Cupriavidus in the intestinal microbiota of Tibet endemic fish Glyptosternum maculatum can help it adapt to habitat of the Qinghai Tibet Plateau.

BACKGROUND: Gut microbes play an important role in the growth and development of fish. The Tibetan Plateau fish Glyptosternum maculatum is a unique species of sisorid catfish living in the river up to 4200 m altitude. RESULTS: To understand the mechanisms underlying the ability of G. maculatum to adapt to the high-altitude habitat, the intestinal microbiota of G. maculatum was studied. We used high-throughput sequencing of the 16S ribosomal RNA gene of intestinal microorganisms of wild and cultured G. maculatum to explore the characteristics of intestinal microorganisms and compared the gut microbial community of wild and cultured G. maculatum. The results showed that the α-diversity and richness of the intestinal microbiome were higher in wild G. maculatum than in cultured fish. The most abundant phylum in both G. maculatum were Fusobacteria, Proteobacteria, Firmicutes, and Bacteroidetes; Cetobacterium and Cupriavidus are the most dominant genus. The membership and structure of intestinal bacterial communities in wild G. maculatum are similar to the cultured fish, suggesting that a core microbiota is present in both G. maculatum intestinal bacterial communities. Metastats analysis showed that six genera were differentially represented between the wild and cultured G. maculatum. CONCLUSIONS: The most interesting characteristic of the intestinal microbial communities of G. maculatum is that there were large numbers of Cupriavidus, which may play an important role in the adaptation of G. maculatum to the water of the Yarlung Zangbo River with a high Cu content. This result, in turn, can guide us on breeding G. maculatum.

Phosphorylated protein modification analysis on normal liver and Exo-celiac liver of Glyptosternum maculatum.

BACKGROUND: This study aimed to reveal the biological function and molecular mechanism of phosphorylated proteins in the normal liver (NG) and Exo-celiac liver (WG) of Glyptosternum maculatum and potential plateau-adaption mechanisms of G. maculatum. METHODS: A multivariate analysis was performed on proteomic quantitative data (label-free group) and phosphorylated proteome data (phosphorylation group) to reveal protein characteristics. The differentially expressed proteins (DEPs) between NG and WG in the two groups were analysed. Enrichment analysis of these DEPs was performed prior to the protein-protein interaction (PPI) analysis. Finally, an integrated interaction network was constructed to reveal the biological mechanism of the DEP-mediated signal transduction process. RESULT: The NG and WG samples in the phosphorylation group were well distinguished compared to the label-free group. A total of 49 and 313 DEPs were identified in the label-free and phosphorylation groups, respectively. These DEPs, including LIM and calponin homology domains-containing protein 1 (LIMCH1) and DEAD(Asp-Glu-Ala-Asp)-Box Helicase 51 (DDX51), were mainly assembled in functions such as cell adhesion. Two PPI networks were constructed using DEPs in the two groups. Finally, an integrated interaction network was constructed using co-DEP Ferredoxin 1 (FDX1) and associated pathways, including RNA transport. CONCLUSION: LIMCH1 and DDX51 might play important roles in the organogenesis of normal liver and Exo-celiac liver in G. maculatum via the cell adhesion function. Moreover, FXD1 might be associated with the plateau-adaption mechanisms of G. maculatum via participation in the RNA transport pathway.

A transcriptome analysis focusing on splenic immune-related mciroRNAs of rainbow trout upon Aeromonas salmonicida subsp. salmonicida infection.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that can regulate the immune responses during pathogen infection. Aeromonas salmonicida (A. salmonicida) subsp. salmonicida is the causative agent of furunculosis in salmon and trout. To identify the miRNAs and investigate the specific miRNAs in rainbow trout upon A. salmonicida subsp. salmonicida infection, we performed high throughput sequencing using the spleens of rainbow trout infected with and without an A. salmonicida subsp. salmonicida clinical isolate. A total of 381 known miRNAs and 926 novel miRNAs were identified. Eleven known and 16 novel miRNAs were found to be differentially expressed upon infection. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that the target genes of the differentially expressed miRNAs were closely associated with immune responses and biological regulations. Additionally, over- and suppressed expression of miR-155-5p significantly enhanced and reduced the IL-2 and IL-1ß expressions in RTG-2 cells induced by A. salmonicida, respectively. To our knowledge, this is the first experimental study on the miRNAs of rainbow trout upon A. salmonicida infection. The results here might lay a foundation for the further understanding of the roles of miRNAs in the immune responses during A. salmonicida infection in rainbow trout.

Heavy Metal Levels in Fish from Heilongjiang River and Potential Health Risk Assessment.

Concentrations of Cu, Zn, Cr, As, Cd, Pb and Hg were determined in 153 samples covering 18 fish species collected from Heilongjiang River, China, and the potential health risks to local residents through fish consumption were estimated. Results revealed that all metals were detected with Cd and Pb having considerably lower detection rate. There were 28.6 % of Lethenteron camtschaticum samples exceeding the limit of inorganic As content established by Chinese legislation. Metal bioaccumulation in fish were poorly correlated with fish sizes, and generally tended to increase with trophic levels. The hazard quotient values of all fish species were far below 1 for general population and fishermen. The carcinogenic risk of As in L. camtschaticum for the local fishermen exceeded the acceptable level of 10(-4) rise in cancer rates, but was considered safe given the migratory habits of the fish.

Heavy metals in sea cucumber juveniles from coastal areas of Bohai and Yellow seas, north China.

The study was undertaken to assess the contents of heavy metals (Cu, Zn, Cr, Pb, Cd, As and Hg) in sea cucumber (Apostichopus japonicus) juveniles from coastal areas of Bohai and Yellow seas in northern China. Sea cucumber juveniles were collected from twenty commercial hatcheries distributed in five coastal cities. The mean concentrations obtained for heavy metals in mg/kg were as follows: Cu (0.179), Zn (2.634), Cr (0.108), Pb (0.065), Cd (0.161), As (0.372), Hg (0.034). All the mean concentrations were below the maximum residual limits set by Chinese legislation, but As in 10 % samples exceeded the safety threshold. Significant differences in contents of Cr, Pb and Hg were found among the five investigated areas. Overall, the heavy metal levels in sea cucumber juveniles were relatively low and more attention should be paid to toxic metals Pb, Cd, As and Hg in future routine monitoring program.

Analysis of free amino acids in Amur sturgeon by ultra-performance liquid chromatography using pre-column derivatization with 6-aminoquinolyl-carbamyl.

A rapid, sensitive, and reliable ultra-performance liquid chromatography (UPLC) coupled with photodiode array detection method was developed for the amino acid analysis of Amur sturgeon (Acipenser schrenckii Brandt). The method uses minimal sample volume and automated online precolumn derivitization of amino acids with fluorescent 6-aminoquinolyl-carbamyl reagent. The chromatographic separation was achieved by UPLC, which used a column with 1.7 µm particle packing that enabled higher speed of analysis, peak capacity, greater resolution, and increased sensitivity. Amino acid derivatives obtained under optimal conditions were separated on a Waters UPLC BEH C(18) column with Acetonitrile-acetate buffer as mobile phase. Matrix effects were investigated and good linearities with correlation coefficients better than 0.9949 were obtained over a wide range of 5-1000 µmol/L for all amino acids. The simple sample preparation and minimal sample volume make the method useful for the quantitation of 17 amino acids in Amur sturgeon samples. It is concluded that a rapid and robust platform based on UPLC was established, and a total of 17 amino acids of Amur sturgeon were tentatively detected. This method showed good accuracy and repeatability that can be used for the quantification of amino acids in real samples.

Comprehensive transcriptome data for endemic Schizothoracinae fish in the Tibetan Plateau.

The Schizothoracinae fishes, endemic species in the Tibetan Plateau, are considered as ideal models for highland adaptation and speciation investigation. Despite several transcriptome studies for highland fishes have been reported before, the transcriptome information of Schizothoracinae is still lacking. To obtain comprehensive transcriptome data for Schizothoracinae, the transcriptome of a total of 183 samples from 14 representative Schizothoracinae species, were sequenced and de novo assembled. As a result, about 1,363 Gb transcriptome clean data was obtained. After the assembly, we obtain 76,602-154,860 unigenes for each species with sequence N50 length of 1,564-2,143 bp. More than half of the unigenes were functionally annotated by public databases. The Schizothoracinae fishes in this work exhibited diversified ecological distributions, phenotype characters and feeding habits; therefore, the comprehensive transcriptome data of those species provided valuable information for the environmental adaptation and speciation of Schizothoracinae in the Tibetan Plateau.

Genome of Tetraploid Fish Schizothorax o'connori Provides Insights into Early Re-diploidization and High-Altitude Adaptation.

Whole-genome duplications (WGDs) of Schizothoracinae are believed to have played a significant role in speciation and environmental adaptation on the Qinghai-Tibet Plateau (QTP). Here, we present a genome for Schizothorax o'connori, a QTP endemic fish and showed the species as a young tetraploid with a recent WGD later than ∼1.23 mya. We exhibited that massive insertions between duplicated genomes caused by transposon bursts could induce mutagenesis in adjacent sequences and alter the expression of neighboring genes, representing an early re-diploidization process in a polyploid genome after WGD. Meanwhile, we found that many genes involved in DNA repair and folate transport/metabolism experienced natural selection and might contribute to the environmental adaptation of this species. Therefore, the S. o'connori genome could serve as a young tetraploid model for investigations of early re-diploidization in polyploid genomes and offers an invaluable genetic resource for environmental adaptation studies of the endemic fish of the QTP.

Genomic characterization of a novel virulent phage infecting the Aeromonas hydrophila isolated from rainbow trout (Oncorhynchus mykiss).

The virulent bacteriophage MJG that specifically infects Aeromonas hydrophila was isolated from a water sample from a river in Harbin, China. The genome of phage MJG was a double-stranded linear DNA with 45,057 bp, possessing 50.11% GC content. No virulence or resistance genes were found in the phage genome. Morphological observation, genomic characterization, and phylogenetic analysis indicated that MJG was closely related to phages belonging to the genus Sp6virus in the Podoviridae family. This phage is a novel member within Sp6virus that could infect and lyse A. hydrophila. This study could serve as a genomic reference of A. hydrophila phages and provide a potential agent for phage therapy.

Global metabolic responses of the lenok (Brachymystax lenok) to thermal stress.

High temperature is a powerful stressor for fish living in natural and artificial environments, especially for cold water species. Understanding the impact of thermal stress on physiological processes of fish is crucial for better cultivation and fisheries management. However, the metabolic mechanism of cold water fish to thermal stress is still not completely clear. In this study, a NMR-based metabonomic strategy in combination with high-throughput RNA-Seq was employed to investigate global metabolic changes of plasma and liver in a typical cold water fish species lenok (Brachymystax lenok) subjected to a sub-lethal high temperature. Our results showed that thermal stress caused multiple dynamic metabolic alterations of the lenok with prolonged stress, including repression of energy metabolism, shifts in lipid metabolism, alterations in amino acid metabolism, changes in choline and nucleotide metabolisms. Specifically, thermal stress induced an activation of glutamate metabolism, indicating that glutamate could be an important biomarker associated with thermal stress. Evidence from Hsp 70 gene expression, blood biochemistry and histology confirmed that high temperature exposure had negative effects on health of the lenok. These findings imply that thermal stress has a severe adverse effect on fish health and demonstrate that the integrated analyses combining NMR-based metabonomics and transcriptome strategy is a powerful approach to enhance our understanding of metabolic mechanisms of fish to thermal stress.

The sequence and de novo assembly of Oxygymnocypris stewartii genome.

Animal genomes in the Qinghai-Tibetan Plateau provide valuable resources for scientists to understand the molecular mechanism of environmental adaptation. Tibetan fish species play essential roles in the local ecology; however, the genomic information for native fishes was still insufficient. Oxygymnocypris stewartii, belonging to Oxygymnocypris genus, Schizothoracinae subfamily, is a native fish in the Tibetan plateau living within the elevation from roughly 3,000 m to 4,200 m. In this report, PacBio and Illumina sequencing platform were used to generate ~385.3 Gb genomic sequencing data. A genome of about 1,849.2 Mb was obtained with a contig N50 length of 257.1 kb. More than 44.5% of the genome were identified as repetitive elements, and 46,400 protein-coding genes were annotated in the genome. The assembled genome can be used as a reference for future population genetic studies of O. stewartii and will improve our understanding of high altitude adaptation of fishes in the Qinghai-Tibetan Plateau.

Draft genome of Glyptosternon maculatum, an endemic fish from Tibet Plateau.

Background: Mechanisms for high-altitude adaption have attracted widespread interest among evolutionary biologists. Several genome-wide studies have been carried out for endemic vertebrates in Tibet, including mammals, birds, and amphibians. However, little information is available about the adaptive evolution of highland fishes. Glyptosternon maculatum (Regan 1905), also known as Regan or barkley and endemic to the Tibetan Plateau, belongs to the Sisoridae family, order Siluriformes (catfishes). This species lives at an elevation ranging from roughly 2,800 m to 4,200 m. Hence, a high-quality reference genome of G. maculatum provides an opportunity to investigate high-altitude adaption mechanisms of fishes. Findings: To obtain a high-quality reference genome sequence of G. maculatum, we combined Pacific Bioscience single-molecule real-time sequencing, Illumina paired-end sequencing, 10X Genomics linked-reads, and BioNano optical map techniques. In total, 603.99 Gb sequencing data were generated. The assembled genome was about 662.34 Mb with scaffold and contig N50 sizes of 20.90 Mb and 993.67 kb, respectively, which captured 83% complete and 3.9% partial vertebrate Benchmarking Universal Single-Copy Orthologs. Repetitive elements account for 35.88% of the genome, and 22,066 protein-coding genes were predicted from the genome, of which 91.7% have been functionally annotated. Conclusions: We present the first comprehensive de novo genome of G. maculatum. This genetic resource is fundamental for investigating the origin of G. maculatum and will improve our understanding of high-altitude adaption of fishes. The assembled genome can also be used as reference for future population genetic studies of G. maculatum.

The complete mitochondrial genome of the Gymnocypris scleracanthus (Cypriniformes: Cyprinidae).

The Gymnocypris scleracanthus, a species of genus Gymnocypris belongs to Schizothoracinaein Cyprinidae. In this study, the complete mitochondrial DNA genome sequence of G. scleracanthus was determined. The 16,771 bp long circular molecule contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and two non-coding regions, showing a typical vertebrate pattern. These genes except ND6 and eight tRNA genes were encoded on the H-strand. Phylogenetic analysis showed that the G. scleracanthus was a part of genus Gymnocypris and had closer relationship with Gymnocypris dobula and was independent from other species of Psilorhynchus and Labeoninae in Cyprinidae.

Trace elements in farmed fish (Cyprinus carpio, Ctenopharyngodon idella and Oncorhynchus mykiss) from Beijing: implication from feed.

Concentrations of 30 trace elements, Li, V, Cr, Mn, Fe, Ni, Cu, Mo, Zn, Se, Sr, Co, Al, Ti, As, Cs, Sc, Te, Ba, Ga, Pb, Sn, Cd, Sb, Ag, Tm, TI, Be, Hg and U in major cultured freshwater fish species (common carp-Cyprinus carpio, grass carp-Ctenopharyngodon idella and rainbow trout-Oncorhynchus mykiss) with the corresponding feed from 23 fish farms in Beijing, China, were investigated. The results revealed that Fe, Zn, Cu, Mn, Sr, Se were the major accumulated essential elements and Al, Ti were the major accumulated non-essential elements, while Mo, Co, Ga, Sn, Cd, Sb, Ag, Tm, U, TI, Be, Te, Pb and Hg were hardly detectable. Contents of investigated trace elements were close to or much lower than those in fish from other areas in China. Correlation analysis suggested that the elemental concentrations in those fish species were relatively constant and did not vary much with the fish feed. In comparison with the limits for aquafeeds and fish established by Chinese legislation, Cd in 37.5% of rainbow trout feeds and As in 20% of rainbow trout samples exceeded the maximum limit, assuming that inorganic As accounts for 10% of total As. Further health risk assessment showed that fish consumption would not pose risks to consumers as far as non-essential element contaminants are concerned. However, the carcinogenic risk of As in rainbow trout for the inhabitants in Beijing exceeded the acceptable level of 10(-)(4), to which more attention should be paid.

[Effect of locomotion and feeding on metabolic mode of juvenile lenok, Brachymystax lenok (Pallas) under different water temperatures].

To investigate the effect of locomotion and feeding on the metabolic mode of juvenile lenok, Brachymystax lenok (Pallas) under different water temperatures, the pre-exercise oxygen consumption rate (MO2p), active oxygen consumption rate (MO2a), metabolic scope (MS), critical swimming speed (Uc) and swimming metabolic rate of both fasting and fed fish were measured at five temperature levels (4 degrees C, 8 degrees C, 12 degrees C, 16 degrees C and 20 degrees C) and ten flow velocities with saturated dissolved oxygen (> 8.0 mg x L(-1)). The results showed that the MO2p and MO2a of the feeding group were significantly higher (P < 0.05) than the fasting group under different temperatures, and the increases in MO2p and MO2a at 4 degrees C, 8 degrees C, 12 degrees C, 16 degrees C and 20 degrees C were 15%, 47%, 30%, 43% and 8%, and 12%, 23%, 21%, 36% and 7%, respectively. No significant differences were observed for Uc and MS between the fasting and the feeding groups (P > 0.05), but the MS showed a trend of decline with increase in water temperature. Swimming metabolic rate of fish was increased with increasing the flow velocity, and further increase of flow velocity resulted in a decline in swimming metabolic rate, and the swimming metabolic rate of the feeding group was significantly higher than that of the fasting group (P < 0.05). The metabolic rate increased with increasing the swimming speed up to 70% Uc, and then decreased with increasing the swimming speed up to Uc. It was concluded that, under certain temperature, the maximum metabolic rate was induced by exercise and feeding; the metabolic rate exhibited the additive metabolic mode before increasing to the maximum and thereafter, the metabolic rate induced by feeding reduced with decreasing the swimming metabolic rate, exhibiting the locomotion prioritized mode.

[Determination of 17 amino acids in fish eggs by ultra performance liquid chromatography coupled with precolumn derivatization].

A rapid quantitative method of ultra performance liquid chromatography (UPLC) has been developed for the analysis of 17 amino acids in fish eggs from Acipenser schrenckii, Huso dauricus and Acipenser ruthenus. The analytes were hydrolyzed with 6 0 mol/L HCI. The extraction solution was concentrated under low pressure and neutralized with NaOH solution before derivatization with 6-aminoquinolyl-N-hydroxy-succinimidyl carbamate (AQC) as derivatization agent in borate buffer solution (pH 8.8). Gradient UPLC separation was performed on a C, column (BEH C18, 100 mm x2.1 mm, 1.7 micro m) with 30 mmol/L ammonium acetate (pH 3. 5 adjusted with formic acid) and acetonitrile (containing 0. 15% (v/v) formic acid and 30 mmol/L ammonium acetate) as the mobile phases at a flow rate of 0. 7 mL/min. The detection was carried out with a photo-diode array (PDA) detector and the wavelength was set at 260 nm. The linear ranges were from 5.0 to 1000 micro mol/L with the correlation coefficients (r2) > or = 0. 995 0. The method was validated by the analysis of seven replicates. The 17 amino aci standards were spiked in fish eggs at three levels of 100, 500 and 750 micro mol/L, and the averag recoveries were 75.4% - 107.3% with the relative standard deviations of 2. 19% - 12.3%. The limits of detection (LOD) for the analytes were from 0.94 micro mol/L to 4.04 micro mol/L. The method was successfully applied to the analysis of the 17 amino acids in fish eggs from Acipenser schrenckii, Huso dauricus and Acipenser ruthenus. The method is simple, rapid, precise and reliable.

Complete mitochondrial genomes of two lenoks, Brachymystax lenok and Brachymystax lenok tsinlingensis.

The circular mitochondrial genomes of Brachymystax lenok and B. lenok tsinlingensis are 16,832 and 16,669 bp in length, respectively. The mitogenomes of two lenoks shared common features with those of other teleosts in terms of gene arrangement, base composition, and transfer RNA structures. The two genomes have an overall nucleotide sequence identity of 98.3%. Through the comparisons between the two lenoks, we found their gene arrangement, composition, and sizes are the same, and the A+T content is identical. As with other freshwater salmonids, a T-type mononucleotide microsatellite and various tandem repeats were identified in the control regions of the lenoks. The low pairwise distance (2.1%) inferred from 12 mitochondrial protein-coding genes on heavy strand showed close proximity of B. lenok and B. lenok tsinlingensis.