Single cell RNA analysis uncovers the cell differentiation and functionalization for air breathing of frog lung.

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.

Why Bufo gargarizans tadpoles grow bigger in Pb-contaminated environments? The gut microbiota matter.

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.

Size matters either way: Differently-sized microplastics affect amphibian host and symbiotic microbiota discriminately.

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 element and host silencing activity in gigantic genomes.

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.

How will warming affect the growth and body size of the largest extant amphibian? More than the temperature-size rule.

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.

Rising floor and dropping ceiling: organ heterogeneity in response to cold acclimation of the largest extant amphibian.

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 of Andrias 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.

Multi-Omics Approaches Revealed the Associations of Host Metabolism and Gut Microbiome With Phylogeny and Environmental Adaptation in Mountain Dragons.

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.

Effects of Habitat River Microbiome on the Symbiotic Microbiota and Multi-Organ Gene Expression of Captive-Bred Chinese Giant Salamander.

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.

From Water to Land: The Structural Construction and Molecular Switches in Lungs during Metamorphosis of Microhyla fissipes.

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 effects of corticosterone and background colour on tadpole physiological plasticity.

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.

Gene Expression Program Underlying Tail Resorption During Thyroid Hormone-Dependent Metamorphosis of the Ornamented Pygmy Frog Microhyla fissipes.

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.

Population genetic diversity of an odorous frog Odorrana grahami (Amphibia: Anura: Ranidae) in relation to conservation based on mitochondrial DNA.

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.

Skin innate immunity of diskless-fingered odorous frogs (Odorrana grahami) with spatial-temporal variations.

The skin innate immunities of diskless-fingered odorous frogs (Odorrana grahami) from three populations were investigated. The antimicrobial capacities of skin secretions against the 60 representative environmental bacterial strains were evaluated using the values of the minimum inhibitory concentration (MIC) equivalents, which were defined as the volumes of antimicrobial solution just inhibiting the tested bacteria per 1 cm2 of surface area, from 0.06 to 9.10 mL/cm2. Our results revealed significantly different skin antimicrobial capacities among the three populations: Mianning < Huili < Kunming. Within the frog population, the skin antimicrobial capacities are highly variable depending on the season: in Mianning frogs, summer < autumn and spring; in Huili frogs, spring < autumn < summer; in Kunming frogs, autumn < spring < summer. The animal density and body mass significantly impacted the skin antimicrobial capacity, while the sex ratio and soil or water bacterial counts did not.

Identification and differential regulation of microRNAs during thyroid hormone-dependent metamorphosis in Microhyla fissipes.

BACKGROUND: Anuran metamorphosis, which is obligatorily initiated and sustained by thyroid hormone (TH), is a dramatic example of extensive morphological, biochemical and cellular changes occurring during post-embryonic development. Thus, it provides an ideal model to understand the actions of the hormone and molecular mechanisms underlying these developmental and apoptotic processes. In addition to transcriptional factors, microRNAs (miRNAs) play key roles in diverse biological processes via post-transcriptional repression of mRNAs. However, the possible role of miRNAs in anuran metamorphosis is not well understood. Screening and identification of TH-responding miRNAs are required to reveal the integrated regulatory mechanisms of TH during metamorphosis. Given the specific role of TRs during M. fissipes metamorphosis and the characteristics of M. fissipes as an ideal model, Illumina sequencing technology was employed to get a full scope of miRNA in M. fissipes metamorphosis treated by T3. RESULTS: Morphological and histological analysis revealed that 24 h T3 treatment M. fissipes tadpoles resembled that at the climax of natural metamorphosis. Thus, small RNA libraries were constructed from control and 24 h T3 treatment groups. A total of 164 conserved miRNAs and 36 predicted novel miRNAs were characterized. Furthermore, 5' first and ninth nucleotides of miRNAs were significantly enriched in U in our study. In all, 21 miRNAs were differentially expressed between the T3 and control groups (p < 0.01). A total of 10,206 unigenes were identified as target genes of these differentially expressed miRNAs. KEGG pathway analysis indicated that the most overrepresented miRNA target genes were enriched in the "PI3k-Akt signaling pathway". In addition, a network associated with the TH signaling pathway provides an opportunity to further understand the complex biological processes that occur in metamorphosis. CONCLUSIONS: We identified a large number of miRNAs during M. fissipes metamorphosis, and 21 of them were differentially expressed in the two groups that represented two different metamorphic stages. These miRNAs may play important roles during metamorphosis. The study gives us clues for further studies of the mechanisms of anuran metamorphosis and provides a model to study the mechanism of TH-affected biological processes in humans.

Mass Spectrometry Approaches for Determining the Structure of Antimicrobial Peptides.

In the past decades, a great amount of antimicrobial peptides (AMPs) has been discovered, the structure identification of which relies heavily on de novo sequencing by Edman degradation or mass spectrometry. Here we outline the basic procedures for the exact mass measurement approaches that use off-line low-energy CID ESI Qq-TOF MS/MS in positive-ion mode, which is typically applied to de novo sequencing of peptides, to elucidate the structure of AMPs. Ambiguity I/L and partial sequence order were elucidated by Edman degradation or/and structural similarity analysis to known sequence. The approaches can determine the structure of peptides composed of as much as 38 amino acids in our practice.

Antimicrobial peptides from the skin of the Asian frog, Odorrana jingdongensis: de novo sequencing and analysis of tandem mass spectrometry data.

Eight intact antimicrobial peptides were identified from the skin of Odorrana jingdongensis by de novo sequencing following low energy ESI CID Q-TOF MS/MS in positive-mode with the help of Edman degradation and structural similarity analysis. We devised exact mass measurements to discriminate the K/Q amino acid residue in the peptides between 2.0 kDa to 3.8 kDa. Moreover, the cleavage at the CS bond at the side chain of Met was observed in all the spectra of the peptides containing Met residue. And we found unusual cleavages within the intramolecular disulfide loop with high frequency. Our data revealed that the cleavage pathways are significantly different from those reported previously which are similar to the cycle peptide cleavage mode followed by the secondary cleavage at the CS bond on oxidized Cys. Thus, our results highly suggest that ion series generated from the cleavages within the intramolecular disulfide loop should be considered in both the top-down sequencing and the disulfide bridge location with the presence of a relatively high intensity of MH(+)-28 ion marker. Furthermore, our activity data implied that different AMPs may use different strategies to kill microbes.

[In vitro activities of Odorrana grahami antimicrobial peptides from skin against 5 pathogenic bacteria].

OBJECTIVE: To evaluate the efficiency of 4 Odorrana grahami antimicrobial peptides from skin against 5 pathogenic bacteria, include 2 wild strains and 7 resistant strains. METHODS: Broth microdilution antimicrobial susceptibility test was used for bacteria that growed aerobically. RESULTS: The 4 Odorrana grahami antimicrobial peptides were basically in vitro efficient agents for inhibition against to methicillin-resistant coagulase negative Staphylococcus (MRSCN, 85460), wild Staphylococcus aureus (24157), penicillin-resistant Streptococcus pneumoniae (PRSP, 84688 and 91452), class I beta-lactamase Enterobacter cloacae (AmpC, 85439 and 93543), wild Escherichia coli (84492), extended-spectrum beta-lactamases Escherichia coli (ESBL, 84492), inhabitor-resistant TEM beta-lactamase Escherichia coli (IRT, 85580). CONCLUSION: The 4 Odorrana grahami antimicrobial peptides from skin have broad spectrum antimicrobial activity; especially have in vitro activity to resistant strains. So it is hopeful to be developed as the antimicrobial agent as well as the disinfectant and the antiseptic.