Focal adhesion signaling pathway involved in skin immune response of tongue sole Cynoglossus semilaevis to Vibrio vulnificus infection.

Focal adhesion (FA) plays a key role in cell adhesion, migration and antibacterial immune, but it remained unclear in fish. In this study, half-smooth tongue sole Cynoglossus semilaevis were infected with Vibrio vulnificus, and then immune-related protein in the skin, especially for FA signaling pathway were screened and identified by iTRAQ analysis. Results showed that the differentially expressed proteins (DEPs) in skin immune response (eg., ITGA6, FN, COCH, AMBP, COL6A1, COL6A3, COL6A6, LAMB1, LAMC1, FLMNA) were firstly found in FA signaling pathway. Furthermore, the validation analysis of FA-related genes were basically consistent with the iTRAQ data at 36 hpi (r = 0.678, p < 0.01), and their spatio-temporal expressions were confirmed by qPCR analysis. The molecular characterization of vinculin of C. semilaevis was described. This study will provide a new perspective for understanding the molecular mechanism of FA signaling pathway in the skin immune response in marine fish.

Single-cell transcriptome, phagocytic activity and immunohistochemical analysis of crucian carp (Carassius auratus) in response to Rahnella aquatilis infection.

In teleost fish, kidney is an important immune and hematopoietic organ with multiple physiological functions. However, the immune cells and cellular markers of kidney require further elucidation in crucian carp (C. auratus). Here we report on the single-cell transcriptional landscape in posterior kidney, immunohistochemical and phagocytic features of C. auratus with R. aquatilis infection. The results showed that a total of 18 cell populations were identified for the main immune cells such as monocytes/macrophages (Mo/Mφ), dendritic cells (DCs), B cells, T cells, granulocytes and hematopoietic progenitor cells (HPCs). Pseudo-time trajectory analysis was reconstructed for the immune cells using Monocle2 to obtain additional insights into their developmental lineage relationships. In the detected tissues (liver, spleen, kidney, intestine, skin, and gills) of infected fish exhibited positive immunohistochemical staining with prepared for antibody to R. aquatilis. Apoptotic cells were fluorescently demonstrated by TUNEL assay, and bacterial phagocytic activity were observed for neutrophils and Mo/Mφ cells, respectively. Moreover, a similar up-ward/down-ward expression trend of the selected immune and inflammatory genes was found in the kidney against R. aquatilis infection, which were significantly involved in TLR/NLR, ECM adhesion, phago-lysosome, apoptosis, complement and coagulation pathways. To our knowledge, this is the first report on the detailed characterization of immune cells and host-R. aquatilis interaction, which will contribute to understanding on the biology of renal immune cells and repertoire of potential markers in cyprinid fish species.

Skin immune response to Aeromonas hydrophila infection in crucian carp Carassius auratus revealed by multi-omics analysis.

Fish skin is an essential protective barrier and functions as the first line of immune defense against pathogens. However, the molecular mechanism at the proteome-level remains unclear in the skin of fish. In this study, the comparative proteomics of skin immune responses of crucian carp Carassius auratus infected with Aeromonas hydrophila was investigated by isobaric tags for relative and absolute quantification (iTRAQ), two-dimensional gel electrophoresis combined with mass spectrometry (2-DE/MS) as well as high-throughput transcriptome (RNA-seq) techniques. A total of 241 and 178 differentially expressed proteins (DEPs) at 6 and 12 h post-infection (hpi) were respectively identified by iTRAQ, and key-DEPs were furtherly verified with 2-DE/MS analysis. GO and KEGG analysis showed that these DEPs were mostly related to metabolism, regulation of the cytoskeleton, stress and immune responses. Co-association results of proteome and transcriptome revealed the lysozyme (LYZ), complement C3, DnaJ (Hsp40) homolog subfamily C member 8 (DNAJC8) and allograft inflammatory factor 1-like (AIF1L) play important roles in skin immune responses of crucian carp. The significantly up-regulated expression of detected immune-related genes (c3, mapk3, f5, nlr, hsp90, itgb2, fnl, flnca, p47, mhc and pros1) were validated by qRT-PCR analysis. To our knowledge, this is first report on multi-omics analysis of the differential proteomics for the skin immune response of C. auratus against A.hydrophila infection, which contribute to the understanding the mechanisms of skin mucosal immunity in cyprinid fish.

First record of isolation and characterization of Vibrio sinaloensis from diseased orange-spotted grouper Epinephelus coioides.

A dominant bacterium, ZYL-12, isolated from the liver of a diseased orange-spotted grouper Epinephelus coioides, was identified as Vibrio sinaloensis, based on phenotypic and molecular analysis. The median lethal dosage of ZYL-12 was calculated as 1.6 × 105 CFU g-1 fish weight. The infection experiment indicated that ZYL-12 caused noticeable histological lesions to the liver, kidney and spleen of the fish. Growth characteristics showed that ZYL-12 possessed strong environmental adaptability. This note is the first report about the pathogenicity of V. sinaloensis isolated from diseased fish.

Characterization of Pathogenic Pseudomonas alcaligenes Isolated from Koi Carp in China.

Pseudomonas alcaligenes infection is rare in aquaculture. In this study, we provide the first report on the characterization of P. alcaligenes from koi (a variant of Common Carp Cyprinus carpio) in China. A gram-negative bacterium was isolated from the diseased koi and was named KCP-516. Morphological and biochemical tests as well as phylogenetic tree analyses derived from 16S ribosomal RNA, gyrase subunit A, and gyrase subunit B gene sequencing all strongly indicated that the isolate KCP-516 was P. alcaligenes. In liquid medium, the optimal growth conditions were 25°C, 2.5% NaCl, and pH 8. The pathogenicity of the isolate was demonstrated in koi, with 7.0 × 104 CFU/g fish weight identified as the dose lethal to 50% of test fish. The results will provide a scientific reference for the diagnosis and treatment of P. alcaligenes infection.

Molecular characterization of carp edema virus disease: An emerging threat to koi Cyprinus carpio in China.

Carp edema virus disease (CEVD) has resulted in great economic losses in koi (Cyprinus carpio koi) and common carp (Cyprinus carpio carpio) populations in the world. In this study, the diseased koi were diagnosed as CEV infection based on 5' untranslated region (5'UTR) and 4a protein genes by the conventional PCR, nested PCR and quantitative PCR (qPCR) analyses. Phylogenetic tree analysis showed that the TJ201708 strain was classified into the genogroup IIa. Furthermore, qPCR of 5'UTR gene revealed that the lowest detection limit was 4.0 fg/µL. The pathogenicity of CEV for koi was demonstrated in the infection experiments. Histopathological examination revealed the petechial hemorrhages of liver and spleen, vacuolization of lamina propria of intestine and swelling and necrosis of respiratory epithelial cells of gills. To our knowledge, this is the first report the qPCR of 5'UTR gene in the detection of carp edema virus.

Skin proteome profiling of tongue sole (Cynoglossus semilaevis) challenged with Vibrio vulnificus.

Vibrio vulnificus is a major pathogen of cultured Cynoglossus semilaevis and results in skin ulceration and haemorrhage, but the proteomic mechanism of skin immunity against V. vulnificus remains unclear. In this study, we investigated the histopathology and skin immune response in C. semilaevis with V. vulnificus infection at the protein levels, the differential proteomic profiling of its skin was examined by using iTRAQ and LC-MS/MS analyses. A total of 951 proteins were identified in skin, in which 134 and 102 DEPs were screened at 12 and 36 hpi, respectively. Selected eleven immune-related DEPs (pvß, Hsp71, MLC1, F2, α2ML, HCII, C3, C5, C8ß, C9 and CD59) were verified for their immune roles in the V. vulnificus infection via using qRT-PCR assay. KEGG enrichment analysis revealed that most of the identified immune proteins were significantly associated with complement and coagulation cascades, antigen processing and presentation, salivary secretion and phagosome pathways. To our knowledge, this study is the first to describe the proteome response of C. semilaevis skin against V. vulnificus infection. The outcome of this study contributed to provide a new perspective for understanding the molecular mechanism of local skin mucosal immunity, and facilitating the development of novel mucosal vaccination strategies in fish.

MicroRNA expression profile analysis of skin immune response in crucian carp (Carassius auratus) infected by Aeromonas hydrophila.

MicroRNAs (miRNAs) are non-coding RNA molecules that regulate gene expression in fish, but its regulatory mechanism of the skin mucosal immune response remains poorly understood. In order to investigate the immunological role of miRNAs, three sRNA libraries (mSC, mST1, mST2) from skin samples of crucian carp (Carassiusauratus) infected with Aeromonas hydrophila at three time points (0, 6 and 12 hpi) were constructed and examined using Illumina Hiseq 2000 platform. All of the identified miRNA, rRNA and tRNA were 69444 (13.39%), 29550 (5.70%) and 10704 (2.06%) in skin, respectively. At 6 and 12 hpi, 829 and 856 miRNAs were differentially expressed, respectively. Among these DEMs, 53 known and 10 novel miRNAs were all significantly differentially expressed during early infection (p < 0.01). GO and KEGG enrichment analyses revealed that 118111 target-genes were primarily involved in cellular process, metabolic process, biological regulation and stress response, such as antigen processing and presentation, complement and coagulation cascades, phagosome, MAPK, TLR, NF-κB and JAK-STAT signaling pathways. These results will help to elucidate the mechanism of miRNAs involved in the skin mucosal immune response of crucian carp against Aeromonas hydrophila infection.

Differentially expressed proteins in the intestine of Cynoglossus semilaevis Günther following a Shewanella algae challenge.

Fish intestine is an important constituent of the mucosal immune system. The gut and gut-associated lymphoid tissue construct a local immune environment. A Shewanella algae strain was previously reported to be a pathogen causing ascitic disease accompanied with intestinal inflammation in Cynoglossus semilaevis. This study aimed to investigate the intestine immune response in C. semilaevis to S. algae infection at the protein level. Two-dimensional electrophoresis coupled with mass spectrometry proteomics was utilized to compare protein expression in the intestines from normal and S. algae-infected C. semilaevis. A total of 70 differentially expressed proteins (DEPs), consisting of 16 upregulated and 54 downregulated proteins, were identified in the intestine tissue of C. Semilaevis. These protein expression changes were further validated using western blot analysis and quantitative real-time PCR. Gene ontology enrichment analysis showed that these 70 DEPs could be assigned across three categories: "cellular components", "molecular function", and "biological process". Forty-one DEPs (six up-regulated and 35 down-regulated proteins) related to metabolic processes were identified. In addition, 20 DEPs (eight up-regulated and 12 down-regulated proteins) related to stress and immune responses were identified. A protein-protein interaction network generated by the STRING (Search Tool for the Retrieval of Interacting Genes/protein) revealed that 30 DEPs interacted with one another to form an integrated network. Among them, 29 DEPs were related to stress, immune, and metabolism processes. In the network, some of the immune related proteins (C9, FGB, KNG1, apolipoprotein A-IV-like, and PDIA3) were up-regulated and most DEPs involved in metabolism processes were down-regulated. These results indicate that the immune defense response of the intestine was activated and the intestinal function associated with metabolism processes was disturbed. This study provides valuable information for further research into the functions of these DEPs in fish.

Isolation and genomic characterization of a pathogenic Providencia rettgeri strain G0519 in turtle Trachemys scripta.

Providencia rettgeri infection has occurred occasionally in aquaculture, but is rare in turtles. Here, a pathogenic P. rettgeri strain G0519 was isolated from a diseased slider turtle (Trachemys scripta) in China, and qPCR assay was established for the RTX toxin (rtxD) gene. Histopathological examination showed that many inflammatory cells were infiltrated into heart, liver and intestine, as well as the necrosis of liver, kidney and spleen. The genome consisted of one circular chromosome (4.493 Mb) and one plasmid (18.8 kb), and predicted to contain 4170 and 19 protein-coding genes, respectively. Multiple pathogenic and virulence factors (e.g., fimbria, adhesion, invasion, toxin, hemolysin, chemotaxis, secretion system), multidrug-resistant genes (e.g., ampC, per-1, oxa-1, sul1, tetR) and a novel genomic resistance island PRI519 were identified. Comparative genome analysis revealed the closest relationship was with P. rettgeri, and with P. heimbachae closer than with other Providencia spp. To our knowledge, this was first report on genomic characterization of multidrug-resistant pathogenic P. rettgeri in cultured turtles.

First report on the characterization of pathogenic Rahnella aquatilis KCL-5 from crucian carp: Revealed by genomic and proteomic analyses.

Rahnella aquatilis is an important pathogen of several aquatic organisms and is found widely distributed in the freshwater, soil, fish and human clinical samples. Our previously published study reported a novel pathogenic R. aquatilis strain KCL-5 to crucian carp (Carassius auratus). To further investigate the characteristics and pathogenesis caused by R. aquatilis, we here report on the pathological changes, bacterial genomic and proteomic analyses of strain KCL-5. Significantly pathological changes in liver, intestine, spleen and gills were observed in infected fish. The genome consists of one circular chromosome 5,062,299 bp with 52.02% GC content and two plasmids (506,827 bp, 52.16%; 173,433 bp, 50.00%) and predicted 5,653 genes, 77 tRNAs and 22 rRNAs. Some virulence factors were characterized, including outer membrane protein, haemolysin, RTX toxin, chemotaxis and T3SS secretion system. Antimicrobial resistance genes such as EmrAB-TolC, MexABC-OpmB and RosAB efflux pump were found in strain KCL-5. KEGG analysis showed that mainly functional modules were ABC transporters, biosynthesis of amino acids, two-component system, quorum sensing, flagellum assembly and chemotaxis, in which most of them were identified by using 2-DE/MS analyses. To our knowledge, this was first report on the molecular characteristics of R. aquatilis by multi-omics approaches, which will provide insights into the pathogenic mechanism of R. aquatilis infection in fish.

Skin transcriptome, tissue distribution of mucin genes and discovery of simple sequence repeats in crucian carp (Carassius auratus).

Crucian carp (Carassius auratus) is one of the major freshwater species and important food fish in China. Fish skin acts as the first line of defence against pathogens, yet its molecular and immune mechanism remains unclear. In this study, a de novo transcriptome assembly of C. auratus skin was performed with the Illumina Hiseq 2000 platform. A total of 49,154,776 clean reads were assembled, among which 60,824 (46.86%), 37,103 (28.59%), 43,269 (33.33%) unigenes were annotated against National Center for Biotechnology Information, Gene Onotology and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. KEGG Orthology categories were significantly involved in immune system (20.50%), signal transduction (18.04%) and mucosal mucin genes (e.g., muc2, muc5AC, muc5B, muc17, muc18). The high expression of muc18 gene was observed in brain; that of muc2 in intestine; and that of muc5AC in skin, liver, spleen, intestine and muscle. Moreover, the potential 28,928 simple sequence repeats with the three most abundant dinucleotide repeat motifs (AC/GT, AG/CT, AT/AT) were detected in C. auratus. To authors' knowledge, this is the first report to describe the transcriptome analysis of C. auratus skin, and the outcome of this study contributed to the understanding of mucosal immune response of the skin and molecular markers in cyprinid species.

Transcriptome analysis in the skin of Carassius auratus challenged with Aeromonas hydrophila.

Skin plays an important role in the innate immune responses of fish, particularly towards bacterial infection. To understand the molecular mechanism of mucosal immunity of fish during bacterial challenge, a de novo transcriptome assembly of crucian carp Carassius auratus skin upon Aeromonas hydrophila infection was performed, the latter with Illumina Hiseq 2000 platform. A total of 118111 unigenes were generated and of these, 9693 and 8580 genes were differentially expressed at 6 and 12 h post-infection, respectively. The validity of the transcriptome results of eleven representative genes was verified by quantitative real-time PCR (qRT-PCR) analysis. A comparison with the transcriptome profiling of zebrafish skin to A. hydrophila with regards to the mucosal immune responses revealed similarities in the complement system, chemokines, heat shock proteins and the acute-phase response. GO and KEGG enrichment pathway analyses displayed the significant immune responses included TLR, MAPK, JAK-STAT, phagosome and three infection-related pathways (ie., Salmonella, Vibrio cholerae and pathogenic Escherichia coli) in skin. To our knowledge, this study is the first to describe the transcriptome analysis of C. auratus skin during A. hydrophila infection. The outcome of this study contributed to the understanding of the mucosal defense mechanisms in cyprinid species.

Transcriptome profiling of immune-responsive genes in the intestine of Cynoglossus semilaevis Günther challenged with Shewanella algae.

To better understand gene expression in the intestine after Shewanella algae infection and provide insights into its immune roles in the tongue sole, Cynoglossus semilaevis, sequencing-based high-throughput RNA analysis (RNA-Seq) for the intestines between the control group and 12 h post-injection group was performed. After assembly, there was an average of 23,957,159 raw sequencing reads, and 23,943,491 clean reads were obtained after filtering out low-quality reads. Then, 383 differentially expressed genes (DEGs) in the intestines in response to S. algae infection were identified. Subsequently, gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs were conducted to further explore their functions. Among all of the pathways involved, sixteen pathways were related to the immune system, among which the complement and coagulation cascades pathway was the most prominent for immunity-related DEGs, followed by the leukocyte transendothelial migration pathway. Furthermore, the expression levels of twelve selected DEGs in the immune-related pathways were identified by quantitative real-time polymerase chain reaction, substantiating the reliability and reproducibility of the RNA-Seq results. In summary, this study represents an important genomic resource for understanding the potential immune role of the tongue sole intestine from the perspective of gene expression.

First report of Rahnella aquatilis infection in crucian carp Carassius auratus in China.

Rahnella aquatilis infection is rare in aquaculture. Here, a Gram-negative rod-shaped bacterium was isolated from diseased crucian carp Carassius auratus in Xuzhou City, Jiangsu Province, eastern China. The isolate was tentatively named strain KCL-5, and subsequently identified as R. aquatilis by biochemical properties and molecular techniques. The results showed that the isolate KCL-5 was most closely related to the type strain ATCC33071 (= DSM4594) of R. aquatilis, which shared 99.67, 96.26 and 99.58% nucleotide sequence identities for 16S rDNA, gyrB and toxin yhaV genes, respectively. Experimental challenges were conducted which demonstrated pathogenicity of the isolate in crucian carp. Antimicrobial susceptibility testing showed that the isolated strain was susceptible to piperacillin, gentamicin, kanamycin, nalidixic acid, norfloxacin, ofloxacin, azithromycin and erythromycin. To our knowledge, this is the first report on R. aquatilis infection in crucian carp, and the first evidence of pathogenicity in fish.

Tissue distribution of olive flounder VDAC2 and its expression in fish cell lines.

Voltage-dependent anion channel (VDAC) is located in the mitochondrial outer membrane, which plays a crucial role in regulating cell life and death. In this study, the tissue distribution of olive flounder Paralichthys olivaceus VDAC2 (PoVDAC2) was detected by quantitative real-time PCR and Western blot analysis. The qRT-PCR results showed that the expression level of PoVDAC2 was abundant in heart, muscle and gill tissues. Western blot analysis revealed a protein of 32 kDa detected in all six tissues. Furthermore, a recombinant eukaryotic expression plasmid pEGFP-N3-PoVDAC2 was successfully constructed and transiently expressed the fusion protein in fish cell lines. Subcellular localization indicated that PoVDAC2-GFP was distributed in a punctate mitochondria-like pattern throughout the cytoplasm in flounder embryonic cells (FEC). The distribution of native VDAC2 in untransfected fish cells was also investigated by immunofluorescence microscopy. The punctate VDAC2 fluorescence signals of both FEC and EPC cells were identically observed in the cytoplasm but not in the nucleus. These results laid a foundation for investigating the functional relevance of VDAC response to pathogens in flounder.

iTRAQ analysis of gill proteins from the zebrafish (Danio rerio) infected with Aeromonas hydrophila.

The gills are large mucosal surfaces and very important portals for pathogen entry in fish. The aim of this study was to determine the gill immune response at the protein levels, the differential proteomes of the zebrafish gill response to Aeromonas hydrophila infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1338 proteins were identified and classified into the categories primarily related to cellular process (15.36%), metabolic process (11.95%) and biological regulation (8.29%). Of these, 82 differentially expressed proteins were reliably quantified by iTRAQ analysis, 57 proteins were upregulated and 25 proteins were downregulated upon bacterial infection. Gene ontology (GO) enrichment analysis showed that approximately 33 (8.8%) of the differential proteins in gills were involved in the stress and immune responses. Several upregulated proteins were observed such as complement component 5, serpin peptidase inhibitor clade A member 7, annexin A3a, histone H4, glyceraldehyde 3-phosphate dehydrogenase, creatine kinase, and peroxiredoxin. These protein expression changes were further validated at the transcript level using microarray analysis. Moreover, complement and coagulation cascades, pathogenic Escherichia coli infection and phagosome were the significant pathways identified by KEGG enrichment analysis. This is first report on proteome of fish gills against A. hydrophila infection, which contribute to understanding the defense mechanisms of the gills in fish.

Multi-omics analysis of miRNA-mediated intestinal microflora changes in crucian carp Carassius auratus infected with Rahnella aquatilis.

Infection by an emerging bacterial pathogen Rahnella aquatilis caused enteritis and septicemia in fish. However, the molecular pathogenesis of enteritis induced by R. aquatilis infection and its interacting mechanism of the intestinal microflora associated with microRNA (miRNA) immune regulation in crucian carp Carassius auratus are still unclear. In this study, C. auratus intraperitoneally injected with R. aquatilis KCL-5 was used as an experimental animal model, and the intestinal pathological changes, microflora, and differentially expressed miRNAs (DEMs) were investigated by multi-omics analysis. The significant changes in histopathological features, apoptotic cells, and enzyme activities (e.g., lysozyme (LYS), alkaline phosphatase (AKP), alanine aminotransferase (ALT), aspartate transaminase (AST), and glutathione peroxidase (GSH-Px)) in the intestine were examined after infection. Diversity and composition analysis of the intestinal microflora clearly demonstrated four dominant bacteria: Proteobacteria, Fusobacteria, Bacteroidetes, and Firmicutes. A total of 87 DEMs were significantly screened, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the potential target genes were mainly involved in the regulation of lipid, glutathione, cytosine, and purine metabolism, which participated in the local immune response through the intestinal immune network for IgA production, lysosome, and Toll-like receptor (TLR) pathways. Moreover, the expression levels of 11 target genes (e.g., TLR3, MyD88, NF-κB, TGF-ß, TNF-α, MHC II, IL-22, LysC, F2, F5, and C3) related to inflammation and immunity were verified by qRT-PCR detection. The correlation analysis indicated that the abundance of intestinal Firmicutes and Proteobacteria was significantly associated with the high local expression of miR-203/NF-κB, miR-129/TNF-α, and miR-205/TGF-ß. These findings will help to elucidate the molecular regulation mechanism of the intestinal microflora, inflammation, and immune response-mediated miRNA-target gene axis in cyprinid fish.

Comprehensive transcriptomics and proteomics analysis of Carassius auratus gills in response to Aeromonas hydrophila.

As one of the mucosal barriers, fish gills represent the first line of defense against pathogen infection. However, the exact mechanism of gill mucosal immune response to bacterial infection still needs further investigation in fish. Here, to investigate pathological changes and molecular mechanisms of the mucosal immune response in the gills of crucian carp (Carassius auratus) challenged by Aeromonas hydrophila, the transcriptomics and proteomics were performed by using multi-omics analyses of RNA-seq coupled with iTRAQ techniques. The results demonstrated gill immune response were mostly related to the activation of complement and coagulation cascades, antigen processing and presentation, phagosome, NOD-like receptor (NLR) and nuclear factor κB (NFκB) signaling pathway. Selected 21 immune-related DEGs (ie., Clam, nfyal, snrpf, acin1b, psme, sf3b5, rbm8a, rbm25, prpf18, g3bp2, snrpd3l, tecrem-2, cfl-A, C7, lysC, ddx5, hsp90, α-2M, C9, C3 and slc4a1a) were verified for their immune roles in the A. hydrophila infection via using qRT-PCR assay. Meanwhile, some complement (C3, C7, C9, CFD, DF and FH) and antigen presenting (HSP90, MHC Ⅱ, CALR, CANX and PSME) proteins were significantly participated in the process of defense against infections in gill tissues, and protein-protein interaction (PPI) network displayed the immune signaling pathways and interactions among these DEPs. The correlation analysis indicated that the iTRAQ and qRT-PCR results was significantly correlated (Pearson's correlation coefficient = 0.70, p < 0.01). To our knowledge, the transcriptomics and proteomics of gills firstly identified by multi-omics analyses contribute to understanding on the molecular mechanisms of local mucosal immunity in cyprinid species.

Multiomics analysis revealed miRNAs as potential regulators of the immune response in Carassius auratus gills to Aeromonas hydrophila infection.

The gill of fish is an important immune organ for pathogen defense, but its microRNA (miRNA) expression and regulatory mechanism remain unclear. In this study, we report on the histopathological and immunohistochemical features of the gills of the crucian carp Carassius auratus challenged with Aeromonas hydrophila. Small RNA libraries of the gills were constructed and sequenced on the Illumina HiSeq 2000 platform. A total of 1,165 differentially expressed miRNAs (DEMs) were identified in gills, of which 539 known and 7 unknown DEMs were significantly screened (p < 0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the potential target genes/proteins were primarily involved in 33 immune-related pathways, in which the inflammatory responses were focused on the Toll-like receptor (TLR), mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB) signaling pathways. Moreover, the expression levels of 14 key miRNAs (e.g., miR-10, miR-17, miR-26a, miR-144, miR-145, and miR-146a) and their target genes (e.g., TNFα, TLR4, NF-κB, TAB1, PI3K, and IRAK1) were verified. In addition, the protein levels based on isobaric tags for relative and absolute quantification (iTRAQ) were significantly associated with the results of the quantitative real-time PCR (qRT-PCR) analysis (p < 0.01). miR-17/pre-miR-17 were identified in the regulation expression of the NF-κB target gene, and the phylogenetic tree analysis showed that the pre-miR-17 of C. auratus with the closest similarity to the zebrafish Danio rerio is highly conserved in teleosts. This is the first report of the multi-omics analysis of the miRNAs and proteins in the gills of C. auratus infected with A. hydrophila, thus enriching knowledge on the regulation mechanism of the local immune response in Cyprinidae fish.