Skin microorganisms are an important component of host innate immunity and serve as the first line of defense against pathogenic infections. The relative abundance of bacterial species, microbial community assembly, and secretion of specific bacterial metabolites are closely associated with host health. In this study, we investigated the association between the skin microbiome and Ranavirus, and compared the bacterial community assemblage, alpha and beta diversity, and functional predictions of the skin bacterial assemblage in cultured healthy Chinese giant salamanders (Andrias davidianus) and individuals infected with Chinese giant salamander iridovirus (GSIV or ADRV). To achieve this, we employed 16S rRNA amplicon sequencing. The results identified Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteriota as the dominant phyla in the diseased and healthy groups. Alpha diversity analysis indicated that the skin bacterial community in the diseased group exhibited no significant differences in bacterial species diversity and lower species richness compared to the healthy group. Beta diversity suggested that the two group bacterial community was quite different. Kyoto encyclopedia of genes and genomes (KEGG) pathway analyze and clusters of orthologous groups of proteins (COG) function predictions revealed that changes and variations occurred in the metabolic pathways and function distribution of skin bacterial communities in two groups.

The Taibai stream salamander (Batrachuperus taibaiensis) is a recently described species of the genus Batrachuperus that occurs in the Zhouzhi Heihe River and is endangered in its native range. Here, we have established a method for water environmental DNA (eDNA) analysis of Batrachuperus using a series of optimizations. We have designed a specific set of primers for the genus Batrachuperus to amplify a 160 bp fragment of Cytb. The sequences were obtained from nested PCR on eDNA extracted from water samples, after which DNA barcoding was performed according to sequence analysis to determine the presence of the target species in the water. The method was validated using water from the Zhouzhi Heihe River with known B. taibaiensis populations and found that B. taibaiensis eDNA can move at least 150 m downstream from its point of origin. This study is the first to establish an optimal method for obtaining the eDNA of Batrachuperus from water samples, which provides a theoretical basis for resource investigation and the protection of B. taibaiensis in future research. It is also an example of the eDNA extraction of other species that live in similar waters and are less genetically diverse between species.

In recent years, the population of Brachymystax lenok tsinlingensis has experienced a rapid decline due to habitat degradation caused by overfishing and dam construction. In the current study, water samples were collected from four tanks containing B. lenok tsinlingensis individuals with wet weights of 5.8 g, 12.3 g, 34.7 g, and 81.7 g, respectively, and the cycle threshold (Ct) values of B. lenok tsinlingensis in each tank were measured using quantitative PCR. Additionally, water samples were collected from six sites along the Zhouzhi Heihe River (Yu Dongquan, Ban Fangzi, Ba Mugou, two sites along Wang Jia, and Qingshui Rivers), and the Ct values of B. lenok tsinlingensis at these sites were quantified. The results show that the relationship between the Ct values measured by the quantitative test and the fish biomass in the corresponding tanks followed an exponential function, with a regression coefficient of 0.9987. The biomass for the six sites was determined using the established relationship, and the results indicate that the biomass of B. lenok tsinlingensis was highest in Yu Dongquan, followed by Ban Fangzi, Ba Mugou, Wang Jia River 2, and Qingshui, with the lowest biomass observed in Wang Jia River 1. These findings are consistent with the species’ adaptive survival distribution across the six sites. This study presents a standardized approach for using an eDNA-based quantitative PCR assay to monitor B. lenok tsinlingensis. It provides an advanced technical method applicable to the protection and monitoring of other aquatic species in future studies.

Amphibians face severe threats from chytridiomycosis, and their skin microbiota plays a crucial role in pathogen defense. However, studies on their mycobiomes are limited. We hypothesized that amphibian cutaneous mycobiomes vary with seasonal variations and host species. To test this hypothesis, we used internal transcribed spacer (ITS) amplicon sequencing to identify the cutaneous fungal communities of two frogs from the Qinling Mountains of China, namely Pelophylax nigromaculatus and Nanorana quadranus. We also compared our ITS amplicon data with those of 30 known anti-Bd fungal ITS sequences to identify Bd-inhibiting fungi in the samples. The results showed that seasonal variation exerted a significantly stronger influence than host species on the fungal community structure (alpha diversity, beta diversity, species composition, abundance, and biomarkers). In the fungal community composition, intergroup consistency was significantly higher at the phylum level than at the genus level; however, one unidentified genus was present in samples from both frogs from different seasons. Anti-Bd fungi were detected in the skin fungal communities of P. nigromaculatus and N. quadranus, although their types and abundances varied seasonally and interspecifically. Overall, this study highlights seasonal dynamics and host-specific variations in amphibian cutaneous fungal ecology and identifies potential Bd-inhibiting fungal taxa.

The Brachymystax lenok tsinlingensis is an endangered endemic salmonid species facing severe threats from habitat fragmentation. Effective conservation requires non-invasive monitoring techniques and the identification of key habitat drivers. This study utilized environmental DNA (eDNA) quantitative PCR to estimate the spatial biomass distri-bution of the Brachymystax lenok tsinlingensis in the Zhouzhi Heihe River, integrating surveys of plankton, benthic macroinvertebrates, and multiple physicochemical factors (e.g., water depth, flow velocity, water temperature) to identify critical environmental influences. The results revealed significant spatial heterogeneity in fish biomass, with the highest values observed at river confluences and high-elevation sites. For instance, the confluence of the Heihe River and the Da Manggou River recorded the maximum biomass (1.5 × 10⁻² g/cm²), followed by the Hua Erping site (5.8 × 10⁻³ g/cm²). Among all measured variables, only water depth showed a significant positive correlation with Brachymystax lenok tsinlingensis biomass (r = 0.57). Spatial variations in plankton and benthic community structures reflected a water quality gradient from oligotrophic to eutrophic conditions, but their diversity was not directly linked to fish abundance. These findings indicate that the complexity of in-stream physical habitats—particularly the availability of deep pools—currently plays a more decisive role in the distribution of the Brachymystax lenok tsinlingensis than prey resource diversity. Therefore, conservation efforts should prioritize the restoration of structurally complex river habitats while maintaining high water quality to ensure the long-term survival of this species.

Brachymystax lenok tsinlingensis is an endangered salmonid endemic to China. Traditional trapping methods frequently fail to detect this rare fish in low-density mountain streams, hampering evidence-based conservation. Here, we employed environmental DNA quantitative PCR (eDNA-qPCR) with species-specific primers to assess the spatial biomass distribution of this species in the Zhouzhi Heihe River. Concurrently, we surveyed plankton, benthic macroinvertebrates, and physicochemical water parameters. eDNA detected the target species at 12 of 14 sites, with reliable quantification achieved at 9 sites, suggesting that the method may be more effective than conventional trapping for detecting this species under the studied low-density conditions. eDNA-derived relative biomass exhibited pronounced spatial heterogeneity, ranging from 6.0 × 10⁻⁴ to 1.5 × 10⁻² g/cm³. Water depth showed a significant positive association with biomass (r = 0.5347), whereas phytoplankton Shannon diversity (a measure of species richness and evenness) was significantly negatively correlated (r = −0.5447). Flow velocity displayed a negative trend that did not reach statistical significance (r = −0.5009). Plankton and benthic communities indicated overall ecological conditions but did not directly explain the observed spatial variation in fish biomass. These findings indicate that the spatial pattern of B. lenok tsinlingensis is primarily shaped by local physical habitat structure, with deeper, hydraulically more complex channel units serving as key microhabitats. eDNA-qPCR thus represents an effective, low-disturbance monitoring tool for this endangered cold-water fish and provides a scientific basis for targeted habitat protection and restoration.