Jianshe Zhang

The draft genome of the large yellow croaker reveals well-developed innate immunity

The large yellow croaker, Larimichthys crocea, is one of the most economically important marine fish species endemic to China. Its wild stocks have severely suffered from overfishing, and the aquacultured species are vulnerable to various marine pathogens. Here we report the creation of a draft genome of a wild large yellow croaker using a whole-genome sequencing strategy. We estimate the genome size to be 728u2009Mb with 19,362 protein-coding genes. Phylogenetic analysis shows that the stickleback is most closely related to the large yellow croaker. Rapidly evolving genes under positive selection are significantly enriched in pathways related to innate immunity. We also confirm the existence of several genes and identify the expansion of gene families that are important for innate immunity. Our results may reflect a well-developed innate immune system in the large yellow croaker, which could aid in the development of wild resource preservation and mariculture strategies.

Transcriptome analysis of the Larimichthys crocea liver in response to Cryptocaryon irritans

The large yellow croaker (Larimichthys crocea) is an economically important marine fish cultured in China and East Asian countries and is facing a serious threat from Cryptocaryon irritans, which is a protozoan ectoparasite that infects most reared marine fish species. To understand the molecular immune mechanisms underlying the response to C.xa0irritans, we first performed a comparative gene transcription analysis using livers from C.xa0irritans-immunized L.xa0croceas and from a control group through RNA-Seq technology. After the removal of low-quality sequences and assembly, 51360xa0contigs were obtained, with an average length of 1066.93xa0bp. Further, a blast analysis indicates that 30747xa0contigs can be annotated based on homology with matches in the NT, NR, gene, and string databases. A gene ontology analysis was used to classify 21598 genes according to three major functional categories: molecular function, cellular component, and biological process. Moreover, 14470 genes were found in 303 KEGG pathways. We used RSEM and EdgeR to determine that 3841 genes were significantly differentially expressed (FDRxa0<xa00.001), including 2129 up-regulated genes and 1712 down-regulated genes. A significant enrichment analysis of these differentially expressed genes and isogenes revealed major immune-related pathways, including the toll-like receptor, complement and coagulation cascades, and chemokine signaling pathways. In addition, 28748 potential simple sequence repeats (SSRs) were detected from 12776 transcripts, and 62992 candidate single nucleotide polymorphisms (SNPs) were identified in the L.xa0croceas liver transcriptome. This study characterized a gene expression pattern for normal and C.xa0irritans-immunized L.xa0croceas for the first time and not only sheds new light on the molecular mechanisms underlying the host-C.xa0irritans interaction but also facilitates future studies on L.xa0croceas gene expression and functional genomics.

Molecular characterization and expression analyses of three RIG-I-like receptor signaling pathway genes (MDA5, LGP2 and MAVS) in Larimichthys crocea

In this study, we sequenced and characterized melanoma differentiation-associated antigen 5 (LcMDA5), laboratory of genetics and physiology 2 (LcLGP2) and mitochondrial antiviral signaling protein (LcMAVS) from large yellow croaker (Larimichthys crocea). The LcMDA5 encodes 969 amino acids and contains two caspase-associated and recruitment domains (CARDs), a DExDc (DExD/H box-containing domain), a HELICc (helicase superfamily C-terminal domain) and a C-terminal regulatory domain (RD). The LcLGP2 encodes 679 amino acids and contains a DExDc, a HELICc and a RD. The LcMAVS encodes 512 amino acids and contains a CARD, a proline-rich domain, a transmembrane helix domain and a putative TRAF2-binding motif ((269)PVQDT(273)). Phylogenetic analyses showed that all the three genes of large yellow croaker are clustered together with their counterparts from other teleost fishes. The Real-time PCR analyses showed that all the three genes were found to be constitutively expressed in all examined tissues in large yellow croaker, but all with relatively low expression levels. Expression analyses showed that the three genes were all rapidly and significantly upregulated inxa0vivo after poly (I:C) challenge in peripheral blood, liver, spleen and head kidney tissues. The results indicate that the LcMDA5, LcLGP2 and LcMAVS might play important roles in antiviral immune responses.

Transcriptome analysis demonstrate widespread differential expression of long noncoding RNAs involve in Larimichthys crocea immune response.

Long noncoding RNAs (lncRNAs) are a class of transcripts that longer than 200xa0bp and do not encode proteins. Recent genome-wide studies of vertebrate transcriptomes have annotated lncRNAs that are expressed in various tissues and development stages. The draft genome and several transcriptome sequencing data sets have been collected for the study of protein-coding genes in large yellow croaker (Larimichthys crocea), but little is known about the expression and functional roles of lncRNAs in this species. In order to obtain a catalog of lncRNAs for large yellow croaker, several RNA-seq datasets were integrated from various tissues including egg, muscle, liver, and spleen. A total of 48,953 high-confidence transcripts were reconstructed in 38,017 loci, recovering the most of expressed reference transcripts while thousands of novel expressed loci have been identified. The tissue expression profile revealed that most lncRNAs were specifically enriched in different tissues. A stringent set of 210 lncRNAs were identified as being specifically expressed in spleen and potentially involved in immune response. Our study first systematically identify lncRNAs in large yellow croaker, benefiting the future genomic study of this species.

Molecular characterization and expression analysis of AMPK α subunit isoform genes from Scophthalmus maximus responding to salinity stress.

AMP-activated protein kinase (AMPK) is a highly conserved and multi-functional protein kinase that plays important roles in both intracellular energy balance and cellular stress response. In the present study, molecular characterization, tissue distribution and gene expression levels of the AMPK α1 and α2 genes from turbot (Scophthalmus maximus) under salinity stress are described. The complete coding regions of the AMPK α1 and α2 genes were isolated from turbot through degenerate primers in combination with RACE using muscle cDNA. The complete coding regions of AMPK α1 (1722xa0bp) and α2 (1674xa0bp) encoded 573 and 557 amino acids peptides, respectively. Multiple alignments, structural analysis and phylogenetic tree construction indicated that S. maximus AMPK α1 and α2 shared a high amino acid identity with other species, especially fish. AMPK α1 and α2 genes could be detected in all tested tissues, indicating that they are constitutively expressed. Salinity challenges significantly altered the gene expression levels of AMPK α1 and α2 mRNA in a salinity- and time-dependent manners in S. maximus gill tissues, suggesting that AMPK α1 and α2 played important roles in mediating the salinity stress in S. maximus. The expression levels of AMPK α1 and α2 mRNA were a positive correlation with gill Na+, K+-ATPase activities. These findings will aid our understanding of the molecular mechanism of juvenile turbot in response to environmental salinity changes.

Cell loss by apoptosis is involved in the intestinal degeneration that occurs during aestivation in the sea cucumber Apostichopus japonicus

The sea cucumber Apostichopus japonicus (Selenka) commonly undergoes aestivation in response to high water temperatures. This process is accompanied by tissue regression and body mass reduction. Previous studies have suggested that apoptosis may play a role in the tissue remodeling that occurs during aestivation, although this has not definitively been shown. To investigate this hypothesis, the present study used A. japonicus as a model organism to examine cell loss through apoptosis in intestinal degeneration during aestivation. Apostichopus japonicus individuals were collected from Yellow Sea (N 36° 05 44.87″, E 120° 31 58.51″), China in April 2016 and split into two groups. Aestivation was induced in the experimental group by incubation at 25°C. This resulted in a significant decrease in body mass and increased evidence of intestinal degeneration in hematoxylin and eosin, Hoechst 33342, and in situ TUNEL analyses of tissue sections. Along with further Hoechst 33342 analysis using intestinal cell smears, these results showed that A. japonicus intestinal cell apoptosis occurred soon after the initial temperature increase, with most apoptotic events completing within 20days. Transcriptional quantification of the Ajcaspase-8 (CASP8) and Ajcaspase-3 (CASP3) apoptotic genes demonstrated that their expression was significantly elevated at the beginning of the experiment but was decreased at later stages of aestivation. The results of this study strongly suggest that apoptosis is involved in the intestinal regression of A. japonicus during aestivation, and play important role in understanding fundamental cellular events in tissue regression under environmental stress.

Pre-acclimation to low copper mitigated immunotoxic effects in spleen and head-kidney of large yellow croaker (Pseudosciaena crocea) when exposed subsequently to high copper

The hypothesis tested in this study was that Cu pre-acclimation would mitigate high Cu induced immunotoxic effects in large yellow croaker Pseudosciaena crocea. To the end, fish were pre-acclimation to 0 and 84μg CuL-1 for 48h and then exposed to 0 and 420μg CuL-1 for another 48h. Survival rate, Cu content, ROS, NO, activities and mRNA levels of inflammatory genes (iNOS and COX-2), and gene expressions of transcription factor NF-κB and its inhibitor IκBα were determined in spleen and head-kidney of large yellow croaker. Cu pre-acclimation significantly reduced mortality of fish exposed to 420μg CuL-1. Cu pre-acclimation triggered the up-regulation of both enzyme activities and express levels of iNOS and COX-2 in spleen under 420μg CuL-1 exposure, resulting in remarkable reduction of Cu content and ROS in this tissue. Contrast to spleen, iNOS activity remained unchanged but the mRNA level of iNOS increased, and the mRNA level of COX-2 remained constant though COX-2 activity enhanced in head-kidney, suggesting iNOS and COX-2 may be modulated by Cu at a post-transcriptional level. In this process, NF-κB/IκBα signaling molecules may play a vital role in the transcriptional activation of inflammatory genes in both spleen and head-kidney. In conclusion, low Cu pre-acclimation alleviated high Cu induced immunotoxicity in spleen and head-kidney of large yellow croaker by enhancing the activities and mRNA levels of inflammatory genes.

Abrupt salinity stress induces oxidative stress via the Nrf2-Keap1 signaling pathway in large yellow croaker Pseudosciaena crocea

The aim of the present study was to evaluate the effects of abrupt salinity stress (12, 26 (control), and 40) on lipid peroxidation, activities and mRNA levels of antioxidant enzymes (Cu/Zn-SOD, CAT, GPx, and GR), and gene expression of the Nrf2-Keap1 signaling molecules at different times (6, 12, 24, and 48xa0h) in the liver of large yellow croaker Pseudosciaena crocea. The results showed that lipid peroxidation was sharply reduced at 6xa0h and increased at 12xa0h before returning to control levels in the hypo-salinity group. Similarly, lipid peroxidation was significantly decreased at 6xa0h followed by a sharp increase towards the end of the exposure in the hyper-salinity group. Negative relationships between lipid peroxidation and antioxidant enzyme activities and positive relationships between activities and gene expression of antioxidant enzymes were observed, suggesting that the changes at molecular levels and enzyme activity levels may provide protective roles against damage from salinity stress. Obtained results also showed a coordinated transcriptional regulation of antioxidant genes, suggesting that Nrf2 is required for regulating these genes. Furthermore, there was a positive relationship between the mRNA levels of Nrf2 and Keap1, indicating that Keap1 plays an important role in switching off the Nrf2 response. In conclusion, this is the first study to elucidate effects of salinity stress on antioxidant responses in large yellow croaker through the Keap1–Nrf2 pathway.

Genomic structure and promoter functional analysis of GnRH3 gene in large yellow croaker (Larimichthys crocea)

Gonadotropin-releasing hormone III (GnRH3) is considered to be a key neurohormone in fish reproduction control. In the present study, the cDNA and genomic sequences of GnRH3 were cloned and characterized from large yellow croaker Larimichthys crocea. The cDNA encoded a protein of 99 amino acids with four functional motifs. The full-length genome sequence was composed of 3797 nucleotides, including four exons and three introns. Higher identities of amino acid sequences and conserved exon-intron organizations were found between LcGnRH3 and other GnRH3 genes. In addition, some special features of the sequences were detected in partial species. For example, two specific residues (V and A) were found in the family Sciaenidae, and the unique 75-72 bp type of the open reading frame 2 and 3 existed in the family Cyprinidae. Analysis of the 2576 bp promoter fragment of LcGnRH3 showed a number of transcription factor binding sites, such as AP1, CREB, GATA-1, HSF, FOXA2, and FOXL1. Promoter functional analysis using an EGFP reporter fusion in zebrafish larvae presented positive signals in the brain, including the olfactory region, the terminal nerve ganglion, the telencephalon, and the hypothalamus. The expression pattern was generally consistent with the endogenous GnRH3 GFP-expressing transgenic zebrafish lines, but the details were different. These results indicate that the structure and function of LcGnRH3 are generally similar to the other teleost GnRH3 genes, but there exist some distinctions among them.

Cloning and functional characterization of thioredoxin genes from large yellow croaker Larimichthys crocea

ABSTRACT Thioredoxin(Trx)with a redox‐active disulfide/dithiol in the active site, is an ubiquitous disulfide reductase majorly responsible for maintaining the balance of reactive oxygen species. In this study, the complete thioredoxin‐like protein 1 (designated as LcTrx) was cloned from large yellow croaker Larimichthys crocea through rapid amplification of cDNA ends. The full‐length cDNA of LcTrx was 1295 bp in length containing a 131 bp 5′ untranslated region (UTR), a 3′UTR of 294bp with a poly (A) tail, and an 870 bp open reading frame (ORF) encoding a polypeptide of 289 amino acids. Protein sequence analysis revealed that LcTrx contains the evolutionarily conserved redox motif CRPC (Cys‐Arg‐Pro‐Cys‐). Multiple alignments revealed that LcTrx is highly identical to Trx from other organisms, especially in the CRPC motifs. The recombinant LcTrx showed obvious insulin reduction activity in vitro. The LcTrx transcripts were constitutively expressed in all examined tissues with the highest levels found in the muscles and the lowest in the head kidney. Results of Vibrio parahaemolyticus infection experiment showed that the expression levels of LcTrx were tissue and time dependent. In the liver and kidney, LcTrx was down‐regulated both at 12 h and 48 h post‐infection. In contrast, LcTrx showed induced expression in the spleen and head kidney at same post‐infection time points. The different responses to pathogen stimulation indicated the diversified physiological function of LcTrx in the four examined tissues. HighlightsClone the full‐length cDNA of Trx from large yellow croaker.Investigate the expression profile of thioredoxin‐like protein 1 (LcTrx) transcript after a bacterial challenge.Recombinant LcTrx protein in vitro and test its antioxidant activity.