Transcriptomic and proteomic strategies to reveal the mechanism of Gymnocypris przewalskii scale development.

BACKGROUND: Fish scales are typical products of biomineralization and play an important role in the adaptation of fish to their environment. The Gymnocypris przewalskii scales are highly specialized, with scales embedded in only specific parts of the dermis, such as the areas around the anal fin and branchiostegite, making G. przewalskii an ideal material for biomineralization research. In this study, we aimed to unveil genes and pathways controlling scale formation through an integrated analysis of both transcriptome and proteome, of which G. przewalskii tissues of the dorsal skin (no scales) and the rump side skin (with scales) were sequenced. The sequencing results were further combined with cellular experiments to clarify the relationship between genes and signaling pathways. RESULTS: The results indicated the following: (1) a total of 4,904 differentially expressed genes were screened out, including 3,294 upregulated genes and 1,610 downregulated genes (with a filtering threshold of |log2Fold-Change|> 1 and p-adjust < 0.05). The identified differentially expressed genes contained family members such as FGF, EDAR, Wnt10, and bmp. (2) A total of 535 differentially expressed proteins (DEPs) were filtered out from the proteome, with 204 DEPs downregulated and 331 DEPs upregulated (with a filtering threshold of |Fold-Change|> 1.5 and p < 0.05). (3) Integrated analyses of transcriptome and proteome revealed that emefp1, col1a1, col6a2, col16a1, krt8, and krt18 were important genes contributing to scale development and that PI3K-AKT was the most important signaling pathway involved. (4) With the use of the constructed G. przewalskii fibroblast cell line, emefp1, col1a1, col6a2, col16a1, krt8, and krt18 were confirmed to be positively regulated by the PI3K-AKT signaling pathway. CONCLUSION: This study provides experimental evidence for PI3K-AKT controlled scale development in G. przewalskii and would benefit further study on stress adaptation, scale biomineralization, and the development of skin appendages.

Integrated miRNA-mRNA analysis uncovers immediate-early response to salinity stress in gill-derived cell line of Gymnocypris przewalskii.

Salinity adaptation is an important issue in aquaculture. Understanding the immediate-early response to salinity stress helps in comprehending this process. In vitro experiments using cell lines can explain cell-independent reactions without the involvement of hormones in vivo. In this study, salinity stress experiments were conducted using cell line derived from the gills of Gymnocypris przewalskii (GPG cell line) to isolate immediate-early response-related genes and miRNAs using transcriptomics, followed by bioinformatics analysis. The results showed that intracellular free Ca2+ appeared to be a key factor in cell sensing and initiating downstream cell signaling in response to external salinity. Additionally, cell apoptosis was the most common feature of salinity stress, with multiple signaling pathways involved in salinity-induced cell apoptosis. Furthermore, MiRNAs played a crucial role in the rapid response to salinity stress by selectively inhibiting the expression of specific genes. Additionally, for the first time in the G. przewalskii genome, Tf2 and TY3 families of transposons were found to have responsive roles to the external salinity stress. This study contributes to a better understanding of osmotic sensing in G. przewalskii and provides theoretical assistance for improving salinity adaptation in aquaculture fish species.

Physiological, metabolomic, and transcriptomic reveal metabolic pathway alterations in Gymnocypris przewalskii due to cold exposure.

Teleost fish have evolved various adaptations that allow them to tolerate cold water conditions. However, the underlying mechanism of this adaptation is poorly understood in Tibetan Plateau fish. RNA-seq combined with liquid chromatography‒mass spectrometry (LC‒MS/MS) metabolomics was used to investigate the physiological responses of a Tibetan Plateau-specific teleost, Gymnocypris przewalskii, under cold conditions. The 8-month G. przewalskii juvenile fish were exposed to cold (4 ℃, cold acclimation, CA) and warm (17 ℃, normal temperature, NT) temperature water for 15 days. Then, the transcript profiles of eight tissues, including the brain, gill, heart, intestine, hepatopancreas, kidney, muscle, and skin, were evaluated by transcriptome sequencing. The metabolites of the intestine, hepatopancreas, and muscle were identified by LC‒MS/MS. A total of 5,745 differentially expressed genes (DEGs) were obtained in the CA group. The key DEGs were annotated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. The DEGs from the eight tissues were significantly enriched in spliceosome pathways, indicating that activated alternative splicing is a critical biological process that occurs in the tissues to help fish cope with cold stress. Additionally, 82, 97, and 66 differentially expressed metabolites were identified in the intestine, hepatopancreas, and muscle, respectively. Glutathione metabolism was the only overlapping significant pathway between the transcriptome and metabolome analyses in these three tissues, indicating that an activated antioxidative process was triggered during cold stress. In combination with the multitissue transcriptome and metabolome, we established a physiology-gene‒metabolite interaction network related to energy metabolism during cold stress and found that gluconeogenesis and long-chain fatty acid metabolism played critical roles in glucose homeostasis and energy supply.

Fatty acid metabolism and antioxidant capacity in Gymnocypris przewalskii (Kessler, 1876) response to thermal stress.

The Qinghai-Tibet Plateau is undergoing a wet-warming transition, which could affect the survival of the native fish. However, the tolerance and physiological response to thermal stress is rarely studied in Gymnocypris przewalskii, a rare native fish in the Tibetan plateau. In this study, first, we detected the thermal tolerance of five groups of six-month G. przewalskii which acclimated at 8, 12, 16, 20, and 24 °C for two weeks, respectively, by critical thermal methodology. Then, through heat challenge, we detected the metabolites, key enzyme activities, and gene expressions involved in metabolism and antioxidant in the hepatopancreas when the temperatures increased from 16 °C to 18, 20, 22, 24, 26, and 28 °C for 12 h, respectively. The results showed that although the fish are sensitive to high temperatures, the quick acclimation at mild high temperatures could significantly improve the tolerance to acute high-temperature stress in juvenile G. przewalskii. During the heat challenge study, blood glucose significantly increased at heat stress (P < 0.05). At the same time, total cholesterol (TC), triglyceride (TG), and free fatty acid (FFA) significantly decreased when the temperature rose continuously to 20 °C. Metabolic enzyme activities of carnitine palmityl transferase I (CPT-Ⅰ), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) significantly decreased at 20 °C (P < 0.05). Superoxide dismutase (SOD) and antioxidant capacity (T-AOC) significantly increased at 20 °C (P < 0.05). The relative transcript levels of genes involved in antioxidant and glycolysis/gluconeogenesis were markedly higher than the control at 20-26 °C (P < 0.05). The genes involved in fatty acid biosynthesis or metabolism showed different expression patterns under heat stress. Heat shock protein 70 (Hsp70) and Hsp90 were significantly higher than the control at 18 °C and 26 °C, respectively. These results confirmed the prediction that G. przewalskii is sensitive to high temperatures, so conservation efforts should pay more attention to the warming damage.

Management strategy of the naked carp (Gymnocypris przewalskii) in the Qinghai lake using matrix population modeling.

Naked carp (Gymnocypris przewalskii) is the only fish species commercially harvested in Qinghai Lake, which is the largest inland saltwater lake in China. Multiple ecological stresses such as long-term overfishing, drying-up of riverine inflows, and decreases in spawning habitat caused the naked carp population to decrease from 320,000 tons before the 1950s to only 3000 tons by the early 2000s. We used matrix projection population modeling to quantitatively simulate the dynamics of the naked carp population from the 1950s to the 2020s. Five versions of the matrix model were developed from the field and laboratory information that represented different population states (high but declining, low abundance, very low abundance, initial recovery, pristine). Equilibrium analysis was applied to density-independent versions of the matrices and population growth rate, age composition, and elasticities were compared among versions. Stochastic, density-dependent version of the most recent decade (recovering) version was used to simulate the time-dependent responses to a range of levels of artificial reproduction (addition of age-1 from hatchery) and of the pristine version to simulate combinations of fishing rate and minimum age of harvest. Results showed the major role of overfishing in the population decline and that the population growth rate was most sensitive to the survival of juveniles and the spawning success of early-age adults. Dynamic simulations showed a rapid population response to artificial reproduction when population abundance was low and that if artificial reproduction continues at its current level, then population biomass would reach 75% of its pristine biomass after 50 years. Simulations with the pristine version identified sustainable fishing levels and the importance of protecting the first few ages of maturity. Overall, modeling results showed that artificial reproduction under conditions of no fishing is an effective approach to restoring the naked carp population. Further effectiveness should consider maximizing survival in the months just after release and maintaining genetic and phenotypic diversity. More information on density-dependent growth, survival, and reproduction, as well as on the genetic diversity and growth and migratory behavior (phenotypic variation) of released and native-spawned fish, would help inform management and conservation strategies and practices going forward.

Establishment of a cell line derived from the gills of Gymnocypris przewalskii, an endemic Schizothoracine fish from Qinghai Lake of Tibet Plateau.

Gymnocypris przewalskii (Naked carp), a native teleost, plays an important role in maintaining the ecological balance of Lake Qinghai (altitude, 3.2 km), the largest saline lake in China. In this study, a new gill cell line from G. przewalskii was developed using the explant technique and named as GPG. This cell line was maintained in Dulbecco's Modified Eagle Medium (DMEM) (high glucose), supplemented with 15% fetal bovine serum (FBS), and was successfully subcultured up to 32 passages. Meanwhile, this cell line was also authenticated by sequencing the mitochondrial cytochrome C oxidase subunit I (COI) and 16S rRNA genes and by chromosome analysis. With the Cytomegalovirus (CMV) promoter, the GPG cell line could express green fluorescent protein (GFP) at about 5% transfection efficiency. MTT test showed that Clostridium botulinum toxin (BTX) was toxic to the cell line. After cryopreservation with 10% dimethyl sulfoxide (DMSO), this cell line could be successfully revived at an efficiency over 70%. This study revealed that the GPG cell line could be used as materials for physio-chemical investigation of G. przewalskii and also provided a tool for gene function study and toxicological reaction in vitro.

Genome-wide identification and expression of the peroxisome proliferator-activated receptor gene family in the Tibetan highland fish Gymnocypris przewalskii.

Peroxisome proliferator-activated receptor (PPAR) plays an important role in the regulation of lipid metabolism and has been widely identified in diverse species. Gymnocypris przewalskii is a native fish of the Qinghai Tibetan Plateau that survives in a chronically cold environment. In the current study, we conducted genome-wide identification of PPAR genes, revealing the existence of seven PPARs in the G. przewalskii genome. Collinearity was observed between two copies of PPARαb and PPARγ in G. przewalskii, suggesting that the additional copy might be gained through whole genome duplication. Both phylogenetic and multiple sequence alignment analyses indicated that PPARs in G. przewalskii were conserved with teleosts. The cold treatment (10 °C and 4 °C) led to the developmental delay of G. przewalskii embryos. Continuous expression of PPARs was observed during the embryonic development of G. przewalskii under normal and cold conditions, with significantly different transcriptional patterns. These results indicated that PPARs participated in the embryonic development of G. przewalskii, and were involved in the cold response during development. The current study proposed a potential role of PPARs in the cold response in the embryonic development of G. przewalskii, which shed light on understanding cold adaptation in Tibetan highland fish.

Ammonia excretion and blood gas variation in naked carp (Gymnocypris przewalskii) exposed to acute hypoxia and high alkalinity.

Naked carp (Gymnocypris przewalskii), endemic to the saline-alkaline Lake Qinghai, have the capacity to tolerate combined hypoxia and high alkalinity. This study evaluated the effect of the interaction between carbonate alkalinity and hypoxia on ammonia excretion and blood gas variation in naked carp. Naked carp were subjected to normoxic, hypoxic and reoxygenation phases at two different carbonate alkalinity levels (CA0 = 0 mmol/L; CA32 = 32 mmol/L) for 4 days. The ammonia excretion rate (JAmm) of the CA0 group rapidly decreased under hypoxia and recovered under normoxia for four consecutive days. The JAmm under CA32 also decreased under hypoxia and recovered to its previous level in the first 2 days. However, the JAmm under CA32 was lower than that under CA0. The blood pO2, sO2 of CA0 and CA32 group was significantly reduced under hypoxia, after which both groups recovered. Blood pCO2 of the CA32 group was lower than CA0 throughout the experiment. There were no changes in haematocrit of the naked carp exposed to carbonate alkalinity and hypoxia. The alkaline water increased the pH of the blood and contributed to increased haemoglobin O2 affinity. Overall, the present findings reveal that naked carp is a tolerant species that can maintain main ionic homeostasis under severe alkalinity and hypoxia. The high alkaline water is beneficial for naked carp to adapt to hypoxic environment.

Transcriptomic profiling of Tibetan highland fish (Gymnocypris przewalskii) in response to the infection of parasite ciliate Ichthyophthirius multifiliis.

Gymnocypris przewalskii is a native cyprinid in the Lake Qinghai of the Qinghai-Tibetan Plateau. G. przewalskii is highly susceptible to the infection of a parasite, Ichthyophthirius multifiliis, in the artificial propagation and breeding. To better understand the host immune reaction to I. multifiliis infection, we characterize the gene expression profiles in the spleen of healthy and I. multifiliis infected G. przewalskii by RNA-seq. Totally, the transcriptomic analysis produces 463,031,110 high quality reads, which are assembled to 213,538 genes with N50 of 1918 bp and the average length of 1205 bp. Of assembled genes, 90.52% are annotated by public databases. The expression analysis shows 744 genes are significantly changed by the infection of I. multifiliis, which are validated by qRT-PCR with the correlation coefficient of 0.896. The differentially expressed genes are classified into 689 GO terms and 230 KEGG pathways, highlighting the promoted innate immunity in I. multifiliis infected G. przewalskii at 2 days post infection. Our results pinpoint that the up-regulated genes are enriched in TLR signaling pathway, inflammatory response and activation of immune cell migration. On the contrary, complement genes are down-regulated, indicating the evasion of host complement cascades by I. multifiliis. The repressed genes are also enriched in the pathways related to metabolism and endocrine, suggesting the metabolic disturbance in I. multifiliis treated G. przewalskii. In summary, the present study profiles the gene expression signature of G. przewalskii in the responses to I. multifiliis infection, and improves our understanding on molecular mechanisms of host-parasite interaction in G. przewalskii, which focuses the crucial function of TLRs, cytokines and complement components in the host defense against I. multifiliis.

Transcriptome-wide identification, molecular evolution and expression analysis of Toll-like receptor family in a Tibet fish, Gymnocypris przewalskii.

Toll-like receptors (TLR) are key components of innate immunity that play significant roles in immune defense against pathogens invasion. Recent frequent outbreaks of the "white spot disease" caused by parasitic infection in farmed Tibetan fishes had resulted in great economic losses. However, to our knowledge, the roles of TLRs in mediating immune response to parasitic infection in Tibetan fishes remain to be determined. Here, we performed data-mining on a widely-farmed Tibetan fish (Gymnocypris przewalskii or Gp) transcriptome to determine the genetic variation and expression pattern of TLRs. We totally obtained 14 GpTLRs and identified 5 with a complete coding sequence. Phylogenetic analysis verified their identities and supported the classification of TLRs into six families as in other vertebrates. The TLR family motifs, such as leucine rich repeat (LRR) and Toll/interleukin (IL)-1 receptor (TIR) domain, are conserved in GpTLR1-5. Selective pressure test demonstrated that all known GpTLRs are under purifying selection, except GpTLR4 underwent positive selection. Further, site model analysis suggested that 11 positively selected sites are found in LRR domain of GpTLR4. Three positively selected sites are located on outside surface of TLR4 3D structure, indicating that function of GpTLR4 may be affected. Tissue specific expression analysis showed all GpTLRs are present in gill, head-kidney and spleen but the relative abundance varied among tissues. In response to parasite Ichthyophthirius multifiliis infection, 5 GpTLR (GpTLR1, -2, -4, -9 and -20) expressions were induced. Intriguingly, GpTLR4 was significantly up-regulated in gills, while GpTLR19 and GpTLR21 unexpectedly showed no any change. In summary, these results revealed the first genomic resources of TLR family and several parasitic infection responsive TLRs in Tibetan fish. These findings provide key information for future studies aiming to understand the molecular mechanisms underlying the immune response to pathogen invasion in Tibetan fishes.

Transcriptome profiling analysis of naked carp (Gymnocypris przewalskii) provides insights into the immune-related genes in highland fish.

The naked carp, Gymnocypris przewalskii, is one of the dominant aquaculture fish species in Qinghai Province, China. Its wild stocks have severely suffered from overfishing, and the farming species are vulnerable to various pathogens infections. Here we report the first immune-related tissues transcriptome of a wild naked carp using a deep sequencing approach. A total of 158,087 unigenes are generated, 2687 gill-specific gene and 3215 kidney-specific genes are identified, respectively. Gene ontology analysis shows that 51,671 unigenes are involved in three major functional categories: biological process, cellular component, and molecular function. Further analysis shows that numerous consensus sequences are homologous to known immune-related genes. Pathways mapping annotate 56,270 unigenes and identify a large number of immune-related pathways. In addition, we focus on the immune-related genes and gene family in Toll-like receptor signaling pathway involved in innate immunity, including toll-like receptors (TLRs), interferon regulatory factors (IRFs), interleukins (ILs) and tumor necrosis factors (TNFs). Eventually, we identify 5 TLRs, 4 IRFs, 3 ILs and 2 TNFs with a completed coding sequence though mining the transcriptome data. Phylogeny analysis shows these genes of naked carp are mostly close to zebrafish. Protein domain and selection pressure analyses together show that all these genes are highly conserved in gene sequence and protein domain structure with other species, and purifying selection underwent in these genes, implied functionally important features are conserved in the genes above. Intriguingly, we detect positive selection signals in naked carp TLR4, and significant divergence occurred among tested species TLR4, suggested that naked carp TLR4 function may be affected. Finally, we identify 23,867 simple sequence repeat (SSR) marks in this transcriptome. Taken together, this study not only contributes a large number of candidate genes in naked carp immunity, and also helps improve current understanding of immunogenetics basis and evolutionary history of immune related genes and gene family in highland fish species.

Transcriptome Analysis Reveals the Molecular Mechanisms Underlying Growth Superiority in a Novel Gymnocypris Hybrid, Gymnocypris przewalskii ♀ × Gymnocypris eckloni ♂.

Artificial hybrid breeding can optimize parental traits to cultivate excellent hybrids with enhanced economic value. In this study, we investigated the growth performance and transcriptomes of Gymnocypris przewalskii (♀) and Gymnocypris eckloni (♂) and their F1 hybrid fishes. Hatched individuals of G. przewalskii (GP) and G. eckloni (GE) of the same size and their F1 hybrids (GH) were separately cultured for eight months in three cement tanks (n = 3). The growth indexes were measured, which showed that the growth rate of the groups was GE > GH > GP, while the survival rate was GH > GE > GP. The RNA-Seq data analysis of the muscles from the three Gymnocypris fish strains revealed that gene transcription has a significant impact on F1 hybrid fish and its parents. The differentially expressed genes (DEGs) in GH show less differences with GP, but more with GE. qRT-PCR was used to confirm the expression profiles of the chosen DEGs, and the results showed positive correlations with the RNA-seq data. KEGG enrichment results indicated that the DEGs were related to a variety of molecular functions, such as glycolysis/gluconeogenesis, arachidonic acid formation, citrate cycle, and the MAPK, PI3K-Akt, or mTOR signal pathways. Subsequent analysis indicated that there may be a significant correlation between the differential expression of IGF2 and a difference in the growth of GE and GP.

Population genomics analysis to identify ion and water transporter genes involved in the adaptation of Tibetan naked carps to brackish water.

Understanding how evolutionary processes shape the genetic variations and influence the response of species to environmental alterations is critical for biodiversity conservation and molecular breeding. Gymnocypris przewalskii przewalskii is the only known cyprinid fish that dwells in the brackish water of Lake Qinghai on the Qinghai-Tibetan Plateau. To reveal the genetic basis of its adaptation to high salinity and alkalinity, whole-genome sequencing was performed in G. p. przewalskii and its freshwater relatives Gymnocypris eckloni and Gymnocypris przewalskii ganzihonensis. Compared with freshwater species, lower genetic diversity and higher linkage disequilibrium were observed in G. p. przewalskii. Selective sweep analysis identified 424 core-selective genes enriched in transport activities. Transfection analysis showed that genetic changes in the positively selected gene aquaporin 3 (AQP3) improved cell viability after salt treatment, suggesting its involvement in brackish water adaptation. Our analysis indicates that ion and water transporter genes experienced intensive selection, which might have contributed to the maintenance of high osmolality and ion content in G. p. przewalskii. The current study identified key molecules involved in the adaptation of fish to brackish water, providing valuable genomic resources for the molecular breeding of salt-tolerant fish.

Cell line derived from muscle of Gymnocypris przewalskii, a species of Schizothoracinae in Qinghai Lake, Qinghai-Tibet Plateau.

Gymnocypris przewalskii (naked carp), a native teleost, plays an important role in the ecosystem of Qinghai Lake (altitude, 3.2 km) on the Qinghai-Tibet Plateau in China. We developed a new cell line from the muscle of G. przewalskii using the explant technique and named the cell line GPM. This cell line was maintained in DMEM medium (high glucose) supplemented with 15% fetal bovine serum (FBS). The cell line was successfully subcultured up to 32 passages and was authenticated by immunofluorescence assay, sequencing the mitochondrial cytochrome C oxidase subunit I (COI) and 16S rRNA genes, and by chromosome analysis. In the medium containing 15% FBS, the cell line could be passaged stably at 25 °C. The GPM cell line could express green fluorescent protein (GFP) with a CMV promoter with about 5% transfection efficiency. MTT tests showed that Clostridium botulinum toxin (BTX) was toxic to the cell line. The cell line could be successfully cryopreserved in liquid nitrogen with a revival efficiency of over 70%. This study demonstrated that the GPM cell line can be used as an important tool for understanding the physiological characteristics of G. przewalskii, and it can provide a resource for studying gene function and toxicological reactions in vitro.

Transcriptomic and proteomic analyses provide insights into the adaptive responses to the combined impact of salinity and alkalinity in Gymnocypris przewalskii.

Gymnocypris przewalskii is the only high-land endemic teleost living in Qinghai Lake, the largest saline-alkaline lake in China. Its osmoregulatory physiology remains elusive due to a lack of precise identification of the response proteins. In the present study, DIA/SWATH was used to identify differentially expressed proteins (DEPs) under alkaline (pH = 10.1, carbonate buffer), saline (12‰, sodium chloride), and saline-alkaline [carbonate buffer (pH = 10.1) plus 11‰ sodium chloride] stresses. A total of 66,056 unique peptides representing 7,150 proteins and 230 DEPs [the false discovery rate (FDR) ≤ 0.05, fold change (FC) ≥ 1.5] were identified under different stresses. Comparative analyses of the proteome and transcriptome indicated that over 86% of DEPs did not show consistent trends with mRNA. In addition to consistent enrichment results under different stresses, the specific DEPs involved in saline-alkaline adaptation were primarily enriched in functions of homeostasis, hormone synthesis and reactions of defense response, complement activation and reproductive development. Meanwhile, a protein-protein interaction (PPI) network analysis of these specific DEPs indicated that the hub genes were ITGAX, MMP9, C3, F2, CD74, BTK, ANXA1, NCKAP1L, and CASP8. This study accurately isolated the genes that respond to stress, and the results could be helpful for understanding the physiological regulation mechanisms regarding salinity, alkalinity, and salinity-alkalinity interactions.

Chromosome-level genome of Tibetan naked carp (Gymnocypris przewalskii) provides insights into Tibetan highland adaptation.

Gymnocypris przewalskii, a cyprinid fish endemic to the Qinghai-Tibetan Plateau, has evolved unique morphological, physiological and genetic characteristics to adapt to the highland environment. Herein, we assembled a high-quality G. przewalskii tetraploid genome with a size of 2.03 Gb and scaffold N50 of 44.93 Mb, which was anchored onto 46 chromosomes. The comparative analysis suggested that gene families related to highland adaptation were significantly expanded in G. przewalskii. According to the G. przewalskii genome, we evaluated the phylogenetic relationship of 13 schizothoracine fishes, and inferred that the demographic history of G. przewalskii was strongly associated with geographic and eco-environmental alterations. We noticed that G. przewalskii experienced whole-genome duplication, and genes preserved post duplication were functionally associated with adaptation to high salinity and alkalinity. In conclusion, a chromosome-scale G. przewalskii genome provides an important genomic resource for teleost fish, and will particularly promote our understanding of the molecular evolution and speciation of fish in the highland environment.

Riverbed Substrate Requirements for Natural Reproduction of Gymnocypris przewalskii.

Gymnocypris przewalskii (i.e., Qinghai Lake naked carp) is a migratory fish species that lives in highland brackish water. It is important to understand the abiotic environment required by this fish to reproduce naturally so that its habitat can be protected and the wild population can be conserved. Here, artificial simulation and spawning ground substrate transformation experiments were conducted to examine the riverbed substrate requirements for G. przewalskii to naturally reproduce. Using various techniques (in vitro markers, videography, and Ethovision XT behavior tracking), this study systematically investigated the riverbed substrate preferences of G. przewalskii as well as the characteristics and effectiveness of natural reproduction induced by pebble riverbed substrate. The findings can be summarized as follows: (1) the habitat preferences of G. przewalskii differed significantly between various riverbed substrate, with pebble substrate being preferred during natural reproduction, and sand substrate being preferred pre- and post-spawning, and (2) the natural reproduction of G. przewalskii was heavily reliant on pebble riverbed substrate. Specifically, pebble substrate significantly improved spawn quantity and fertilization rate. These findings provide scientific evidence for the improvement and restoration of G. przewalskii spawning grounds, and insights regarding the artificial bionic reproduction of G. przewalskii.

Characterization and complexity of transcriptome in Gymnocypris przewalskii using single-molecule long-read sequencing and RNA-seq.

The Tibetan Schizothoracinae fish Gymnocypris przewalskii has the ability to adapt to the extreme plateau environment, making it an ideal biological material for evolutionary biology research. However, the lack of well-annotated reference genomes has limited the study of the molecular genetics of G. przewalskii. To characterize its transcriptome features, we first used long-read sequencing technology in combination with RNA-seq for transcriptomic analysis. A total of 159,053 full-length (FL) transcripts were captured by Iso-Seq, having a mean length of 3,445 bp with N50 value of 4,348. Of all FL transcripts, 145,169 were well-annotated in the public database and 134,537 contained complete open reading frames. There were 4,149 pairs of alternative splicing events, of which three randomly selected were defined by RT-PCR and sequencing, and 13,293 long non-coding RNAs detected, based on all-vs.-all BLAST. A total of 118,185 perfect simple sequence repeats were identified from FL transcripts. The FL transcriptome might provide basis for further research of G. przewalskii.

Identification and characterization of microRNAs involved in scale biomineralization in the naked carp Gymnocypris przewalskii.

The mineralized scale derived from skin plays a protective role for the fish body and also possesses important application values in the biomaterial field. However, little is known about fish scale biomineralization and related molecular regulatory mechanisms. Here, we used a comparative microRNA sequencing approach to identify and characterize differentially expressed microRNAs (DEMs) involved in scale biomineralization in the naked carp Gymnocypris przewalskii. A total of 18, 43, and 66 DEMs were obtained from skin tissues covered with initial, developing, and mature scales (IS, DS, and MS) compared with scale-uncovered skin. The target genes of these DEMs were significantly enriched in a sole biomineralization-related sphingolipid signaling pathway. Seven DEMs (dre-miR-124-3p, dre-miR-133a-2-5p, dre-miR-184, dre-miR-206-3p, novel_33, novel_56 and novel_75) were common in IS, DS, and MS. Dre-miR-124-3p, dre-miR-206-3p, and novel_33 were predicted to be able to target biomineralization-related genes. Stem-loop real-time quantitative PCR further confirmed that the common DEMs had higher expression levels in scale-covered skin tissues than that in the gill, intestine, and brain, except for dre-miR-133a-2-5p. Our results suggest that these identified microRNAs may play a role in scale biomineralization in G. przewalskii, and the obtained microRNAs are expected to be candidates in understanding the molecular mechanism of scale biomineralization in fish species.

Characterization of two paralogous myostatin genes and evidence for positive selection in Tibet fish: Gymnocypris przewalskii.

Myostatin (mstn) is an important member of TGF-ß superfamily, a muscle growth inhibitor. Though mstn has been identified in many organisms, little is known about this gene in highland fish, Gymnocypris przewalskii endemic to the Qinghai-Tibetan Plateau. In this study, we first cloned two paralogous mstn genes (mstn1 and mstn2) from G. przewalskii through homologue cloning. The 3D structures of both Mstn proteins varied in the numbers of ß-sheets and conformations of α-helices. The branch-site model showed that mstn1 has undergone positive selection, and two positively selected sites (107M and 181T) were located on the random coils of the 3D protein structure. Expression patterns indicated that the mstn1 expressed widely, while the mstn2 only expressed in the muscle and brain. During the early stage of embryo development, the expression levels of both mstn paralogous genes showed different increasing trends. These results suggest that it is diverging in two mstn paralogues of G. przewalskii via specific differences in gene structure, protein structure, selection pressure and gene expression patterns. Taken together, this study provides novel contribution on the research topics of growth related gene function and mechanism of highland fish in extreme aquatic environment on the Qinghai-Tibetan Plateau.