Schizothorax prenanti is an economically important cold-water fish in China. Lipopolysaccharide (LPS) can induce an immune response in S. prenanti; however, little is known about the effects of LPS on oxidative stress (OS) and apoptosis in S. prenanti. In this study, S. prenanti fish were stimulated with LPS at a dose of 10 mg/kg of body weight. After 0 h, 12 h and 24 h, the tissue samples were collected. The OS- and apoptosis-related genes and enzymatic activities in the liver, head kidney (HK), and spleen of S. prenanti were analyzed by a two-way repeated-measures analysis of variance (ANOVA). Hematoxylin and eosin and terminal transferase uridyl nick end labeling staining were also performed. In S. prenanti, LPS administration downregulated the catalase (CAT) and B-cell lymphoma/Leukemia-2 (Bcl-2) expression levels, and upregulated BCL2-associated X (Bax) and cysteine-aspartic-specific protease-3 (caspase-3) expression levels. Meanwhile, superoxide dismutase and CAT enzymatic activities were inhibited and malondialdehyde (MDA) content was increased by LPS treatment. Additionally, LPS treatment induced OS damage and apoptosis in tissue sections. These results indicated that apoptosis in the liver, HK, and spleen of LPS-administered S. prenanti may be mediated by OS via the mitochondrial apoptotic signaling pathway. Our findings are expected to contribute to a better understanding of the responses of different tissues to bacterial challenges. In addition, we can increase the tolerance of fish to the OS through dietary manipulation in the future.

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.

Toll-like receptor (TLR) signaling is conserved between fish and mammals, except for TLR4, which is absent in most fish. In the present study, we aimed to evaluate whether TLR4 is expressed in Schizothorax prenanti (SpTLR4). The SpTLR2 and SpTLR4 were cloned and identified, and their tissue distribution was examined. The cDNA encoding SpTLR4 and SpTLR2 complete coding sequences (CDS) were identified and cloned. Additionally, we examined the expression levels of seven SpTLRs (SpTLR2, 3, 4, 18, 22-1, 22-2, and 22-3), as well as SpMyD88 and SpIRF3 in the liver, head kidney, hindgut, and spleen of S. prenanti, after intraperitoneal injection of polyinosinic-polycytidylic acid (poly (I:C)). The SpTLR2 and SpTLR4 shared amino acid sequence identity of 42.15–96.21% and 36.21–93.58%, respectively, with sequences from other vertebrates. SpTLR2 and SpTLR4 were expressed in all S. prenanti tissues examined, particularly in immune-related tissues. Poly (I:C) significantly upregulated most of the genes evaluated in the four immune organs compared with the PBS-control (p < 0.05); expression of these different genes was tissue-specific. Our findings demonstrate that TLR2 and TLR4 are expressed in S. prenanti and that poly (I:C) affects the expression of nine TLR-related genes, which are potentially involved in S. prenanti antiviral immunity or mediating pathological processes with differential kinetics. This will contribute to a better understanding of the roles of these TLR-related genes in antiviral immunity.

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.

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.