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.

Effects of plant-derived protein and rapeseed oil on growth performance and gut microbiomes in rainbow trout.

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) is becoming popular with the increased demand for fish protein. However, the limited resources and expense of fish meal and oil have become restrictive factors for the development of the rainbow trout related industry. To solve this problem, plant-derived proteins and vegetable oils have been developed as alternative resources. The present study focuses on evaluating the effects of two experimental diets, FMR (fish meal replaced with plant-derived protein) and FOR (fish oil replaced with rapeseed oil), through the alteration of the gut microbiota in triploid rainbow trout. The commercial diet was used in the control group (FOM). RESULTS: Amplicon sequencing of the 16S and 18S rRNA genes was used to assess the changes in gut bacteria and fungi. Our analysis suggested that the α-diversity of both bacteria and fungi decreased significantly in the FMR and FOR groups, and ß-diversity was distinct between FOM/FMR and FOM/FOR based on principal coordinate analysis (PCoA). The abundance of the Planctomycetota phylum increased significantly in the FMR group, while that of Firmicutes and Bacteroidetes decreased. We also found that the fungal phylum Ascomycota was significantly increased in the FMR and FOR groups. At the genus level, we found that the abundance of Citrobacter was the lowest and that of pathogenic Schlesneria, Brevundimonas, and Mycoplasma was highest in the FMR and FOR groups. Meanwhile, the pathogenic fungal genera Verticillium and Aspergillus were highest in the FMR and FOR groups. Furthermore, canonical correspondence analysis (CCA) and network analysis suggested that the relatively low-abundance genera, including the beneficial bacteria Methylobacterium, Enterococcus, Clostridium, Exiguobacterium, Sphingomonas and Bacteroides and the fungi Papiliotrema, Preussia, and Stachybotrys, were positively correlated with plant protein or rapeseed oil. There were more modules that had the above beneficial genera as the hub nodes in the FMR and FOR groups. CONCLUSIONS: Our study suggested that the FMR and FOR diets could affect the gut microbiome in rainbow trout, which might offset the effects of the dominant and pathogenic microbial genera. This could be the underlying mechanism of explaining why no significant difference was observed in body weight between the different groups.

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.

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.

Duplication of toll-like receptor 22 in teleost fishes.

The TLRs of teleost fishes have distinct features and are highly diverse, but the duplication characteristics and expression patterns of the tlr22 gene remain unclear. Here, we identified paralogous tlr22 genes in 13 teleost fishes by screening available fish genomic resources and using molecular cloning. We then conducted comprehensive bioinformatics analyses and investigated spatiotemporal differences in the expression patterns of the tlr22 genes in G. eckloni. The results indicated that more than three paralogous tlr22 genes were possessed by some teleost fishes. Of these, tlr22c is specific to some subfamilies of the Cyprinidae (e.g., Barbinae, Cyprininae, Schizothoracinae, and Leuciscinae). Phylogenetic and syntenic analyses showed that the paralogous tlr22 genes originated from two single-gene duplication events. Molecular clock calculations dated the two gene duplication events at 49.5 and 39.3 MYA, which is before the common carp-specific genome duplication event and well after the fish-specific genome duplication. Gene duplication of tlr22 was followed by gene loss or pseudogene events in certain lineages. Spatiotemporal expression differences between the three duplicated tlr22 genes from G. eckloni suggested that these genes diverged functionally after gene duplication.

Molecular characterization and expression changes of cytoglobin genes in response to hypoxia in a Tibetan schizothoracine fish, Schizopygopsis pylzovi.

Schizopygopsis pylzovi, an endemic fish of the subfamily Schizothoracinae, is comparatively well adapted to dissolved oxygen fluctuations in the aqueous environments of the Qinghai-Tibetan Plateau. Here, we cloned the complete cDNA of cytoglobin 1 and 2 (Cygb1 and Cygb2) from S. pylzovi and then investigated transcriptional changes of both genes in the selected tissues in response to hypoxia. Both the two genes had the standard exon-intron structure of vertebrate Mb genes but lacked an exon at downstream of the H helix (HC11.2) as seen in mammals. We applied severe hypoxia (4 h at PO2 = 3.6% saturation) and moderate hypoxia (72 h at PO2 = 36.0% saturation) to adult S. pylzovi. Under severe hypoxia, the Cygb1 mRNA levels decreased significantly in the liver, kidney, and brain, but increased significantly in the heart, while the Cygb2 mRNA levels downregulated significantly in the muscle and liver. But, the transcriptional activity of Cygb1 in muscle and that of Cygb2 in the kidney, brain, and heart remained almost unchanged. Under moderate hypoxia, the transcriptional activities of both genes in muscle and brain were turned down quickly after onset hypoxia, while in the liver, kidney, and heart, the transcriptional activities of both genes showed a short-term upregulation in different time periods of hypoxia exposure. Our data suggest that both the Cygb1 and Cygb2 in S. pylzovi are hypoxia-induced genes, and the responses of the transcription regulation of Cygb1 and Cygb2 genes to hypoxia are tissue specific and also depend on the hypoxia regime, which are different from that of other fish species.

Adaptive evolution of interferon regulatory factors is not correlated with body scale reduction or loss in schizothoracine fish.

Body scales in teleost fish are the first line of defense in protecting the fish from invading pathogens. However, the relationship between the adaptive evolution of immune-related genes and changes in the body scale-covering of fish has not been previously studied. Schizothoracine fish, characterized by progressive reduction of body scales from the primitive to the highly specialized species, are a good group to investigate this relationship. We obtained 11 IRF genes (IRF1-11) from 14 of schizothoracine fish representing primitive, specialized, and highly specialized species, of which seven IRF genes (IRF2, IRF3, IRF5, IRF6, IRF7, IRF8 and IRF9) contained the complete CDS. Sequence analysis demonstrated the deletion or insertion of 4-7 amino acids in IRF2, IRF3, IRF6 and IRF9, which seems to be a common phenomenon in the schizothoracine fish. Selection pressure analysis supported the hypothesis that positive selection has been driving the rapid evolution of IRFs in specific lineages of the schizothoracine fish. This, however, is not correlated with body scale reduction or loss in the evolution of these IRFs. Remarkably, the deletion or insertion found in IRF protein sequences presented a regular pattern corresponding to the scale-covering changes in schizothoracine fish. Our study provides evidence for positive selection in the IRF family, contributing to a better understanding of the adaptive evolution of immune-related genes in schizothoracine fish in response to environmental changes of the Qinghai-Tibetan Plateau.

Comparison of Phenols Content and Antioxidant Activity of Fruits from Different Maturity Stages of Ribes stenocarpum Maxim.

Differences in the content of nine phenols and the antioxidant capacity of Ribes stenocarpum Maxim (RSM) fruits at different stages of maturity were investigated, and the extraction process of polyphenols from RSM was also optimized using Box-Behnken design method. Results showed that the content of the nine phenols varied considerably at different ripening stages; catechin, chlorogenic acid, coumaric acid, and ferulic acid were abundant in immature fruits but decreased with fruit ripening, whereas the levels of rosemary acid and querctin acid were low in immature fruits and increased with time, reaching the highest value after the fruit was completely mature. The phenols extracted from RSM fruits possessed good antioxidant activities for effective and rapid scavenging of DPPH and ABTS free radicals, as well as intracellular ROS. Analysis of the phenols content at different maturity stages indicated that the unripe fruits had significantly higher polyphenols content than mature fruits. Consequently, unripe fruits possessed higher antioxidant activities. According to the overall results of the extraction process optimization, the selected optimal conditions for extracting polyphenols from RSM were as follows: extraction time, 95 min; solvent concentration, 60%; ratio of sample to solvent, 1:25.

Molecular evolution of myoglobin in the Tibetan Plateau endemic schizothoracine fish (Cyprinidae, Teleostei) and tissue-specific expression changes under hypoxia.

Myoglobin (Mb) is an oxygen-binding hemoprotein that was once thought to be exclusively expressed in oxidative myocytes of skeletal and cardiac muscle where it serves in oxygen storage and facilitates intracellular oxygen diffusion. In this study, we cloned the coding sequence of the Mb gene from four species, representing three groups, of the schizothoracine fish endemic to the Qinghai-Tibetan Plateau (QTP), then conducted molecular evolution analyses. We also investigated tissue expression patterns of Mb and the expression response to moderate and severe hypoxia at the mRNA and protein levels in a representative of the highly specialized schizothoracine fish species, Schizopygopsis pylzovi. Molecular evolution analyses showed that Mb from the highly specialized schizothoracine fish have undergone positive selection and one positively selected residue (81L) was identified, which is located in the F helix, close to or in contact with the heme. We present tentative evidence that the Mb duplication event occurred in the ancestor of the schizothoracine and Cyprininae fish (common carp and goldfish), and that the Mb2 paralog was subsequently lost in the schizothoracine fish. In S. pylzovi, Mb mRNA is expressed in various tissues with the exception of the intestine and gill, but all such tissues, including the liver, muscle, kidney, brain, eye, and skin, expressed very low levels of Mb mRNA (< 8.0%) relative to that of the heart. The trace levels of Mb expression in non-muscle tissues are perhaps the major reason why non-muscle Mb remained undiscovered for so long. The expression response of the Mb gene to hypoxia at the mRNA and protein levels was strikingly different in S. pylzovi compared to that found in the common carp, medaka, zebrafish, and goldfish, suggesting that the hypoxia response of Mb in fish may be species and tissue-specific. Notably, severe hypoxia induced significant expression of Mb at the mRNA and protein levels in the S. pylzovi heart, which suggests Mb has a major role in the supply of oxygen to the heart of Tibetan Plateau fish.

Identification, molecular evolution of toll-like receptors in a Tibetan schizothoracine fish (Gymnocypris eckloni) and their expression profiles in response to acute hypoxia.

Hypoxia plays an important role in regulating a variety of physiological responses as well as in pathological situations, but to date the roles of Toll-like receptors (TLRs) in fish in response to hypoxia are still poorly understood. Here, we sequenced the transcriptome of G. eckloni and identified the members of TLR family by scanning transcriptome, and then investigated the expression profiles of a complete set of TLRs in G. eckloni in response to acute hypoxia (4 h at DO = 0.3 ± 0.1 mg/L). The de novo-assembled transcriptome consisted of a total of 162,235 transcripts, further clustered into 110,231 unigenes. Based on the transcriptome, a total of 18 TLRs were identified in G. eckloni, and of them three TLRs (TLR5, TLR8 and TLR22) possessed two distinct paralogous genes. The duplicated genes of TLR22 were discovered for the first time in cyprinid fish, but did not origin from a recent duplication event. Of them TLR22b may be specific for schizothoracine fish, at least for G. eckloni. Phylogenetic analysis supported the classification of TLRs into six families as in other vertebrates but was partly different from the previous study. The sliding window analysis showed strong signals of positive selection in TLR2, TLR 4, TLR 5a, TLR 7, TLR 19, TLR 20, TLR 21, TLR 22a and TLR 22b, but most codons under positive selection were located in the putative LRR regions. The mRNA expression of most TLRs in head kidney, spleen and gill decreased significantly or remained unchanged under acute hypoxia, whereas acute hypoxia increased expressions of TLR2 and TLR3 in head kidney, of TLR8a, TLR12 and TLR19 in spleen, and of TLR1 in gill, suggesting tissues-specific expressions of TLRs play important roles in mediating innate immune responses for host defense against tissue damages or physiological changes induced by hypoxia.

Inhibition of inducible nitric oxide synthase expression and nitric oxide production in plateau pika (Ochotona curzoniae) at high altitude on Qinghai-Tibet Plateau.

Nitric oxide (NO), a potent vasodilator, plays an important role in preventing hypoxia induced pulmonary hypertension. Endogenous NO is synthesized by nitric oxide synthases (NOSs) from l-arginine. In mammals, three different NOSs have been identified, including neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Plateau pika (Ochotona curzoniae) is a typical hypoxia tolerant mammal that lives at 3000-5000 m above sea level on the Qinghai-Tibet Plateau. The aim of this study was to investigate whether NOS expression and NO production are regulated by chronic hypoxia in plateau pika. Quantitative real-time PCR and western blot analyses were conducted to quantify relative abundances of iNOS and eNOS transcripts and proteins in the lung tissues of plateau pikas at different altitudes (4550, 3950 and 3200 m). Plasma NO metabolites, nitrite/nitrate (NO(x)⁻) levels were also examined by Ion chromatography to determine the correlation between NO production and altitude level. The results revealed that iNOS transcript levels were significantly lower in animals at high altitudes (decreased by 53% and 57% at altitude of 3950 and 4550 m compared with that at 3200 m). Similar trends in iNOS protein abundances were observed (26% and 41% at 3950 and 4550 m comparing with at 3200 m). There were no significant differences in eNOS mRNA and protein levels in the pika lungs among different altitudes. The plasma NO(x)⁻ levels of the plateau pikas at high altitudes significantly decreased (1.65±0.19 µg/mL at 3200 m to 0.44±0.03 µg/mL at 3950 m and 0.24±0.01 µg/mL at 4550 m). This is the first evidence describing the effects of chronic hypoxia on NOS expression and NO levels in the plateau pika in high altitude adaptation. We conclude that iNOS expression and NO production are suppressed at high altitudes, and the lower NO concentration at high altitudes may serve crucial roles for helping the plateau pika to survive at hypoxic environment.

Response of phytoplankton composition to environmental stressors under humidification in three alpine lakes on the Qinghai-Tibet Plateau, China.

Owning to their extreme environmental conditions, lakes on the Qinghai-Tibet Plateau have typically displayed a simplistic food web structure, rendering them more vulnerable to climate change compared to lakes in plains. Phytoplankton, undergoing a changing aquatic environment, play a crucial role in the material cycle and energy flow of the food chain, particularly important for the unique fish species of the Tibetan Plateau. To identify the changing environment indexes and determine the response of phytoplankton composition to the environment change in alpine lakes, three lakes-Lake Qinghai, Lake Keluke and Lake Tuosu-were selected as study areas. Seasonal sampling surveys were conducted in spring and summer annually from 2018 to 2020. Our findings revealed there were significant changes in physicochemical parameters and phytoplankton in the three lakes. Bacillariophyta was the predominant phytoplankton in Lake Qinghai from 2018 to 2020, with the genera Synedra sp., Navicula sp., Cymbella sp. and Achnanthidium sp. predominated alternately. Lake Keluke alternated between being dominated by Bacillariophyta and cyanobacteria during the same period. Dolichospermum sp., a cyanobacteria, was prevalent in the summer of 2018 and 2019 and in the spring of 2020. In Lake Tuosu, Bacillariophyta was the predominant phytoplankton from 2018 to 2020, except in the summer of 2019, which was dominated by cyanobacteria. Synedra sp., Oscillatoria sp., Pseudoanabaena sp., Chromulina sp. and Achnanthidium sp. appeared successively as the dominant genera. Analysis revealed that all three lakes exhibited higher phytoplankton abundance in 2018 that in 2019 and 2020. Concurrently, they experienced higher average temperatures in 2018 than in the subsequent years. The cyanobacteria, Bacillariophyta, Chlorophyta and overall phytoplankton increased with temperature and decreased with salinity and NH4-N. Besides, the ratios of cyanobacteria, and the ratios of Bacillariophyta accounted in total phytoplankton increased with temperature. These findings suggest that cyanobacteria and phytoplankton abundance, especially Bacillariophyta, may have an increase tendency in the three alpine lakes under warm and wet climate.

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.

Genome-wide characterization of the Elovl gene family in Gymnocypris przewalskii and their potential roles in adaptation to cold temperature.

The elongase of the very long-chain fatty acids (Elovls) gene family in fish has more diversity than in other vertebrates, which plays several critical roles in fatty acid synthesis and low-temperature stress adaptation. Gymnocypris przewalskii settles in plateau lakes with cold and resource-poor settings, and the evolution and function of Elovl genes in this fish are unknown. In the study, to identify the Elovl genes in G. przewalskii, the genome-wide identification and phylogenetic analysis of the gene members have been conducted with the expression profile of different tissues under cold stress. Fatty acid compositions, meanwhile, were detected in both the hepatopancreas and skeletal muscle during cold adaptation. A total of 21 Elovl members have been identified from the genome of G. przewalskii, belonging to Elovl1, Elovl2, Elovl4, Elovl5, Elovl6, Elovl7, and Elovl8 subgroups, with conserved ELO domain and four common motifs. Phylogenetic analysis revealed that subfamilies Elovl1 and Elovl7, Elov2, and Elovl5 have a closer genetic relationship, while the Elovl6 class was classed into an independent clade. Synteny analysis showed that whole-genome duplication, tandem duplicates, and gene conversion could drive the Elovls family expansion in G. przewalskii. The Ka/Ks and RELAX analysis showed distinguishing positive selection traces in ORF sequences of gpElovl2. Transcriptional data showed that different gpElovl subtypes exhibited a tissue-specific expression. Subtypes gpElovl1a, gpElovl2 and gpElovl6l were highly expressed induced by cold stress, as well as fatty acid metabolism-related genes, including Acyl-CoA synthetase long-chain gene (Ascl1a-1) and Stearyl-CoA desaturase gene (Scd1a-1). In addition, monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) contents of the hepatopancreas and skeletal muscle were significantly increased under 15-day cold stress. These results provide a better understanding of fish Elovl genes and their roles in cold adaptation.

Physiological, morphological and transcriptomic responses of Tibetan naked carps (Gymnocypris przewalskii) to salinity variations.

Gymnocypris przewalskii is a native cyprinid fish that dwells in the Lake Qinghai with salinity of 12-13‰. It migrates annually to the freshwater rivers for spawning, experiencing the significant changes in salinity. In the present study, we performed the physiological, morphological and transcriptomic analyses to understand the osmoregulation in G. przewalskii. The physiological assay showed that the osmotic pressure of G. przewalskii was almost isosmotic to the brackish lake water. The low salinity reduced its ionic concentrations and osmotic pressure. The plasticity of gill microstructure was linked to the salinity variations, including the presence of mucus and intact tight junctions in brackish water and the development of the mitochondria-rich cells and the loosened tight junctions in freshwater. RNA-seq analysis identified 1926 differentially expressed genes, including 710 and 1216 down- and up-regulated genes in freshwater, which were enriched in ion transport, cell-cell adhesion, and mucus secretion. Genes in ion uptake were activated in low salinity, and mucus pathways and tight junction showed the higher transcription in brackish water. The isosmoticity between the body fluid and the environment suggested G. przewalskii was in the metabolic-saving condition in the brackish water. The decreased salinity disrupted this balance, which activated the ion uptake in freshwater to maintain osmotic homeostasis. The gill remodeling was involved in this process through the development of the mitochondria-rich cells to enhance ion uptake. The current finding provided insights into the potential mechanisms of G. przewalskii to cope with salinity alteration.

Insights Into miRNA-mRNA Regulatory Mechanisms of Cold Adaptation in Gymnocypris eckloni: Ubiquitin-Mediated Proteolysis Is Pivotal for Adaptive Energy Metabolism.

This study aimed to understand cold stress adaptations mechanism in fish. Thus, the transcriptional response to cold conditions in Gymnocypris eckloni was evaluated using RNA-seq and microRNA (miRNA)-seq analyses. Low-temperature (LT) group G. eckloni was cultivated outdoors in waters cooled to 2-4°C for 3 weeks, while individuals in the control temperature (CT) group were exposed to 14-16°C. Significantly different responses were observed in both mRNA and miRNA expression profiles, with more mRNAs (1,833 and 1,869 mRNAs were up- and downregulated, respectively) and fewer miRNAs (15 and 6 were up- and downregulated, respectively) observed in the LT group individuals relative to the CT group individuals. A miRNA-mRNA network involved in the regulation of G. eckloni responses to cold stress was constructed; this network included ubiquitin-mediated proteolysis, protein processing, and oxidative phosphorylation. These results provided new insights into mechanisms of cold tolerance by fish, including decreased metabolic activity in addition to proteolysis.

A homotetrameric hemoglobin expressed in alveolar epithelial cells increases blood oxygenation in high-altitude plateau pika (Ochotona curzoniae).

The plateau pika (Ochotona curzoniae) is native to the Qinghai-Tibet Plateau. In this study, the gene that encodes a heme-binding protein in the pulmonary surfactant (PS) of the pika is identified. The protein is a homotetrameric hemoglobin (δ4) encoded by HBD (δ). HBD is expressed in alveolar epithelial type II (ATII) and type I (ATI) cells, upregulated by hypoxia. δ4 is secreted into alveolar cavities through osmiophilic multilamellar bodies. HBD expression is downregulated by RNAi, which significantly increases hypoxia-inducible factor 1α expression in lung tissue and red blood cells and hemoglobin and blood lactate concentrations but significantly decreases arterial partial pressure of oxygen (PaO2). Our results indicate that plateau pikas physiologically show hypoxemia when HBD expression is downregulated. Therefore, specific HBD expression in the lungs helps plateau pikas to obtain oxygen under hypoxia by maintaining higher PaO2. These findings provide insights into the adaptive mechanisms of plateau pikas to withstand high-altitude environments.

A New Homotetramer Hemoglobin in the Pulmonary Surfactant of Plateau Zokors (Myospalax Baileyi).

The plateau zokor (Myospalax baileyi) is a native species to the Qinghai-Tibetan Plateau, inhabiting hypoxia and hypercapnia sealed subterranean burrows that pose several unique physiological challenges. In this study, we observed a novel heme-containing protein in the pulmonary surfactant (PS) of plateau zokor, identified the encoding gene of the protein, predicted its origination and structure, verified its expression in alveolar epithelial cells, and determined the protein's affinity to oxygen and its effect on the oxygen-dissolving capability in the PS of plateau zokors. The protein is an unusual homotetramer hemoglobin consisting of four γ-like subunits, and the subunit is encoded by a paralog gene of γ, that is γ-like. The divergence time of γ-like from γ is estimated by the molecular clock to be about 2.45 Mya. The generation of γ-like in plateau zokors might well relate to long-time stress of the high land hypoxia. Unlike γ, the γ-like has a hypoxia response element (HRE) and a lung tissue-specific enhancer in its upstream region, and it is expressed specifically in lung tissues and up-regulated by hypoxia. The protein is named as γ4-like which is expressed specifically in Alveolar epithelial type II (ATII) cells and secreted into the alveolar cavities through the osmiophilic multilamellar body (LBs). The γ4-like has a higher affinity to oxygen, and that increases significantly oxygen-dissolving capability in the PS of plateau zokors by its oxygenation function, which might be beneficial for the plateau zokors to obtain oxygen from the severe hypoxia environments by facilitating oxygen diffusion from alveoli to blood.

Chromosome-level assembly of Gymnocypris eckloni genome.

Gymnocypris eckloni is widely distributed in isolated lakes and the upper reaches of the Yellow River and play significant roles in the trophic web of freshwater communities. In this study, we generated a chromosome-level genome of G. eckloni using PacBio, Illumina and Hi-C sequencing data. The genome consists of 23 pseudo-chromosomes that contain 918.68 Mb of sequence, with a scaffold N50 length of 43.54 Mb. In total, 23,157 genes were annotated, representing 94.80% of the total predicted protein-coding genes. The phylogenetic analysis showed that G. eckloni was most closely related to C. carpio with an estimated divergence time of ~34.8 million years ago. For G. eckloni, we identified a high-quality genome at the chromosome level. This genome will serve as a valuable genomic resource for future research on the evolution and ecology of the schizothoracine fish in the Qinghai-Tibetan Plateau.

Prediction of water positions in the binding sites of proteins based on collections of multi-source heterogeneous atoms.

Water molecules play an important role in mediating the interactions between proteins and ligands. However, it is difficult to distinguish the key water molecules directly because they are widely and irregularly distributed. Based on the results of statistical analysis, a composite tetrahedral model is proposed to predict the potential hydration sites in the binding sites of crystal structures. By analyzing the different protein atoms and ligand atoms that interact with water molecules, the unified representation and measurement of these multi-source heterogeneous atoms in the multi-dimensional feature space were adopted. The potential hydration sites could be predicted based on the results of the preference analysis and the shape-matching method. A test set was used to evaluate the model performance and extensive comparison with the tetrahedral-water-cluster model and Dowser++ revealed that the composite tetrahedral model can not only predict the potential sites of multiple key water molecules in the binding sites but also has a better prediction accuracy.