The Chinese giant salamander (CGS, Andrias davidianus), a flagship amphibian species, is highly vulnerable to high temperatures, posing a significant threat under future climate change. Previous research linked this susceptibility to liver energy deficiency, accompanied by shifts in gut microbiota and reduced food conversion rates, raising questions about the role of the gut-liver axis in mediating heat sensitivity. This study investigated the responses of Chinese giant salamander larvae to a temperature gradient (10–30 °C), assessing physiological changes alongside histological, gut metagenomic, and tissue transcriptomic analyses. Temperatures above 20 °C led to mortality, which resulted in delayed growth. Histological and transcriptomic data revealed metabolic exhaustion and liver fibrosis in heat-stressed salamanders, underscoring the liver’s critical role in heat sensitivity. While heat stress altered the gut microbiota’s community structure, their functional profiles, especially in nutrient absorption and transformation, remained stable. Both gut and liver showed temperature-dependent transcriptional changes, sharing some common variations in actins, heat shock proteins, and genes related to transcription and translation. However, their energy metabolism exhibited opposite trends: it was downregulated in the liver but upregulated in the gut, with the gut showing increased activity in the pentose phosphate pathway and oxidative phosphorylation, potentially countering metabolic exhaustion. Our findings reveal that the liver of the larvae exhibits greater thermal sensitivity than the gut, and the gut-liver axis plays a limited role in mediating thermal intolerance. This study enhances mechanistic understanding of CGS heat susceptibility, providing a foundation for targeted conservation strategies in the face of climate change.

Polyploidization plays a pivotal role in vertebrate evolution and diversification. However, the effects of polyploidization on animals across various biological levels, and how these differences drive ecological shifts, remain unclear. Through karyotype analysis and whole-genome sequencing, we identified an autotetraploid Microhyla fissipes from Hainan Island, which shows reproductive isolation and geographic differentiation from its diploid counterpart. Tetraploids exhibited larger cell size, improved tadpole growth rates, and greater whole-body size, along with reduced cell cycle activity. Rather than being simple scaled-up diploids, tetraploids showed shifts in physiological performance, organ allometry, gene expression profiles, and metabolic patterns. Tetraploid adults demonstrated superior jumping ability and increased reproductive investment (e.g. larger gonads and steeper slopes in the relationship between gonadal weight and body weight), suggesting a potential competitive advantage over diploids. However, tetraploids exhibited higher energy expenditure at elevated temperatures, reduced hepatic energy storage, and altered pulmonary regulatory metabolites at 25 °C. Males had smaller relative heart sizes, and females showed flatter slopes in the relationship between heart and lung weight and body weight, indicating reduced investment in cardiopulmonary system. These variations suggest an increased risk of metabolic constraints under heat stress, putting tetraploids at a disadvantage in warmer regions. Importantly, the physiological tradeoffs associated with polyploidization help explain the geographical differentiation between diploids and tetraploids, which reflects a climatic boundary, with tetraploids occupying cooler northeastern areas. Our findings identify an autotetraploid frog, report the first autotetraploid genome in amphibians, and demonstrate how vertebrate polyploids physiologically and ecologically diverge from their diploid counterparts.

The widespread use of antimicrobial agent triclosan (TCS) poses significant health risks to both aquatic organisms and humans. The research on its neurotoxicity and underlying mechanisms is, however, limited. Here we first conducted a 32-day exposure experiment with five TCS concentrations (10, 30, 60, 90 and 120 µg/L) to investigate its impact on overall gene expression in Rana omeimontis larvae. Transcriptomics analysis unveiled a strong dose-dependent pattern of gene expression alterations, with a distinct transcriptomic shift observed in the T030 (30 µg/L) group. In addition, neurodegenerative disease pathway and oxidative stress response GO (gene ontology) terms were found to be highly enriched across the regulated genes in all TCS-exposed groups, suggesting potential TCS-induced neurotoxicity. To further explore this, we performed a 40-day experiment with a low (30 µg/L) or high (90 µg/L) TCS concentration. Morphological assessments revealed that TCS-exposed larvae exhibited developmental and growth inhibition. Using RT-qPCR and immunohistochemical analysis, we confirmed that TCS exposure induced neurotoxicity and triggered neurodegenerative diseases as suggested by Tau protein aggregation in the midbrain. Consistent with these findings, TCS-exposed larvae displayed abnormal behaviors. Our study thus for the first time presents a comprehensive assessment of the adverse effects of TCS exposure on amphibian larvae, encompassing morphological, biochemical, and physiological aspects. Notably, we identified RNF112 and Tau as potential molecular targets that may mediate TCS-induced neurotoxicity. These findings advance the knowledge on how organisms respond to environmental changes and highlight the importance of further investigation into the potential neurotoxicity of TCS within aquatic ecosystems and its implications for human health.

Diet can influence the thermal performance of ectotherms, providing potential strategies for biological conservation in the context of global warming. The endangered Andrias davidianus is susceptible to heat stress due to energy deficiency in the liver when fed a worm-based diet rich in carbohydrates. A fish-based diet, rich in protein and lipids, improves their thermal performance, but the underlying physiological mechanisms remain unclear. In this study, we used metabolomics and metagenomics to examine the combined effects of temperature (15, 20, and 25°C) and diet (fish-based and worm-based) on liver metabolism and gut microbiota. Our results show that both temperature and diet shape liver metabolism, with several vital metabolic pathways (e.g., TCA cycle and sulfate metabolism) regulated by their combined effects. Notably, diet-dependent thermal responses in energy metabolism were observed, with fish-fed salamanders exhibiting a marked upregulation of the TCA cycle intermediates under heat stress, a response absent in worm-fed individuals. Given the role of TCA cycle in heat susceptibility of A. davidianus, these findings suggest that the TCA cycle likely mediates the interactive effects of temperature and diet on thermal performance. We then examined whether the gut microbiota is also a target of interactive effects or a mediator of the diet’s influence on liver metabolism. While both temperature and diet shape microbiota composition, functional shifts occur only in response to temperature, indicating that the microbiota is not a major link between diet and liver metabolism. However, several bacterial groups (e.g., Thiosulfatimonas and Alcanivorax), jointly regulated by temperature and diet, correlate with liver metabolites, suggesting alternative, function-independent pathways through which dietary-related microbial changes may influence liver metabolism and even thermal tolerance. Overall, this study provides molecular insights into the dietary modulation of thermal performance in A. davidianus and highlight the potential of dietary microbial management strategies for amphibian conservation.

Bufotes taxkorensis and B. zamdaensis are toads endemic to China. Both species inhabit highelevation and saline environments, and so provide a unique opportunity to investigate the adaptive responses of amphibians to extreme habitats. In the present study, we first observed and measured the histological structures of their skin and kidney in B.taxkorensis and B. zamdaensis. We then compared these results with similar findings for the low-elevation seawater dweller Fejervarya cancrivora and, finally,contrasted them with available data on species living in diverse habitats. Our results revealed the following adaptations:(1) In the Bufotes species, the epidermis and dermis both contain capillary vessels, facilitating blood-gas exchange and promoting adaptation to high-elevation hypoxia. The thick pigment layers present in the ventral skin of B. taxkorensis also help in adaptation to lower temperatures. In addition, the relative epidermis thickness of the dorsal and ventral skin is significantly greater in Bufotes species than in F.cancrivora, indicating adaptation to a terrestrial habitat.Comparing the terrestrial and semiaquatic species, we find that total dorsal and ventral thicknesses are both significantly greater in terrestrial than in semiaquatic species, helping to reduce water evaporation and damage from crawling, and thus promoting adaptation to terrestrial life.(2) The relative kidney filtration area of the Bufotes species is not significantly different from that of F. cancrivora, but accounts for only half that of terrestrial Bufo species. This lower total relative filtration area prevents excessive urea from being filtered out, avoiding excessive loss of body water and promoting adaptation to saline conditions. At the same time, the relative diameter of the proximal segment of Bufotes species is found to be similar to that of the semiaquatic F. cancrivora,facilitating the resorption of water and ions to increase plasma osmolality and promoting adaptation to saline environments. The findings of the present study offer insights into the adaptive mechanisms of amphibians in extreme environments and different habitats.

Endogenous secretory peptides (ESPs) play crucial roles in amphibian skin, yet their identification remains challenging in species lacking genomic data. This study developed an innovative pipeline integrating transcriptomics and peptidomics to identify ESPs in the skin of Odorrana grahami. This approach enhances endogenous secretory protein prediction accuracy by rescuing candidates erroneously discarded during SignalP-based screening. Such false negatives typically result from inaccurate annotation of N-terminal start sites within 5′-UTRs by protein prediction tools like TransDecoder. Our approach enhanced potential endogenous secretory protein identification rates by 61.6

The evolution and development of vertebrate lungs have been widely studied due to their significance in terrestrial adaptation. Amphibians possess the most primitive lungs among tetrapods, underscoring their evolutionary importance in bridging the transition from aquatic to terrestrial life. However, the intricate process of cell differentiation during amphibian lung development remains poorly understood. Using single-cell RNA sequencing, we identify 13 cell types in the developing lungs of a land-dwelling frog (Microhyla fissipes). We elucidate the differentiation trajectories and mechanisms of mesenchymal cells, identifying five cell fates and their respective driver genes. Using temporal dynamics analyses, we reveal the gene expression switches of epithelial cells, which facilitate air breathing during metamorphosis. Furthermore, by integrating the published data from another amphibian and two terrestrial mammals, we illuminate both conserved and divergent cellular repertoires during the evolution of tetrapod lungs. These findings uncover the frog lung cell differentiation trajectories and functionalization for breathing in air and provide valuable insights into the cell-type evolution of vertebrate lungs. Single-cell atlas of Microhyla fissipes lungs reveals cell differentiation and functionalization during metamorphosis, further cross-species comparison sheds light vertebrate lung cell type evolution.

The Chinese giant salamander (CGS, Andrias davidianus), a flagship amphibian species, is highly vulnerable to high temperatures, posing a significant threat under future climate change. Previous research linked this susceptibility to liver energy deficiency, accompanied by shifts in gut microbiota and reduced food conversion rates, raising questions about the role of the gut-liver axis in mediating thermal intolerance. This study investigated CGS responses to a temperature gradient (10–30°C), assessing physiological changes alongside histological, gut metagenomic, and tissue transcriptomic analyses. Temperatures above 20°C led to mortality and delayed growth. Histological and transcriptomic data revealed metabolic exhaustion and liver fibrosis in heat-stressed salamanders, underscoring the liver's critical role in thermal intolerance. While heat stress altered the gut microbiota's community structure, their functional profiles, especially in nutrient absorption and transformation, remained stable. Both gut and liver showed temperature-dependent transcriptional changes, sharing some common variations in actins, heat shock proteins, and genes related to transcription and translation. However, their energy metabolism exhibited opposite trends: it was downregulated in the liver but upregulated in the gut, with the gut showing increased activity in the pentose phosphate pathway and oxidative phosphorylation, potentially countering metabolic exhaustion. These findings suggest that the gut and its microbiota are less sensitive to high temperatures than the liver, and the gut-liver axis may not be central to CGS thermal sensitivity. This study enhances mechanistic understanding of CGS heat susceptibility, providing a foundation for targeted conservation strategies in the face of climate change.

Concerns about the implications of microplastics (MPs) on aqueous animals have gained widespread attention. It has been postulated that the magnitude of MPs can influence its toxicity. However, little is known about how MPs toxicity changes with particle size. Amphibians are reliable bioindicators of ecosystem health due to their complex life cycles. In this study, we compared the influences of two sizes nonfunctionalized polystyrene microspheres (1 and 10 μm) on the metamorphosis of Asiatic toad (Bufo gargarizans). Acute exposure to MPs at high concentrations led to bioaccumulation in the digestive track and internal organs (i.e., liver and heart) of tadpoles. Long-term exposure to either size, at environmentally-related concentrations (1 and 4550 p/mL), led to growth and development delay in pro-metamorphic tadpoles. Remarkably, developmental plasticity mitigated these deleterious effects prior to the onset of metamorphic climax without compromising survival rate in later stages. MPs with a diameter of 10 μm dramatically altered the gut microbiota (e.g., abundance of Catabacter and Desulfovibrio) of pro-metamorphic tadpoles, whereas MPs with a diameter of 1 μm induced much more intensive transcriptional responses in the host tissues (e.g., upregulation of protein synthesis and mitochondrial energy metabolism, and downregulation of neural functions and cellular responses). Given that the two MPs sizes induced similar toxic outcomes, this suggests that their principal toxicity mechanisms are distinct. Small-sized MPs can travel easily across the intestinal mucosa and cause direct toxicity, while large-sized MPs accumulate in gut and affect the host by changing the homeostasis of digestive track. In conclusion, our findings indicate that MPs can affect the growth and development of amphibian larvae, but their developmental plasticity determines the ultimate detrimental effects. Multiple pathways of toxicity may contribute to the size-dependent toxicity of MPs. We anticipate that these findings will increase our understanding of the ecological effects of MPs.

Transposable elements (TEs) and the silencing machinery of their hosts are engaged in a germline arms-race dynamic that shapes TE accumulation and, therefore, genome size. In animal species with extremely large genomes (>10 Gb), TE accumulation has been pushed to the extreme, prompting the question of whether TE silencing also deviates from typical conditions. To address this question, we characterize TE silencing via two pathways—the piRNA pathway and KRAB-ZFP transcriptional repression—in the male and female gonads of Ranodon sibiricus, a salamander species with a ∼21 Gb genome. We quantify 1) genomic TE diversity, 2) TE expression, and 3) small RNA expression and find a significant relationship between the expression of piRNAs and TEs they target for silencing in both ovaries and testes. We also quantified TE silencing pathway gene expression in R. sibiricus and 14 other vertebrates with genome sizes ranging from 1 to 130 Gb and find no association between pathway expression and genome size. Taken together, our results reveal that the gigantic R. sibiricus genome includes at least 19 putatively active TE superfamilies, all of which are targeted by the piRNA pathway in proportion to their expression levels, suggesting comprehensive piRNA-mediated silencing. Testes have higher TE expression than ovaries, suggesting that they may contribute more to the species’ high genomic TE load. We posit that apparently conflicting interpretations of TE silencing and genomic gigantism in the literature, as well as the absence of a correlation between TE silencing pathway gene expression and genome size, can be reconciled by considering whether the TE community or the host is currently “on the attack” in the arms race dynamic.

The impacts of lead/Pb2+ on ecosystems have received widespread attention. Growth suppression is a major toxic effect of Pb compounds on aquatic animals, however, some studies have also reported their growth-promoting effects. These complex outcomes may be explained by anions that accompany Pb2+ or by the multiple toxic mechanisms/pathways of Pb2+. To examine these hypotheses, we tested how Bufo gargarizans tadpoles responded to Pb(NO3)2 (100 and 200 μg/L Pb2+) using transcriptomics and microbiomics, with NaNO3 and blank groups as controls. Tadpoles exposed to Pb(NO3)2 showed delayed development while increased somatic growth in a dose-dependent manner, which can be attributed to the effects of NO3- and Pb2+, respectively. Tadpole transcriptomics revealed that exposure to NO3- downregulated the MAPK pathway at transcriptional level, explaining the development-suppressing effect of NO3-; while Pb2+ upregulated the transcription of detoxification pathways (e.g., xenobiotics metabolism by cytochrome P450 and glutathione metabolism), indicating cellular stress and thus contradicting the growth advantage of Pb2+-exposed tadpoles. Pb2+ exposure changed the tadpole gut microbiota drastically, characterized by increased polysaccharides and carbohydrate utilization while decreased fatty acid and amino acid consumption according to microbial functional analysis. Similar gut microbial variations were observed in field-collected tadpoles from different Pb2+ environments. This metabolic shift in gut microbiota likely improved the overall food utilization efficiency and increased the allocation of fatty acids and amino acids to the host, explaining the growth advantage of Pb2+-exposed tadpoles. In summary, our results suggest multiple toxic pathways of Pb2+, and the gut microbiota may affect the pollution outcomes on animals.

Declining body size is a universal ecological response to global warming in ectotherms. Ectotherms grow faster but mature at a smaller size at higher temperatures. This phenomenon is known as the temperature-size rule (TSR). However, we know little about the details of the relationship between temperature and size. Here, this issue was studied in the Chinese giant salamander (Andrias davidianus), one of the largest extant amphibians and a flagship species of conservation in China. Warm-acclimated A. davidianus larvae (25 °C) had accelerated development but little superiority in body growth when compared to their 15 °C counterparts when fed with red worm. This predicts a drastic decrease in adult body size with warming. However, a fish diet (more abundant lipid and protein) improved the growth performance at 25 °C. The underlying mechanism was studied. Warm-acclimated larvae had enlarged livers but shortened tails (fat depot). Their livers suffered from energy deficiencies and decreased protein levels, even when protein synthesis and energy metabolism were transcriptionally upregulated. This could be a direct explanation for their poor growth performance. Further analyses revealed a metabolic disorder resembling mammal glycogen storage disease in warm-acclimated larvae, indicating deficiency in glycogen catabolism. This speculation is consistent with their increased lipid and amino acid catabolism and explained the poor energy conditions of the warm-acclimated larvae. Additionally, a deficiency in glycogen metabolism explains the different efficiency of worm and fish diets in supporting the growth of warm-acclimated larvae, even when both diets were provided sufficiently. In conclusion, our results suggest that the relationship between temperature and body size can be flexible, which is a significant finding in terms of the TSR. The underlying metabolic and nutrient mechanisms were revealed. This knowledge can help deepen our understanding of the consequences of warming and can contribute to the conservation of A. davidianus.

The molecular basis enabling the adaptation of animals to spatially heterogeneous environments is a critical clue for understanding the variation, formation, and maintenance of biodiversity in the context of global climate change. Mountain dragons (Agamidae: Diploderma) thrive in the Hengduan Mountain Region, a biodiversity hotspot and a typical spatially heterogeneous environment. Here, we compare the liver and muscle metabolome and gut microbiome of 11 geographical populations from three Diploderma species (D. iadinum, D. yulongsense, and D. vela) after 7 days acclimation in the same laboratory conditions. Amino acid metabolism, particularly the products of the glutathione cycle, accounted for major interspecies variations, implying its significance in genetic differentiation among mountain dragons. Notably, the cold-dwelling D. vela and D. yulongense populations tended to have higher glycerophosphate, glycerol-3-phosphocholine, and kinetin levels in their liver, higher carnosine levels in their muscle, and higher Lachnospiraceae levels in their gut. Phylogeny, net primary productivity (NPP), and the temperature had the highest explanation rate to the variations in muscle metabolome, liver metabolome, and gut microbiome, respectively, suggesting heterogeneity of biological systems in response to climatic variations. Therefore, we suggested that the organ heterogeneity in environmental responsiveness might be substantial for mountain dragons to thrive in complicated environments.

Most anurans must undergo metamorphosis to adapt to terrestrial life. This process enhances the air-breathing ability of the lungs to cope with the change in oxygen medium from water to air. Revealing the structural construction and molecular switches of lung organogenesis is essential to understanding the realization of the air-breathing function. In this study, histology and transcriptomics were conducted in combination to explore these issues in Microhyla fissipes’ lungs during metamorphosis. During the pro-metamorphic phase, histological structural improvement of the alveolar wall is accompanied by robust substrate metabolism and protein turnover. The lungs, at the metamorphic climax phase, are characterized by an increased number of cilia in the alveolar epithelial cells and collagenous fibers in the connective tissues, corresponding to the transcriptional upregulation of cilia and extracellular matrix-related genes. Post-metamorphic lungs strengthen their contracting function, as suggested by the thickened muscle layer and the upregulated expression of genes involved in muscle contraction. The blood–gas barrier is fully developed in adult lungs, the transcriptional features of which are tissue growth and regulation of differentiation and immunity. Importantly, significant transcriptional switches of pulmonary surfactant protein and hemoglobin facilitate air breathing. Our results illuminated four key steps of lung development for amphibians to transition from water to land.

The reintroduction of captive-bred individuals is a primary approach to rebuild the wild populations of the Chinese giant salamander (Andrias davidianus), the largest extant amphibian species. However, the complexity of the wild habitat (e.g., diverse microorganisms and potential pathogens) potentially threatens the survival of reintroduced individuals. In this study, fresh (i.e., containing environmental microbiota) or sterilized river sediments (120°C sterilized treatment) were added to the artificial habitats to treat the larvae of the Chinese giant salamander (control group—Cnt: 20 individuals, treatment group 1 with fresh river sediments—T1: 20 individuals, and treatment group 2 with sterilized river sediments—T2: 20 individuals). The main objective of this study was to test whether this procedure could provoke their wild adaptability from the perspective of commensal microbiotas (skin, oral cavity, stomach, and gut) and larvae transcriptomes (skin, spleen, liver, and brain). Our results indicated that the presence of habitat sediments (whether fresh or sterilized) reshaped the oral bacterial community composition. Specifically, Firmicutes decreased dramatically from ~70% to ~20–25% (mainly contributed by Lactobacillaceae), while Proteobacteria increased from ~6% to ~31–36% (mainly contributed by Gammaproteobacteria). Consequently, the proportion of antifungal operational taxonomic units (OTUs) increased, and the function of oral microbiota likely shifted from growth-promoting to pathogen defense. Interestingly, the skin microbiota, rather than the colonization of habitat microbiota, was the major source of the pre-treated oral microbiota. From the host perspective, the transcriptomes of all four organs were changed for treated individuals. Specifically, the proteolysis and apoptosis in the skin were promoted, and the transcription of immune genes was activated in the skin, spleen, and liver. Importantly, more robust immune activation was detected in individuals treated with sterilized sediments. These results suggested that the pathogen defense of captive-bred individuals was improved after being treated, which may benefit their survival in the wild. Taken together, our results suggested that the pre-exposure of captive-bred Chinese giant salamander individuals to habitat sediments could be considered and added into the reintroduction processes to help them better adapt to wild conditions.

Low temperature imposes strong selective pressure on ectotherms. To maximize their overall fitness under cold conditions, the ectotherms may either try to maintain their physiological activities through metabolic compensation or enter into metabolic depression; however, some species adopt both strategies to cope with different degrees of cold. Nevertheless, how these two seemingly opposite strategies are coordinated has rarely been elucidated. Here, we investigated the molecular strategy underlying the cold acclimation ofAndrias davidianus, the largest extant amphibian, using multi-organ metabolomics and transcriptomics. The results showed remarkable organ heterogeneity in response to cold. While most organs showed transcriptional upregulation of metabolic processes, the heart exhibited downregulation. This heterogeneity explained the adaptive reorganization in resource allocation, which compensates for metabolic maintenance by compromising growth. Importantly, the cardiac function might constitute a ‘ceiling’ to constrain the space for compensation, especially under colder conditions. Additionally, the opposite transcriptional regulation of oxidative phosphorylation and other pathways might also shape the overall metabolic capacity under cold conditions. The heterogeneity in cold responses may have directed a shift in cold adaptive strategy from compensation to depression with a drop in temperature. These results provide a novel insight into the regulatory mechanisms underlying cold survival strategies of ectotherms.

Corticosterone (CORT)-mediated adaptive plasticity improves animal fitness in stressful environments. Although it brings ecological benefits, the cost potentially constrains its expression and evolution. Revealing the factors affecting plasticity costs is of great ecological and evolutionary significance. Evidence indicates that both CORT and background colour can induce metabolic changes in animals, which in turn determine phenotypic plasticity. However, whether and/or how CORT and background colour jointly act on plastic responses has not been studied. Here, this question has been investigated in amphibian tadpoles (Microhyla fissipes) exposed to CORT at different background colours (white or black) using integrated morphological, histological, and transcriptomic analyses. The results showed that CORT exposure increased relative tail length, immune function, and metabolic maintenance (i.e., transcription of substrate catabolism and oxidative phosphorylation) at the expense of reduction in growth rate and skin melanin level. The black background also increased relative tail length and metabolic maintenance (i.e., transcription of oxidative phosphorylation) at the cost of reduction in growth rate, but increased skin melanin level. The expression of critical pigmentation genes indicated that black background activated a distinct and opposite pigmentation regulating route to CORT. Although there was no interactive effect of background colour and CORT on phenotypic and metabolic variations, their additive effects further impact the trade-off between somatic growth, metabolic maintenance, and pigmentation in terms of resource allocation. In conclusion, the individual and additive effects of background colour and CORT exposure on tadpole plasticity were revealed. These results likely provide new insights into the environmental adaptation of animals.

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

Thyroid hormone (T3) is essential for vertebrate development, especially during the so-called postembryonic development, a period around birth in mammals when plasma T3 level peaks and many organs mature into their adult form. Compared to embryogenesis, postembryonic development is poorly studied in mammals largely because of the difficulty to manipulate the uterus-enclosed embryos and neonates. Amphibian metamorphosis is independent of maternal influence and can be easily manipulated for molecular and genetic studies, making it a valuable model to study postembryonic development in vertebrates. Studies on amphibian metamorphosis have been largely focused on the two highly related species Xenopus laevis and Xenopus tropicalis. However, adult X. laevis and X. tropicalis animals remain aquatic. This makes important to study metamorphosis in a species in which postmetamorphic frogs live on land. In this regard, the anuran Microhyla fissipes represents an alternative model for developmental and genetic studies. Here we have made use of the advances in sequencing technologies to investigate the gene expression profiles underlying the tail resorption program during metamorphosis in M. fissipes. We first used single molecule real-time sequencing to obtain 67, 939 expressed transcripts in M. fissipes. We next identified 4,555 differentially expressed transcripts during tail resorption by using Illumina sequencing on RNA samples from tails at different metamorphic stages. Bioinformatics analyses revealed that 11 up-regulated KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and 88 Gene Ontology (GO) terms as well as 21 down-regulated KEGG pathways and 499 GO terms were associated with tail resorption. Our findings suggest that tail resorption in M. fissipes and X. laevis shares many programs. Future investigations on function and regulation of these genes and pathways should help to reveal the mechanisms governing amphibian tail resorption and adaptive evolution from aquatic to terrestrial life. Furthermore, analysis of the M. fissipes model, especially, on the changes in other organs associated with the transition from aquatic to terrestrial living, should help to reveal important mechanistic insights governing mammalian postembryonic developments.

The diskless-fingered odorous frog Odorrana grahami is widely distributed in the high-altitude mountains in the southwestern China and northern Indochina regions. In this study, a comparative analysis of the mitochondrial COI gene sequences was performed to examine the population genetic diversity of 76 individuals from 10 localities across the distributional range of the species. Haplotype diversity and nucleotide diversity were 0.605 and 0.00199, respectively, in the total population. An AMOVA indicated that 47.3% of the total variation originated from variation within individual populations and 52.7% came from variation between the 10 geographic populations. Tests of neutral evolution indicated that a recent expansion occurred in total population. The findings provide useful information for the conservation of this species.