The first complete chloroplast genome of Thalictrum fargesii: insights into phylogeny and species identification.

Introduction: Thalictrum fargesii is a medicinal plant belonging to the genus Thalictrum of the Ranunculaceae family and has been used in herbal medicine in the Himalayan regions of China and India. This species is taxonomically challenging because of its morphological similarities to other species within the genus. Thus, herbal drugs from this species are frequently adulterated, substituted, or mixed with other species, thereby endangering consumer safety. Methods: The present study aimed to sequence and assemble the entire chloroplast (cp) genome of T. fargesii using the Illumina HiSeq 2500 platform to better understand the genomic architecture, gene composition, and phylogenetic relationships within the Thalictrum. Results and discussion: The cp genome was 155,929 bp long and contained large single-copy (85,395 bp) and small single-copy (17,576 bp) regions that were segregated by a pair of inverted repeat regions (26,479 bp) to form a quadripartite structure. The cp genome contains 133 genes, including 88 protein-coding genes (PCGs), 37 tRNA genes, and 8 rRNA genes. Additionally, this genome contains 64 codons that encode 20 amino acids, the most preferred of which are alanine and leucine. We identified 68 SSRs, 27 long repeats, and 242 high-confidence C-to-U RNA-editing sites in the cp genome. Moreover, we discovered seven divergent hotspot regions in the cp genome of T. fargesii, among which ndhD-psaC and rpl16-rps3 may be useful for developing molecular markers for identifying ethnodrug species and their contaminants. A comparative study with eight other species in the genus revealed that pafI and rps19 had highly variable sites in the cp genome of T. fargesii. Additionally, two special features, (i) the shortest length of the ycf1 gene at the IRA-SSC boundary and (ii) the distance between the rps19 fragment and trnH at the IRA-LSC junction, distinguish the cp genome of T. fargesii from those of other species within the genus. Furthermore, phylogenetic analysis revealed that T. fargesii was closely related to T. tenue and T. petaloidium. Conclusion: Considering all these lines of evidence, our findings offer crucial molecular and evolutionary information that could play a significant role in further species identification, evolution, and phylogenetic studies on T. fargesii.

Roles of ß-catenin in innate immune process and regulating intestinal flora in Qi river crucian carp (Carassius auratus).

In mammals, ß-catenin participates in innate immune process through interaction with NF-κB signaling pathway. However, its role in teleost immune processes remains largely unknown. We aimed to clarify the function of ß-catenin in the natural defense mechanism of Qi river crucian carp (Carassius auratus). ß-catenin exhibited a ubiquitous expression pattern in adult fish, as indicated by real-time PCR analysis. Following lipopolysaccharide (LPS), Polyinosinic-polycytidylic acid (polyI: C) and Aeromonas hydrophila (A. hydrophila) challenges, ß-catenin increased in gill, intestine, liver and kidney, indicating that ß-catenin likely plays a pivotal role in the immune response against pathogen infiltration. Inhibition of the ß-catenin pathway using FH535, an inhibitor of Wnt/ß-catenin pathway, resulting in pathological damage of the gill, intestine, liver and kidney, significant decrease of innate immune factors (C3, defb3, LYZ-C, INF-γ), upregulation of inflammatory factors (NF-κB, TNF-α, IL-1, IL-8), and downregulation of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities, increase of Malondialdehyde (MDA) content. Following A. hydrophila invasion, the mortality rate in the FH535 treatment group exceeded that of the control group. In addition, the diversity of intestinal microflora decreased and the community structure was uneven after FH535 treatment. In summary, our findings strongly suggest that ß-catenin plays a vital role in combating pathogen invasion and regulating intestinal flora in Qi river crucian carp.

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.

Characteristics of the complete chloroplast genome of Saxifragaceae species Bergenia purpurascens (Hook. f. et Thoms.) Engl.

Bergenia purpurascens (Hook. f. et Thoms.) Engl. is one species of traditional Chinese medicinal plant. This is the first publication of its complete chloroplast (cp) genome. The whole cp genome has 157,246 base pairs in length with 132 annotated genes, of which were 87 protein-coding genes, 37 tRNAs, and 8 rRNAs. According to the phylogenetic study, B. purpurascens and Bergenia scopulosa T. P. Wang. 1974 had a sister relationship. This genomic data and conclusions from B. purpurascens phylogenetic research will provide useful information and throw light on more in-depth investigations of the systematics and evolutionary patterns of Saxifragaceae.

De novo gonad transcriptome analysis of elongate loach (Leptobotia elongata) provides novel insights into sex-related genes.

Elongate loach (Leptobotia elongata) is endemic in middle and upper reaches of the Yangtze River in China. Because of many anthropogenic factors such as overfishing and dam construction, the loach has become a highly endangered species. So far, the genomic resources which benefit for species conservation and utilization are still lacking in elongate loach. Therefore, the first gonad transcriptome analysis of the loach was conducted in this study, providing novel insights into sex-related genes. A total of 286,800,660 clean reads with a total length of 42.02 Gb were obtained. 18,975 differentially expressed genes (DEGs) were identified, where 12,976 DEGs, especially Sox9a, Sox9b, Igf2 and Fgfr2, were upregulated in the testis, and 5999 DEGs, especially Zp3, Eg2, Plk1, Ccnb1, Cdc20 and Mos, were upregulated in the ovary. Meanwhile, some testis-specific genes (i.e. Cald1, Atp1a, Muc2 and Ca2) and ovary-specific genes (i.e. Ca4, Tuba, Acp5, Ccna, Larp6 and Nop4) were identified and verified. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs, we found a series of enrichment pathways related to reproduction in elongate loach, such as the MAPK signaling pathway, oxytocin signaling pathway and oocyte meiosis pathway. Twelve DEGs were randomly selected to verify RNA-seq results by qPCR. In conclusion, this study provides a data source to study the molecular characteristics and regulatory mechanisms of sex-related genes in elongate loach, which has a potential to improve the resource protection and aquaculture production of the loach.

Tartrazine exposure results in histological damage, oxidative stress, immune disorders and gut microbiota dysbiosis in juvenile crucian carp (Carassius carassius).

Tartrazine (TZ) is an azo dye widely used in foods, cosmetics, beverages, textile, and leather. In recent years, there are reports on detecting azo dyes in the aquatic environment, so the impact of these compounds on aquatic organisms could not be ignored. In this study, we aimed to evaluate the adverse effects of TZ exposure on teleosts' embryo development and juvenile's health by using crucian carp (Carassius carassius) as the experimental fish. The results showed that embryos exposed to TZ (0.19, 0.76 and 1.5 mM) exhibited a deformity, delayed egg resorption and decreased fertilization and hatching rate. When the juvenile fish were exposed to TZ at a level higher than those present in water for 30 days caused severe histopathological damages of the gill, intestine, kidney and liver. Antioxidant enzymes (CAT, SOD and GSH-Px) activities in the gill, intestine and liver, exhibited a decreasing trend after TZ exposure, while MDA contents elevated. TZ exposure also resulted in the upregulation of pro-inflammatory cytokines (il1 and il6), lysozymes (lyz), complement component 3 (c3), and ß-defensin 3 (defb3). In addition, TZ exposure also affected the intestinal microbiota structure. In summary, the data in the present study indicated that TZ exposure  reduce the embryo fertilization and hatching rate; cause histopathological damage of tissues, trigger oxidative stress, innate immune disorders and dysbiosis of gut microbiota in juvenile crucian carp. Therefore, it is necessary to be informed about the hazards of TZ exposure and the discharge of the dye into waters should be strictly administrated to prevent environmental pollution.

MRAP2 Interaction with Melanocortin-4 Receptor in SnakeHead (Channa argus).

The melanocortin-4 receptor (MC4R) plays an important role in the regulation of food intake and energy expenditure. Melanocortin-2 receptor accessory protein 2 (MRAP2) modulates trafficking, ligand binding, and signaling of MC4R. The Northern snakehead (Channa argus) is an economically important freshwater fish native to East Asia. To explore potential interaction between snakehead MC4R and MRAP2, herein we cloned snakehead mc4r and mrap2. The snakehead mc4r consisted of a 984 bp open reading frame encoding a protein of 327 amino acids, while snakehead mrap2 contained a 693 bp open reading frame encoding a protein of 230 amino acids. Synteny analysis indicated that mc4r was highly conserved with similar gene arrangement, while mrap2 contained two isoforms in teleost with different gene orders. Snakehead mc4r was primarily expressed in the brain, whereas mrap2 was expressed in the brain and intestine. Snakehead mc4r and mrap2 expression was modulated by fasting and refeeding. Further pharmacological experiments showed that the cloned snakehead MC4R was functional, capable of binding to peptide agonists and increasing intracellular cAMP production in a dose-dependent manner. Snakehead MC4R exhibited high constitutive activity. MRAP2 significantly decreased basal and agonist-stimulated cAMP signaling. These findings suggest that snakehead MC4R might be involved in energy balance regulation by interacting with MRAP2. Further studies are needed to elucidate MC4R in regulating diverse physiological processes in snakehead.

Complete mitochondrial genome of a kind of snakehead fish Channa siamensis and its phylogenetic consideration.

The snakehead fish, Channa siamensis, belongs to the genus of Channa (perciformes: Channidae) and was first reported by Günther in 1861. Despite it has been described approximately for 15 decades, the genetic information is limited and the taxon status of this kind of fish is still unclear. The primary objective of this study is to get more genomic data and calculate the taxon location of this kind of fish. The next generation sequencing method was used to obtain the whole mitochondrial DNA information, and bioinformatic analysis was performed to investigate the evolutionary status and taxon location of C. siamensis. The circular mitochondrial DNA was 16,570 bp in length, and which showed typical piscine structure and arrangement. The overall nucleotide composition was 29.28% A, 24.72% T, 30.71% C, 15.29% G, with 54.1% AT, respectively. Phylogenetic analyses using concatenated amino acid and nucleotide sequences of the 13 protein-coding genes with two different methods (Maximum likelihood and Bayesian analysis) both highly supported C. siamensis belongs to the genus Channa and shows a close relationship with C. micropeltes. These data will provide more useful information for a better understanding of the mitochondrial genomic diversities and evolution in fish as well as novel genetic markers for studying population genetics and species identification.

Identification and analysis of the complete mitochondrial genome of Acheilognathus omeiensis (Cypriniformes, Cyprinidae).

Acheilognathus omeiensis is a small-size freshwater ornamental fish. In this study, the complete mitochondrial genome sequence of A. omeiensis was first determined. This mitogenome was 16,774 bp in length and contained 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, and 2 main noncoding regions. Most mitochondrial genes were encoded on the H-strand, except for ND6 and eight tRNA genes. The base composition was 28.97% A, 27.72% T, 26.02% C, and 17.29% G, with 56.69% AT, respectively. Phylogenetic tree was constructed based on the nucleotide sequence of 13 PCGs of A. omeiensis and closely related 12 species ticks to assess their phylogenic relationship and evolution. These data should be helpful for a better understanding of the mitochondrial genomic diversities and evolution in fish as well as novel genetic markers for studying population genetics and species identification.

Complete mitochondrial genome and phylogenetic analysis of Sinogastromyzon szechuanensis (Teleostei, Cypriniformes, Homalopteridae).

Sinogastromyzon szechuanensis is endemic to the Upper Yangtze River in China. In this study, we first determined the complete mitochondrial genome of S. szechuanensis. The circular mitogenome was 16,565 bp in length, including 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and 2 main noncoding regions. Most mitochondrial genes were encoded on the H-strand, except for ND6 and eight tRNA genes. The overall nucleotides composition was 30.38% A, 25.26% T, 27.74% C, and 16.62% G, with AT bias of 55.64%, respectively. Phylogenetic analysis using concatenated nucleotide sequences of the 13 PCGs with two different methods (ML and NJ) both highly supported that S. szechuanensis showed a close relationship with S. puliensis and S. sichangensis. These data would be useful for further studies on genetic diversity and molecular phylogenetic relationship of the family Homalopteridae.

The complete mitochondrial genome of Ancherythroculter wangi and its phylogeny.

Ancherythroculter wangi, is a unique freshwater fish and mainly distributed in the upper stream of Yangtze River and its tributary. In this study, the complete mitochondrial genome of A. wangi was first determined. The total length of the complete mitochondrial genome is 16,622 bp, contained 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, one D-loop locus, and an origin of replication on the light-strand (OL). The overall nucleotide composition was 31.19% A, 24.84% T, 27.79% C, 16.18% G, with 56.03% AT, respectively. Phylogenetic analysis both highly supported that A. wangi showed a close relationship with Culter mongolicus and A. kurematsui. These data would contribute to elucidate the evolutionary mechanisms and biogeography of Ancherythroculter and is useful for the conservation of genetics and stock evaluation for A. wangi.

The complete mitochondrial genome of Sarcocheilichthys davidi and its phylogeny.

Sarcocheilichthys davidi is a small-size ornamental freshwater species. In the present study, the complete mitochondrial genome of S. davidi was first determined using the next-generation sequencing technology. The result showed that the circular mitogenome was 16,675 bp in length, contained 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and two non-coding regions: origin of light-strand replication (OL) and displacement loop locus (D-loop). In addition to ND6 and eight tRNA genes, most mitochondrial genes were encoded on the heavy strand. Furthermore, the overall base composition was 29.97% A, 26.12% T, 26.86% C, 17.04% G, respectively, and showed AT-rich feature (56.09%). Moreover, phylogenetic analysis based on the tandem 13 PCGs nucleotide sequences indicated that S. davidi had a close relationship with S. snigripinnis and S. variegatus wakiyae. The complete mitogenome sequence of S. davidi would contribute to the better understanding of molecular systematics, selection, conservation, species identification and evolution of this species.

Sequence identification and phylogenetic analysis of the mitochondrial genome of Microphysogobio kiatingensis (Cypriniformes: Cyprinidae).

Microphysogobio kiatingensis is endemic freshwater fish of China and mainly distributed in the middle and upper reaches of the Yangtze River. The complete mitogenome of M. kiatingensis was first determined by using the next-generation sequencing in present study. The circular mitogenome followed the expected pattern for vertebrates, being 16,603 bp in length. It contained 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, one displacement loop (D-loop) locus and an origin of replication on the light-strand (OL). Most of the genes are encoded on the heavy strand except ND6 and eight tRNA genes. The overall base composition was 30.78% A, 26.09% T, 26.59% C, 16.54% G, with 56.87% AT. Thirteen intergenic spacers and nine gene overlaps exist in the complete mitochondrial genome. Phylogenetic analysis based on the tandem 13 coding protein genes nucleotide sequences with two different methods (maximum likelihood and neighbour-joining analysis) indicated that M. kiatingensis was grouped in one clade with the same genus. The complete mitogenome of M. kiatingensis can provide a useful data for the further research in Gobioninae. Meanwhile, it also provides help for biological genetics and classification of species.

Molecular cloning, tissue distribution, and effect of fasting and refeeding on the expression of neuropeptide Y in Channa argus.

Neuropeptide Y (NPY) is a 36 amino-acid amidated peptide of the pancreatic polypeptide (PP) family, which plays an important role in appetite regulation and energy expenditure in mammals. Although several teleost NPY have been identified, its roles remain unclear in fish. We herein reported on the molecular cloning, tissue distribution and the effect of fasting on the expression of NPY in Channa argus, and designated as CaNPY. It consisted of a 300 bp open reading frame predicted to encode a prepro-NPY of 99 amino acids. Sequence analysis revealed that CaNPY was highly conserved (>60%) with other vertebrate NPY. Phylogenetic analysis highly supported CaNPY was closely related to piscine NPY. In addition, except for muscle and spleen tissues, CaNPY was found to extensively expressed in all other detected tissues, with the highest level in brain. Futhermore, the CaNPY transcript was found to significantly increase after short-term and long-term food deprivation, and dramatically decrease following refeeding. These findings suggested that CaNPY might be involved in food intake regulation and it could be as a potential target locus to improve commercial production of this kind of fish.

Comparative analysis of the liver transcriptome of Pelteobagrus vachellii with an alternative feeding time.

Pelteobagrus vachellii, an important freshwater fish in China, shows predominantly nocturnal behavior. To better understand the growth and molecular mechanisms underlying altered feeding times in this species, we studied the growth and liver transcriptome of P. vachellii with shifted feeding times. In this study, a 9-week growth trial was conducted on male P. vachellii (mean weight±S.E.=1.05±0.36g) with commercial feed. Two triplicate groups of fish were fed either at 0800 (day group, control) or at 2000 (night group) with the same amount of feed. After nine weeks, a significant increase in growth was observed in the night group, demonstrated by the specific growth rate (SGR). Using high-throughput RNA-seq, 70,793,844 and 67,930,610 paired-end clean reads were obtained from six cDNA libraries of P. vachellii liver, and 60,069 unigenes were assembled. Gene expression comparison revealed that 122 genes were significantly up-regulated and 59 genes were significantly down-regulated in the night group. Gene pathway and GO enrichment analyses revealed metabolic responses of genes and gene networks related to protein, lipid and carbohydrate metabolism and rhythms. This study indicates that an alternative feeding time can improve growth and create metabolic alterations in the liver of P. vachellii.

The complete mitochondrial genome of Pseudogyrinocheilus prochilus (Cypriniformes: Cyprinidae).

The complete mitochondrial sequence of Pseudogyrinocheilus prochilus, an endemic species to China and mainly distributed in upper Yangtze River and its tributaries, was obtained by PCR. It was 16,595 bp in length and comprised 37 mitochondrial genes (22 transfer RNA genes, 13 protein-coding genes, 2 ribosomal RNA genes) and 2 non-coding regions. The overall base composition of H-strand was 31.40% A, 26.78% C, 16.18% G, and 25.64% T. Most of the genes are encoded on heavy strand, except for eight tRNA and ND6 genes. The composition and order of mitochondrial gene have no unique characteristics compared to most of other fishes.

The complete mitochondrial genome sequence of Schizothorax lissolabiatus (Cypriniformes: Cyprinidae).

Schizothorax lissolabiatus is a widely distributed fish species in Lancang River, Yuanjiang River, Pearl River, Nujiang River in China. The complete mitochondrial genome sequence of S. lissolabiatus from Yuanjiang River (the upper Red River) has been sequenced, which is 16,583 bp in length and contains 22 tRNA genes, 13 protein-coding genes, 2 rRNA genes and 2 main non-coding regions: origin of light-strand replication and control region. Except for eight tRNA and ND6 genes, most of the genes are encoded on the heavy strand. The length of mitochondrial genome, the order, composition and anticodons of all genes are exactly similar to that of S. dolichonema. The phylogenetic tree suggested S. prenanti, S. dolichonema and S. lissolabiatus may have closer affinities than other fish of the genus Schizothorax.

The complete mitochondrial genome sequence of Schizothorax dolichonema (Cypriniformes: Cyprinidae).

The complete mitochondrial genome sequence of Schizothorax dolichonema has been sequenced, which contains 22 tRNA genes, 13 protein-coding genes, 2 rRNA genes and 2 non-coding regions: origin of light-strand replication and control region, with the total length of 16,583 bp. The gene order and composition are similar to most of other vertebrates. Most of the genes are encoded on heavy strand, except for eight tRNA and ND6 genes. The mitogenome sequence of S. dolichonema would contribute for better understanding of biogeography and evolution of Schizothoracine fishes.

Effects of pathogenic bacterial challenge after acute sublethal ammonia-N exposure on heat shock protein 70 expression in Botia reevesae.

The objective of this study was to investigate the effects of pathogenic bacterial challenge after acute sublethal ammonia-N exposure on heat shock protein 70 expression in Botia reevesae. After ammonia-N exposure at a constant concentration of 7.21 ± 0.10 mg L(-1) for 96 h, B. reevesae was challenged with Aeromonas hydrophila. Quantitative PCR analysis showed predominant and significant expression of HSP70 in liver, gill, skin, spleen and kidney (P < 0.05), with significantly upregulated expression of the mRNA transcript in these tissues after sublethal ammonia-N exposure and A. hydrophila challenge. Furthermore, following A. hydrophila challenge after ammonia-N exposure, HSP70 mRNA expression was significantly upregulated in kidney and gill tissues, although its expression levels were significantly lower than those detected following A. hydrophila challenge or ammonia-N exposure individually. These results indicate that B. reevesae HSP70 is involved in resistance to pathogenic bacteria. It is hypothesized that ammonia-N results in the downregulation of HSP70 mRNA in immune organs after an A. hydrophila challenge, thus lowering their resistance to pathogenic stress.