Comprehensive identification and expression profiling of immune-related lncRNAs and their target genes in the intestine of turbot (Scophthalmus maximus L.) in response to Vibrio anguillarum infection.

Long non-coding RNA (lncRNA) play vital regulatory roles in various biological processes. Intestine is one of the most sensitive organs to environmental and homeostatic disruptions for fish. However, systematic profiles of lncRNAs in the intestine of teleost in responses to pathogen infections is still limited. Turbot (Scophthalmus maximus L.), an important commercial fish species in China, has been suffering with Vibrio anguillarum infection, resulted in dramatic economic loss. Hereinto, the intestinal tissues of turbot were sampled at 0 h, 2 h, 12 h, and 48 h following V. anguillarum infection. The histopathological analysis revealed that the pathological trauma was mainly present in intestinal tunica mucosal epithelium. After high-throughput sequencing and bioinformatic analysis, a total of 9722 lncRNAs and 21,194 mRNAs were obtained, and the average length and exon number of lncRNAs were both less than those of mRNAs. Among which, a set of 158 lncRNAs and 226 mRNAs were differentially expressed (DE-lncRNAs and DEGs) in turbot intestine at three time points, related to many immune-related genes such as complement, interleukin, chemokine, lysosome, and macrophage, indicating their potential critical roles in immune responses. In addition, 2803 and 1803 GO terms were enriched for DEGs and co-expressed target genes of DE-lncRNAs, respectively. Moreover, 127 and 50 KEGG pathways including cell adhesion molecules (CAMs), phagosome, JAK-STAT signaling pathway, cytokine-cytokine receptor interaction, and intestinal immune network for IgA production, were enriched for DEGs and co-expressed target genes of DE-lncRNAs, respectively. Finally, qRT-PCR was conducted to confirm the reliability of sequencing data. The present study will set the foundation for the future exploration of lncRNA functions in teleost in response to bacterial infection.

A novel full-length transcriptome resource from multiple immune-related tissues in turbot (Scophthalmus maximus) using Pacbio SMART sequencing.

Turbot (Scophthalmus maximus) is an important cold-water economic fish. However, the production and development of turbot industry has been constantly hindered by the frequent occurrence of some diseases. Lacking full-length transcriptome for turbot limits immune gene discoveries and gene structures analysis. Therefore, we generated a full-length transcriptome using mixed immune-related tissues of turbot with PacBio Sequel platform. In this study, a total of 31.7 Gb high quality data were generated with the average subreads length of 2618 bp. According to the presence of 5' and 3' primers as well as poly (A) tails, FL (Full-length) and NFL (Non-full-length) isoforms were obtained. Meanwhile, we identified 32,003 non-redundant transcripts, 76.02% of which was novel isoforms of known genes. In addition, 12,176 alternative splicing (AS) events, 6614 polyadenylation (APA) events, 1905 transcription factors, and 2703 lncRNAs were identified. This work is a comprehensive report on the full-length transcriptome of immune-related tissues of turbot, and it also provides valuable molecular resources for future research on the adaptation mechanisms and functional genomics of turbot.

CC and CXC chemokines in turbot (Scophthalmus maximus L.): Identification, evolutionary analyses, and expression profiling after Aeromonas salmonicida infection.

Chemokines are a superfamily of structurally related cytokines, which exert essential roles in guiding cell migration in development, homeostasis, and immunity. CC and CXC chemokines are the two major subfamilies in teleost species. In this study, a total of seventeen CC and CXC chemokines, with inclusion of twelve CC and five CXC chemokines, were systematically identified from the turbot genome, making turbot the teleost harboring the least number of CC and CXC chemokines among all teleost species ever reported. Phylogeny, synteny, and genomic organization analyses were performed to annotate these genes, and multiple chemokine genes were identified in the turbot genome, due to the tandem duplications (CCL19 and CCL20), the whole genome duplications (CCL20, CCL25, and CXCL12), and the teleost-specific members (CCL34-36, CCL44, and CXCL18). In addition, chemokines were ubiquitously expressed in nine examined healthy tissues, with high expression levels observed in liver, gill, and spleen. Moreover, most chemokines were significantly differentially expressed in gill and spleen after Aeromonas salmonicida infection, and exhibited tissue-specific and time-dependent manner. Finally, protein-protein interaction network (PPI) analysis indicated that turbot chemokines interacted with a few immune-related genes such as interleukins, cathepsins, stats, and TLRs. These results should be valuable for comparative immunological studies and provide insights for further functional characterization of chemokines in teleost.

The CC and CXC chemokine receptors in turbot (Scophthalmus maximus L.) and their response to Aeromonas salmonicida infection.

Chemokines are crucial regulators of cell mobilization for development, homeostasis, and immunity. Chemokines signal through binding to chemokine receptors, a superfamily of seven-transmembrane domain G-coupled receptors. In the present study, eleven CC chemokine receptors (CCRs) and seven CXC chemokine receptors (CXCRs) were identified from turbot genome. Phylogenetic and syntenic analyses were performed to annotate these genes, indicating the closest relationship between the turbot chemokine receptors and their counterparts of Japanese flounders (Paralichthys olivaceus). Evolutionary analyses revealed that the tandem duplications of CCR8 and CXCR3, the whole genome duplications of CCR6, CCR9, CCR12, and CXCR4, and the teleost-specific CCR12 led to the expansion of turbot chemokine receptors. In addition, turbot chemokine receptors were ubiquitously expressed in nine examined healthy tissues, with high expression levels observed in spleen, gill, and head kidney. Moreover, most turbot chemokine receptors were significantly differentially expressed in spleen and gill after Aeromonas salmonicida infection, and exhibited general down-regulations at early time points and then gradually up-regulated. Finally, protein-protein interaction network (PPI) analyses indicated that chemokine receptors interacted with a few immune-related genes such as interleukins, Grk genes, CD genes, etc. These results should be valuable for comparative immunological studies and provide insights for further functional characterization of chemokine receptors in turbots.

Genome-wide characterization of Toll-like receptors in black rockfish Sebastes schlegelii: Evolution and response mechanisms following Edwardsiella tarda infection.

As one of the key components of pattern recognition receptors, Toll-like receptors (TLRs) play pivotal roles in the innate immune system. However, little information is available about the TLR genes in Sebastes schlegelii. In the present study, 17 TLR genes were identified and classified based on the whole genome database. Tandem duplication events in TLR1, TLR2, TLR5 and TLR13 played major role in the expansion of S. schlegelii TLR genes; both TLR2-3 and TLR2-4 had the same largest number of introns/exons, 11 exons and 10 introns. The syntenic analysis showed neighboring genes of TLR genes were most conserved in S. schlegelii and in L. crocea. Phylogenetic and evolutionary analysis showed that these TLR genes were divided into five subfamilies and exhibited different selection pressures. Meanwhile, the expression patterns of TLR genes in the intestine after E. tarda infection were investigated by qRT-PCR. Finally, protein and protein interaction (PPI) network analysis indicated that TLR genes interacted with IFN-inducible genes, inflammatory cytokines, and participated in MyD88-dependent pathway. In summary, this study provided valuable information for further functional characterization of TLR genes in S. schlegelii.

Genome-wide identification, expression signature and immune functional analysis of two cathepsin S (CTSS) genes in turbot (Scophthalmus maximus L.).

Cathepsins, a superfamily of hydrolytic enzymes produced and enclosed within lysosomes, play multiple roles at physiological and pathological states. Cathepsin S is a lysosomal cysteine endopeptidase of the papain family, and exerts critical roles in the regulation of MHC class II immune responses. In the present study, we captured two Cathepsin S genes in turbot (SmCTSS1 and SmCTSS2.1), characterized their expression patterns following V. anguillarum and S. iniae infections, and explored their binding ability and agglutination capability. Firstly, the SmCTSS1 contained a 990 bp ORF encoding 329 amino acids, while SmCTSS2.1 contained a 1,014 bp ORF encoding 337 amino acids. The phylogenetic analysis revealed that both genes showed the closest relationship to their counterparts of Japanese flounder (Paralichthys olivaceus). In addition, both genes were ubiquitously expressed in all examined healthy tissues, with the highest expression level observed in spleen and intestine, respectively, while the lowest expression level both observed in liver. Both SmCTSS1 and SmCTSS2.1 were significantly differentially expressed, and exhibited general down-regulations at most time points in skin and intestine after two bacterial infections. Finally, both rSmCTSS1 and rSmCTSS2.1 showed significant binding ability to three examined microbial ligands (LPS, PGN and LTA), and strong agglutination effect to different bacteria (E. tarda, S. agalactiae, S. aureus and V. anguillarum). Collectively, this study provided valuable data for understanding the roles of CTSS in the host defense against bacterial infections in turbot, and indicated the potential vital roles of CTSS in innate immune responses of teleost species.