Huanling Wang

Spring viraemia of carp virus induces autophagy for necessary viral replication

Outbreaks of spring viraemia of carp virus (SVCV) in several carp species and other cultivated fish can cause significant mortality and jeopardize the billion‐dollar worldwide fish industry. Spring viraemia of carp virus, also known as Rhabdovirus carpio, is a bullet‐shaped RNA virus that enters and amplifies in gill epithelium and later spreads to internal organs. Young fish under stressed conditions (spring cold water, etc.) are more vulnerable to SVCV‐induced lethality because of their lack of a mature immune system. Currently, the host response of SVCV remains largely unknown. Here, we observed that autophagy is activated in SVCV‐infected epithelioma papulosum cyprini (EPC) cells. We demonstrated that the SVCV glycoprotein, rather than viral replication, activates the autophagy pathway. In addition, SVCV utilized the autophagy pathway to facilitate its own genomic RNA replication and to enhance its titres in the supernatants. Autophagy promoted the survival of SVCV‐infected cells by eliminating damaged mitochondrial DNA generated during viral infection. We further showed that SVCV induces autophagy in EPC cells through the ERK/mTOR signalling pathway. Our results reveal a connection between autophagy and SVCV replication and propose autophagy suppression as a novel means to restrict SVCV viral replication.

Isolation and expression of a cold-responsive gene PtCBF in Poncirus trifoliata and isolation of citrus CBF promoters

C-repeat/dehydration-responsive element binding factor (CBF) plays important roles in cold response network in plants. Here, one member of CBF coding gene family in trifoliate orange (Poncirus trifoliata), designated as PtCBF, was isolated. Semi-quantitative reverse transcription-polymerase chain reactions showed up-regulation of PtCBF not only under low temperature but also induced by abscisic acid. Additionally, the CBF genomic fragments in four citrus species including trifoliate orange, sweet orange (Citrus sinensis), pummel (Citrus grandis) and rough lemon (Citrus jambhiri) were isolated with complete open reading frames. According to the results of alignment analysis between full length cDNA and genomic DNA sequences in trifoliate orange, there were no introns in PtCBF. Moreover, the results of multiple sequence alignment analysis and phylogenetic analysis on putative protein sequences suggested that the AP2 DNA binding domains and CBF signature sequences were highly conserved in four citrus CBF proteins. Finally, the CBF promoters in above citrus species were isolated, which provides some information concerning promoter function.

Molecular characterization of trypsinogens and development of trypsinogen gene expression and tryptic activities in grass carp (Ctenopharyngodon idellus) and topmouth culter (Culter alburnus)

This study examined the gene structures and expression of trypsinogens, as well as the trypsin activities of the grass carp Ctenopharyngodon idellus (herbivorous) and the topmouth culter Culter alburnus (carnivorous), which are commercially important freshwater species of the family Cyprinidae in China. Isolated full-length trypsinogen cDNA clones were 869 bp and 857 bp. The deduced amino acid sequences were 242aa and 247aa long, both containing the highly conserved residues essential for serine protease catalytic and conformational maintenance. The results from isoelectric and phylogenetic analyses suggest that grass carp trypsinogen is grouped with teleost trypsinogen group I, while topmouth culter trypsinogen is grouped with group II. The expression pattern of trypsinogen mRNA was similar between these two species, appearing 2 days post-hatching (dph) and reaching peaks at 11 and 23 dph. The trypsin-specific activities in both species were detected 2 dph and reached the major peaks at 8 dph, however the minor peaks were observed at 20 dph in the grass carp and 17 dph in the topmouth culter. The trypsin-specific activity was significantly higher in the grass carp than in the topmouth culter, which may be attributed to the nature of their different nutritional habits.

cDNA cloning and expression analysis of a Poncirus trifoliata CBF gene.

The transcription factors CBF/DREB play an important role during low temperature, drought and high-salt stress in higher plants. A new CBF (CRT/DRE binding factor) gene was cloned from trifoliate orange [Poncirus trifoliata (L.) Raf.] by RT-PCR with degenerate primers and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of CBF gene from trifoliate orange (designated as Ptcbfb) was 847 bp containing a 732 bp open reading frame (ORF), encoding a 243 amino acid protein. The predicted protein (designated as PtCBFb) had over 60 % identity to CBFs from some other plant species. Bioinformatical analysis showed that PtCBFb contained N-terminal bipartite nuclear targeting sequence, potential C-terminal acid domain and high conserved AP2 domain. Some other loci such as phosphorylation sites of several protein kinases, N-myristoylation site, tyrosine sulfation site and amidation site were also conserved in PtCBFb. Predicted three-dimentional structure of PtCBFb was similar to CBF from Arabidopsis thaliana. Expression pattern analysis revealed Ptcbfb expression in every tested organ, and Ptcbfb was cold induced.

The draft genome of blunt snout bream (Megalobrama amblycephala) reveals the development of intermuscular bone and adaptation to herbivorous diet

Abstract The blunt snout bream Megalobrama amblycephala is the economically most important cyprinid fish species. As an herbivore, it can be grown by eco-friendly and resource-conserving aquaculture. However, the large number of intermuscular bones in the trunk musculature is adverse to fish meat processing and consumption. As a first towards optimizing this aquatic livestock, we present a 1.116-Gb draft genome of M. amblycephala, with 779.54 Mb anchored on 24 linkage groups. Integrating spatiotemporal transcriptome analyses, we show that intermuscular bone is formed in the more basal teleosts by intramembranous ossification and may be involved in muscle contractibility and coordinating cellular events. Comparative analysis revealed that olfactory receptor genes, especially of the beta type, underwent an extensive expansion in herbivorous cyprinids, whereas the gene for the umami receptor T1R1 was specifically lost in M. amblycephala. The composition of gut microflora, which contributes to the herbivorous adaptation of M. amblycephala, was found to be similar to that of other herbivores. As a valuable resource for the improvement of M. amblycephala livestock, the draft genome sequence offers new insights into the development of intermuscular bone and herbivorous adaptation.

The zebrafish miR-462/miR-731 cluster is induced under hypoxic stress via hypoxia-inducible factor 1α and functions in cellular adaptations

Hypoxia, a unique and essential environmental stress, evokes highly coordinated cellular responses, and hypoxia‐inducible factor (HIF) 1 in the hypoxia signaling pathway, an evolutionarily conserved cellular signaling pathway, acts as a master regulator of the transcriptional response to hypoxic stress. MicroRNAs (miRNAs), a major class of posttranscriptional gene expression regulators, also play pivotal roles in orchestrating hypoxia‐mediated cellular adaptations. Here, global miRNA expression profiling and quantitative real‐time PCR indicated that the up‐regulation of the miR‐462/miR‐731 cluster in zebrafish larvae is induced by hypoxia. It was further validated that miR‐462 and miR‐731 are upregulated in a Hif‐1α‐mediated manner under hypoxia and specifically target ddx5 and ppm1da, respectively. Overexpression of miR‐462 and miR‐731 represses cell proliferation through blocking cell cycle progress of DNA replication, and induces apoptosis. In situ detection revealed that the miR‐462/miR‐731 cluster is highly expressed in a consistent and ubiquitous manner throughout the early developmental stages. Additionally, the transcripts become restricted to the notochord, pharyngeal arch, liver, and gut regions from postfertiliztion d 3 to 5. These data highlight a previously unidentified role of the miR‐462/miR‐731 cluster as a crucial signaling mediator for hypoxia‐mediated cellular adaptations and provide some insights into the potential function of the cluster during embryonic development.—Huang, C.‐X., Chen, N., Wu, X.‐J., Huang, C.‐H., He, Y., Tang, R., Wang, W.‐M., Wang, H.‐L. The zebrafish miR‐462/miR‐731 cluster is induced under hypoxic stress via hypoxia‐inducible factor 1α and functions in cellular adaptations. FASEB J. 29, 4901–4913 (2015). www.fasebj.org

Comparative and evolutionary analysis in natural diploid and tetraploid weather loach Misgurnus anguillicaudatus based on cytochrome b sequence data in central China

To obtain the phylogenetic relationship between diploid and tetraploid Misgurnus anguillicaudatus, the mitochondrial cyt b gene in the diploid and tetraploid weather loach were isolated and sequenced. The DNA sequences were analyzed using MEGA 3.0 software to determine the phylogenetic relationship. Forty-five variable sites among cyt b gene sequences and 18 amino acid substitutions occurred within the diploid and tetraploid loaches as deduced from the nucleotide sequences analysis of the cyt b gene. The nucleotide pairwise distance between diploid and tetraploid loach ranged from 0.001 to 0.025. Phylogenetic analysis revealed evolutionary relationships between diploid and tetraploid loach. Our results indicated a significant difference between diploid and tetraploid loach about the cyt b gene. AMOVA analysis indicated that there were no significant genetic variations within diploid loaches (Fst = 0.2529, P > 0.05) and within tetraploid loaches (Fst = 0.0564, P > 0.05), neither. However, significant genetic differences were found between diploid and tetraploid loaches (Fst = 0.7634, P < 0.05). Thus, it is concluded that no reproductive isolation was found within the same cytotypes of different localities, but there was reproductive isolation between these two cytotypes. The diploid loach existed before the tetraploid loach in nature. The present study is the first to describe the phylogenetic relationships of natural polyploidy weather loach using mtDNA cyt b gene.

Characterization of muscle morphology and satellite cells, and expression of muscle-related genes in skeletal muscle of juvenile and adult Megalobrama amblycephala.

Hyperplasia and hypertrophy are two distinct processes of skeletal muscle growth regulated by four myogenic regulatory factors (MRFs, contains MyoD, Myf5, Mrf4 and myogenin (MyoG)) and myostatin (MSTN). In this study, characterization of muscle morphology and satellite cells in juvenile (1-year-old) and adult (2-year-old) Megalobrama amblycephala was described. Compared with 1-year-old M. amblycephala, the diameter scope of epaxial, horizontal septum and hypaxial muscle fibers including red and white muscle in 2-year-old fish exhibited broader with dramatic reduction in frequency distribution of <20 μm diameter, nevertheless observable increase in frequency distribution of >50 μm diameter. Intermyofibrillar (IM) nuclei were also found except numerous subsarcolemmal (SS) nuclei in 2-year-old fish, whereas only SS nuclei were observed in 1-year-old fish. Immunofluorescence results showed that more satellite cells existed in red muscle than white muscle in 1-year-old fish, rather than 2-year-old fish. Moreover, we observed predominant increase in the mRNA levels of MyoD, Myf5, Mrf4, and MSTN during muscle development of fish in 2-year-old fish except MyoG.

Characterization of a novel CC chemokine CCL4 in immune response induced by nitrite and its expression differences among three populations of Megalobrama amblycephala

A novel CC chemokine gene, chemokine CC motif ligand 4 (CCL4), was isolated from Megalobrama amblycephala. The full-length cDNA was 913 bp, encoding 94 amino acid residues. The deduced amino acid sequence possessed the typical arrangement of four cysteines as found in other known CC chemokines. The expression of M. amblycephala CCL4 during the early development showed the mRNA levels before hatching and at 62 h post fertilized (hpf) were significantly higher than other post-hatching stages (P < 0.05). Besides, it was widely expressed in all detected tissues with the highest transcription in liver, followed by intestine, spleen and gill, where a larger number of immune cells including lymphocytes and macrophages are present. Our findings had fully confirmed that CCL4 expression was strongly induced in vitro and quickly up-regulated after nitrite stress, then substantially altered in all tested tissues, supporting a potential pro-inflammatory function. We also indicated that inflammation effect might firstly happen in blood after nitrite stress. Furthermore, the tissue expression differences of CCL4 among three natural populations revealed that CCL4 mRNA in Yuni Lake population was obviously higher than the other two populations, Liangzi Lake population and Poyang Lake population, which will provide valuable insights into breeding strategies for selecting population with better immune property of M. amblycephala.

Expression profiles of PtrLOS2 encoding an enolase required for cold-responsive gene transcription from trifoliate orange

Low expression of osmotically responsive genes 2 (LOS2) encodes an enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11) that converts 2-phospho-D-glycerate (PGA) to phosphoenolpyruvate (PEP) in the glycolytic pathway in Arabidopsis. Meanwhile, it is a transcriptional activator of cold-responsive gene, negatively controlling the expression of STZ/ZAT10, a zinc finger transcriptional repressor of cold-responsive gene from Arabidopsis. A novel LOS2 gene, designated PtrLOS2 (GenBank accession number GQ144341), was isolated from trifoliate orange [Poncirus trifoliata (L.) Raf.]. The PtrLOS2 cDNA is 1 662 bp in length with a 1 338 bp open reading frame (ORF), encoding a deduced 445 amino acid residue protein with a predicted molecular mass of 47.79 kDa and an isoelectric point of 5.54. The deduced protein of the PtrLOS2 gene shares high identity (over 86 %) with other plant species enolase, which suggests that the PtrLOS2 probably encodes an enolase. Sequence alignment showed that PtrLOS2 protein has a conserved DNA-binding and a repression domain. Moreover, a conserved start site of alternative translation for the c-myc promoter binding protein (MBP-1) was also found in PtrLOS2 protein. PtrLOS2 was constitutively expressed in leaves, stems and roots. PtrLOS2 expression in roots and stems was much higher than that in leaves under normal conditions, however, the expression of PtrLOS2 was up-regulated in leaves, but down-regulated in roots after cold treatments. The PtrLOS2 expression in stems was firstly up-regulated and then down-regulated after cold treatments. Meanwhile, after ABA treatment, the expression of PtrLOS2 was up-regulated in leaves but in stems and roots firstly down-regulated followed with up-regulation.