Sperm cryopreservation is pivotal for conserving fish germplasm, yet cryodamage-induced quality decline limits its application. This study focused on Sichuan bream (Sinibrama taeniatus), an endemic and economically important fish species in the upper Yangtze River. Based on an established cryopreservation protocol, we evaluated sperm quality using computer-assisted sperm analysis (CASA) and fertility assays, followed by a systematic assessment of structural and functional damage via flow cytometry (membrane integrity, mitochondrial potential, reactive oxygen species, and DNA fragmentation), enzymatic assays (energy metabolism and antioxidant enzymes), Western blotting, and ultrastructural observation. Finally, integrated proteomic and metabolomic analyses were employed to elucidate the underlying physiological mechanisms. The results demonstrated that freeze-thawing significantly impaired sperm motility, fertility, and ultrastructure, concurrently disrupting energy metabolism and the antioxidant system. Crucially, multi-omics revealed that these functional declines were linked to dysregulation in key pathways involving cytoskeleton organization, lipid metabolism, energy homeostasis, and oxidative stress, forming a coherent network from initial molecular perturbation to phenotypic dysfunction. This study provides a comprehensive characterization of sperm cryodamage in Sichuan bream, advancing the understanding of fish sperm cryobiology and informing targeted cryoprotection strategy development.

Resolving species relationships in rapidly radiating lineages remains a major challenge in evolutionary biology, particularly when hybridization obscures phylogenetic signals. Here, we present a chromosome-level, haplotype-resolved genome assembly for Triplophysa pseudoscleroptera, a species residing at the Qinghai-Tibet Plateau, and integrate it with eight other Triplophysa genomes and resequencing data from 57 Triplophysa individuals to reconstruct a robust phylogeny of the genus. We uncovered extensive discordance between mitochondrial and nuclear phylogenies, driven by both ancient and recent introgression. Notably, the individual selected for genome assembly was found to have undergone a recent hybridization event, retaining ∼ 22% introgressed genomic segments. These introgressed segments are phylogenetically closer to T. dalaica, and their inclusion in concatenated whole-genome alignments was sufficient to mislead species tree inference. Empirical genomic resampling analyses demonstrate that as little as 14% introgression is sufficient to result in incorrect phylogenetic inference in our focal system. Our findings provide a cautionary example that reliance on a single individual genome can lead to erroneous phylogenetic conclusions, when a moderate proportion of introgressed segments is present. We therefore advocate chromosome-scale and window-based phylogenomic approaches as essential practices for reconstructing species relationships in systems shaped by reticulate evolution.

Channa asiatica is an economically valuable fish species and excellent model for studying hypoxic tolerance. However, the underlying genetic and molecular mechanisms are poorly understood. In this study, we assembled a high-quality C. asiatica genome (23 chromosomes, totaling 722 Mb) using a combination of Illumina short-read, PacBio long-read, and Hi-C sequencing. Repetitive elements accounted for 28.39%of the C. asiatica genome, and 23,949 protein-coding genes were predicted, with 96.63 % of these functionally annotated. Moreover, a comparative genomic analysis of 12 fish genomes showed that gene families associated with oxygen binding and transport were expanded in C. asiatica. In addition, transcriptome analysis revealed that multiple oxidative stress pathways were activated when C. asiatica was exposed to air. In conclusion, this study provided high-quality genome assembly and transcriptome data, both serving as critical resources for researching the genetic basis of hypoxic tolerance in C. asiatica.

IntroductionBody color is a prominent phenotypic trait and a significant economic characteristic in fish. While current research mainly examines the effects of genetic factors, less emphasis has been placed on the role of endogenous hormones in fish body color.MethodsIn this study, we investigated the changes in phenotype and pigment content in juvenile Sinibrama taeniatus after a 30-day treatment with gradient concentrations of L-thyroxine. To further understanding of the regulatory roles of thyroid hormone on pigment synthesis, we investigated the expression changes in genes involved in melanin-based and non-melanin-based pigmentation under the different hormonal regimes.ResultsCompared to the control group, the body color of the low-concentration L-thyroxine treatment group (20 nM) was lighter, while that of the high-concentration group (40 nM and 80 nM) was darker. Thyroid hormone treatment simultaneously affected melanophores, xanthophores, and iridophores in different ways, leading to an increase of melanin and guanine, and a reduction of pteridine and β-carotene. These findings suggest that thyroid hormone regulates fish body color pigment synthesis through both melanin-based and non-melanin-based mechanisms. Additionally, expression of genes involved in melanin-based (e.g. ctnnb1, wnt10b, and adcy3) and non-melanin-based pigmentation (e.g. xdh, ltk, pnp4a, and mpv17) were modified by thyroid hormone treatment.DiscussionOur results enhanced the understanding of the role of thyroid hormone in fish pigmentation and provide useful information for cultivating fish with desirable body colors in aquaculture.

The Qinghai -Tibetan Plateau has exhibited heightened sensitivity to global warming than any other region. G. ekloni, an endemic fish of the Qinghai -Tibetan Plateau, holds ecological significance and is a key highland cold -water species in Chinese aquaculture. However, the warming of its habitat poses a significant threat to its survival. The intestine serves as the primary line of external immune defense. There are no reports available on alterations in the intestine of G. eckloni in response to varying levels of heat stress. In this study, G. ekloni was categorized into the optimum temperature culture group (CO, control group), the 22 degrees C heat stress culture group (HT -22) and the 28 degrees C heat stress culture group (HT-28).The adaptability of G. ekloni intestine to varying levels of heat stress was investigated through a comprehensive analysis involving tissue sections, blood biochemical indexes, sequencing of 16S and RNA-Seq. Tissue sections showed severe intestinal damage in the HT -28, characterized by mucosal layer necrosis, swelling of thrush cells, and erosion of villi. In the HT -28 group, the significantly increased plasma levels of inflammatory markers (DAO, LPS, IL-1 beta, IL -6, and TNF-alpha) also corroborated the substantial impairment of intestinal barrier function. Meanwhile, the RNA-Seq analysis revealed a significant upregulation of most genes in the HIF-1 signaling pathway. This suggests that the HIF-1 signaling pathway likely plays a crucial role in safeguarding the intestinal tract from injuries caused by extreme temperature stress. Intriguingly, there was very little change in the intestinal histological structure and no significant changes in biochemical indicators of stress and inflammation in the blood in the HT -22 group, and 16S amplification sequencing also revealed a greater abundance of intestinal microbial species in the HT -22 group. Through coanalysis with the up -regulated DEGs in HT -22, it was determined that the augmentation of intestinal flora taxa in the HT -22 group positively contributing to enhancing intestinal immunity and stabilizing intestinal barrier. This comprehensive histomorphological and multi-omics study showcased the resilience of G. ekloni intestine to environmental warming. The findings offer fundamental data for future research on the artificial culture of highland economic fishes, and their environmental adaptations.

Global warming has heightened the severity and frequency of extreme high-temperature events, presenting challenges to fish survival and negatively affecting the production of fishery. Gymnocypris eckloni, native to the Tibetan Plateau, is widely cultured in southwest China. Its sensitivity to heat makes it a suitable model for studying the impact of high temperatures on fish. The heart is a vital organ for survival, while is relatively understudied concerning the impact of high temperatures on fish hearts. In this study, G. eckloni was subjected to a seven-day exposure to 27 °C followed by a transfer to 18 °C for a seven-day recovery period. Behavioral tests revealed a significant increase in locomotor speed, distance, and respiratory rate of G. eckloni at 27 °C. Serum indices exhibited significantly elevated stress indicators (cortisol) and kinases associated with heart damage (lactate dehydrogenase, lactate dehydrogenase isoenzyme 1, creatine kinase, creatine kinase isoenzyme, and aspartate transaminase) at 27 °C, and histological analyses have confirmed the occurrence of heart damage subjected to high temperatures. After 7 days of recovery, most of these indicators showed a significant decrease compared to the heat stress group, with no significant differences observed compared to the control group. Transcriptomic analyses have revealed the widespread involvement of ferroptosis, P53 signaling pathway, oxidative phosphorylation, myocardial contraction, and DNA repair pathway in the response of G. eckloni to high temperatures. Our study offers a systematic exploration of the effects of heat stress on G. eckloni, providing valuable insights for the cultivation of high-altitude fish.

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Body color is one of the most noticeable phenotypic and economic characteristics in fish. Fish exhibit a diverse array of body color mutations influenced by factors such as artificial selection and natural mutations. Elucidating the genetic mechanisms of these color variants in fish is crucial for developing new breeds with higher ornamental value. In this study, we identified a nonsense mutation in csf1ra (c. 1249 C>T) with whole genome resequencing technology, and which may potentially contribute to the albino phenotype in Channa asiatica. The albino individuals exhibited a substantial decrease in the expression level of csf1ra compared to that of the wild individuals. To unravel the underlying mechanism of csf1ra, we delved into comparative transcriptomic and gene expression analyses. Our findings suggest that csf1ra may impact tyrosine kinase activity. Furthermore, csf1ra may not only regulate the expression levels of genes (sox9, ephb6, and wnt9) involved in melanin-based pigmentation but also influence genes (ampd3, ak1, and bco1) associated with non-melanin-based pigmentation. Our study investigated the genetic basis of the albino mutant of C. asiatica and provided new insights into the function of csf1ra in fish pigmentation.

OBJECTIVE:To investigate the protective effects of total saponins from Panax japonicus (TSPJ) against high-fat dietinduced testicular Sertoli cell junction damage in mice.METHODS:Forty male C57BL/6J mice were randomized into normal diet group, high-fat diet group, and low-dose (25 mg/kg) and high-dose (75 mg/kg) TSPJ treatment groups (n=10). The mice in the normal diet group were fed a normal diet, while the mice in the other groups were fed a high-fat diet. After TSPJ treatment via intragastric administration for 5 months, the testes and epididymis of the mice were collected for measurement of weight, testicular and epididymal indices and sperm parameters. HE staining was used for histological evaluation of the testicular tissues and measurement of seminiferous tubule diameter and seminiferous epithelium height. The expression levels of ZO-1, occludin, claudin11, N-cadherin, E-cadherin and β-catenin in Sertoli cells were detected with Western blot, and the localization and expression levels of ZO-1 and β-catenin in the testicular tissues were detected with immunofluorescence assay. The protein expressions of LC3B, p-AKT and p-mTOR in testicular Sertoli cells were detected using double immunofluorescence assay.RESULTS:Treatment with TSPJ significantly improved high-fat diet-induced testicular dysfunction by reducing body weight (P < 0.001), increasing testicular and epididymal indices (P < 0.05), and improving sperm concentration and sperm viability (P < 0.05). TSPJ ameliorated testicular pathologies and increased seminiferous epithelium height of the mice with high-fat diet feeding (P < 0.05) without affecting the seminiferous tubule diameter. TSPJ significantly increased the expression levels of ZO-1, occludin, N-cadherin, E-cadherin and β-catenin (P < 0.05) but did not affect claudin11 expression in the testicular tissues. Immunofluorescence assay showed that TSPJ significantly increased ZO-1 and β-catenin expression in the testicular tissues (P < 0.001), downregulated LC3B expression and upregulated p-AKT and p-mTOR expressions in testicular Sertoli cells.CONCLUSION:TSPJ alleviates high-fat diet-induced damages of testicular Sertoli cell junctions and spermatogenesis possibly by activating the AKT/mTOR signaling pathway and inhibiting autophagy of testicular Sertoli cells.

IntorductionColoration is a prominent trait in fish that is closely linked to their market value. Fish exhibit a diverse range of body colors, making them an excellent model for studying molecular mechanisms underlying pigmentation. Although regulatory pathways involved in pigmentation have been extensively studied in model fish such as zebrafish and medaka, the presence of varying chromatophore types across different fish species suggests that fish pigmentation mechanisms are not fixed. Therefore, more studies should be conducted on non-model fish. Sinibrama taeniatus is an endemic fish in the upper Yangtze River that is highly valued for its ornamental and edible properties. MethodsIn this study, we identified three chromatophore types in S. taeniatus and investigated changes in body color, chromatophores, and pigments at different developmental stages. Subsequently, RNA-seq analysis revealed that retinol metabolism, thyroid hormone synthesis, purine metabolism, and pyrimidine metabolism pathway are closely associated with pigmentation. ResultsWeighted gene co-expression network analysis (WGCNA) identified several hub genes, including mitf, wnt 9a, wnt10b, wnt11, adcy5, edn1, adcy8, and rdh8, that may play an important role in pigmentation. DiscussionOverall, our findings provide valuable insights into the role of genes and pathways in fish pigmentation and offer useful information for body color-based molecular breeding programs in aquaculture.

Vitellogenesis is the most important stage of ovarian maturation in fish, involving the synthesis and transport of essential yolk substances and their complex mechanisms and coordination process. The liver is the main tissue involved in the vitellogenesis of oviparous animals, but studies of vitellogenesis in fish rarely include the liver and ovary as a whole. The aim of this study was to explore the molecular mechanism and associated regulatory factors behind vitellogenesis in Sichuan bream (Sinibrama taeniatus). The different stages of oogenesis were first identified by successive histological observations. Then, ovary and liver tissues that developed to 115 days (stage II, previtellogenesis stage), 165 days (stage III, vitellogenesis stage) and 185 days (stage IV, late-vitellogenesis stage) were collected for transcriptomic and metabolomic analyses, and serum testosterone (T), 17β-estradiol (E2), vitellogenin (Vtg), triiodothyronine (T3), and thyroxine (T4) levels were measured at the corresponding stages. We found that energy redistribution during vitellogenesis is mainly regulated through glycolysis, fatty acid biosynthesis and the citrate cycle pathway. In the liver, energy metabolism was promoted by activating glucolipid metabolic pathways to provide sufficient ATP, but at the same time, the ovary tends to retain nutrients rather than decompose them to produce energy. In addition, we have identified several key factors involved in the metabolism of neutral lipids, polar lipids, amino acids and vitamins, which are involved in the assembly and transport of important yolk nutrients. The initiation of vitellogenesis was found to be associated with a surge in serum E2 levels, but the sustained increase in Vtg levels in the late stage may be due more to upregulation of the estrogen receptor. These results provide valuable information about the regulation of ovarian development in cultured fish.

Northern snakehead, Channa argus ( C. argus ), is an important economic and ecological fish species. The wild population of the species was sharply declined in the last decade. A high-quality reference genome could lay a solid foundation for the genetic and conservation studies for C. argus . In this work, we report a chromosomal genome assembly with PacBio and Hi-C technology using the albino northern snakehead, a color variety of C. argus . A 644.1-Mb genome with 24 chromosomes was obtained with a contig and scaffold N50 of 11.78 and 27.8 Mb, respectively. We inferred that C. argus diverged from A. testudineus around 85.6 million years ago. 514 expanded gene families and 214 positively selected genes were identified in the C. argus genome. The chromosome-level genome provides a valuable high-quality genomic resource for population, as well as genetic and evolutionary studies for C. argus and other species in Channidae.

Fish skin is mainly composed of the epidermis, dermis, and its derivative scales. There is a wide diversity in scale number in fishes, but the diversity of skin structure lacks systematic histological comparison. This research aimed to improve our understanding of the functional relationship between the scale number and the skin structure in freshwater fishes and to determine which ecological factors affect the scale number and skin structure. First, we presented a method to quantify skin structure in fish and histologically quantified the skin structure of 54 freshwater fishes. Second, we collected the scale number and habitat information of 509 Cyprinidae fishes in China and explored which ecological factors were related to their scale number. Third, common carp and scaleless carp were used as models to study the effects of scale loss on swimming. We found a strong negative correlation between scale thickness and scale number. The main factor affecting the skin structure of fishes was the species' water column position, and the skin of benthic fishes was the most well-developed (thicker skin layers (dermis, epidermis) or more/larger goblet cells and club cells). The scale number was related to two factors, namely, temperature and water column position, and cold, benthic and pelagic adaptation may have contributed to increased scale numbers. Only in benthic fishes, the more well-developed their skin, the more scales. In common carp, scale loss did not affect its swimming performance. In summary, we suggest that there is a rich diversity of skin structure in freshwater fishes, and the scales of fish with well-developed skin tend to degenerate (greater number/smaller size/thinner, or even disappear), but the skin of fish with degenerated scales is not necessarily well developed.

Ultraviolet radiation (UVR) is a ubiquitous environmental factor and with complex and diverse effects on organisms, and the UVR tolerance of species varies due to selection, adaptation and evolution. This study aimed to improve our understanding of the interspecific differences in UVR tolerance of fishes. First, we compared skin histology between high-altitude fishes and their low-altitude relatives. Second, we assessed the skin histological changes of 22 fish species after UVR exposure and quantified their damage levels. Finally, the factors affecting the difference in UVR tolerance among fishes were studied. Thin or absent scales was a common characteristic of high-altitude fishes in different taxonomic groups, but other skin structure parameters (the thickness of the dermis and epidermis, the size and number of mucous and club cells) did not show convergence. After UVR exposure, the size and number of mucous cells and club cells in the experimental fishes were generally reduced, but the changes in epidermal and dermal thickness varied among species. The change rata of melanin area was strong negatively correlated with the level of damage, indicating that increased melanin was helpful to reduce UVR damage. On the whole, the skin histological changes of high-altitude fishes after UVR exposure did not differ from those of low-altitude fishes. However, the epidermis and dermis of Gymnocypris eckloni and Oncorhynchus mykiss, which had the highest UVR tolerance, were nonpathologically thickened, indicating that they may contribute to UVR tolerance. There were significant interspecific differences in UVR tolerance among the experimental fishes, with high-altitude and cold water fishes showing stronger UVR tolerance. Interestingly, some low-altitude cold water fish (e.g., O. mykiss) also showed high UVR tolerance, suggesting that besides high-altitude adaptation, low-temperature adaptation also may play an important role in organismal adaptation to UVR.

The effect of thermal changes on the physiology and behavior of fish is a major research focus in the face of ongoing global warming. There is little information about the effects of temperature increase on fish in the wild. However, the consequences of temperature increase on fish in controlled laboratory conditions can provide insights into what can be expected in the wild. Triplophysa bleekeri, a high-plateau fish, exhibits high sensitivity to high temperatures, suggesting it to be a good model to investigate the impact of temperature increase on fish. In this study, we analyzed the effect of gradual temperature increase on transcriptional and metabolic levels of T. bleekeri subjected to a gradual temperature change of 0.5°C/day until temperatures of 10°C, 13°C, 16°C, and 19°C were reached. Transcriptomics results of the liver, gut, spleen, and trunk kidney showed that metabolic pathways are widely involved in the response to increased temperatures in T. bleekeri. Lipidomics results further indicated that the lipid composition was altered by increased temperatures, and three lipids (PC 14:0e/22:1, PC 18:0e/22:5, and TAG 14:3-21:2-21:2) were identified as potential biomarkers of heat stress in T. bleekeri. Moreover, a decline in unsaturated fatty acid levels was observed in T. bleekeri under high temperatures. These results suggest that high temperatures modify the metabolomic pathways. Overall, our results help improve the understanding of physiological responses in fish to increased temperatures, and provide valuable information predicting the consequences of global warming on fish.

To investigate the physiological features of the melanocortin-4 receptor (MC4R) gene, which was demonstrated to be strictly related to food regulation in vertebrates, the coding DNA sequence (CDS) of MC4R in gibel carp (Carassius auratus gibelio) was cloned and characterized (designated as CgMC4R). In this study, the CDS of the CgMC4R gene was 981 bp long that predicted to encode a protein of 327 putative amino acids with seven potential hydrophobic transmembrane domains (TMD1-TMD7). Bioinformatic analysis revealed that CgMC4R shared 99% identity with that in goldfish and exhibited high similarity with that in Cyprinidae species. In addition, CgMC4R was predominantly expressed in the hypothalamus. Furthermore, hypothalamic CgMC4R mRNA expression exhibited altered responses under fed/fasting conditions. In the postprandial experiment, the expression of CgMC4R in the unfed group was upregulated at 1 and 3 h postfeeding compared to that in the fed fish, respectively. During long-term fasting, CgMC4R mRNA transcript levels dramatically increased within 1, 3, 5, and 7 days of fasting. After refeeding, the CgMC4R gene presented a marked decrease in expression after refeeding at 9 days and finally returned to the level in control group. Taken together, these results indicated that the MC4R gene plays a significant role in the feed intake of gibel carp and provides basic evidence to explore the appetite-related roles of MC4R in gibel carp.

Leptin is a multifunctional hormone that serves as a feeding regulator in mammals. However, the effect of leptin on fish remains unclear. We sequenced the leptin gene from gibel carp (Carassius auratus gibelio) and designated it gLEP. The length of the gLEP cDNA sequence was 562 bp, including an open reading frame (ORF) of 516 bp. The ORF putatively encodes a peptide of 171 amino acids, including a signal peptide of 20 amino acids. gLEP shared low primary amino acid sequence homology with leptin genes in vertebrates, whereas three-dimensional (3D) structural modeling revealed strong identity with the structures in other vertebrates. gLEP mRNA was widely distributed in all of the tissue that we examined, with the highest levels of expression in the hepatopancreas. Hepatopancreas gLEP mRNA expression levels showed no changes following postprandial treatment. However, hepatopancreas gLEP mRNA expression levels greatly decreased (P < 0.05) after fasting but substantially increased (P < 0.05) after refeeding in the long-term fasting treatment. In summary, these results indicate that leptin expression could be influenced by the regulation of food intake. These results provide the initial step toward elucidating the appetite regulatory systems associated with leptin in gibel carp.

Schizothorax davidi is a rare fish in Southwest China and is considered a promising species for aquaculture. Compared with other teleosts, little is known about the endocrine regulation of feeding in this species. In this study, we identified the CRH, apelin, and GnRH2 genes in S. davidi and assessed the effects of different energy statuses on CRH, apelin, and GnRH2 expression. Our results showed that the full-length cDNA sequences of CRH, apelin, and GnRH2 of S. davidi were 995, 905, and 669 bp long, respectively. Furthermore, CRH was mainly expressed in the hypothalamus, telencephalon, and myelencephalon; apelin was highly expressed in the spleen and heart; and GnRH2 mRNA was widely distributed in all examined tissues, with the highest level in the hypothalamus. Notably, the levels of CRH and GnRH2 increased in the hypothalamus at 1 h and 3 h post-feeding, while hypothalamic apelin levels decreased. Conversely, CRH and GnRH2 expression in the hypothalamus significantly decreased after fasting for 7 days and returned to the control levels after re-feeding for 3 or 5 days. In contrast, fasting increased apelin levels in the hypothalamus. Overall, this study suggests that CRH, apelin, and GnRH2 play critical roles in appetite regulation in S. davidi. These results provide an essential groundwork to elucidate the appetite regulatory systems in S. davidi as well as in other teleosts.

NUCB1 and NUCB2, two novel nucleobindins, have attracted extensive attention for their role in the appetite regulation in mammals. However, little is known about the appetite regulation of NUCB1 and NUCB2 in fish species. Therefore, we investigated the role of these peptides in the regulation of feeding in Schizothorax davidi (S. davidi). In this study, full-length cDNA sequences of nucb1 and nucb2A of S. davidi were obtained for the first time. Additionally, the tissue distribution and the effects of different energy status on nucb1 and nucb2A mRNAs abundance were assessed, showing that nucb1 and nucb2A are widely distributed in 18 detected tissues, with the highest expression in the cerebellum. The abundances of nucb1 and nucb2A increased in the hypothalamus at 1 h and 3 h post-feeding. Furthermore, fasting and re-feeding experiments showed that the expressions of nucb1 and nucb2A in hypothalamus significantly decreased after fasting for 7 days, and returned to the control level after re-feeding for 3 or 5 days. In conclusion, the present study suggests that both NUCB1 and NUCB2A are involved in the short-term and long-term appetite regulation, as an anorexigenic factor, in S. davidi. These results can provide a basis for further investigation into the appetite regulatory role of NUCB family in teleost.

Abstract Background Intense stresses caused by high-altitude environments may result in noticeable genetic adaptions in native species. Studies of genetic adaptations to high elevations have been largely limited to terrestrial animals. How fish adapt to high-elevation environments is largely unknown. Triplophysa bleekeri, an endemic fish inhabiting high-altitude regions, is an excellent model to investigate the genetic mechanisms of adaptation to the local environment. Here, we assembled a chromosomal genome sequence of T. bleekeri, with a size of ∼628 Mb (contig and scaffold N50 of 3.1 and 22.9 Mb, respectively). We investigated the origin and environmental adaptation of T. bleekeri based on 21,198 protein-coding genes in the genome. Results Compared with fish species living at low altitudes, gene families associated with lipid metabolism and immune response were significantly expanded in the T. bleekeri genome. Genes involved in DNA repair exhibit positive selection for T. bleekeri, Triplophysa siluroides, and Triplophysa tibetana, indicating that adaptive convergence in Triplophysa species occurred at the positively selected genes. We also analyzed whole-genome variants among samples from 3 populations. The results showed that populations separated by geological and artificial barriers exhibited obvious differences in genetic structures, indicating that gene flow is restricted between populations. Conclusions These results will help us expand our understanding of environmental adaptation and genetic diversity of T. bleekeri and provide valuable genetic resources for future studies on the evolution and conservation of high-altitude fish species such as T. bleekeri.