Fish diversity, an important indicator of aquatic ecosystem health, is declining due to water pollution, overfishing, climate change, and invasive species. Effective surveying and monitoring are required to protect fish diversity. Here, a high-sensitivity environmental DNA (eDNA) metabarcoding technique was used to investigate fish diversity in the Danjiang River, Shaanxi Province, China. In total, 59 species were identified in eight orders, 19 families, and 40 genera. Cypriniformes and Perciformes were the main groups in the survey area, while Cyprinidae accounted for 50.85% of the total fish species. Rhinogobius similis (19%), Hemibarbus umbrifer (11%), Gnathopogon herzensteini (10%), Triplophysa stewarti (8%), and Zacco platypus (7%) were the dominant species. Eight rare and two exotic fish species were identified. Combined with analysis of historical data, the richness of fish identified using eDNA metabarcoding was significantly higher than that of fish captured in ground cages. Temperature, pH, and oxidation-reduction potential are the main environmental factors that affect the spatial distribution of fish communities. These results suggest that eDNA metabarcoding could be a new tool with broad application prospects; however, local databases must be improved. This study provides theoretical data and a methodological reference for protecting and managing fish diversity in the Qinling Mountains.

The Chinese giant salamander (Andrias davidianus), an endangered amphibian species endemic to China, has been previously evaluated with regards to its phyletic evolution, zooecology, and ethology, but molecular mechanisms underlying its skin pigmentation remain unknown. Herein, a skin transcriptome database of different colored salamanders was established using RNA-seq, and a total of 47,911 unigenes were functionally annotated. Among these unigenes, a total of 1252 differentially expressed genes (DEGs) were annotated in the seven public databases, and six DEGs were validated by qPCR between five different skin colors and eight tissues. The results showed that TYR, TYRP1, and ASIP were significantly differentially expressed between different body colors, while TYR, TYRP1, and DCT were highly expressed in skin tissue. The full-length complementary DNA of TYR was cloned and analyzed between normal and yellow phenotypes. Three nucleotide sequence deletion sites were identified in the coding region of TYR, leading to premature termination of transcription and translation in yellow individuals. Our study provides useful data for the further study of the molecular mechanisms of melanin formation, and a valuable reference for the breeding of specific skin colors in other salamanders.

Environmental DNA (eDNA) techniques have emerged as a cost-effective and non-invasive strategy for monitoring the distribution of endangered aquatic species. Despite their numerous advantages, operational uncertainty at each step of the process represents a significant challenge that can impact the reliability of results. Hence, the optimization of the eDNA analytic protocol is of utmost importance. In this study, the rare and endangered fish species Sichuan taimen was chosen as the target species to evaluate the concordance between different approaches (tissue, pond, and field water samples) for eDNA detection. The results showed that membrane filtration, collection of a 2-L water sample, and utilization of the DNeasy Tissue and Blood DNA extraction kit were suitable for the analysis of DNA in water samples. Furthermore, the mtDNA D-loop region demonstrated potential as a specific molecular marker for the precise identification of Sichuan taimen. Our results indicated that TaqMan fluorescence PCR can serve as the optimal detection method for reliable identification of target DNA even at extremely low concentrations in the field. This study established an optimized eDNA analysis protocol for Sichuan taimen detection, which will provide the basis for future resource investigations aimed at protection of this endangered aquatic species.

Gram-negative bacteria are the main bacterial pathogens infecting Chinese giant salamanders (Andrias davidianus; CGS) in captivity and the wild, causing substantial economic losses in the CGS industry. However, the molecular mechanisms underlying pathogenesis following infection remain unclear. Spleen-derived macrophages from healthy CGS were isolated, cultured, and identified using density gradient centrifugation and immunofluorescence. A macrophage transcriptome database was established 0, 6, and 12 h post lipopolysaccharide stimulation using RNA-sequencing. In the final database 76,743 unigenes and 4,698 differentially expressed genes (DEGs) were functionally annotated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results showed that DEGs were concentrated in toll-like receptor–nuclear factor kappa B-related immune pathways. Ten DEGs were validated 12 h after lipopolysaccharide (LPS) stimulation. Although the common LPS recognition receptor toll-like receptor 4 was not activated and the key adaptor protein MyD88 showed no significant response, we observed significant up-regulation of the following adaptors: toll/interleukin-1 receptor domain-containing adaptor inducing interferon-β, tumour necrosis factor receptor-associated factor 6, and transforming growth factor-β activated kinase 1, which are located downstream of the non-classical MyD88 pathway. In contrast to that in other species, macrophage activation in CGS could depend on the non-classical MyD88 pathway in response to bacterial infection. Our study provides insights into the molecular mechanisms regulating CGS antibacterial responses, with implications for disease prevention and understanding immune evolution in amphibians.

Sichuan taimen (Hucho bleekeri Salmonidae) populations are declining, and the status of remnant populations in the Qinling Mountains is poorly known. In this study, eDNA and creel netting were used to estimate the distributions of Sichuan taimen in two upper tributaries of the Hanjiang River. A qPCR assay was applied to detect the eDNA of H. bleekeri, and the presence of Sichuan taimen in the Taibai River, but not in the Xushui River, was confirmed. Sampling during summer achieved a relatively higher detection rate. The utility of eDNA techniques for detecting low-density Sichuan taimen was demonstrated in intricate stream ecosystems. The eDNA method can increase the efficiency of the management of endangered freshwater species, such as Sichuan taimen, by providing reliable distribution data.