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.

We identified and cloned cDNA encoding the heat shock protein (Hsp) 27 gene from Schizothorax prenanti (SpHsp27), and compared its expression with that of SpHsp60, SpHsp70, and SpHsp90 in the liver, head kidney, hindgut, and spleen of S. prenanti that were injected with polyinosinic-polycytidylic acid [Poly (I:C)]. The SpHsp27 partial cDNA (sequence length, 653 bp; estimated molecular mass, 5.31 kDa; theoretical isoelectric point, 5.09) contained an open reading frame of 636 bp and a gene encoding 211 amino acids. The SpHsp27 amino acid sequence shared 61.0–92.89% identity with Hsp27 sequences from other vertebrates and SpHsp27 was expressed in seven S. prenanti tissues. Poly (I:C) significantly upregulated most SpHsps genes in the tissues at 12 or 24 h (p < 0.05) compared with control fish that were injected with phosphate-buffered saline. However, the intensity of responses of the four SpHsps was organ-specifically increased. The expression of SpHsp27 was increased 163-fold in the head kidney and 26.6-fold SpHsp27 in the liver at 24 h after Poly (I:C) injection. In contrast, SpHsp60 was increased 0.97–1.46-fold in four tissues and SpHsp90 was increased 1.21- and 1.16-fold in the liver and spleen at 12 h after Poly (I:C) injection. Our findings indicated that Poly (I:C) induced SpHsp27, SpHsp60, SpHsp70, and SpHsp90 expression and these organ-specific SpHsps are potentially involved in S. prenanti antiviral immunity or mediate pathological process.

Minimally invasive sampling was used to determine the sex of Chinese giant salamanders (Andrias davidianus). Urine samples (n = 25) were collected from 6 adults in the breeding season and from 19 individuals (7 adults and 12 juveniles) in the non-breeding season. The hormone testosterone (T) and estrone-3-glucuronide (E1G) in urine were collected from Chinese giant salamanders (CGSs), and the hormone extracts were analyzed by enzyme immunoassays (EIA). The data demonstrated that the urine T concentration of the male CGSs was significantly higher than that of the females during the breeding season (p < 0.05) and even more pronounced during the non-breeding season (p < 0.01). The urine E1G concentration of the males was less pronounced than that of the females during the breeding season (p < 0.01) and significantly lower during the non-breeding season (p < 0.05). The urine T/E1G values of all the male salamanders were significantly higher than those of the females (p < 0.01) during both the breeding season and the non-breeding season. An interesting pattern was found in this study: the value of urine log₁₀(T/E1G) of the male CGSs was higher than 1, whereas the value for the females was lower than 1, during both the breeding and non-breeding seasons, and in the adult and sub-adult age groups of CGSs. There were 25 salamanders in this study and the accuracy rate reached 100% by using a log₁₀(T/E1G) value of 1. The results of the log₁₀(T/E1G) value provide new insight into the future development of the sex identification of CGSs and also lay the foundation for accurate sex identification in the preparation for artificial release. This is the first study to show that the T/E1G ratio in urinary hormones is reliable for the sex identification of CGSs. Additionally, urinary hormone T/E1G measures are promising sex identification tools for amphibian or monomorphic species and for those whose secondary sex characteristics are visible only during the breeding season.

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.