetobacterium might play an important role on easing the neurotoxicity caused by IMI. In addition, LVFX might have a laxation effect on the increased relative abundance of Bacteroidota caused by IMI. In conclusion, IMI not only affected oxidative stress and hormone levels in the brain, but also affected the synthesis of neurotransmitters in the intestine by regulating intestinal microbiota. In LVFX and IMI co-exposed groups, LVFX alleviated the neurotoxicity caused by IMI through regulating the intestinal microbiota, showing as an antagonistic effect. Our results provided a new perspective for aquatic ecological risk assessment under co-exposure of antibiotics and pesticides.

Perfluorooctanoic acid and perfluorooctanesulfonic acid induce immunotoxicity through the NF-κB pathway in black-spotted frog (Rana nigromaculata)

Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are widely detected in the environment and wild animals, thus posing a threat to wildlife and public health; however, knowledge about their immunotoxicity and the underlying mechanism remains limited. In the present study, male black-spotted frogs (Rana nigromaculata) were exposed to environmentally relevant concentrations (0, 1, and 10 μg/L) of PFOA or PFOS for 21 days; subsequently, biochemical analysis, molecular docking, and gene expression determination were conducted. The results indicated that exposure to 10 μg/L PFOA decreased the serum levels of immunoglobulin A. PFOS exposure significantly increased the hepatic levels of interleukin-1β, interleukin-6, tumor necrosis factor-α, interferon-γ, and nitric oxide; but PFOA significantly increased the levels of only tumor necrosis factor-α. Furthermore, PFOA and PFOS exposure significantly decreased the activity of inducible nitric oxide synthase and total nitric oxide synthase. IBRv2 analysis indicated that PFOA and PFOS had a similar effect on these immune indicators, but PFOS was more toxic than PFOA. Molecular docking revealed that PFOA and PFOS can bind to nuclear factor-κB (NF-κB) by forming stable hydrogen bonds. PFOA and PFOS exposure upregulated the gene expression of NF-κB and its downstream genes. Significant correlations between the expression of genes involved in the NF-κB pathway and immune-related indicators suggests that PFOA- and PFOS-induced immunotoxicity was associated with the activation of NF-κB. Our findings provide novel insights into the potential role of NF-κB in immunotoxicity induced by PFOA and PFOS in frogs.

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.

Effects of water level variations on survival, morphological phenotype and responsiveness of tadpoles to phytohemagglutinin

To investigate the responses of morphological and physiological characteristics in tadpoles of Pelophylax nigromaculatus to various water levels, a total of 480 tadpoles with similar sizes were treated with rapid, moderate and slow desiccation. Both the metamorphosis duration and average metamorphosis survival rate of specific developmental stages were counted. Their body and internal organ sizes, as well as the responsiveness to phytohemagglutinin (PHA-P) of froglets were measured after complete metamorphosis. The results showed that the metamorphosis duration of tadpoles in the control group was the longest. The average metamorphosis survival rate of different treatment groups ranged from 72.5% to 90.8%, which was the highest in the control group and the lowest in the rapid desiccation group. Body weight (BW) and body length (BL) of froglets in control and slow desiccation groups were significantly higher than those of moderate or rapid desiccation groups. Body width, ratio of BW to BL, carcass wet weight and the wet weight index of lung and fat body were the lowest in rapid desiccation group. There was no significant difference in the wet weight index of heart, spleen, kidneys and digestive organs, as well as the length index of different segments of whole digestive tract between groups. The maximal response value of froglets to PHA-P appeared at 3 h after injection in different treatments, which was significantly higher in moderate and slow desiccation groups than the control group. There was no significant difference between rapid desiccation group and any other groups. The tadpoles of P. nigromaculatus might accelerate the metamorphosis developmental process to cope with desiccation stress, accompanying with decreased atrophic body size and weakened cell-mediated immunity, which would harm tadpoles to successfully land.

Male wasp genitalia as an anti-predator defense

Predators can cause selection that drives the evolution of various anti-predator defenses in prey1,2,3. Some prey species have evolved specific defensive devices, while others simply use body parts that evolved for reasons other than defense to repel predators1. For example, many animal species have strong canine teeth or mandibles to kill prey and to counterattack their enemies1,2,3. Bees and wasps (Insecta: Hymenoptera) use their ovipositors as stings to repel attackers, including humans4. Stinging by injecting venom can cause intense pain and allergic reactions in humans4. Therefore, stinging hymenopterans that are recognized as dangerous have evolved warning signals, such as conspicuous body coloration4. Hymenopteran males, which lack ovipositors, are believed to be harmless, and the males avoid predators by mimicking the females' coloration and behavior4. Here, we report that males of the mason wasp Anterhynchium gibbifrons (Hymenoptera: Vespidae: Eumeninae) use sharp genital spines to counterattack predators. A potential predator, the tree frog Dryophytes japonica (= Hyla japonica) (Anura: Hylidae) or the pond frog Pelophylax nigromaculatus (Anura: Ranidae), was housed with male A. gibbifrons under laboratory conditions. All tree and pond frogs attacked the male wasps. Although all of the pond frogs ate the male wasps, 35.3% of the tree frogs ultimately rejected them. Male wasps were frequently observed to pierce the mouth or other parts of frogs with their genitalia while being attacked. The tree frogs were also housed with male wasps from which the genitalia were removed. All these frogs ate the genitalia-less males. Therefore, male wasps used their genitalia to prevent tree frogs from swallowing them. This study highlights the importance of male genitalia as an anti-predator defense and provides a new perspective for understanding the ecological roles of male genitalia in animals.

Characterization and Dynamics of the Gut Microbiota in Rice Fishes at Different Developmental Stages in Rice-Fish Coculture Systems.

The rice-fish system (RFS), a traditional coculture farming model, was selected as a "globally important agricultural heritage system." Host-associated microbiota play important roles in development, metabolism, physiology, and immune function. However, studies on the gut microbiota of aquatic animals in the RFS are scarce, especially the lack of baseline knowledge of the dynamics of gut microbial communities in rice fish during different developmental stages. In this study, we characterized the microbial composition, community structure, and functions of several sympatric aquatic animals (common carp (Cyprinus carpio), crucian carp (Carassius carassius), and black-spotted frogs (Pelophylax nigromaculatus)), and the environment (water) in the RFS using 16S rRNA gene sequencing. Moreover, we investigated stage-specific signatures in the gut microbiota of common carp throughout the three developmental stages (juvenile, sub-adult, and adult). Our results indicated that the Fusobacteriota, Proteobacteria, and Firmicutes were dominant gut microbial phyla in rice fish. The differences in gut microbial compositions and community structure between the three aquatic species were observed. Although no significant differences in alpha diversity were observed across the three developmental stages, the microbial composition and community structure varied with development in common carp in the RFS, with an increase in the relative abundance of Firmicutes in sub-adults and a shift in the functional features of the community. This study sheds light on the gut microbiota of aquatic animals in the RFS. It deepens our understanding of the dynamics of gut microflora during common carp development, which may help improve aquaculture strategies in the RFS.

Transcriptomic Analysis Reveals the Hepatotoxicity of Perfluorooctanoic Acid in Black-Spotted Frogs (Rana nigromaculata)

Perfluorooctanoic acid (PFOA) is frequently detected in the environment and accumulates in amphibians such as black-spotted frogs (Rana nigromaculata) with toxic effects; however, the mechanism underlying this toxicity is unclear. In this study, male black-spotted frogs were exposed to 10 μg/L waterborne PFOA for 21 days. Subsequently, the effect of PFOA exposure on gene expression in liver tissue was investigated using transcriptomic techniques. In total, 754 differentially expressed genes (DEGs; 389 up-regulated and 365 down-regulated) were identified. According to Kyoto Encyclopedia of Genes and Genomes enrichment and Gene Ontology functional enrichment analyses, the DEGs were mainly involved in lipid metabolism, endocrine functions, and immunity. Quantitative real-time polymerase chain reaction analysis of 15 selected DEGs revealed a high correlation (R2 = 0.9917) with the transcriptomic results. These results indicated that the PFOA hepatotoxicity in frogs is mediated mainly by lipid metabolism dysregulation, endocrine system disruption, and immunotoxicity. This study provides insights into the hepatotoxic mechanism of PFOA and other perfluorinated compounds in amphibians.

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.

Per- and Polyfluoroalkyl Substances (PFASs) Impair Lipid Metabolism in Rana nigromaculata: A Field Investigation and Laboratory Study.

Per- and polyfluoroalkyl substances (PFASs) are ubiquitous environmental pollutants, causing environmental threats and public health concerns, but information regarding PFAS hepatotoxicity remains elusive. We investigated the effects of PFASs on lipid metabolism in black-spotted frogs through a combined field and laboratory study. In a fluorochemical industrial area, PFASs seriously accumulate in frog tissues. PFAS levels in frog liver tissues are positively related to the hepatosomatic index along with triglyceride (TG) and cholesterol (TC) contents. In the laboratory, frogs were exposed to 1 and 10 μg/L PFASs, respectively (including PFOA, PFOS, and 6:2 Cl-PFESA). At 10 μg/L, PFASs change the hepatic fatty acid composition and significantly increase the hepatic TG content by 1.33 to 1.87 times. PFASs induce cross-talk accumulation of TG, TC, and their metabolites between the liver and serum. PFASs can bind to LXRα and PPARα proteins, further upregulate downstream lipogenesis-related gene expression, and downregulate lipolysis-related gene expression. Furthermore, lipid accumulation induced by PFASs is alleviated by PPARα and LXRα antagonists, suggesting the vital role of PPARα and LXRα in PFAS-induced lipid metabolism disorders. This work first reveals the disruption of PFASs on hepatic lipid homeostasis and provides novel insights into the occurrence and environmental risk of PFASs in amphibians.

Assessing the hepatotoxicity of PFOA, PFOS, and 6:2 Cl-PFESA in black-spotted frogs (Rana nigromaculata) and elucidating potential association with gut microbiota

Pollution caused by per- and polyfluoroalkyl substances (PFASs) has become a major global concern. The association between PFAS-induced hepatotoxicity and gut microbiota in amphibians, particularly at environmentally relevant concentrations, remains elusive. Herein we exposed male black-spotted frogs (Rana nigromaculata) to 1 and 10 μg/L waterborne perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), and 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA) for 21 days; subsequently, liver histopathological, oxidative stress, molecular docking, gene/protein expression, and gut microbiome analyses were conducted. PFOS and 6:2 Cl-PFESA exposure enhanced serum alanine aminotransferase and aspartate aminotransferase activities, and markedly increased hepatic area of vacuoles and inflammatory cell infiltration, while PFOA exposure increased serum alanine aminotransferase but not aspartate aminotransferase activities and affected hepatic area of vacuoles and inflammatory cell infiltration to a lesser extent. All three PFASs elevated catalase, glutathione S-transferase, and glutathione peroxidase activities and glutathione and malondialdehyde contents in the liver, suggesting the induction of oxidative stress. Further, PFASs could bind to mitogen-activated protein kinases (p38, ERK, and JNK), upregulating not only their expression but also the expression of downstream oxidative stress-related genes and that of P-p38, P-ERK, and Nrf2 proteins. In addition, PFAS exposure significantly increased the relative abundance of Proteobacteria and Delftia and decreased that of Firmicutes and Dietzia, Mycoplasma, and Methylobacterium–Methylorubrum in the order of PFOS ≈ 6:2 Cl-PFESA > PFOA. Altogether, it appears that PFOS and 6:2 Cl-PFESA are more toxic than PFOA. Finally, microbiota function prediction, microbiota co-occurrence network, and correlation analysis between gut microbiota and liver indices suggested that PFAS-induced hepatotoxicity was associated with gut microbiota dysbiosis. Our data provide new insights into the role of gut microbiota in PFAS-induced hepatotoxicity in frogs.

Brevinin-2PN, an antimicrobial peptide identified from dark-spotted frog (Pelophylax nigromaculatus), exhibits wound-healing activity

Brevinins exhibit a wide range of structural features and strong biological activities. Brevinin-2, derived from several amphibians, has shown antimicrobial activities. However, little is known about the wound-healing activity of brevinin-2. In this study, brevinin-2 cDNA was identified from the skin transcriptome of the dark-spotted frog (Pelophylax nigromaculatus) and it comprises a signal peptide, a propeptide, and a mature peptide. Sequence alignment with brevinin-2 derived from other amphibians showed variability of the mature peptide, and the presence of a C-terminal cyclic heptapeptide domain (Cys-Lys-Xaa4-Cys) in the mature peptide. Dark-spotted frog brevinin-2 belonged to the brevinin-2 cluster and was closely related to brevinin-2HB1 from Pelophylax hubeiensis. Synthetic dark-spotted frog brevinin-2 mature peptide (brevinin-2PN) exhibited antibacterial activity against several pathogens by destroying cell membrane integrity and hydrolysis of genomic DNA. Brevinin-2PN exhibited significant wound-healing activity by accelerating the healing of human skin fibroblast cell scratches, influencing cell migration, and stimulating gene expression of growth factors.

The toxic effects of chronic atrazine exposure on the intestinal microbiota, metabolism and transcriptome of Pelophylax nigromaculatus larvae

Atrazine, a widely used herbicide, is a potential threat to the intestines of animals due to its long-term persistence in water and accumulation in animal tissues, but the underlying mechanism is unclear. In this study, we evaluated the toxicity of atrazine (50, 100, and 500 µg/L) on the intestine of Pelophylax nigromaculatus larvae from Gosner stages 18–42 (about 90 days). We conducted a 16S rRNA gene amplification study, which showed that chronic exposure to atrazine changed the diversity and composition of intestinal microbiota of P. nigromaculatus larvae. Chronic exposure to atrazine (50, 100, and 500 µg/L) significantly decreased the relative abundance of Fusobacteria and U114, while 50 µg/L and 100 µg/L atrazine resulted in a significant increase of Clavibacter. The LC-MS/MS metabolomics indicated that there were 145 significantly changed metabolites. Furthermore, atrazine led to metabolic abnormalities related to purine metabolism and biosynthesis of amino acids in the intestine. The transcriptomic results showed that atrazine exposure caused 21,120 differentially expressed genes between the control and the atrazine exposure groups. Atrazine exposure primarily disturbed the pathways related to the cytokine-cytokine receptor interaction and cell adhesion molecules. Moreover, 500 µg/L atrazine caused a significant up-regulation in the gene expression of apolipoprotein (ApoA1, ApoB, and ApoA4), and fatty acid binding protein (FABP1), as well as a significant down-regulation in the gene expression of superoxide dismutase (SOD). Our work provides insight into the ecotoxicity mechanisms of atrazine in amphibians at the level of intestinal microbiota response, metabolome and transcriptome, and serves as a basis for future research on pesticide pollutants.

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.

Effects of short-term continuous and pulse cadmium exposure on gut histology and microbiota of adult male frogs (Pelophylax nigromaculatus) during pre-hibernation

Cadmium (Cd) is an environmental endocrine-disrupting pollutant which mainly occurs in pulsed manner in natural waters, while traditional toxicology experiments have less examined the effects of pulsed exposure. Here, we studied the effects of short-term (7 days) continuous and pulse exposure to 100 μg/L Cd on gut morphology and microbiota of frogs (Pelophylax nigromaculatus) during pre-hibernation. Compared to continuous exposure, Cd pulse exposure significantly increased individual mortality and decreased the villi height and the ratio of villi height to crypt depth of the gut. Cd continuous and pulse exposure both changed the community structure and relative abundance of intestinal microbiota. Compared to continuous exposure, Cd pulse exposure significantly decreased the relative abundance of beneficial bacteria (e.g., Cetobacterium and Aeromonas genus), and significantly increased the relative abundance of harmful bacteria (e.g., Parabacteroides, Odoribacter, and Acinetobacter genus). This study shows that the gut histology and microbiota of amphibians during pre-hibernation are more susceptible to Cd pulse exposure than continuous exposure.

Insight into the toxic effects, bioconcentration and oxidative stress of acetamiprid on Rana nigromaculata tadpoles

Pesticide pollution has been identified as a factor in the amphibian population decrease. Acetamiprid is a common neonicotinoid pesticide that poses a risk to amphibians due to its high water solubility and inability to be digested. However, there is little research on acetamiprid's toxicity in amphibians, particularly on its biochemical toxic effects. In this study, we investigated the acute toxicity, bioenrichment-elimination, biochemical parameters and metabolism of acetamiprid in Rana nigromaculata tadpoles. The results indicated that acetamiprid is harmful to Rana nigromaculata tadpoles, with an LC50 = 18.49 mg L−1 of 96 h for acute toxicity. Acetamiprid showed rapid accumulation and low bioconcentration levels in tadpoles, with bioconcentration factors (BCFs) < 1. In the elimination process, the concentration of acetamiprid decreased rapidly, with the elimination half-life t1/2 values < 1 d. Additionally, oxidative stress was observed in tadpoles, with biochemical parameters such as superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) being significantly altered. Nontargeted metabolomics revealed significant changes in biomolecules such as lipids, organic acids and nucleotides in tadpoles, and these metabolites influence pathways including serine and threonine metabolism, histidine metabolism, linoleic acid metabolism and sphingolipid metabolism. These results indicate that acetamiprid caused toxic effects on Rana nigromaculata tadpoles. Our study provides a better understanding of the fate and risk of acetamiprid in amphibians, as well as guidelines for its rational use.

A novel antimicrobial peptide found in Pelophylax nigromaculatus

BackgroundMany active peptides have been found in frog skin secretions. In this paper, our research focused on Pelophylax nigromaculatus and found a broad-spectrum antimicrobial peptide Nigrocin-PN based on the molecular cloning technique. Thereafter, the “Rana box” function was briefly studied by two mutated peptides (Nigrocin-M1 and Nigrocin-M2). Furthermore, in vitro and in vivo assays were used to characterize the peptide’s biofunctions, and the peptide’s function in treating multidrug-resistant pathogens was also studied. ResultsNigrocin-PN not only displayed potent antimicrobial abilities in vitro but also significantly ameliorated pulmonary inflammation induced by Klebsiella pneumoniae in vivo. By comparing, leucine-substituted analogue Nigrocin-M1 only displayed bactericidal abilities towards gram-positive bacteria, while the shorter analogue Nigrocin-M2 lost this function. More strikingly, Nigrocin-PN exhibited synergistic effects with commonly used antibiotics; in vitro evolution experiments revealed that coadministration between Nigrocin-PN and ampicillin could delay Staphylococcus aureus antibiotic resistance acquisition. Kinetics and morphology studies indicate that antibacterial mechanisms involved membrane destruction. Furthermore, toxicities and anticancer abilities of these peptides were also studied; compared to two analogues, Nigrocin-PN showed mild haemolytic activity and indistinctive cytotoxicity towards normal cell lines HMEC-1 and HaCaT. ConclusionsA broad-spectrum antimicrobial peptide Nigrocin-PN was discovered from the skin secretion of Pelophylax nigromaculatus. Structurally, “Rana box” played a crucial role in reducing toxicities without compromising antibacterial abilities, and Nigrocin-PN could be a desired therapeutic candidate. Graphical abstract▪ Highlights•For AMPs, disulphide bond can affect their biofunction and cytotoxicity.•Frog skin secretion is a reservoir to delve valuable peptides.•AMPs-antibiotics coadministration could be a strategy to delay drug resistance.

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.

Short-term continuous and pulse Pb exposure causes negative effects on skin histomorphological structure and bacterial composition of adult Pelophylax nigromaculatus.

Lead (Pb) is a toxic heavy metal often present in the environment as a pulse in water. Traditional toxicity tests are usually carried out under conditions of continuous concentration, without considering the impact of pulse exposure on aquatic organisms. This study aimed to evaluate the effects of short-term continuous and pulse Pb exposures on the skin bacteria and histomorphological structure ofPelophylax nigromaculatus. Results showed that compared to the control (CON) and Pb continuous exposure group (CEPb), the Pb pulse exposure group (PEPb) showed the smallest size of granular glands, which would interfere with the permeability and secretory function of skin, making the individual more sensitive to external pollution. Lead exposure significantly changed the composition and diversity of skin bacteria. Compared to the CON and CEPb groups, the PEPb group showed a significant increase in the abundance of harmful bacteria (e.g., Bacteroidetes and Chryseobacterium)and a decrease in the abundance of beneficial bacteria (e.g., Pseudomonas).PICRUSt software showed that there were differences in the metabolic pathway of skin bacteria among the three groups (CON, CEPb, and PEPb). Overall, this study indicates that Pb pulse exposure can aggravate the toxicity of Pb for frog skin, providing a new framework for simulating short-term heavy metal exposure in the context of frog health.