Rana Nigromaculata Research Articles

Seasonality influences skin bacterial community structure and anti-Bd function in two anuran species

Microorganisms on amphibian skin reduce disease susceptibility and play an important role in pathogen defense. We hypothesized that anuran skin bacterial communities would change in response to seasonal variation and host species. To test this hypothesis, we used 16S rRNA amplicon sequencing to identify cutaneous bacterial communities of two frogs from the Qinling Mountains of China, Pelophylax nigromaculatus and Nanorana quadranus. We matched the amplicon sequence variants (ASVs) of microbes exhibiting protective effects against the pathogen Batrachochytrium dendrobatidis (Bd), using a database containing over 1900 16S rRNA gene sequences from amphibian skin bacteria. The results showed that seasonal variation had a stronger effect than host species on the structure (alpha-diversity, beta-diversity, species composition and abundance, and biomarkers) and anti-Bd function of cutaneous bacterial communities. These communities were highly dynamic but varied similarly between hosts. Their structural similarities were more consistent at the phylum level, but markedly less so at finer taxonomic levels. The highest relative abundance of anti-Bd reads was observed in P. nigromaculatus during summer, but anti-Bd reads were present in both frog species during different seasons. Therefore, the protective function of cutaneous microbial communities appears to be continuous despite between-species differences in anti-Bd ASV abundance. This observation does not directly explain why Bd infections have not been recorded in the region, butprovides important insight on anuran pathogen defense mechanisms. Our findings also suggest that specific seasons can be periods of high infection risk, with major implications for research on amphibian ecology and conservation.

Environmental concentrations of 6PPD and 6PPD-quinone induce hepatic lipid metabolism disorders in male black-spotted frogs

Aquatic environments are generally contaminated with N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) and its oxidation product 6PPD-quinone (6PPD-Q). Recently, 6PPD-Q was found lethally toxic to some specific species, especially salmonid silverfish. This study investigated male black-spotted frogs (Pelophylax nigromaculatus) exposed to 6PPD and 6PPD-Q with different environmental concentrations (0, 1, and 10 μg/L) for 21 days, after which biochemical, metabolomic, gene expression analyses, and molecular docking were conducted. 6PPD and 6PPD-Q were both found to bioaccumulate in frogs’ livers in a dose-dependent manner and produce a significant reduction of the hepatosomatic index. Metabolomics data showed that 6PPD and 6PPD-Q induced distinct alterations in metabolite expression, predominantly within pathways associated with the biosynthesis of unsaturated fatty acids as well as the metabolism of arachidonic and linoleic acids. Exposure to 10 μg/L 6PPD and 6PPD-Q increased the cholesterol level by 2.22 and 4.35 folds, and the triglyceride level by 1.90 and 2.25 folds, respectively. 6PPD-Q inhibited the enzyme activity and gene expression involved in lipolysis, and promoted the lipid synthesis. Moreover, 6PPD and 6PPD-Q bound to peroxisome proliferators-activated receptors of α and γ. In conclusion, 6PPD and 6PPD-Q with environmental concentrations induced frogs’ lipid metabolism disorders. These findings contribute to our understanding of 6PPD and 6PPD-Q health risks in amphibians.

Effects of Breeding Habitat on the Abundance and Body Condition of Two <i>Pelophylax</i> Species in Agriculture and Urban Landscapes

Amphibians play a key role in structuring biological assemblages of agricultural landscapes, but they are threatened globally by agricultural intensification. Numerous studies have considered the morphology of animals to be an indicator of the health of a population, but data about the differences in amphibian morphology in different breeding habitats in agricultural landscapes are still missing. Here, we investigated the differences in the abundance and morphology of Pelophylax plancyi and Pelophylax nigromaculatus in the three waterbody types: fields, forests, and parks in agricultural landscapes in Nanjing, China. Our results emphasize that both frog species in forests and parks had significantly higher body condition indexes than those in fields, whereas their abundance in fields habitat was higher than those of the two other habitat types. Our results demonstrated the importance of non-agricultural habitats (such as forests and parks) in agricultural landscapes to the persistence of both Pelophylax species. We recommend preserving field ecosystems by constructing diverse waterbodies other than fields in rural areas and maintaining microhabitats in parks and forests to improve amphibian biodiversity in Nanjing.

Synthesis of a novel β-cyclodextrin chiral stationary phase and its application to the evaluation of the enantioselective bioaccumulation and elimination behavior of tebuconazole in Rana nigromaculata tadpoles

BackgroundThe increased production and use of chiral pesticides will enhance their exposure in the environment. Chiral pesticides typically exhibit varied biological effects among these enantiomers. Therefore, it is very essential to develop and validate chiral analytical methods to investigate their potential ecological risks from a stereoselective perspective. Current separation of pesticides enantiomers relies extensively on chiral stationary phases (CSPs), while the development of β-Cyclodextrin derivatives CSPs become the research focus due to their great modifiability and excellent chiral recognition capabilities. ResultsA novel chiral stationary phase, 3,5-dichlorophenylaminomethyl-6-phenylenediamine-β-cyclodextrin chemically bonded silica gel (MPDCDA), was successfully prepared. Based on that, a stereoselective HPLC-MS/MS method was developed and validated for the determination of tebuconazole enantiomers in Rana nigromaculata tadpoles. After extraction by QuEChERS, the tebuconazole enantiomers were completely separated with the resolutions of 1.63 using the mobile phase of methanol-water (70/30, v/v). Good linearity (r > 0.9990) for both enantiomers over a concentration range of 0.20–500.0 ng/mL was obtained with the accuracy ranged from 6.7 % to 9.3 % and the intra-day and inter-day precisions below 6.2 % at three quality control levels. The proposed method was successfully applied in evaluating the enantioselective bioaccumulation and elimination profiles of tebuconazole in tadpoles. At the tested conditions, there was no significantly enantioselective difference in the bioaccumulation process for S- tebuconazole and R-tebuconazole. However, the elimination process of tebuconazole enantiomers was enantioselective with R-tebuconazole preferentially degraded. SignificanceThis work provided an accurate risk assessment of chiral pesticides to non-target aquatic organisms from a stereoselective perspective. These findings would deepen our understanding of the potential ecological risks of chiral pesticides on aquatic organisms and provide scientific support for the protection of aquatic organisms and their ecological environments, as well as sustainable development.

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.

Combined effect and mechanism of microplastic with different particle sizes and levofloxacin on developing Rana nigromaculata: Insights from thyroid axis regulation and immune system

Microplastics (MPs) usually appear in the aquatic environment as complex pollutants with other environmental pollutants, such as levofloxacin (LVFX). After 45-day exposure to LVFX and MPs with different particle sizes at environmental levels, we measured the weight, snout-to-vent length (SVL), and development stages of Rana nigromaculata. Furthermore, we analyzed proteins and genes related to immune system and thyroid axis regulation, intestinal histological, and bioaccumulation of LVFX and MPs in the intestine and brain to further explore the toxic mechanism of co-exposure. We found MPs exacerbated the effect of LVFX on growth and development, and the order of inhibitory effects is as follows: LVFX-MP3>LVFX-MP1>LVFX-MP2. 0.1 and 1 μm MP could penetrate the blood–brain barrier, interact with LVFX in the brain, and affect growth and development by regulating thyroid axis. Besides, LVFX with MPs caused severer interference on thyroid axis compared with LVFX alone. However, 10 μm MP was prone to accumulating in the intestine, causing severe histopathological changes, interfering with the intestinal immune system and influencing growth and development through immune enzyme activity. Thus, we concluded that MPs could regulate the thyroid axis by interfering with the intestinal immune system.

Metabolomics reveals the lipid metabolism disorder in Pelophylax nigromaculatus exposed to environmentally relevant levels of microcystin-LR

Cyanobacterial blooms have emerged as a significant environmental issue worldwide in recent decades. However, the toxic effects of microcystin-LR (MC-LR) on aquatic organisms, such as frogs, have remained poorly understood. In this study, frogs (Pelophylax nigromaculatus) were exposed to environmentally relevant concentrations of MC-LR (0, 1, and 10 μg/L) for 21 days. Subsequently, we assessed the impact of MC-LR on the histomorphology of the frogs' livers and conducted a global MS-based nontarget metabolomics analysis, followed by the determination of substances involved in lipid metabolism. Results showed that MC-LR significantly induced histological alterations in the frogs' hepatopancreas. Over 200 differentially expressed metabolites were identified, primarily enriched in lipid metabolism. Biochemical analysis further confirmed that MC-LR exposure led to a disorder in lipid metabolism in the frogs. This study laid the groundwork for a mechanistic understanding of MC-LR toxicity in frogs and potentially other aquatic organisms.

UV radiation-induced peptides in frog skin confer protection against cutaneous photodamage through suppressing MAPK signaling.

Overexposure to ultraviolet light (UV) has become a major dermatological problem since the intensity of ultraviolet radiation is increasing. As an adaption to outside environments, amphibians gained an excellent peptide-based defense system in their naked skin from secular evolution. Here, we first determined the adaptation and resistance of the dark-spotted frogs (Pelophylax nigromaculatus) to constant ultraviolet B (UVB) exposure. Subsequently, peptidomics of frog skin identified a series of novel peptides in response to UVB. These UV-induced frog skin peptides (UIFSPs) conferred significant protection against UVB-induced death and senescence in skin cells. Moreover, the protective effects of UIFSPs were boosted by coupling with the transcription trans-activating (TAT) protein transduction domain. In vivo, TAT-conjugated UIFSPs mitigated skin photodamage and accelerated wound healing. Transcriptomic profiling revealed that multiple pathways were modulated by TAT-conjugated UIFSPs, including small GTPase/Ras signaling and MAPK signaling. Importantly, pharmacological activation of MAPK kinases counteracted UIFSP-induced decrease in cell death after UVB exposure. Taken together, our findings provide evidence for the potential preventive and therapeutic significance of UIFSPs in UV-induced skin damage by antagonizing MAPK signaling pathways. In addition, these results suggest a practicable alternative in which potential therapeutic agents can be mined from organisms with a fascinating ability to adapt.

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.

Cytochrome P450 enzymes in the black-spotted frog (Pelophylax nigromaculatus): molecular characterization and upregulation of expression by sulfamethoxazole.

Cytochrome P450 (CYP) enzymes are crucial for the detoxification of xenobiotics, cellular metabolism, and homeostasis. This study investigated the molecular characterization of CYP enzymes in the black-spotted frog, Pelophylax nigromaculatus, and examined the regulation of CYP expression in response to chronic exposure to the antibiotic sulfamethoxazole (SMX) at various environmental concentrations (0, 1, 10, and 100μg/L). The full-length cDNA of Pn-CYP26B1 was identified. The sequence included open reading frames of 1,536 bp, encoding proteins comprising 511 amino acids. The signature motif, FxxGxxxCxG, was highly conserved when compared with a number of selected animal species. SMX significantly upregulated the expression of the protein CYP26B1 in frog livers at concentrations of 1 and 10μg/L. SMX showed an affinity for CYP26B1 of -7.6kcal/mol, indicating a potential mechanism for SMX detoxification or adaptation of the frog. These findings contributed to our understanding of the environmental impact of antibiotics on amphibian species and underscored the importance of CYP enzymes in maintaining biochemical homeostasis under exposure to xenobiotic stress.

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.

An esculentin-1 homolog from a dark-spotted frog (Pelophylax nigromaculatus) possesses antibacterial and immunoregulatory properties

BackgroundEsculentin-1, initially discovered in the skin secretions of pool frogs (Pelophylax lessonae), has demonstrated broad-spectrum antimicrobial activity; however, its immunomodulatory properties have received little attention.ResultsIn the present study, esculentin-1 cDNA was identified by analysing the skin transcriptome of the dark-spotted frog (Pelophylax nigromaculatus). Esculentin-1 from this species (esculentin-1PN) encompasses a signal peptide, an acidic spacer peptide, and a mature peptide. Sequence alignments with other amphibian esculentins-1 demonstrated conservation of the peptide, and phylogenetic tree analysis revealed its closest genetic affinity to esculentin-1P, derived from the Fukien gold-striped pond frog (Pelophylax fukienensis). Esculentin-1PN transcripts were observed in various tissues, with the skin exhibiting the highest mRNA levels. Synthetic esculentin-1PN demonstrated antibacterial activity against various pathogens, and esculentin-1PN exhibited bactericidal activity by disrupting cell membrane integrity and hydrolyzing genomic DNA. Esculentin-1PN did not stimulate chemotaxis in RAW264.7, a murine leukemic monocyte/macrophage cell line. However, it amplified the respiratory burst and augmented the pro-inflammatory cytokine gene (TNF-α and IL-1β) expression in RAW264.7 cells.ConclusionsThis novel finding highlights the immunomodulatory activity of esculentin-1PN on immune cells.

Effects of atrazine on movement, metabolism and gene expression in Pelophylax nigromaculatus larvae under global warming

Global warming and environmental pollutants both pose a threat to the behavior and physiology of animals, but research on the combined effects of the two is limited. Atrazine, a widely used herbicide, has toxic effects on organisms. In this study, the effects of environmental concentrations of atrazine exposure (100 μg/L) for seven days on the movement, metabolism and gene expression related to motility of Pelophylax nigromaculatus larvae (GS8) were investigated under global warming. The results showed that compared to the optimal growth temperature (18 °C), atrazine treatment under global warming (21 °C) significantly increased the average speed (about 11.2 times) and maximum acceleration (about 1.98 times) of P. nigromaculatus larvae, altered the relative abundance of 539 metabolites, including Formyl-5-hydroxykynurenamine, 2,4-Dihydroxybenzophenone, and FAPy-adenine, and changed the nucleotide metabolism, pyrimidine metabolism, glycerophospholipid metabolism, and purine metabolism, as well as increased the gene expression of SPLA2 (about 6.46 times) and CHK (about 3.25 times). In summary, atrazine treatment under global warming caused metabolic disorders in amphibian larvae and increased the expression of some movement-related genes in the brain, resulting in abnormally active.

First Report of Endemic Frog Virus 3 (FV3)-like Ranaviruses in the Korean Clawed Salamander (Onychodactylus koreanus) in Asia.

Frog virus 3 (FV3) in the genus Ranavirus of the family Iridoviridae causes mass mortality in both anurans and urodeles worldwide; however, the phylogenetic origin of FV3-like ranaviruses is not well established. In Asia, three FV3-like ranaviruses have been reported in farmed populations of amphibians and reptiles. Here, we report the first case of endemic FV3-like ranavirus infections in the Korean clawed salamander Onychodactylus koreanus, caught in wild mountain streams in the Republic of Korea (ROK), through whole-genome sequencing and phylogenetic analysis. Two isolated FV3-like ranaviruses (Onychodactylus koreanus ranavirus, OKRV1 and 2) showed high similarity with the Rana grylio virus (RGV, 91.5%) and Rana nigromaculata ranavirus (RNRV, 92.2%) but relatively low similarity with the soft-shelled turtle iridovirus (STIV, 84.2%) in open reading frame (ORF) comparisons. OKRV1 and 2 formed a monophyletic clade with previously known Asian FV3-like ranaviruses, a sister group of the New World FV3-like ranavirus clade. Our results suggest that OKRV1 and 2 are FV3-like ranaviruses endemic to the ROK, and RGV and RNRV might also be endemic strains in China, unlike previous speculation. Our data have great implications for the study of the phylogeny and spreading routes of FV3-like ranaviruses and suggest the need for additional detection and analysis of FV3-like ranaviruses in wild populations in Asian countries.

Integrated transcriptome and SNP analysis reveals sex-related genetic insights in the black-spotted frog (Pelophylax nigromaculatus)

Integrated transcriptome and SNP analysis reveals sex-related genetic insights in the black-spotted frog (Pelophylax nigromaculatus)

Impact of an environment-friendly rice-production method on the population density of Pelophylax nigromaculatus frogs

Impact of an environment-friendly rice-production method on the population density of Pelophylax nigromaculatus frogs

Defenses of whirligig beetles against native and invasive frogs.

Many native insects have evolved defenses against native predators. However, their defenses may not protect them from non-native predators due to a limited shared history. The American bullfrog, Aquarana catesbeiana (Anura: Ranidae), which has been intentionally introduced to many countries, is believed to impact native aquatic animals through direct predation. Adults of whirligig beetles (Coleoptera: Gyrinidae), known for swimming and foraging on the water surface of ponds and streams, reportedly possess chemical defenses against aquatic predators, such as fish. Although whirligig beetles potentially encounter both bullfrogs and other frogs in ponds and lakes, the effectiveness of their defenses against frogs has been rarely studied. To assess whether whirligig beetles can defend against native and non-native frogs, we observed the behavioral responses of the native pond frog, Pelophylax nigromaculatus (Anura: Ranidae), and the invasive non-native bullfrog, A. catesbeiana, to native whirligig beetles, Gyrinus japonicus and Dineutus orientalis, in Japan. Adults of whirligig beetles were provided to frogs under laboratory conditions. Forty percent of G. japonicus and D.orientalis were rejected by P. nigromaculatus, while all whirligig beetles were easily consumed by A. catesbeiana. Chemical and other secondary defenses of G. japonicus and D. orientalis were effective for some individuals of P. nigromaculatus but not for any individuals of A. catesbeiana. These results suggest that native whirligig beetles suffer predation by invasive non-native bullfrogs in local ponds and lakes in Japan.

Exploration of the Antibacterial and Anti-Inflammatory Activity of a Novel Antimicrobial Peptide Brevinin-1BW.

Antibiotic resistance has emerged as a grave threat to global public health, leading to an increasing number of treatment failures. Antimicrobial peptides (AMPs) are widely regarded as potential substitutes for traditional antibiotics since they are less likely to induce resistance when used. A novel AMP named Brevinin-1BW (FLPLLAGLAASFLPTIFCKISRKC) was obtained by the Research Center of Molecular Medicine of Yunnan Province from the skin of the Pelophylax nigromaculatus. Brevinia-1BW had effective inhibitory effects on Gram-positive bacteria, with a minimum inhibitory concentration (MIC) of 3.125 μg/mL against Enterococcus faecalis (ATCC 29212) and 6.25 μg/mL against both Staphylococcus aureus (ATCC 25923) and multidrug-resistant Staphylococcus aureus (ATCC 29213) but had weaker inhibitory effects on Gram-negative bacteria, with a MIC of ≥100 μg/mL. Studies using scanning electron microscopy (SEM) and flow cytometry have revealed that it exerts its antibacterial activity by disrupting bacterial membranes. Additionally, it possesses strong biofilm inhibitory and eradication activities as well as significant lipopolysaccharide (LPS)-binding activity. Furthermore, Brevinin-1BW has shown a significant anti-inflammatory effect in LPS-treated RAW264.7 cells. In conclusion, Brevinin-1BW is anticipated to be a promising clinical agent with potent anti-Gram-positive bacterial and anti-inflammatory properties.

Whole-genome sequencing and analysis of Chryseobacterium arthrosphaerae from Rana nigromaculata

Chryseobacterium arthrosphaerae strain FS91703 was isolated from Rana nigromaculata in our previous study. To investigate the genomic characteristics, pathogenicity-related genes, antimicrobial resistance, and phylogenetic relationship of this strain, PacBio RS II and Illumina HiSeq 2000 platforms were used for the whole genome sequencing. The genome size of strain FS91703 was 5,435,691 bp and GC content was 37.78%. A total of 4,951 coding genes were predicted; 99 potential virulence factors homologs were identified. Analysis of antibiotic resistance genes revealed that strain FS91703 harbored 10 antibiotic resistance genes in 6 categories and 2 multidrug-resistant efflux pump genes, including adeG and farA. Strain FS91703 was sensitive to β-lactam combination drugs, cephem, monobactam and carbapenems, intermediately resistant to phenicol, and resistant to penicillin, aminoglycosides, tetracycline, fluoroquinolones, and folate pathway inhibitors. Phylogenetic analysis revealed that strain FS91703 and C. arthrosphaerae CC-VM-7T were on the same branch of the phylogenetic tree based on 16 S rRNA; the ANI value between them was 96.99%; and the DDH values were 80.2, 72.2 and 81.6% by three default calculation formulae. These results suggested that strain FS91703 was a species of C. arthrosphaerae. Pan-genome analysis showed FS91703 had 566 unique genes compared with 13 other C. arthrosphaerae strains, and had a distant phylogenetic relationship with the other C. arthrosphaerae strains of the same branch in phylogenetic tree based on orthologous genes. The results of this study suggest that strain FS91703 is a multidrug-resistant and highly virulent bacterium, that differs from other C. arthrosphaerae strains at the genomic level. The knowledge about the genomic characteristics and antimicrobial resistance of strain FS91703 provides valuable insights into this rare species, as well as guidance for the treatment of the disease caused by FS91703 in Rana nigromaculata.

Effects of ramp slope and substrate type on the climbing success of Pelophylax nigromaculatus in agricultural landscapes

Habitat fragmentation has become a serious threat to amphibian populations worldwide. Concrete anti-seepage, widely applied in waterway construction, has posed a threat by causing amphibians to easily fall into concrete irrigation channels and struggle to escape. However, our understanding of the impact of irrigation channels on frog movement and survival remains limited. In this study, we evaluated the effects of concrete-lined irrigation channels on the escape ability of a common Chinese frog (Pelophylax nigromaculatus). We also analyzed the correlation between the morphology and locomotion ability of the frogs, tested the impact of irrigation channel slope gradient and substrate material on their escape ability, and proposed an optimized design for frog ramps to assist trapped frogs in escaping. The results showed that the frogs have easily fallen into these channels and encountered difficulties in escaping during field activities. As body size was positively related to the frogs’ ability to climb the slope of irrigation channels, the design parameters of the frog ramps should focus on the movement ability of the frogs. The maximum climbing gradients for most frogs on concrete, reverse slope (the protruding structure is placed in the opposite direction of the channel slope), grass and crushed stone are 40°, 50°, 60° and 65°, respectively. Based on experimental tests of the ability of the frogs to climb on various slopes and substrates, we suggest that the slope gradient of concrete irrigation channels should be less than 40°. The findings offer references for scholars and engineers to improve ecological engineering designs of irrigation channels throughout the world.