Integrated metabolomic and transcriptomic responses to heat stress in a high-altitude fish, Triplophysa siluroides.

Species in Triplophysa display strong adaptability to the extreme environment of the plateau, thus offering an ideal model to study the molecular mechanism of fish adaptation to environmental stress. In the present study, we conducted integrated analysis of the transcriptome and metabolism of liver tissue in Triplophysa siluroides under heat stress (28 °C) and control (10 °C) conditions to identify heat stress-induced genes, metabolites and pathways. RNA-Seq identified 2373 differentially expressed genes, which consisted of 1360 upregulated genes and 1013 downregulated genes, in the heat stress group vs. the control group. Genes in the heat shock protein (Hsp) family, including Hsp40, Hsp70, Hsp90 and other Hsps, were strongly upregulated by heat stress. Pathway enrichment analysis revealed that the PI3K/AKT/mTOR and protein processing in the endoplasmic reticulum (ER) pathways were significantly affected by heat stress. Metabolism sequencing identified a total of 155 differentially abundant metabolites, including 118 significantly upregulated metabolites and 37 downregulated metabolites. Combined analysis of the transcriptome and metabolism results showed that ubiquitin-dependent proteolysis and purine metabolism pathways were enhanced in response to acute heat stress to protect cells from damage under stress conditions. The results of this study may contribute to our understanding of the underlying molecular mechanism of the heat stress response in cold-water fish.

Combination of metabolome and proteome analyses provides insights into the mechanism underlying growth differences in Acipenser dabryanus.

To analyze the differences between different-sized Acipenser dabryanus, we randomly selected 600 3-month-old A. dabryanus juveniles. Four months later, the blood and white muscle of these fish were analyzed. The results showed no significant difference in the length-weight relationship (LWR) b value between the large and small A. dabryanus. The levels of serum growth hormone (gh) and insulin-like growth factor 1 (igf1) in the large A. dabryanus were significantly lower than those in the small, whereas the activity levels of Total superoxide dismutase (T-sod) and catalase (cat) were opposite to the results of gh and igf1. A total of 212 and 245 metabolites showed significant changes in the positive and negative polarity mode, respectively. Among 3,308 proteins identified, 69 proteins showed upregulated expression, and 185 proteins showed downregulated expression. These results indicated that the growth advantage of A. dabryanus was closely related to glycolysis, protein synthesis, and antioxidant function.

Integrated biochemical, transcriptomic and metabolomic analyses provide insight into heat stress response in Yangtze sturgeon (Acipenser dabryanus).

Temperature fluctuations caused by climate change and global warming pose a great threat to various species. Most fish are particularly vulnerable to elevated temperatures. Understanding the mechanism of high-temperature tolerance in fish can be beneficial for proposing effective strategies to help fish cope with global warming. In this study, we systematically studied the effects of high temperature on Acipenser dabryanus, an ancient living fossil and flagship species of the Yangtze River, at the histological, biochemical, transcriptomic and metabolomic levels. Intestinal and liver tissues from the control groups (18 °C) and acute heat stress groups (30 °C) of A. dabryanus were sampled for histological observation and liver tissues were assessed for transcriptomic and metabolomic profiling. Histopathological analysis showed that the intestine and liver tissues were damaged after heat stress. The plasma cortisol content and the levels of oxidative stress markers (catalase/glutathione reductase) and two aminotransferases (aspartate aminotransferase/alanine aminotransferase) increased significantly in response to acute heat stress. Transcriptomic and metabolomic methods showed 6707 upregulated and 4189 downregulated genes and 64 upregulated and 78 downregulated metabolites in the heat stress group. Heat shock protein (HSP) genes showed striking changes in expression under heat stress, with 21 genes belonging to the HSP30, HSP40, HSP60, HSP70 and HSP90 families significantly upregulated by short-term heat stress. The majority of genes associated with ubiquitin and various immune-related pathways were also markedly upregulated in the heat stress group. In addition, the combined analysis of metabolites and gene profiles suggested an enhancement of amino acid metabolism and glycometabolism and the suppression of fatty acid metabolism during heat stress, which could be a potential energy conservation strategy for A. dabryanus. To the best of our knowledge, the present study represents the first attempt to reveal the mechanisms of heat stress responses in A. dabryanus, which can provide insights into improved cultivation of fish in response to global warming.

Biochemical, transcriptomic and metabolomic responses to total dissolved gas supersaturation and their underlying molecular mechanisms in Yangtze sturgeon (Acipenser dabryanus).

With the rapid development of hydropower facility construction, the total dissolved gas (TDG) generated by dam discharge is seriously threatening the survival of fish and has become an ecological environmental issue of global concern. However, how TDG affects fish physiology and the underlying molecular mechanism remain poorly known. In this study, Acipenser dabryanus, an ancient living fossil that is a flagship species of the Yangtze River, was exposed to water supersaturated with TDG at a level of 116% for 48 h. A comprehensive analysis was performed to study the effect of TDG supersaturation stress on A. dabryanus, including histopathological, biochemical, transcriptomic and metabolomic analyses. The histopathological results showed that mucosal-associated lymphoid tissues were seriously damaged after TDG supersaturation stress. Plasma catalase levels increased significantly under TDG supersaturation stress, while superoxide dismutase levels decreased significantly. Transcriptomic analysis revealed 289 upregulated genes and 162 downregulated genes in gill tissue and 535 upregulated and 104 downregulated genes in liver tissue. Metabolomic analysis revealed 63 and 164 differentially abundant metabolites between the control group and TDG group in gill and liver, respectively. The majority of heat shock proteins and genes related to ubiquitin and various immune-related pathways were significantly upregulated by TDG supersaturation stress. Integrated transcriptomic and metabolomic analyses revealed the upregulation of amino acid metabolism and glycometabolism pathways under TDG supersaturation stress. Glycerophospholipid metabolism was increased which might be associated with maintaining cell membrane integrity. This is the first study revealing the underlying molecular mechanisms of effects of TDG supersaturation on fish. Our results suggested that acute TDG supersaturation stress could enhance immune and antioxidative functions and activate energy metabolic pathways as an adaptive mechanism in A. dabryanus.

Full-length transcriptome and analysis of bmp-related genes in Platypharodon extremus.

Platypharodon extremus is an endemic species on the Qinghai-Tibet Plateau. As a secondary protected species in China, the basic genomic information of this species has not yet been reported. Here, through third-generation sequencing, the full-length transcriptome of P. extremus was obtained. We identified 323,290 CCS sequences, and a total of 50,083 unigenes were extracted after correction with second-generation sequencing data and the removal of redundant reads. A total of 50,067 transcripts were annotated with the various databases. Based on the sequence information, three members in the bone morphogenetic proteins (bmps) family and their receptors, were identified. We found that the special structures of these proteins (zinc-dependent metalloproteinase domain, CUB domains, EGF-like domains and TGF-ß domain) are highly conserved in fish and that they are closely evolutionarily related to the bmps and bmp receptors of Cyprinidae fishes. This is the first study to sequence the full-length transcriptome of P. extremus, which will help us to further understand its biology.

Comprehensive transcriptome analysis reveals the effect of feeding rhythm on the immunity and metabolism of Acipenser dabryanus.

Acipenser dabryanus is a species endemic to Yangtze River drainage in China and is listed as a critical endangered species on the IUCN Red List. In the present study, the liver and spleen transcriptomes were analyzed by comparing the data of A. dabryanus that experienced nine different feeding rhythms (once a day diurnal, T1; two times a day diurnal, T2; three times a day diurnal, T3; four times a day, T4; five times a day, T5; six times a day, T6; once a day nocturnal, Tn1; two times a day nocturnal, Tn2; and three times a day nocturnal, Tn3). Transcriptome sequencing generated 1,901,236,482 clean reads, encompassing 570.4 Gb of sequence data. The reads were assembled into 287,372 unigenes with an average length of 803 bp and an N50 of 1004 bp. KEGG analysis showed that 1,080, 1,030, and 1216 unigenes were annotated to lipid metabolism, amino acid metabolism and carbohydrate metabolism, respectively, and 2549 unigenes were annotated to the immune system category. Differentially expressed genes (DEGs) between different feeding frequency groups or between nighttime and daytime feeding were obtained and functionally enriched. Importantly, DEGs participating in nutrition metabolism and various immunoregulation pathways and their expression profiles in A. dabryanus were discussed. Interestingly, the majority of key genes related to lipid metabolism or in immunodependent gene families, such as antimicrobial peptides, Toll-like receptors, chemokines, NOD-like receptors, B cell receptors and the major histocompatibility complex, were all significantly upregulated in animals in the T6 group compared to the characteristics of animals in the T2 group that had a normal feeding frequency. In addition, light/dark rhythm also affected the immunity of A. dabryanus, and fish fed at night possessed an improved immune response than fish fed at daytime. Our study suggested that feeding six times a day is optimal for A. dabryanus juvenile growth as it enhances the organism's nutrition metabolism and immune function.

Molecular characterization and tissue distribution of cholecystokinin and its receptor in Yangtze sturgeon (Acipenser dabryanus) and their response to different feeding conditions.

Yangtze sturgeon (Acipenser dabryanus) is a species endemic to Yangtze River drainage in China and is listed as a critically endangered species on the IUCN Red List. In the present study, cholecystokinin (CCK), one of the most important neuroregulatory digestive genes, and its receptor (CCKr) were identified from the full-length transcriptome analysis of A. dabryanus. The deduced amino acid sequences of CCK and CCKr from A. dabryanus showed structural features common to those in other vertebrates. Gene expression profile analysis showed that CCK and CCKr were universally expressed in different tissues, and both had the highest expression in the brain. Starvation and refeeding significantly regulated the expression levels of CCK and CCKr in the brain, suggesting that CCK and CCKr were involved in feed intake regulation in A. dabryanus as in mammals. In addition, the expression levels of CCK and CCKr under different feeding frequencies were studied. Compared with the control group (fed two times a day), the expression levels of CCK and CCKr in the intestine and brain did not change significantly in the other groups after 8 weeks of rearing, indicating that the feeding frequency might not influence the appetite of A. dabryanus. The present work provides a basis for further investigation into the regulation of feeding in A. dabryanus.

Characterization of MRF genes and their tissue distributions and analysis of the effects of starvation and refeeding on the expression of these genes in Acipenser dabryanus muscle.

The purpose of the study was to clone the sequences of myogenic regulatory factors in Acipenser dabryanus and explore the changes in their expression during starvation and refeeding in A. dabryanus muscle. One hundred twenty fish (60.532 ± 0.284 g) were randomly assigned to four groups (fasted for 0, 3, 7 or 14 d and then refed for 14 d). Our predictions showed that the coding sequences of myod1, myf5, myog and myf6 in A. dabryanus encoded 275, 248, 248 and 243 amino acids, respectively, and the expression of the four genes was the highest in muscle. During fasting, the expression of myod1 in muscle was significantly decreased in the 14 d group. The expressions of myf5 and myf6 were increased significantly at first and then decreased with prolonged starvation time. The expression of myog in the 14 d group was significantly decreased compared with other groups (P < 0.05). During refeeding, the highest values of myod1 and myf6 expression were found in the 3 d group (P < 0.05).The expressions of myf5 and myog in 0 d and 3 d group were significantly higher than those in 7 d and 14 d group (P < 0.05). These results indicate that myogenic regulatory factors (MRFs) play important roles in muscle growth and development in A. dabryanus. The inhibition of long-term starvation (14 d) on the expression of myogenic regulatory factors is probably one of the reasons why it can not achieve full compensation for growth.

Effects of starvation and refeeding on growth performance, appetite, growth hormone-insulin-like growth factor axis levels and digestive function of Acipenser dabryanus.

The aim of this study was to explore the effects and mechanisms of different starvation treatments on the compensatory growth of Acipenser dabryanus. A total of 120 fish (60·532 (sem 0·284) g) were randomly assigned to four groups (fasting 0, 3, 7 or 14 d and then refed for 14 d). During fasting, middle body weight decreased significantly with prolonged starvation. The whole-body and muscle composition, serum biochemical indexes, visceral indexes and digestive enzyme activities had been effected with varying degrees of changes. The growth hormone (GH) level in serum was significantly increased in 14D; however, insulin-like growth factor-1 (IGF-1) showed the opposite trend. The neuropeptide Y (npy) mRNA level in brain was significantly improved in 7D; peptide YY (pyy) mRNA level in intestine was significantly decreased during fasting. After refeeding, the final body weight, percentage weight gain, specific growth rate, feed intake, feed efficiency and protein efficiency ratio showed no difference between 0D and 3D. The changes of whole-body and muscle composition, serum biochemical indexes, visceral indexes and digestive enzyme activities had taken place in varying degrees. GH levels in 3D and 7D were significantly higher than those in the 0D; the IGF-1 content decreased significantly during refeeding. There was no significant difference in npy and pyy mRNA levels. These results indicated that short-term fasting followed by refeeding resulted in full compensation and the physiological and biochemical effects on A. dabryanus were the lowest after 3 d of starvation and 14 d of refeeding. Additionally, compensation in A. dabryanus may be mediated by appetite genes and GH, and the degree of compensation is also affected by the duration of starvation.

Full-length transcriptome sequencing and identification of immune-related genes in the critically endangered Hucho bleekeri.

Hucho bleekeri is a glacial relict and critically endangered fish restricted to the Yangtze River drainage in China. The lack of basic genomic information and immune characteristics will hinder the way toward protecting this species. In the present study, we conducted the first transcriptome analysis of H. bleekeri using the combination of SMRT and Illumina sequencing technology. Transcriptome sequencing generated a total of 93,330 non-redundant full-length unigenes with a mean length of 3072 bp. A total of 92,472 (99.08%) unigenes were annotated in at least one of the Nr protein, Swiss-Prot, KEGG, KOG, GO, Nt and Pfam databases. KEGG analysis showed that a total of 7240 unigenes belonging to 28 immune pathways were annotated to the immune system category. Meanwhile, differentially expressed genes between mucosa-associated tissues (skin, gill and hindgut) and systemic-immune tissues (spleen, head kidney and liver) were obtained. Importantly, genes participating in diverse immune signalling pathways and their expression profiles in H. bleekeri were discussed. In addition, a large number of long non-coding RNAs (lncRNAs) and simple sequence repeats (SSRs) were obtained in the H. bleekeri transcriptome. The present study will provide basic genomic information for H. bleekeri and for further research on analysing the characteristics of both the innate and adaptive immune systems of this critically endangered species.

Transcriptome analysis identifies candidate genes associated with skin color variation in Triplophysa siluroides.

In vertebrates, skin pigmentation is the most diverse phenotypic trait, and it is produced by a complex biological process that is often genetically controlled. Recently, two different colors (the typical brown and orange varieties) of Triplophysa siluroides, a species restricted to Yellow River drainage in China, were discovered. In the present study, the skin, brain and liver transcriptomes of T. siluroides of both colors were sequenced to search for genes related to skin pigmentation. Transcriptome sequencing generated 1,484,197,774 clean reads, resulting in a total of 222.6 Gb of sequence. The reads were assembled into 470,788 unigenes with a mean length of 1550 bp and an N50 size of 2944 bp. Functional annotation of the unigene dataset showed that 214,507, 304,161, 112,886, 179,074, 180,064, 184,837 and 82,081 unigenes were significantly matched to entries in the Nr protein, Nt, KO, Swiss-Prot, Pfam, GO and KOG databases, respectively. A differential expression analysis revealed that 2774, 3552 and 1529 unigenes were upregulated and 2720, 2663 and 1103 unigenes were downregulated in the skin, brain and liver of orange-skinned T. siluroides, respectively. Several genes that play key roles in pigmentation, i.e., Agouti, Slc45a2, Cbs, Mift and Slc7a11, showed significantly differential expression between brown and orange fish. In addition, we detected 158,863 simple sequence repeats (SSRs) in the T. siluroides transcriptome, and a total of 201,338 single-nucleotide polymorphisms (SNPs) were discovered in the different transcriptomes. The present results will facilitate further study of the molecular mechanisms of skin pigmentation and marker-assisted breeding of fish with valuable skin colors.

Molecular polymorphism and expression of MHC I α, II α, II ß and II invariant chain in the critically endangered Dabry's sturgeon (Acipenser dabryanus).

The major histocompatibility complex (MHC) is a key player in the regulation of immune responses through presenting foreign antigens to T lymphocytes. In this study, three MHC genes, namely, MHC I α, II α, II ß and the II invariant chain (Ii), were identified and characterized in the critically endangered Dabry's sturgeon (Acipenser dabryanus). A tissue distribution study showed that the MHC and Ii transcripts were widely expressed in various tissues. The highest expression levels of MHC I α, II α and Ii were found in the gill, while MHC II ß was primarily expressed in the spleen. Challenge of A. dabryanus with a pathogenic bacterium in vivo resulted in significant upregulation of both MHC and Ii expression, indicating potential roles of these genes in immune response. Phylogenetic analysis showed that A. dabryanus MHC grouped with other teleost MHC genes and sequences from Polyodon spathula and A. dabryanus had an intermingling of alleles. According to the split time between paddlefishes and sturgeons, this result indicated that trans-species MHC lineages in Chondrostei were much older than those in tetrapods. The molecular polymorphisms of the complete open reading frame regions of the MHC genes were analysed in several A. dabryanus individuals. MHC II α and II ß were highly polymorphic in different individuals, while MHC I α was more conserved. The ratio of non-synonymous substitution occurred at a significantly higher frequency than synonymous substitution in peptide-binding regions (PBR) of MHC II α and II ß, demonstrating the existence of positive selection at peptide-binding sites. Our study suggested potential roles of the MHC chains in immune response to pathogen microbial infection, and the numerous alleles identified in this study will help further genetic management and molecular marker-assisted selective breeding programmes in A. dabryanus.

Identification and characterization of three novel antimicrobial peptides from Acipenser dabryanus.

Antimicrobial peptides (AMPs) play essential roles in the innate immune system to protect against a wide variety of pathogens in aquatic environments. In this study, three very important AMPs, cathelicidin, hepcidin and defensin, were identified in the critically endangered Acipenser dabryanus. The full-length cDNA sequences of these three AMPs were identified from transcriptome sequencing and the rapid amplification of cDNA ends (RACE) technique. Phylogenetic analysis showed that cathelicidin formed a clade with the other members of the cathelicidin family, and similar results were obtained for hepcidin. The A. dabryanus ß-defensin belonged to the fish class 2 ß-defensins. A tissue distribution study showed that the three AMP transcripts could be detected constitutively in various tissues. The highest expression levels of cathelicidin and hepcidin were found in the liver, while defensin was primarily expressed in the skin. Bacterial challenge in vivo revealed significant changes in the gene expression of the three AMPs at both mucosal sites and systemic sites. Striking upregulation of cathelicidin and hepcidin was observed in the skin at 12 h post-challenge, with increases of more than 7000-fold and 1000-fold, respectively, compared to the control, and the expression of defensin mRNA was remarkably elevated in the hindgut (by 230-fold at 6 h post-challenge). Moreover, according to the expression profiles of the AMPs post-challenge, we found that the mucosal immune response occurred earlier than the systemic immune response following bacterial infection. Our results suggest that these three novel AMPs may play important roles in the innate immune system of A. dabryanus to protect against invading pathogens, especially during the mucosal immune response.

Gonadal transcriptome sequencing of the critically endangered Acipenser dabryanus to discover candidate sex-related genes.

BACKGROUND: Acipenser dabryanus, an endemic Chinese species, has been listed as a first-class protected animal in China. Sturgeons are among the oldest and most primitive group of existing fish in the world and occupy a special place in the evolutionary history of fish. Thus, a study of the reproduction and sex differentiation of sturgeon will be of great value for fish as well as the whole vertebrate group. METHODS: In this study, we conducted comparative analysis of the testes and ovaries transcriptomes of A. dabryanus to screen for sex-differentiation and sexual development-related genes. RESULTS: The transcriptome sequencing of six cDNA libraries generated 265 million clean reads, encompassing 79 Gb of sequences. The N50 and mean length of the identified 91,375 unigenes were 1,718 and 989 bp, respectively. A total of 6,306, 9,961, 13,170, 15,484, and 23,588 unigenes were annotated in the clusters of orthologous groups, gene ontology categories, Kyoto Encyclopedia of Genes and Genomes Pathway, euKaryotic orthologous groups, and NCBI non-redundant protein databases, respectively. A total of 5,396 differentially expressed genes were found between the two sexes, with 1,938 predicted to be up-regulated in ovaries and 3,458 in testes. A total of 73 candidate genes known to be involved in sex differentiation and sexual development were searched in the transcriptome of A. dabryanus of which 52 showed significant similarity. We highlighted six genes that are differentially expressed between the two sexes and may play important roles in sex differentiation and gonad maintenance. In addition, 24,271 simple sequence repeats (SSRs) and 550,519 single-nucleotide polymorphisms (SNPs) were detected. DISCUSSION: This work represents the first transcriptome study comparing the ovary and testis in A. dabryanus. The putative differentially expressed genes between the gonads provide an important source of information for further study of the sex-differentiation related genes and the sex-differentiation mechanism in sturgeons. The SSRs or SNPs identified in this study will be helpful in the discovery of sex-related markers in A. dabryanus.