Molecular characterization of a teleost-specific toll-like receptor 22 (tlr22) gene from yellow catfish (Pelteobagrus fulvidraco) and its transcriptional change in response to poly I:C and Aeromonas hydrophila stimuli.

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that can recognize pathogen-associated molecular patterns (PMPs) and play important roles in the innate immune system in vertebrates. In this study, we identified a teleost-specific tlr22 gene from yellow catfish (Pelteobagrus fulvidraco) and its immune roles in response to different pathogens were also determined. The open reading frame (ORF) of the tlr22 was 2892 bp in length, encoding a protein of 963 amino acids. Multiple protein sequences alignment, secondary and three-dimensional structure analyses revealed that TLR22 is highly conserved among different fish species. Phylogenetic analysis showed that the phylogenetic topology was divided into six families of TLR1, TLR3, TLR4, TLR5, TLR7 and TLR11, and TLR22 subfamily was clustered into TLR11 family. Meanwhile, synteny and gene structure comparisons revealed functional and evolutionary conservation of the tlr22 gene in teleosts. Furthermore, tlr22 gene was shown to be widely expressed in detected tissues except barbel and eye, with highest expression level in liver. The transcription of tlr22 was significantly increased in spleen, kidney, liver and gill tissues at different timepoints after Poly I:C infection, suggesting TLR22 plays critical roles in defensing virus invasion. Similarly, the transcription of tlr22 was also dramatically up-regulated in spleen, kidney and gill tissues with different patterns after Aeromonas hydrophila infection, indicating that TLR22 is also involved in resisting bacteria invasion. Our findings will provide a solid basis for the investigation the immune functions of tlr22 gene in teleosts, as well as provide useful information for disease control and treatment for yellow catfish.

Transcriptomics analysis provides new insights into the fish antiviral mechanism and identification of interferon-stimulated genes in grass carp (Ctenopharyngodon idella).

Grass carp is an economically important freshwater fish in China, and haemorrhagic disease caused by GCRV has seriously restricted its farming scale. To understand the host molecular basis for antiviral defence and explore the effector molecules, a global transcriptional profiling of four major immune tissues (liver, spleen, head kidney, and trunk kidney) of GCRV-infected grass carp was established. A total of 192.65 Gb clean data was obtained with 6.11 Gb per sample and stored in the NCBI Sequence Read Archive (with accession number PRJNA759556). Based on the GO and KEEG analyses, 108 unique GO terms were enriched in the four tissues. Thirty-five enriched pathways were obtained, with 21 metabolism-related pathways mainly gained in the liver and trunk kidney, and 14 immune response pathways were enriched in the spleen and head kidney. Also demonstrated was that GCRV stimulates not only the expression of interferon-stimulated genes (ISGs) but also proinflammatory cytokines. 27 ISGs were screened from the candidate DEGs, and eight ISGs were identified for the first time in grass crap. These ISGs were classified into three categories by their function found in mammals: (i) positively regulates the IFN signalling pathway (RIG-I, MDA5, IRF7, IRF9, STAT2, and TRIM25); (ii) negatively regulates the IFN signalling pathway (usp18 and SOCS1); and (iii) exerts direct antiviral activity such as Mx1, ISG15, ISG56, ISG58, viperin, and PKR. Eight major ISGs and four typical differentially inflammatory cytokines were used for further expression analysis with prominent expression in the liver, spleen and kidney. The onset time of IFN-mediated antiviral response was trunk kidney (12-24 h) > liver (48 h) > spleen (96-120 h), and the intensity was liver > spleen > trunk kidney. Notably, the inflammatory reaction occurs early in the liver and trunk kidney. This result implies that ISGs may act synergistically and that the IFN response is closely related to the inflammatory response against GCRV infection. The transcriptomic profiles obtained and the function of ISGs predicted in this study provide new insights into fish antiviral mechanisms and developing effective therapeutic directions.

Comprehensive comparison of thirteen kinds of cytokine receptors from the endangered fish Chinese sturgeon (Acipenser sinensis).

The interferon receptor system in teleost fish is more complex than that in mammals. In the present study, we identified 13 cytokine receptor genes (10 interferon receptor genes and 3 IL10R2-like genes) from Chinese sturgeon (Acipenser sinensis) using RNA-sequencing. Sequence analysis indicated that these receptors had conserved domains, including signal peptides, FNⅢ, and transmembrane domains. Phylogenetic analysis suggested that they belonged to the cytokine receptor family. In the present study, we named them IFNAR1-like (CRFB5a, CRFB5b), IFNAR2-like (CRFB3a, CRFB3b), IFNGR1-like (IFNGR1), IFNGR2-like (CRFB6a, CRFB6b/IFNGR2-1, CRFB6c/IFNGR2-2, CRFB6d/IFNGR2-3, CRFB6e/IFNGR2-4) and IL10R2-like (CRFB4a, CRFB4b, CRFB4c), respectively. Constitutive expression analysis revealed that these receptor genes had potential functions in immune and non-immune tissue compartments. After stimulating with Poly (I:C), the expression fold changes of CRFB3a, CRFB4a, CRFB4b, CRFB5b, and CRFB6e/IFNGR2-4 in Chinese sturgeon were higher than those of other receptor genes, which revealed that these five genes had important functions in the immune process to resist virus invasion in the host. After stimulating with IFN gamma, the expression fold changes of CRFB3a, CRFB4a, and CRFB6b/IFNGR2-1 were higher than those other receptor genes. Based on other teleost fish interferon receptor models, we speculated that IFNAR1-like (CRFB5a, CRFB5b) and IFNAR2-like (CRFB3a, CRFB3b), comprised Chinese sturgeon type Ⅰ IFN receptors; and IFNGR1-like (IFNGR1) and IFNGR2-like (CRFB6/IFNGR2) comprised Chinese sturgeon type Ⅱ IFN receptors.

Characterization of the mitochondrial genome of Microphysogobio tungtingensis (Cypriniformes) and its taxonomic status.

In the present study, the complete mitochondrial genome of Microphysogobio tungtingensis has been amplified with 16 pairs of primers. There are 16 627 base pairs has been identified and deposited in the GenBank with accession numbers MN970213. The arrangement was similar to typical vertebrate mitochondrial, including 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs genes and a noncoding control region. The overall base composition of M. tungtingensis was G + C: 42.9%, A + T: 57.1%, apparently with a slight AT bias. Phylogenetic analysis showed that M. tungtingensis was close to M. fukiensis.

Characterization the mitochondrial genome of Acrossocheilus Labiatus (Cypriniformes, Acrossocheilus) and its phylogeny.

The first complete mitochondrial genome of Acrossocheilus Labiatus from the Qingshui River were reported in this study with accession number MG878098. The overall nucleotide composition was 31.13% A, 25.10% T, 27.52% C, 16.24% G, respectively. Phylogenetic analysis shows that the A. parallens and A. Hemispinus showed a closest phylogenetic relationship, then clustal with A. Labiatus.

Complete mitochondrial genome of javeline goby (Synechogobius hasta).

In this study, we cloned and sequenced the complete mitochondrial DNA (mtDNA) of Synechogobius hasta to characterize and compare their mitochondrial genomes. The total length of the mitochondrial genome is 16,655 bp with an accession number KM891736. The organization of the mitochondrial genomes was similar to those reported from other fish mitochondrial genomes containing 37 genes (13 protein-coding genes, 2 ribosomal RNA and 22 transfer RNAs) and a major non-coding control region. Except for ND6 and 8 tRNAs, all other genes are encoded on the heavy strand. The base composition of the 13 mitochondrial protein-coding genes in the third position was relatively low (9.7%). The complete mitogenome may provide important date set for the study of genetic mechanism of S. hasta.

Molecular characterization and expression regulation of Smyd1a and Smyd1b in skeletal muscle of Chinese perch (Siniperca chuatsi).

Smyd1 is a SET and MYND domain-containing protein, which functions as a histone methyltransferase for control of gene expression and regulates the skeletal and cardiac muscle differentiation. In this study, the full-length cDNA sequences of Smyd1a and Smyd1b were cloned from Chinese perch, and their molecular structure and expression profile in response to nutrition supply and in vivo IGF treatments were also analyzed. The cDNA sequence of Smyd1a and Smyd1b consists of 1862 and 1802 base pairs (bp), encoding 479 and 476 amino acids, respectively. The SET domains of the two proteins were split into two segments by the MYND domain. Furthermore, the amino acid sequence of Smyd1a contains an extra 13-aa insertion in the SET domain in comparison with Smyd1b. The two genes apparently exhibited temporal and spatial differential expression status. In adults, the two genes showed the higher expression level in the muscle and heart than in other testing tissues. During the post-embryonic developmental stages, the higher expression of Smyd1a was detected at 150 days post-hatching (dph), whereas the expression of Smyd1b peaked at 50 dph. It was indicated that they have potential function in muscle developmental regulation. The mRNA levels of Smyd1a and Smyd1b were sharply up-regulated within one day after refeeding in the Chinese perch juveniles following fasting for a week. Moreover, IGF-1 treatments in vivo significantly stimulated their expression in the skeletal muscle. Together, these data provide us with further understanding of the molecular characterization and expression regulation of the two genes upon internal and external stimuli.

Characteristics and phylogenetic studies of complete mitochondrial DNA based on the ricefield eel (Monopterus albus) from four different areas.

In this study, we cloned and sequenced the complete mitochondrial DNA of ricefield eel populations from four different areas (Guizhou province, Guangxi province, Hunan province, and Guangdong province) in China. The mitochondrial genome was 16,622 bp in length and deposited in the GenBank with accession numbers KP779622-KP779625. The organizations of the complete mitogenomes of the four ricefield eel were similar to those of other teleost species which contained 13 protein-coding genes, two rRNA genes, and 22 tRNA genes, as well as a putative control region (CR). Most of these genes were encoded on the H-strand, except for the ND6 and eight tRNA genes, all other genes were encoded on the H-strand. Phylogenetic analyses using N-J computational algorithms showed that the ricefield eel was clustered with Mastacembelus armatus (KJ184553) and they belong to Synbranchiformes. The four ricefield eel populations could be divided into two groups: Guangxi province and the other population cluster together.

Complete mitochondrial genome of the hybrid of Ctenopharyngodon idellus (♀) × Elopichthys bambusa (♂).

In this study, we determined the complete mitochondrial DNA sequence of the hybrid of Ctenopharyngodon idellus (♀) × Elopichthys bambusa (♂). The total length of the mitochondrial genome was 16,609 bp (accession number KM401549), with the base composition of 31.89% A, 26.33% C, 15.68% G, 26.11% T. The genome contained 2 ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes, and a major non-coding control region (D-loop region). Except for ND6 and 8 tRNAs, all other genes were encoded on the heavy strand. The arrangements of these genes were similar to that found in the other teleosts. The complete mitogenome may provide important date set for the study of genetic mechanism in the hybrid fish of Cyprinidae.

The complete mitochondrial genome sequence of Cirrhinus mrigala (♀) × Labeo rohita (♂).

We cloned and sequenced the complete mitochondrial DNA (mtDNA) of Cirrhinus mrigala (♀) × Labeo rohita (♂) by PCR-based method. The total of the mtDNA was 16,595 bp. It consisted of 13 protein-coding genes, 22 tRNAs, 2 rRNA genes, and one putative control region (D-loop region). The overall base composition on strand was as follows A: 32.09%, G: 15.51%, C: 27.95%, T: 24.45%, and the A + T content 56.54%. All the protein-coding genes initiated by typical ATG codon, and ten genes ended with the complete stop codon TAA or TAG, while COII2, ND4 and Cytb genes terminated with GCC, TAT and GCT. The control region contains a thiolases active site, EGF-like domain signature and two anaphylatoxin domain signature. This mitogenome sequence data would play an important role in population genetics and phylogenetics of the hybrid fish in the Cyprinidae family.

Complete mitochondrial genome sequence of Procypris merus.

In this study, the complete mitochondrianl genome of Procypris merus was sequenced. It was determined to be 16,581 bp and included 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes and 1 non-coding region (D-loop). The descending order of the base composition on heavy strand was 31.91% A, 24.95% T, 27.39% C, 15.75% G, which is similar to other cyprinid fish mitochondrial genomes. All protein-coding genes had ATG as the start codon but the stop codons have three types. Night genes end with TAA or TAG, and COII, ND4, ND6 and Cytb genes terminate with an incomplete - -T. The complete mitochondrial genome may provide important DNA molecular data for further phylogenetic analyses for higher taxa of Cyprinidae.

Selection of reference genes for microRNA quantitative expression analysis in Chinese perch, Siniperca chuatsi.

Real-time quantitative reverse transcription PCR (RT-qPCR) is one of the most effective and sensitive techniques in gene expression assay, for which selection of reference genes is a prerequisite. In teleost species, such as Chinese perch, the expression profiling of miRNAs as reference genes for RT-qPCR has not been intensively studied. In the present study, the expression profiles of six miRNAs (miR-101a, miR-146a, miR-22a, miR-23a, miR-26a and let-7a) and one small nuclear RNA (U6) were assayed with RT-qPCR in different adult tissues, developmental stages and growth conditions of Chinese perch, Siniperca chuatsi. The analyses revealed that embryonic developmental stage is an important variability factor in the expression stability of miRNAs. All six miRNAs exhibited better expression consistency than U6 in most of the conditions examined, and therefore, they may be more suitable as a reference gene for miRNA quantification. When different tissues and developmental stages were considered, miR-22a demonstrated the most consistent expression pattern, and the best combination of reference genes was miR-22a and miR-23a. Our study offers useful data for selecting miRNAs as reference genes for RT-qPCR analysis of miRNAs in teleost fishes under different conditions.

The microRNA signature in response to nutrient restriction and refeeding in skeletal muscle of Chinese perch (Siniperca chuatsi).

The Chinese perch (Siniperca chuatsi) is one of the most commercially important carnivorous fish species in aquaculture with its large-scale culture in China. Increasing evidence suggests that microRNAs (miRNAs) play an important role in muscle cell proliferation and differentiation. However, the knowledge of the identity of myogenic miRNAs and the effect of nutrient status on miRNA expression in teleost remains limited. In the present study, among the 21 miRNAs identified with high abundance in the fast muscle of adult Chinese perch, 19 miRNAs were differentially expressed in the adults and juveniles. The postprandial changes in the transcript abundance were determined for the 21 miRNAs following a single satiating meal in the juveniles after fasting for 1 week. The results showed that the seven miRNAs (miR-10c, miR-107a, miR-133a-3p, miR-140-3p, miR-181a-5p, miR-206, and miR-214) were sharply upregulated or downregulated within 1 h after refeeding. These miRNAs may be the promising candidate miRNAs involved in a fast-response signaling system that regulates fish skeletal muscle growth. Target prediction and expressional analysis suggested that four miRNAs (miR-10c, miR-107a, miR-140-3p, and miR-181a-5p) might play a role in regulating the translation of target gene transcripts such as myostatin following acute anabolic stimuli.

The phylogenetic placement of Siniperca obscura base on complete mitochondrial DNA sequence.

Abstract The extant freshwater sinipercids represent a group of 12 species and they are endemic to East Asia. In this study, we cloned and sequenced the complete mitochondrial DNA of Siniperca obscura from the Lijiang River. The size of the complete mitochondrial genome is 16,492 bp. The organization of the mitochondrial contained 37 genes (13 protein-coding genes, 2 ribosomal RNA and 22 transfer RNAs) and a major non-coding control region as well as those reported sinipercid fishes. Among the 13 protein-coding genes, three reading-frame overlaps were found: ATP8 and ATP6 overlap by 10 nucleotides and ND4 and ND4L overlap by 7 nucleotides and ND5 and ND6 overlap by 5 nucleotides. Phylogenetic analyses using N-J and maximum parsimony (MP) computational algorithms showed that S. chuatsi and S. kneri are sister species, next joined by S. Obscura, based on combined 12 protein-coding genes (excluding DN6).

The complete mitochondrial genome of Culter dabryi (Cyprinidae: Cultrinae).

Culter dabryi, a medium-sized economic cyprinid fish, is widely distributed in East Asia. We sequenced the complete mitochondrial genome of this species. The genome is 16,622 bp in length, and consists of 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes and the non-coding control region with a structural organization conserved relative to that of other fishes. The overall base composition of C. dabryi in descending order is A 31.51%, C 27.60%, T 24.95% and G 15.94%, with a slight A + T bias. The mitogenome sequence data may provide useful information to the population genetics analysis of C. dabryi and the elucidation of evolutionary mechanisms in the endemic clade of East Asian Cyprinidae.

Analysis of the variable sites and phylogenetic studies of complete mitochondrial DNA based on the Siniperca scherzeri (Perciformes: Sinipercidae) from four different areas.

In this study, we cloned and sequenced the complete mitochondrial DNAs of Chinese mandarin fish, Siniperca scherzeri populations from four different areas of the Yangtze River and the Pearl River systems in China. In the Yangtze River system, fish samples were taken from three regions, Dongting Lake (DT), Poyang Lake (PL) and Xiangjiang River (XR). In the Pearl River system, fish samples were collected from the Lijiang River (LR). The sizes of their complete mitochondrial genome were 16,502 bp (LR), 16,508 bp (PL), 16,502 bp (XR) and 16,508 bp (DT), respectively. The organization of the S. scherzeri mitochondrial genomes was similar to those reported from other fish mitochondrial genomes. Phylogenetic analyses using N-J computational algorithms showed that the Sinipercidaes are monophyletic and can be divided into two genera, Siniperca and Coreoperca. The S. scherzeri could be divided into two groups along the Yangtze River system and the Pearl River.

Complete mitochondrial genome of the blind cave barbel Sinocyclocheilus furcodorsalis (Cypriniformes: Cyprinidae).

Sinocyclocheilus furcodorsalis, a typical cavefish, has evolved some striking characters, for example loss of its eyes and reduction in melanin pigmentation, and can serve as a good model system in evolutionary adaptation developmental mechanisms. So we cloned the complete mitochondrial DNA of S. furcodorsalis (16,581 bp), which is similar to those reported from other fish mitochondrial genomes, containing 37 genes (13 protein-coding genes, 2 ribosomal RNAs, and 22 transfer RNAs) and a major noncoding control region. The complete mitogenome of the S. furcodorsalis provides an additional important data-set for the study in evolutionary adaptation developmental mechanisms.

Phylogenetic studies of three sinipercid fishes (Perciformes: Sinipercidae) based on complete mitochondrial DNA sequences.

The sinipercids are a group of 12 species of freshwater percoid fish endemic to East Asia and their phylogenetic placements have perplexed generations of taxonomists. We cloned and sequenced the complete mitochondrial DNA (mtDNA) of three sinipercid fishes (Siniperca chuatsi, S. kneri, and S. scherzeri) to characterize and compare their mitochondrial genomes. The mitochondrial genomes of S. chuatsi, S. kneri, and S. scherzeri were 16,496, 17,002, and 16,585 bp in length, respectively. The organization of the three mitochondrial genomes is similar to those reported from other fish mitochondrial genomes, which contains 37 genes (13 protein-coding genes, 2 ribosomal RNAs, and 22 transfer RNAs) and a major non-coding control region. Among the 13 protein-coding genes of all the three sinipercid fishes, three reading-frame overlaps were found on the same strand. There is an 81-bp tandem repeat cluster at the end of CSB-3 in the S. scherzeri control region. The complete mitochondrial genomes of the three sinipercids should be useful for the evolutionary studies of sinipercids and other vertebrate species.