Rongquan Zheng

Acrossocheilus fasciatus is an economically important freshwater fish species in China, and infection with Citrobacter freundii poses a serious threat to the sustainable development of its aquaculture industry. In this study, histopathological examination, TUNEL staining, immune physiological assays, and whole-transcriptome sequencing were integrated to systematically characterize the expression profiles of lncRNAs, circRNAs, miRNAs, and mRNAs in the spleen of A. fasciatus following C. freundii infection. The results showed that infection caused marked tissue damage in the intestine, spleen, and kidney, significantly increased splenic cell apoptosis, and induced pronounced changes in immune physiological parameters. Whole-transcriptome analysis identified 88 differentially expressed lncRNAs, 193 differentially expressed circRNAs, 140 differentially expressed miRNAs, and 1935 differentially expressed mRNAs. KEGG enrichment analysis revealed that the differentially expressed mRNAs were mainly enriched in immune-related pathways, including neutrophil extracellular trap formation, complement and coagulation cascades, antigen processing and presentation, cytokine–cytokine receptor interaction, phagosome, chemokine signaling pathway, and IL-17 signaling pathway. Furthermore, a bioinformatically predicted lncRNA-miRNA-mRNA ceRNA network was constructed, comprising 10 mRNAs, 14 lncRNAs, and 22 miRNAs, which formed 44 putative regulatory interactions. In this network, miR-212-y may mediate the regulation of Il12rb2 and PIK3R1, whereas miR-8-y may be involved in the regulation of Ccr9 and PIK3R1. The qRT-PCR validation results were consistent with the RNA-seq expression trends. This study provides the first comprehensive characterization of non-coding RNA and mRNA expression profiles in the spleen of A. fasciatus after C. freundii infection and identifies potential ceRNA regulatory networks, providing a valuable reference for elucidating antibacterial immune mechanisms and screening candidate molecular markers for disease resistance research in A. fasciatus.

Male spiny frogs ( Quasipaa spinosa) develop seasonal chest spines during the breeding season, but the structural basis, molecular changes, and reproductive role of this trait remain unclear. Therefore, to clarify how structural and molecular remodeling of the chest skin work together to form this important reproductive trait, we performed AB-PAS histological staining, quantitative proteomic analysis, and real-time quantitative PCR validation on male skin tissues. We then combined these analyses with spine blunting, reproductive competition experiments, and SSR-based paternity testing to examine chest spine formation and its relationship with male reproductive success. We found that Q. spinosa chest skin has specialized glandular structures and a collagen-rich dermal framework, suggesting that it provides a structural basis for later spine development. During the reproductive cycle, chest spine formation is accompanied by significant changes in extracellular matrix (ECM)-related proteins and pathways, including focal adhesions and ECM–receptor interactions. Moreover, the expression of genes related to the TGF-β/Smad signaling pathway and matrix remodeling was dynamically altered during this process. Reproductive experiments revealed that males with better-developed chest spines produced more offspring, whereas artificial blunting of the spines reduced reproductive success. Together, these results suggest that seasonal chest spine formation in Q. spinosa is closely related to extracellular matrix remodeling and contributes to male reproductive success during the breeding season. These findings provide practical implications for broodstock selection, conservation breeding, and the management of reproductive fitness in amphibian populations. In particular, chest spine development may serve as a practical indicator of male reproductive performance, linking an evolutionarily selected secondary sexual trait to reproductive management.

Pangolin-associated viruses have received considerable attention because of their diversity and potential zoonotic relevance, yet the evidence remains fragmented. This review evaluates the diversity, evolutionary adaptation, and zoonotic spillover risks of pangolin-associated viruses. Diverse RNA and DNA viruses, such as coronaviruses, paramyxoviruses, astroviruses, parvoviruses, and papillomaviruses, have been detected in pangolins, showing substantial taxonomic and genomic variation. Studies of selected pangolin-associated viral groups have documented sequence variation and identified signals of recombination or reassortment. Some pangolin coronaviruses can use human receptors and replicate in human-derived cells, organoids, or transgenic animal models. Several viruses known to infect humans have also been detected in pangolin samples, although conclusive evidence of natural spillover from pangolins to humans is lacking. Most studies examine Sunda and Chinese pangolins from illegal trade or rescue centers, relying largely on nucleic acid detection and sequence analysis. Habitat disruption, wildlife trafficking, long-distance transport and captive-rescue operations may increase cross-host–pathogen exposure. Future work should prioritize surveillance of wild pangolin populations of known provenance and integrate virus isolation, tissue localization, serology, and functional assays to support pangolin conservation and reliable zoonotic-risk evaluation.

The Chinese giant salamander (CGS, Andrias davidianus) is an endangered aquatic amphibian in China. Using environmental DNA (eDNA), this study aimed to monitor the spatial distribution of this species in Zhejiang Province, providing a scientific basis for species assessment, evaluation of reintroduction effectiveness, and non-invasive monitoring. Based on historical records of species presence, 18 representative water localities were selected for sampling. Among these localities, eDNA signals of the CGS were detected at nine. After excluding sampling sites affected by historical CGS releases and surrounding breeding farms, Jinzi Peak and Huyuan were inferred as candidate potentially natural stream habitats within the province. Additionally, eDNA monitoring was conducted at two release sites, Gutian Mountain and Banshanyuan, for two consecutive years. Results revealed lower detection rates in both areas in the second year of monitoring, potentially attributed to low survival rates, dispersal and disappearance, or sampling variability. A principal component analysis (PCA) of water quality factors (including parameters such as NH₃-N, TN, TP, COD) revealed distinct geographic clustering of CGS-positive and CGS-negative sampling sites that corresponded to their separation in topographic space, suggesting that the measured physicochemical conditions may be associated with the CGS eDNA detection probability. This study demonstrates that eDNA is a feasible and efficient method for surveying CGS in Zhejiang Province. Further, it provides an efficient non-invasive monitoring approach for endangered amphibians, with broad application prospects.

Variations in sexual size dimorphism (SSD) have important consequences for animal ecology, behavior, population dynamics, and the evolution of life‐history traits, and can be explained by many intraspecific and interpopulation hypotheses. Urbanization is an important factor that affects anuran phenotypic characteristics (including morphology), but how it impacts anuran SSD has been explored less widely. In this study, we aimed to investigate the differences in SSD in two dominant amphibian species populations; that is, the Hong Kong rice‐paddy frog ( Fejervarya multistriata ) and Beijing gold‐striped pond frog ( Pelophylax plancyi ), across an urban–rural gradient in Shanghai (including urban, suburban, and rural areas). We also tested whether these variations in SSD supported Rensch's rule or not. Our results showed that in both sexes, the snout‐vent length (SVL) in F. multistriata was higher in urban areas compared with suburban and rural areas. In addition, only male P. plancyi exhibited a higher SVL in urban areas compared with those in rural areas. Furthermore, we found that the SSD in SVL was significantly higher for urban populations than suburban and rural populations of F. multistriata . All SSD results in terms of SVL, head width, forelimb length, and hindlimb length in these two frog species supported the inverse Rensch's rule based on reduced major axis regression. Moreover, the size scaling rates in these two frog species were higher in urban and suburban populations than rural populations. Thus, we concluded that urbanization shapes SSD in these two anurans according to the inverse of Rensch's rule, and that variations in morphological characteristics, SSD, and size scaling rates differed between these two anuran species regarded as an “urban‐associated species” ( P. plancyi ) and “urban‐sensitive species” ( F. multistriata ).

Gut microbiota play a crucial regulatory role in host energy metabolism and environmental adaptation, with their composition and function significantly influenced by dietary shifts. This study employed high‐throughput sequencing to analyze the dietary composition of Myotis chinensis and the seasonal dynamics of its gut microbiota, with functional predictions used to explore adaptive changes in metabolic pathways. Results indicated notable seasonal variations in dietary composition: Lepidoptera constituted the majority in spring (60.13%), and the diet transitioned to Orthoptera dominance in early summer (78.19%), with Odonata becoming the leading prey in late summer (79.99%), and Arachnida (47.93%) became the dominant taxon in autumn. Gut microbial diversity reached its maximum in late summer and was at its minimum during autumn. Functional predictions indicated significant upregulation of carbohydrate and amino acid metabolism pathways in summer, while lipid metabolism and hibernation‐related pathways were enriched in autumn. These synergistic dietary and microbial functional adjustments help Myotis chinensis adapt to seasonal energy demands and climatic extremes. This study provided novel insights into the ecological adaptation mechanisms of insectivorous bats.

The accumulation of heavy metals in rice (Oryza sativa L.) compromises food safety and endangers public health. Previous studies have postulated that ecological co-cultivation systems can potentially improve soil quality and reduce crop absorption of heavy metals. Herein, three treatment groups, rice mono-culture (CG), low-density rice–frog co-culture (LRF), and high-density rice–frog co-culture (HRF), were employed to evaluate the effects of rice–frog co-culture on the physicochemical properties of soils in reclaimed rice fields and heavy metal accumulation in rice. Notably, the rice–frog co-culture markedly increased levels of soil organic matter (SOM), dissolved organic carbon (DOC), cation exchange capacity (CEC), pH, and redox potential (Eh) (p < 0.05), particularly under high-density conditions, compared to the mono-culture system. These changes significantly reduced the bioavailable fractions of Cd, As, and Hg in the soil and substantially diminished their uptake in the roots, stems, leaves, and grains of rice. Conversely, the co-cultivation systems increased the bioavailable content and plant uptake of Pb, particularly under high-density conditions. These findings highlight the feasibility of the rice–frog co-cropping systems in improving soil conditions and reducing the accumulation of specific toxic metals within rice, thereby enhancing the safety of rice grown in reclaimed fields. However, increased Pb accumulation warrants further investigation.

The black-spotted frog (Pelophylax nigromaculatus) is a common economic species in the rice–frog ecological cropping mode. The present study investigated microbial community structures in paddy water and black-spotted frog’s guts across rice monoculture and low-/high-density rice–frog co-cropping systems at four rice growth stages. Proteobacteria dominate in paddy water, while frog guts are enriched in Firmicutes and Actinobacteriota. The frog density shows no impact on the α-diversity, but rice growth stages significantly alter the Shannon, Simpson, and Pielou_e indices (p < 0.01). Co-cropping may promote amino acid synthesis, elemental cycling, and stress tolerance in paddy water microbiota, which are more diverse than gut microbiota. Strong correlations exist between paddy water and gut microbiotas, with Limnohabitans being linked to gut diversity (p < 0.05). Low-density co-cropping enhances Xenorhabdus, which is beneficial for pest control and stabilizes gut microbiota. The results of this study offer insights for managing rice–frog systems based on rice growth stages.

Reclaimed fields have a low soil fertility and low productivity compared to conventional arable land, necessitating research on productivity enhancement. The rice–frog co-culture model is an ecologically intensive practice that combines biodiversity objectives with agricultural production needs, offering high ecological and economic value. However, there is a lack of research on this model that has focused on factors other than soil nutrient levels. The present study evaluated the rice–frog co-culture model in a reclaimed paddy field across three experimental plots with varying frog stocking densities: a rice monoculture (CG), low-density co-culture (LRF), and high-density co-culture (HRF). We investigated the effects of the frog density on greenhouse gas emissions throughout the rice growth. The rice–frog co-culture model significantly reduced methane (CH₄) emissions, with fluxes highest in the CG plot, followed by the LRF and then HRF plots. This reduction was achieved by altering the soil pH, the cation exchange capacity, the mcrA gene abundance, and the mcrA/pmoA gene abundance ratio. However, there was a contrasting nitrous oxide (N₂O) emission pattern. The co-culture model actually increased N₂O emissions, with fluxes being highest in the HRF plots, followed by the LRF and then CG plots. The correlation analysis identified the soil nosZ gene abundance, redox potential, urease activity, nirS gene abundance, and ratio of the combined nirK and nirS abundance to the nosZ abundance as key factors associated with N₂O emissions. While the co-cultivation model increased N₂O emissions, it also significantly reduced CH₄ emissions. Overall, the rice–frog co-culture model, especially at a high density, offers a favorable sustainable agricultural production model.

As a mammal with unique ecological habits, the Chinese pangolin, Manis pentadactyla, exhibits nocturnal behavior and has a hidden, cave-dwelling lifestyle. Severe habitat degradation and fragmentation have led to a rapid decline in its wild population. Moreover, the immature state of artificial breeding technology restricts the availability of research samples, particularly with regard to behavioral and ecological data. This makes it extremely difficult to continuously monitor the pangolin’s ecological habits and population dynamics for a long time. The comprehensive research archive is yet to be established, greatly limiting the in-depth understanding of this species’ ecological behavior. To mitigate this data shortage, this study installed GPS positioning and accelerometer technology devices on five Chinese pangolins that were rescued from Zhejiang and released into the wild. Activity rhythms and home ranges were then analyzed based on the collected data. The results showed that all the five released Chinese pangolins were active in the wild and survived throughout the tracking period (21–72 days). The pangolins were mainly active at night, with activity starting after 18:00 and being mainly concentrated between 22:00 and 4:00. The home range of the Chinese pangolins based on MCP was 777.41 ± 426.26 hm², and the core range based on KDE was 596.21 ± 265.58 hm² (50% KDE). This result confirms that high-frequency GPS positioning combined with accelerometer data can effectively determine the home range sizes, habitat utilization, and fine-scale activity rhythms of wild-released pangolins.