Heat shock proteins (HSPs) play a key role in anti-stress and immune processes and are associated with autoimmune diseases. In order to explore the immunological role of HSPs from Schizothorax prenanti (S. prenanti), SpHSP60 was cloned for the first time in this study, and the gene expressions of SpHSP27, SpHSP60, SpHSP70 and SpHSP90 in the hepatopancreas, head kidney, hindgut and spleen were analyzed by quantitative real-time PCR (qPCR) after treatment with lipopolysaccharide (LPS). The open reading frame of the SpHSP60 gene (GenBank accession number ON245159) is 1728 bp. It encodes a protein of 575 amino acids. Its C-terminus is a highly conserved and repeated glycine sequence, which is an important cofactor in ATP binding. Compared with the control group, most of the SpHSPs were significantly upregulated in the tissues examined at 12 or 24 h after LPS challenge. The most abundant expression of SpHSP70 was found in the head kidney at 24 h after LPS injection, followed by SpHSP27 in the spleen at 24 h; both of these SpHSPs displayed strong expression under the LPS stresses, about 20–70 fold more than that of SpHSP60 and SpHSP90. The temporal expression patterns of the four SpHSP genes were different in the four tissues examined. Taken together, the results suggest that SpHSP27, SpHSP60, SpHSP70 and SpHSP90 participate in innate immunity stimulated by LPS, and the response intensity of the SpHSPs was organ-specific, indicating they could provide early warning information against bacterial infection. The findings in our study will contribute to better understanding the biological processes and important roles of SpHSPs involved in defending against pathogenic bacterial challenge.

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.

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.

The Chinese giant salamander (Andrias davidianus) is the biggest extant and endangered amphibian, which has an incongruous sex ratio during the artificial breeding process and is sex indistinguishable in appearance. The sex development mechanism of this rare species thus needs to be explored. We therefore conducted this study to explore the molecular mechanisms of sex hallmarks or gonad maintenance genes for CGS. In this study, we sequenced and analyzed the sexually mature male and female gonads of Chinese giant salamanders (CGSs) using RNA-seq technology and found a total of 15,063 differentially expressed genes (DEGs) in the testicular and ovarian tissues, of which 7973 (52.93%) were expressed in testicular and 7090 (47.07%) in ovarian tissues. Thirteen gonad DEGs were selected based on gene expression abundance and quantified in the muscle and skin tissues of both male and female CGSs. The results showed that these 13 DEGS, out of the 20 analyzed DEGs, were expressed in both the muscle and skin tissues and only two of them (Bmp15 and ZP3) were consistent in the muscle and skin compared to the gonads of CGSs of the two sexes. Overall, our study should provide an important reference for the sex-characteristics-related genes in this type of species, shedding new light on future research on this topic.

Toll-like receptor 3 (SpTLR3) from Schizothorax prenanti (S. prenanti) was cloned and identified, and the tissue distribution of the SpTLR3 gene was examined in this study. Moreover, the relative mRNA expression levels of myeloid differentiation factor 88 gene (SpMyD88) and seven TLR genes (SpTLR2, SpTLR3, SpTLR4, SpTLR18, SpTLR22-1, SpTLR22-2 and SpTLR22-3) from S. prenanti after lipopolysaccharide (LPS) challenge were analyzed through quantitative real-time polymerase chain reaction (qRT-PCR). The full length of SpTLR3 gene is 3097 bp, and complete coding sequence (CDS) is 2715 bp, which encodes 904 amino acids. The SpTLR3 amino acid sequence shared 43.94–100% identity with TLR3 sequences from other vertebrates; SpTLR3 was expressed in all eight tissues examined; and the highest level appeared in the liver, which was significantly higher than in all other tissues (p < 0.05), followed by the levels in the heart and muscles. LPS significantly up-regulated all eight genes in the S. prenanti tissues at 12 or 24 h (p < 0.05). Compared with the PBS control group, no significant transcripts changes were found in SpTLR2 or SpTLR3 at 12 h after LPS induction, but they were significantly up-regulated at 24 h (p < 0.001). The most abundant transcripts were found in the head kidney SpTLR22 genes after 24 h LPS induction, with high to low levels, which were SpTLR22-1 (564-fold), SpTLR22-3 (508-fold) and SpTLR22-2 (351-fold). Among these eight genes, the expression level of SpTLR4 was the least up-regulated. Overall, SpTLR4 in the head kidney was involved in the antibacterial immune response earlier, and the level was increased at 12 h with extreme significance after LPS stimulation (p < 0.001), while the other seven genes were the most significantly up-regulated at 24 h post injection. Taken together, the results suggest that SpMyD88, SpTLR2, SpTLR3, SpTLR4, SpTLR18, SpTLR22-1, SpTLR22-2 and SpTLR22-3 participate in an innate immune response stimulated by LPS, and the response intensity of the genes was organ-specific, with differing kinetics. Our findings will contribute to a more complete understanding of the roles of these TLR genes in antibacterial immunity.

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.