Microbial diversity in mountain-dwelling amphibians: The combined effects of host and climatic factors.

Comprehending the determinants of host-associated microbiota is pivotal in microbial ecology. Yet, the links between climatic factors and variations in host-associated microbiota necessitate further clarification. Mountain-dwelling amphibians, with limited dispersal abilities, serve as valuable models for addressing these questions. Our study, using 126 amphibian-associated microbial samples (64 gut and 62 skin) and 101 environmental microbial samples (51 soil and 50 water) from the eastern Tibetan Plateau, revealed host factors as primary drivers of the variations in host-associated microbiota. However, climatic factors contributed to additional variations in gut microbial beta-diversity and skin microbial function. Water microbiota were identified as a significant contributor to the amphibian-associated microbiomes, with their climate-driven variations mediating an indirect association between the variations in climatic factors and host-associated microbiota. These findings extend our understanding of the assembly of host-associated microbiota in amphibians, emphasizing the significance of microbiota in evaluating the impact of climate change on animals.

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.

Identification of thyroid hormone response genes in the remodeling of dorsal muscle during Microhyla fissipes metamorphosis.

Introduction: Extensive morphological, biochemical, and cellular changes occur during anuran metamorphosis, which is triggered by a single hormone, thyroid hormone (TH). The function of TH is mainly mediated through thyroid receptor (TR) by binding to the specific thyroid response elements (TREs) of direct response genes, in turn regulating the downstream genes in the cascade. The remodeling of dorsal skeletal muscle during anuran metamorphosis provides the perfect model to identify the immediate early and direct response genes that are important during apoptosis, proliferation, and differentiation of the muscle. Methods: In our current study, we performed Illumina sequencing combined with single-molecule real-time (SMRT) sequencing in the dorsal muscle of Microhyla fissipes after TH, cycloheximide (CHX), and TH_CHX treatment. Results and Discussion: We first identified 1,245 differentially expressed transcripts (DETs) after TH exposure, many of which were involved in DNA replication, protein processing in the endoplasmic reticulum, cell cycle, apoptosis, p53 signaling pathway, and protein digestion and absorption. In the comparison of the TH group vs. control group and TH_CHX group vs. CHX group overlapping gene, 39 upregulated and 6 downregulated genes were identified as the TH directly induced genes. Further analysis indicated that AGGTCAnnTnAGGTCA is the optimal target sequence of target genes for TR/RXR heterodimers in M. fissipes. Future investigations on the function and regulation of these genes and pathways should help to reveal the mechanisms governing amphibian dorsal muscle remodeling. These full-length and high-quality transcriptomes in this study also provide an important foundation for future studies in M. fissipes metamorphosis.

Cascading effects of Pb on the environmental and symbiotic microbiota and tadpoles' physiology based on field data and laboratory validation.

Heavy metal pollution poses a serious threat to ecosystems. Currently, there is a lack of field data that would enable us to gain a systematic understanding of the influences of heavy metals on aquatic ecosystems, especially the interactions between environments and animals. We studied the relationships between the variations in heavy metal concentrations (10 species including Pb in sediments and surface water), the community structure of environmental and symbiotic microbiota, and the gut traits of Bufo gargarizans tadpoles across 16 sampling sites on the Chengdu Plain through rigorous statistical analysis and laboratory validation. The results show that heavy metal concentrations, especially the Pb concentration of the sediment, are linked to the variations in sediment and tadpoles' gut microbiomes but not to water microbiota. For the sediment microbiota, Pb causes a trade-off between the proportions of Burkholderiales and Verrucomicrobiae and affects the methane, sulfide, and nitrate metabolisms. For tadpoles, a high sediment Pb content leads to a low abundance of gut aerobic bacteria and a large relative gut weight under both field and laboratory conditions. In addition, Pb promotes the growth of B. gargarizans tadpoles under laboratory conditions. These effects seem to be beneficial to tadpoles. However, a high Pb content leads to a low abundance of probiotic bacteria (e.g., Verrucomicrobiae, Eubacteriaceae, and Cetobacterium) and a high abundance of pathogenic bacteria in the gut and environment, suggesting potential health risks posed by Pb. Interestingly, there is a causal relationship between Pb-induced variations in sediment and symbiotic microbiotas, and the latter is further linked to the variation in relative gut weight of tadpoles. This suggests a cascading effect of Pb on the ecosystem. In conclusion, our results indicate that among the heavy metals, the Pb in sediment is a critical factor affecting the aquatic ecosystem through an environment-gut-physiology pathway mediated by microbiota.

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.

Ti-substituted O3-type layered oxide cathode material with high-voltage stability for sodium-ion batteries.

O3-type layered transition metal oxides (NaxTMO2) have attracted extensive attention as a promising cathode material for sodium-ion batteries because of their high capacity. However, the irreversible phase transition especially cycled under high voltage remains a concerning challenge for NaxTMO2. Herein, a Ti-substituted NaNi0.5Co0.2Mn0.3O2 cathode with strongly suppressed phase transition and enhanced storage stability is investigated. The Ti substitution effectively inhibits the irreversible phase transition and alleviates the structural change even charged to 4.3 V during the repeated Na+ deintercalation process. After storing in air or water, the original O3 phase structure of the material is integrally maintained without the generation of impurity phase. As a result, the as-prepared material shows excellent long-term cycle stability and rate performance when charged to a high voltage of 4.3 V.

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.

Molecular and Cytogenetic Identification of Wheat-Thinopyrum intermedium Double Substitution Line-Derived Progenies for Stripe Rust Resistance.

Thinopyrum intermedium (2n = 6x = 42, JJJSJSStSt) has been hybridized extensively with common wheat and proven to be a valuable germplasm source for improving disease resistance and yield potential of wheat. A novel disease-resistant wheat-Th. intermedium double substitution line X479, carrying 1St(1B) and 4St-4JS (4B), was identified using multi-color non-denaturing fluorescence in situ hybridization (ND-FISH). With the aim of transferring Thinopyrum-specific chromatin to wheat, a total of 573 plants from F2 and F3 progenies of X479 crossed with wheat cultivar MY11 were developed and characterized using sequential ND-FISH with multiple probes. Fifteen types of wheat-Thinopyrum translocation chromosomes were preferentially transmitted in the progenies, and the homozygous wheat-1St, and wheat-4JSL translocation lines were identified using ND-FISH, Oligo-FISH painting and CENH3 immunostaining. The wheat-4JSL translocation lines exhibited high levels of resistance to stripe rust prevalent races in field screening. The gene for stripe rust resistance was found to be physically located on FL0-0.60 of the 4JSL, using deletion lines and specific DNA markers. The new wheat-Th. intermedium translocation lines can be exploited as useful germplasms for wheat improvement.

Physical location of tandem repeats in the wheat genome and application for chromosome identification.

MAIN CONCLUSION: A general distribution of tandem repeats (TRs) in the wheat genome was predicted and a new web page combined with fluorescence in situ hybridization experiments, and the newly developed Oligo probes will improve the resolution for wheat chromosome identification. Comprehensive sequence analysis of tandem repeats (TR) in the wheat reference genome permits discovery and application of TRs for chromosome identification. Genome-wide localization of TRs was identified in the reference sequences of Chinese Spring using Tandem Repeat Finder (TRF). A database of repeats unit size, array number, and physical coverage length of TRs in the wheat genome was built. The distribution of TRs occupied 3-5% of the wheat chromosomes, with non-random dispersal across the A, B, and D genomes. Three classes of TRs surrounding the predicted genes were compared. An optimized computer-assisted website page B2DSC was constructed for the general distribution and chromosomally enriched zones of TR sequences to be displayed graphically. The physical distribution of predicted TRs in the wheat genome by B2DSC matched well with the corresponding hybridization signals obtained with fluorescence in situ hybridization (FISH). We developed 20 oligonucleotide probes representing 20-60 bp lengths of high copy number of TRs and verified by FISH. An integrated physical map of TR-Oligo probes for wheat chromosome identification was constructed. Our results suggest that the combination of both molecular cytogenetics and genomic research will significantly benefit wheat breeding through chromosome manipulation and engineering.

Precise identification of wheat - Thinopyrum intermedium translocation chromosomes carrying resistance to wheat stripe rust in line Z4 and its derived progenies.

The wheat - Thinopyrum intermedium derived line Z4 has displayed novel and effective stripe rust resistance for over 40 years. This study aimed to precisely identify the chromosome constitution of line Z4 and determine the stripe rust resistance contribution using multicolor fluorescent in situ hybridization (FISH) and molecular marker analysis. The results indicated that the Z4 line (2n = 44) contained two pairs of non-Robertsonian translocations without the 3A chromosomes of wheat. FISH karyotypes of F3 progenies derived from crosses between Z4 and MY11 indicated that the transmission of the translocated chromosomes appeared normal and the number of wheat chromosomes 3A and 3D frequently varied. The FISH signal distribution of a new repetitive probe, named Oligo-3A1, confirmed the physical breakage points on chromosome 3AL incorporated into translocated chromosomes. PLUG markers revealed the breakage points on chromosomes 3A, 7JS, and 3D invloved in the translocated chromosomes, and they were designated as T3DS-3AS.3AL-7JSS and T3AL-7JSS.7JSL. Stripe rust resistances surveys indicated that the proximal region of 7JSS or 7JSL may confer the resistance at the adult plant stage. The precise characterization of the chromosome complements of wheat - Th. intermedium Z4 and derived progenies has demonstrated the importance of combining cytogenetic and molecular approaches in the genomics era for further wheat genetic manipulation and breeding purposes.