Search Results (29)

Nr5a2 (nuclear receptor subfamily 5, group a, member 2) is involved in gonad development and sex hormone synthesis. In this study, the full length of Nr5a2f and Nr5a2m were obtained by Nr5a2 variable splicing from Andrias davidianus, and the tissue distribution was detected. We identified Nr5a2f of 2455 bp and Nr5a2m of 2150 bp length, encoding 479 and 325 amino, respectively. We first characterized Nr5a2f and Nr5a2m gene expression in developing gonads. Results showed that Nr5a2f had significantly high expression in the ovary and little expression in other tissues, during the sex differentiation and sex reversal, Nr5a2f expression was gradually decreased in the ovary and the expression in the testis was significantly lower than in the ovary from 1 year to 6 year old. Significantly high expression was observed in the ovary and reversal ovary, while low expression was in the testis and reversal testis. While Nr5a2m expression exhibited the opposite profile, high expression was observed in the brain and testis. During sex differentiation and sex reversal, high expression was shown in the testis and low expression in the ovary from one year to six years old and significantly higher expression emerged in testis and reversal testis than in ovary and reversal ovary. In situ hybridization, results showed that Nr5a2f began to express in female undifferentiated gonads and the expression level increased from 48 dpf to 91, while Nr5a2m was expressed in male undifferentiated gonads. Three RNA interference sites were designed and we detected that site 293 exhibited a significant inhibitory effect in ovary cells. After Nr5a2f expression was inhibited by site 293, we observed that female-based gene Nr5a2f, foxl2 and cyp19 expression were decreased, while the male-based gene dmrt1 and cyp17 expression was increased. These results suggested that Nr5a2f and Nr5a2m exhibited different expression patterns in the process of sex differentiation, which provided a foundation for further functional characterizations. Full article

The circadian rhythm of an animal’s body color change is crucial for its survival and adaptation to the environment, with photoperiod serving as a primary “zeitgeber” that significantly affects the circadian rhythm of color change. To explore the circadian body color change in juvenile Chinese giant salamanders (Andrias davidianus), four distinct photoperiod regimens were established, including the DD group (constant darkness), the LD12:12 group (12 h photophase, 12 h scotophase), the LD16:8 group (16 h photophase, 8 h scotophase), and the LD8:16 group (8 h photophase, 16 h scotophase). Reflectance spectra of dorsal skin were measured every 4 h over a 24 h cycle, with data collected every other day for three consecutive days, totaling 72 h. Reflectance spectra were converted into colorimetric variables to analyze circadian patterns. The results demonstrated that juvenile A. davidianus exhibited highly significant circadian rhythmicity under different photoperiods, with their body color becoming darker during the photophase (mean brightness: 14.650 ± 0.629 to 16.385 ± 1.301) and lighter during the scotophase (mean brightness: 16.473 ± 0.875 to 34.422 ± 2.692). Photoperiod alterations significantly affected the rhythm parameters (mesor, amplitude, and acrophase) of mean brightness variation. Compared with the LD12:12 group, the LD16:8 group (extended photophase) reduced rhythm mesor by 1.415, decreased amplitude by 0.988, and induced a 1.510 h acrophase delay. Conversely, the LD8:16 group (extended scotophase) increased rhythm mesor by 2.141 and amplitude by 2.919. These findings indicate that the circadian rhythm of body color change in juvenile A. davidianus is coordinately regulated by endogenous circadian rhythms and exogenous photoperiodic signals. The findings of this study provide a theoretical foundation for the conservation and management of A. davidianus. Full article

Amid the global rise in type 2 diabetes, natural peptide-based therapies provide a safer alternative to synthetic drugs with fewer side effects. This study highlights the antidiabetic potential of peptides derived from cultured Chinese giant salamander (CGS) meat hydrolyzed by Alcalase 2.4L FG through DPP4 inhibitory activity. The hydrolysate exhibited significant DPP4 inhibitory activity, with an IC₅₀ value of 1317.0 ± 29.5 μg/mL. Using activity-guided modified-SPE fractionation, the most potent DPP4 inhibitory peptide, PPAAQLL (PL7), was identified from CGS alcalase hydrolysate, with an IC₅₀ of 230.1 ± 4.9 μM. PL7 was identified as a non-competitive inhibitor through enzyme kinetic studies, and intermolecular docking simulations suggested that it does not interact with the active site of DPP4. Additionally, PL7’s stability against DPP4 and simulated gastrointestinal protease digestion suggested that its inhibitory activity remains intact, indicating potential for effective oral administration. SRM quantification analysis revealed nearly a six-fold enrichment of PL7 in the RP-SPE fraction S1 compared to the crude hydrolysate, underscoring the effectiveness of the fractionation method. These findings highlight CGS alcalase hydrolysate as a promising source of DPP4 inhibitory peptides for managing type 2 diabetes. Future studies should focus on the in vivo efficacy, bioavailability, and pharmacokinetics of PL7. Full article

This study explored the effects of replacing live fish feed with artificial compound feed on the growth performance, nutritional composition, digestive performance, and gut microbiota of Chinese giant salamander (Andrias davidianus). The experiment was divided into three groups, each with three biological replicates. The salamander juveniles were fed artificial compound feed (S), live fish (H), and frozen fish (D) for 184 days. Results showed no significant differences in growth performance among the three groups (p > 0.05). The water content in the S group was significantly higher than that in the H group (p < 0.05), and the crude fat content was slightly higher, whereas other nutritional components showed no significant differences (p > 0.05). The analysis of amino acid composition found no significant differences among the three groups of Chinese giant salamanders (p > 0.05). The analysis of fatty acid composition revealed that the feed group had significantly lower levels of polyunsaturated fatty acids compared with the other two groups (p < 0.05), and significantly higher levels of saturated fatty acids (p < 0.05). There were no significant changes in digestive enzyme activity among the three salamander groups (p > 0.05). There were also no significant differences in the remaining antioxidant indicators in the intestine and liver among the three groups of salamanders (p > 0.05). The S group had significantly higher Pielou_e and Shannon indices (p < 0.05), thereby indicating a greater diversity and evenness of the gut microbiota. In conclusion, replacing live feed with artificial compound feed does not affect the growth performance, nutritional composition, antioxidant capacity, and digestive function of Chinese giant salamander juveniles. Moreover, it enhances the diversity and richness of the gut microbiota, providing effective data to support the development of artificial feeds for giant salamanders. Full article

Optimizing the breeding techniques and increasing the hatching rate of Andrias davidianus offspring necessitates a thorough understanding of its parental care behaviors. However, A. davidianus’ nocturnal and cave-dwelling tendencies pose significant challenges for direct observation. To address this problem, this study constructed a dataset for the parental care behavior of A. davidianus, applied the target detection method to this behavior for the first time, and proposed a detection model for A. davidianus’ parental care behavior based on the YOLOv8s algorithm. Firstly, a multi-scale feature fusion convolution (MSConv) is proposed and combined with a C2f module, which significantly enhances the feature extraction capability of the model. Secondly, the large separable kernel attention is introduced into the spatial pyramid pooling fast (SPPF) layer to effectively reduce the interference factors in the complex environment. Thirdly, to address the problem of low quality of captured images, Wise-IoU (WIoU) is used to replace CIoU in the original YOLOv8 to optimize the loss function and improve the model’s robustness. The experimental results show that the model achieves 85.7% in the mAP50-95, surpassing the YOLOv8s model by 2.1%. Compared with other mainstream models, the overall performance of our model is much better and can effectively detect the parental care behavior of A. davidianus. Our research method not only offers a reference for the behavior recognition of A. davidianus and other amphibians but also provides a new strategy for the smart breeding of A. davidianus. Full article

The Chinese giant salamander (Andrias davidianus), listed as an endangered species under “secondary protection” in China, faces significant threats due to ecological deterioration and the expansion of human activity. Extensive field investigations are crucial to ascertain the current status in the wild and to implement effective habitat protection measures to safeguard this species and support its population development. Traditional survey methods often fall short due to the elusive nature of the A. davidianus, presenting challenges that are time-consuming and generally ineffective. To overcome these obstacles, this study developed a real-time monitoring method that uses environmental DNA (eDNA) coupled with recombinase polymerase amplification and lateral flow strip (RPA-LFD). We designed five sets of species-specific primers and probes based on mitochondrial genome sequence alignments of A. davidianus and its close relatives. Our results indicated that four of these primer/probe sets accurately identified A. davidianus, distinguishing it from other tested caudata species using both extracted DNA samples and water samples from a tank housing an individual. This method enables the specific detection of A. davidianus genomic DNA at concentrations as low as 0.1 ng/mL within 50 min, without requiring extensive laboratory equipment. Applied in a field survey across four sites in Huangshan City, Anhui Province, where A. davidianus is known to be distributed, the method successfully detected the species at three of the four sites. The development of these primer/probe sets offers a practical tool for field surveying and monitoring, facilitating efforts in population recovery and resource conservation for A. davidianus. Full article

The stimulator of interferon genes (STING) is a central and multifaceted mediator in innate immunity, and plays vital roles in defending against pathogen infection. In this study, we identified and functionally characterized the STING homolog from the Chinese giant salamander Andrias davidianus (AdSTING) for the first time. The open reading frame of AdSTING encodes a 362 amino acid protein with a predicted molecular mass of 41.6 kDa, which shares 31.1–46.7% of its sequence identity with STING homologs in other vertebrates. Structural analysis revealed that AdSTING possesses four predicted transmembrane domains (TMs) at the N-terminal, and a C-terminal domain (CTD) featuring a dimerization domain (DD), a c-di-GMP-binding domain (CBD), and a short C-terminal tail (CTT). Tissue distribution analysis showed that AdSTING mRNA was ubiquitously expressed in all examined tissues, with abundant expression in muscles, intestine, and thymus. During Andrias davidianus ranavirus (ADRV) infection, significant up-regulation of AdSTING expression was observed in the thymus, spleen, and kidney. Upon different stimuli in vitro, the expression of AdSTING was significantly induced by ADRV infection or polyinosin-polycytidylic acid (poly I:C) stimulation, but no obvious changes were observed during lipopolysaccharide (LPS) stimulation. Subcellular localization analysis revealed that AdSTING mainly localized in the cytoplasm in the Chinese giant salamander thymus cell line (GSTC) and co-localized with the endoplasmic reticulum (ER). Luciferase reporter assays confirmed the ability of AdSTING to activate the interferon-stimulated response element (ISRE) and interferon (IFN) promoter. Furthermore, overexpression of AdSTING effectively decreased ADRV infection, as evidenced by the reduction of virus titers and viral gene expression. Collectively, our findings underscore the pivotal role of AdSTING in the antiviral innate immunity of the Chinese giant salamander, offering insights into the functional evolution of STING in amphibians. Full article

The PAE (Posture-Act-Environment) coding system is a behavior coding system that divides the study of animal behavior into postures, actions, and the corresponding environmental factors, and they are coded correspondingly. It determines the analysis dimension to standardize the study of behavior. To investigate the behavior of A. davidianus during the breeding period, as well as their related postures, actions, and required environmental conditions, this study monitored the behavior of four pairs of A. davidianus in a simulated natural breeding pool using an infrared image monitoring system and recorded the changes in water quality during this process using a water quality monitoring system. The process of reproductive behaviors was observed and recorded with the random sampling method and the focal animal sampling method to classify and code the behaviors, and the ethogram of A. davidianus during the breeding period was constructed based on the PAE coding system. The result showed that 10 postures, 33 actions, 11 environments, and 45 behavioral patterns were differentiated and defined, which were classified into 9 categories of behaviors according to the behavioral function. Among these categories, five were distinguished as behaviors unique to the reproductive period, which include sand pushing, showering, courtship, oviposition, and parental care. The remaining four categories were daily behaviors: exercise, feeding, rest, and miscellaneous behaviors. The quantitative data on water quality and habitat factors that had a significant impact on the behavior of A. davidianus, such as water temperature (WT), pH, and dissolved oxygen (DO), were included in the coding framework, which more accurately expresses the environmental conditions and thresholds required for the breeding behavior. Full article

The Chinese giant salamander (Andrias davidianus) has been increasingly popular in the aquaculture market in China in recent years. In the breeding process of Andrias davidianus, we found that some albino individuals were extremely rare and could not be inherited stably, which severely limits their commercialization in the aquaculture market. In this study, we performed transcriptome and small RNA (sRNA) sequencing analyses in the skin samples of wild-type (WT) and albino (AL) Andrias davidianus. In total, among 5517 differentially expressed genes (DEGs), 2911 DEGs were down-regulated in AL, including almost all the key genes involved in melanin formation. A total of 25 miRNAs were differentially expressed in AL compared to WT, of which 17 were up-regulated. Through the integrated analysis, no intersection was found between the target genes of the differentially expressed miRNAs and the key genes for melanin formation. Gene Ontology (GO) and KEGG pathway analyses on DEGs showed that these genes involved multiple processes relevant to melanin synthesis and the key signal pathway MAPK. Interestingly, the transcription factors SOX10 and PAX3 and the Wnt signaling pathway that play a key role in other species were not included, while the other two transcription factors in the SOX family, SOX21 and SOX7, were included. After analyzing the key genes for melanin formation, it was interesting to note an alternative splicing form of the MITF in WT and a critical mutation of the SLC24A5 gene in AL, which might be the main reason for the skin color change of Andrias davidianus. The results contributed to understanding the molecular mechanism of skin pigmentation in Andrias davidianus and accelerating the acquisition process of individuals with specific body colors by genetic means. Full article

Ranavirus is a large nucleocytoplasmic DNA virus. Chinese giant salamander iridovirus (CGSIV) belongs to the ranavirus genus, and its replication involves a series of essential viral genes. Viral PCNA is a gene closely associated with viral replication. CGSIV−025L also encodes PCNA−like genes. We have described the function of CGSIV−025L in virus replication. The promoter of CGSIV−025L is activated during viral infection, and it is an early (E) gene that can be effectively transcribed after viral infection. CGSIV−025L overexpression promoted viral replication and viral DNA replication. siRNA interfered with CGSIV−025L expression and attenuated viral replication and viral DNA replication. The Δ025L−CGSIV strain with the deletion of CGSIV−025L could not replicate normally and could be rescued by the replenishment of 025L. CGSIV−025L was proven to be an essential gene for CGSIV by overexpression, interference, and deletion mutation experiments. CGSIV−025L was found to interact with CGSIV−062L by yeast two−hybrid, CoIP, and GST pulldown. Thus, the current study demonstrated that CGSIV−025L is an essential gene of CGSIV, which may be involved in viral infection by participating in viral DNA replication and interacting with replication−related proteins. Full article

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. Full article

The DNAJ family, a class of chaperone proteins involved in protein folding, assembly, and transport, plays an essential role in viral infections. However, the role of DNAJA4 (DnaJ Heat Shock Protein Family (Hsp40) Member A4) in the ranavirus infection has not been reported. This study demonstrates the function of the epithelial papilloma of carp (EPC) DNAJA4 in Chinese giant salamander (Andrias davidianus) iridovirus (CGSIV) replication. DNAJA4 consists of 1479 base pairs and encodes a 492 amino acid polypeptide. Sequence analysis has shown that EPC DNAJA4 contains a conserved J domain and shares 84% homology with Danio rerio DNAJA4 and 68% homology with Homo sapiens DNAJA4. EPC DNAJA4 was localized in the cytoplasm, and its expression was significantly upregulated after CGSIV infection. Overexpression of EPC DNAJA4 promotes CGSIV replication and CGSIV DNA replication. siRNA knockdown of DNAJA4 expression attenuates CGSIV replication and viral DNA replication. Overexpression and interference experiments have proved that EPC DNAJA4 is a pro-viral factor. Co-IP, GST–pulldown, and immunofluorescence confirmed the interaction between EPC DNAJA4 and CGSIV proliferating cell nuclear antigen (PCNA). Our results demonstrate for the first time that EPC DNAJA4 is involved in viral infection by promoting viral DNA replication and interacting with proteins associated with viral replication. Full article

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. Full article

Amphibians, including Andrias davidianus, are declining worldwide partly due to infectious diseases. The Myxovirus resistance (Mx) gene is a typical interferon (IFN)-stimulated gene (ISG) involved in the antiviral immunity. Therefore, knowledge regarding the antiviral immunity of A. davidianus can be used for improved reproduction in captivity and protection in the wild. In this study, we amplified and characterized four different A. davidianus Mx genes (adMx) and generated temporal mRNA expression profiles in healthy and Chinese giant salamander iridovirus (GSIV) infected A. davidianus by qualitative real-time PCR (qPCR). The four adMx genes ranged in length from 2008 to 2840 bp. The sequences revealed conserved protein domains including the dynamin superfamily signature motif and the tripartite guanosine-5-triphosphate (GTP)-binding motif. Gene and deduced amino acid sequence alignment revealed relatively high sequence identity with the Mx genes and proteins of other vertebrates. In phylogenetic analysis, the adMx genes clustered together, but also clustered closely with those of fish species. The four adMx genes were broadly expressed in healthy A. davidianus, but were differentially expressed in the spleen during the GSIV infection. Our results show that the adMx genes share major structural features with their homologs, suggesting similar functions to those in other species. Full article

Glycosylphosphatidylinositol mannosyltransferase I (GPI-MT-I) is an essential glycosyltransferase of glycosylphosphatidylinositol-anchor proteins (GPI-APs) that transfers the first of the four mannoses in GPI-AP precursors, which have multiple functions, including immune response and signal transduction. In this study, the GPI-MT-I gene that regulates GPI-AP biosynthesis in Andrias davidianus (AdGPI-MT-I) was characterized for the first time. The open reading frame (ORF) of AdGPI-MT-I is 1293 bp and encodes a protein of 430 amino acids that contains a conserved PMT2 superfamily domain. AdGPI-MT-I mRNA was widely expressed in the tissues of the Chinese giant salamander. The mRNA expression level of AdGPI-MT-I in the spleen, kidney, and muscle cell line (GSM cells) was significantly upregulated post Chinese giant salamander iridovirus (GSIV) infection. The mRNA expression of the virus major capsid protein (MCP) in AdGPI-MT-I-overexpressed cells was significantly reduced. Moreover, a lower level of virus MCP synthesis and gene copying in AdGPI-MT-I-overexpressed cells was confirmed by western blot and ddPCR. These results collectively suggest that GSIV replication in GSM cells was significantly reduced by the overexpression of the AdGPI-MT-I protein, which may contribute to a better understanding of the antiviral mechanism against iridovirus infection. Full article