Slow stripe rusting in Chinese wheat Jimai 44 conferred by Yr29 in combination with a major QTL on chromosome arm 6AL.

KEY MESSAGE: YrJ44, a more effective slow rusting gene than Yr29, was localized to a 3.5-cM interval between AQP markers AX-109373479 and AX-109563479 on chromosome 6AL. "Slow rusting" (SR) is a type of adult plant resistance (APR) that can provide non-specific durable resistance to stripe rust in wheat. Chinese elite wheat cultivar Jimai 44 (JM44) has maintained SR to stripe rust in China since its release despite exposure to a changing and variable pathogen population. An F2:6 population comprising 295 recombinant inbred lines (RILs) derived from a cross between JM44 and susceptible cultivar Jimai 229 (JM229) was used in genetic analysis of the SR. The RILs and parental lines were evaluated for stripe rust response in five field environments and genotyped using the Affymetrix Wheat55K SNP array and 13 allele-specific quantitative PCR-based (AQP) markers. Two stable QTL on chromosome arms 1BL and 6AL were identified by inclusive composite interval mapping. The 1BL QTL was probably the pleiotropic gene Lr46/Yr29/Sr58. QYr.nwafu-6AL (hereafter named YrJ44), mapped in a 3.5-cM interval between AQP markers AX-109373479 and AX-109563479, was more effective than Yr29 in reducing disease severity and relative area under the disease progress curve (rAUDPC). RILs harboring both YrJ44 and Yr29 displayed levels of SR equal to the resistant parent JM44. The AQP markers linked with YrJ44 were polymorphic and significantly correlated with stripe rust resistance in a panel of 1,019 wheat cultivars and breeding lines. These results suggested that adequate SR resistance can be obtained by combining YrJ44 and Yr29 and the AQP markers can be used in breeding for durable stripe rust resistance.

CRISPR/Cas9-mediated knock-in of masu salmon (Oncorhyncus masou) elongase gene in the melanocortin-4 (mc4r) coding region of channel catfish (Ictalurus punctatus) genome.

Channel catfish, Ictalurus punctatus, have limited ability to synthesize Ω-3 fatty acids. The ccßA-msElovl2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, ß-actin promoter was inserted into the channel catfish melanocortin-4 receptor (mc4r) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish containing both the ccßA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6 months post-hatch (p = 0.005). Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and mc4r knockout mutants (MC4R) were 121.6% and 94.1% higher than in controls, respectively (p = 0.045; p = 0.025). Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA + DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 transgenic channel catfish than controls (p = 0.368; p = 0.025). To our knowledge this is the first example of genome engineering to simultaneously target transgenesis and knock-out a gene in a commercially important aquaculture species for multiple improved performance traits. With a high transgene integration rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 transgenic channel catfish appears beneficial for application on commercial farms.

Characterization and the potential immune role of class A scavenger receptor member 4 (SCARA4) in bacterial infection in turbot (Scophthalmus maximus L.).

The class A scavenger receptors play important roles in innate immunity and are distributed on plasma membrane of macrophages and other cell types. Notably, the class A scavenger receptor 4 (SCARA4) contains a typical C-type (calcium-dependent) lectin domain, which belongs to the collectin family of pattern recognition receptors and is involved in the immune response against infection. Here, one turbot SCARA4 gene was identified with a 2,292 bp open reading frame (ORF) encoding 763 amino acid residues. Multiple sequence analysis and phylogenetic analysis confirmed that SmSCARA4 gene was more close to that of P. olivaceus. Gene structure and syntenic analysis showed conserved exon/intron organization pattern and syntenic pattern across selected vertebrate species. Tissue distribution analysis showed SmSCARA4 was expressed in all the tested healthy tissues with the relative high expression levels in skin, gill and spleen. Following both E. tarda and V. anguillarum challenge in vivo, SmSCARA4 was significantly repressed in gill and intestine. Remarkably, SmSCARA4 showed the strongest binding ability to LPS and strongest upregulation in turbot head kidney macrophages in response to LPS. Knockdown and overexpression of SmSCARA4 revealed its interactions with the two pro-inflammatory cytokines, TNF-α and IL-1ß. Finally, repression of SmSCARA4 via combined treatment of LPS and overexpression of SmSCARA4 construct in turbot head kidney macrophages further indicated an inhibitory role of SmSCARA4 in LPS-stimulated inflammation. Taken together, turbot SmSCARA4 plays an important role in turbot immunity, especially in the mucosa-related systems; SmSCARA4 possesses strong binding specificity to LPS, and exerts protective roles in response to LPS infection by reducing the release of pro-inflammatory cytokines. The mechanisms of inhibitory role of SmSCARA4 in LPS-elicited inflammation await further investigation.

Identification and characterization of a C-type lectin in turbot (Scophthalmus maximus) which functioning as a pattern recognition receptor that binds and agglutinates various bacteria.

C-type lectins (CTLs) are important pathogen pattern recognition receptors that recognize carbohydrate structures. In present study, a C-type lectin domain family 4 member E-like gene from turbot, which tentatively named SmCLEC4E-like (SmCLEC4EL), was identified, and the expressional and functional analyses were performed. In our results, SmCLEC4EL showed conserved synteny with CLEC4E-like genes from several fish species in genome, and possessed a typical type II transmembrane CTL architecture: an N-terminal intracellular region, a transmembrane domain and a C-terminal extracellular region which contained a predicted carbohydrate recognition domain (CRD). In addition, SmCLEC4EL exhibited the highest expression level in spleen in healthy fish, and showed significantly induced expression in mucosal tissues, intestine and skin, under bacteria challenge. Finally, the recombinant SmCLEC4EL protein combined with LPS, PGN, LTA and five different kinds of bacteria in a dose-dependent manner, and agglutinated these bacteria strains in the presence of calcium. These findings collectively demonstrated that SmCLEC4EL, a calcium-dependent CTL, could function as a pattern recognition receptor in pathogen recognition and participate in host anti-bacteria immunity.

Characterization of toll-like receptor 1 (TLR1) in turbot (Scophthalmus maximus L.).

TLRs are the first and best-characterized pattern recognition receptors conserved across all the species. Different from mammals, the TLRs in teleost fishes are very diversified due to various evolutionary mechanisms. Here, we characterized one TLR1 gene in turbot, with a 2,415 bp open reading frame (ORF), that encoding 804 amino acid residues, and have the highest similarity and identity both to Paralichthys olivaceus with 88.9% and 79.9%. In phylogenetic analysis, it was firstly clustered with P. olivaceus, and then clustered with Takifugu rubripes. TLR1 was widely expressed in all the examined healthy tissues with the highest expression level in spleen, followed by head-kidney. In addition, it was significantly regulated in gill, skin and intestine following Edwardsiella tarda and Vibrio anguillarum challenge with different expression patterns. In in vitro stimulation with pathogen-associated molecular patterns, TLR1 showed significantly strong and elevated responses to LPS, but only responded to LTA and Poly(I:C) at the highest evaluated concentration, while no response was detected using PGN stimulation. Moreover, in subcellular localization analysis, TLR1 was distributed in the cytoplasm, membrane and nucleus. Taken together, TLR1 played vital roles for host immune response to bacterial infection, only with strong binding ability to LPS and involved in the production of inflammatory cytokines. However, the specific ligand for TLR1 and its functional association with other TLRs should be further characterized in fish species.

Characterization of the immune roles of cathepsin L in turbot (Scophthalmus maximus L.) mucosal immunity.

Cathepsin L (CTSL) is one of the crucial enzymes in cathepsin family, which has been widely known for its involvement in the innate immunity. However, it still remains poorly understood how CTSL modulates the immune system of teleosts. In this study, we captured three cathepsin L genes (SmCTSL, SmCTSL.1 and SmCTSL1) from turbot (Scophthalmus maximus). The coding sequences of SmCTSL, SmCTSL.1 and SmCTSL1 are 1,026 bp, 1,005 bp and 1,017 bp in length and encode 341, 334 and 338 amino acids, respectively. In details, transcripts of CTSL genes share same domains as other CTSL genes, one signal peptide, one propeptide and one papain family cysteine protease domain. Protein interaction network analysis indicated that turbot CTSL genes may play important roles in apoptotic signaling and involve in innate immune response. Evidence from subcellular localization demonstrated that the three Cathepsin L proteins were ubiquitous in nucleus and cytoplasm. The cathepsin L genes were widely expressed in all the tested tissues with the highest expression level of SmCTSL in spleen, and SmCTSL.1 and SmCTSL1 in intestine. Following Vibrio anguillarum, Edwardsiella tarda and Streptococcus iniae challenge, these cathepsin L genes were significantly regulated in mucosal tissues in all the challenges, especially significant down-regulation occurred rapidly in intestine in all the three challenges. In addition, the three cathepsin L genes showed strong binding ability to all the examined microbial ligands (LPS, PGN and LTA). Further studies should be used to analyze the specific function of these three cathepsin L genes. By then, we can use their function to maintain the integrity of the mucosal barrier, thereby promoting the disease resistance line and family selection in turbot.

Full length transcriptome profiling reveals novel immune-related genes in black rockfish (Sebastes schlegelii).

Lacking full-length transcriptome for black rockfish (Sebastes schlegelii) limits novel gene discoveries and gene structures analysis. Therefore, we constructed the full-length transcriptome of black rockfish using Single-Molecule Real-Time Sequencing technology. Totally, we produced 21.73 Gb raw reads containing 298,904 circular consensus sequence (CCS) reads. Full-length (FL) and Non-full-length (NFL) isoforms were obtained based on the presence of 5' and 3' primers as well as poly (A) tails. The results showed 70.71% reads were identified as FL isoforms. Moreover, the average length of these PacBio isoforms is 2,632 bp, which is much longer than the length of the unigenes with the average length of 589 bp which generated from Illumina platform. Meanwhile, we identified 43,068 non-redundant transcripts, 12,485 alternative splicing (AS), 6,320 polyadenylation (APA) and 499 gene fusions as well as numerous long non-coding RNAs based on mapped FL isoforms. In addition, we identified 147 and 528 immune-related genes from novel genes and unmapped transcripts. The provided dataset can be utilized to discover novel genes and construct a comprehensive transcript dataset for black rockfish.

Characterization of class B scavenger receptor type 1 (SRB1) in turbot (Scophthalmus maximus L.).

Class B scavenger receptor type 1 (SRB1) serves as a high-density lipoprotein (HDL) receptor essential for HDL metabolism, and plays vital roles in innate immunity. In this study, the turbot (Scophthalmus maximus) SRB1 was cloned and characterized. The gene structure consists of a coding region of 1,527 bp nucleotides dividing into 13 exons and 12 introns. Such genome structure is highly conserved among teleost fishes. The deduced SRB1 encodes 508 amino acids that mainly has a CD36 transmembrane domain. Tissue distribution of SRB1 showed the lowest expression in liver, while the highest expression was found in intestine. Significantly down-regulation pattern of SmSRB1 expression in intestine was shared after infection with Vibrio anguillarum and Streptococcus iniae. Brach and site models in CODEML program showed that SmSRB1 underwent a conservative evolutionary and three potential positive selected sites 470K, 496E, and 501Y were detected, which requires further investigation and confirmation using base-editing technologies. Subcellular localization demonstrated that turbot SRB1 was distributed in the membrane and cytoplasm. rSmSRB1 showed binding ability in vitro to bacteria, LPS, PGN, LTA and virus. Protein-protein interaction network agrees the function of SRB1 as lipoprotein receptor. Our results indicated SmSRB1 might act as co-receptors to TLRs and NLRs to modulate the immune response to pathogens. Further studies should pay attention to evaluate the specific co-receptor for SRB1 in recognition of different pathogens and selective mechanisms involved.

Analysis of specific mRNA gene expression profiles as markers of egg and embryo quality for hybrid catfish aquaculture.

Despite best efforts to optimize reproduction, egg incubation, and larval performance in captivity, inconsistencies in hatchery fish production are still created by high variations in egg quality from individual females. In some hatchery species, egg quality and generation of viable embryos are correlated to abundances of specific mRNAs. Channel catfish females show considerable extremes in egg quality, causing inconsistencies in channel catfish, Ictalurus punctatus, female × blue catfish, Ictalurus furcatus, male hybrid fry production. The objectives of this study were to examine relative transcripts linked to egg and embryo quality and determine expression between low-hatch and high-hatch egg batches through early development (0, 24, 48, and 96 h post-fertilization; HPF). RNA was extracted from eggs/embryos of nine females (n = 4 high-quality, n = 5 low-quality) and Real-Time PCR was used to quantify relative gene expression. The transcripts assessed in this study perform critical cellular functions, including tubulin ß (tubb), cathepsin D (ctsd), cathepsin Z (ctsz), cathepsin B (ctsb), cyclin B (ccnb1), exportin-1 (xpo1), ring finger protein 213 (rnf213), glucocorticoid receptor-1 (GR-1), and heat shock protein 70 (hsp70). Relative gene expression of all transcripts except GR-1 and hsp70 were up-regulated in the high-hatch group and peaked at 48 HPF (neurulation stage), indicating the importance of these gene products at this threshold to normally progress until hatch. Due to lack of expression during earlier stages, maternally derived mRNAs for these genes do not seem to impact early embryonic development. Using mRNA markers as a selection mechanism for hatchery broodstock may lead to more high-hatch egg batches by reducing problems associated with poor egg quality.

Deep Transcriptomic Analysis Reveals the Dynamic Developmental Progression during Early Development of Channel Catfish (Ictalurus punctatus).

The transition from fertilized egg to larva in fish is accompanied with various biological processes. We selected seven early developmental stages in channel catfish, Ictalurus punctatus, for transcriptome analysis, and covered 22,635 genes with 590 million high-quality RNA-sequencing (seq) reads. Differential expression analysis between neighboring developmental timepoints revealed significantly enriched biological categories associated with growth, development and morphogenesis, which was most evident at 2 vs. 5 days post fertilization (dpf) and 5 vs. 6 dpf. A gene co-expression network was constructed using the Weighted Gene Co-expression Network Analysis (WGCNA) approach and four critical modules were identified. Among candidate hub genes, GDF10, FOXA2, HCEA and SYCE3 were involved in head formation, egg development and the transverse central element of synaptonemal complexes. CK1, OAZ2, DARS1 and UBE2V2 were mainly associated with regulation of cell cycle, growth, brain development, differentiation and proliferation of enterocytes. IFI44L and ZIP10 were critical for the regulation of immune activity and ion transport. Additionally, TCK1 and TGFB1 were related to phosphate transport and regulating cell proliferation. All these genes play vital roles in embryogenesis and regulation of early development. These results serve as a rich dataset for functional genomic studies. Our work reveals new insights of the underlying mechanisms in channel catfish early development.

Characterization and initial functional analysis of cathepsin K in turbot (Scophthalmus maximus L.).

Cathepsins are the best-known group of proteases in lysosomes, playing a significant role in immune responses. Cathepsin K (CTSK) is abundantly and selectively expressed in osteoclasts, dendritic cells and monocyte-derived macrophages, where it is involved in ECM degradation and bone remodeling. A growing body of evidences have indicated the vital roles of cathepsin K in innate immune responses. Here, one CTSK gene was captured in turbot (SmCTSK) with a 993 bp open reading frame (ORF). The genomic structure analysis showed that SmCTSK had 7 exons similar to other vertebrate species. The syntenic analysis revealed that CTSK had the same neighboring genes across all the selected species, which suggested the synteny encompassing CTSK region was conserved during vertebrate evolution. Subsequently, SmCTSK was widely expressed in all the examined tissues, with the highest expression level in spleen and the lowest expression level in liver. In addition, SmCTSK was significantly down-regulated in intestine following Gram-negative bacteria Vibrio anguillarum immersion challenge, but up-regulated in three tissues (gill, skin and intestine) following Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmCTSK showed strong binding ability to all the examined microbial ligands. Taken together, our results suggested SmCTSK played vital roles in fish innate immune responses against infection. However, the knowledge of SmCTSK is still limited in teleost species, further studies should be carried out to better characterize its comprehensive roles in teleost mucosal immunity.

Expression profiling and microbial ligand binding analysis of high-mobility group box-1 (HMGB1) in turbot (Scophthalmus maximus L.).

High-mobility group box 1 (HMGB1), a highly conserved DNA-binding protein, was involved in nucleosome formation and transcriptional regulation, and could also act as an extracellular cytokine to trigger inflammation and immune responses. In this study, we identified a HMGB1 gene in turbot (Scophthalmus maximus L.). The full-length SaHMGB1 cDNA includes an open reading frame of 615 bp which encoded a 204 amino acid polypeptide with an estimated molecular mass of 23.19 kDa. SaHMGB1 was closely related to several fish HMGB1 and shared 74.4% overall identity with human. In addition, phylogenetic analyses revealed SaHMGB1 showed the closest relationship to Larimichthys crocea. Furthermore, QPCR analysis showed that SaHMGB1 was expressed in all examined tissues with abundant expression levels in brain, gill, intestine, and head kidney, and showed different expression patterns following different bacterial challenge. The significant quick regulation of SaHMGB1 in mucosal surfaces against infection suggest that HMGB1 might play critical roles in mucosal immunity against bacterial challenge. Finally, the in vitro binding assay showed that SaHMGB1 had strong binding ability to LPS, LTA, and PGN. Functional studies should further characterize HMGB1 function to understand the importance of the integrity of the mucosal barriers against infection, and to facilitate selection of the disease resistant family/strain in turbot.

l-rhamnose-binding lectins (RBLs) in turbot (Scophthalmus maximus L.): Characterization and expression profiling in mucosal tissues.

Rhamnose-binding lectin (RBL) were mostly identified from egg cortex and ovary cells from vertebrates and invertebrates, with the specific binding activities to l-rhamnose or d-galactose. Previously, we found that a RBL gene was dramatically down-regulated (-11.90 fold at 1 h, -48.95 fold at 4 h, -905.94 fold at 12 h) in the intestine of turbot following Vibrio anguillarum challenge using RNA-seq expression analysis. In this regard, we sought here to identify RBLs in turbot, as well as the analysis of genomic structure, phylogenetic relationships, basal tissue distribution and the expression patterns following different bacteria challenge in mucosal tissues. In this study, two RBLs were captured in turbot with two conserved type 5 CRD5s, which were belong to type IIIc RBL. In phylogenetic tree analysis, turbot RBLs were clustered with tilapia, European sea bass and snakehead. In addition, in comparison of genomic architecture of turbot RBLs with the available published RBL genes revealed a high degree of conservation in the exon/intron organization among the teleost species. Moreover, both RBLs were significantly up-regulated in mucosal tissues following V. anguillarum and Streptococcus iniae challenge, indicated their critical roles in turbot mucosal immunity. Further studies are needed to expand functional characterization of detailed mechanisms of RBLs in fish innate immunity.

Testicular germ line cell identification, isolation, and transplantation in two North American catfish species.

Our aim was to transplant blue catfish germ line stem cells into blastulae of triploid channel catfish embryos to produce interspecific xenogenic catfish. The morphological structure of the gonads of blue catfish (Ictalurus furcatus) in ~ 90- to 100-day-old juveniles, two-year-old juveniles, and mature adults was studied histologically. Both oogonia (12-15 µm, diameter with distinct nucleus 7-8 µm diameter) and spermatogonia (12-15 µm, with distinct nucleus 6-7.5 µm diameter) were found in all ages of fish. The percentage of germ line stem cells was higher in younger blue catfish of both sexes. After the testicular tissue was trypsinized, a discontinuous density gradient centrifugation was performed using 70, 45, and 35% Percoll to enrich the percentage of spermatogonial stem cells (SSCs). Four distinct cell bands were generated after the centrifugation. It was estimated that 50% of the total cells in the top band were type A spermatogonia (diameter 12-15 µm) and type B spermatogonia (diameter 10-11 µm). Germ cells were confirmed with expression of vasa. Blastula-stage embryos of channel catfish (I. punctatus) were injected with freshly dissociated blue catfish testicular germ cells as donor cells for transplantation. Seventeen days after the transplantation, 33.3% of the triploid channel catfish fry were determined to be xenogenic catfish. This transplantation technique was efficient, and these xenogenic channel catfish need to be grown to maturity to verify their reproductive capacity and to verify that for the first time SSCs injected into blastulae were able to migrate to the genital ridge and colonize. These results open the possibility of artificially producing xenogenic channel catfish males that can produce blue catfish sperm and mate with normal channel catfish females naturally. The progeny would be all C × B hybrid catfish, and the efficiency of hybrid catfish production could be improved tremendously in the catfish industry.

Phenoscape: Identifying Candidate Genes for Evolutionary Phenotypes.

Phenotypes resulting from mutations in genetic model organisms can help reveal candidate genes for evolutionarily important phenotypic changes in related taxa. Although testing candidate gene hypotheses experimentally in nonmodel organisms is typically difficult, ontology-driven information systems can help generate testable hypotheses about developmental processes in experimentally tractable organisms. Here, we tested candidate gene hypotheses suggested by expert use of the Phenoscape Knowledgebase, specifically looking for genes that are candidates responsible for evolutionarily interesting phenotypes in the ostariophysan fishes that bear resemblance to mutant phenotypes in zebrafish. For this, we searched ZFIN for genetic perturbations that result in either loss of basihyal element or loss of scales phenotypes, because these are the ancestral phenotypes observed in catfishes (Siluriformes). We tested the identified candidate genes by examining their endogenous expression patterns in the channel catfish, Ictalurus punctatus. The experimental results were consistent with the hypotheses that these features evolved through disruption in developmental pathways at, or upstream of, brpf1 and eda/edar for the ancestral losses of basihyal element and scales, respectively. These results demonstrate that ontological annotations of the phenotypic effects of genetic alterations in model organisms, when aggregated within a knowledgebase, can be used effectively to generate testable, and useful, hypotheses about evolutionary changes in morphology.

Identification and expression analysis of fetuin B (FETUB) in turbot (Scophthalmus maximus L.) mucosal barriers following bacterial challenge.

Fetuin B (FETUB), a recently described cysteine proteinase inhibitor, has numerous conserved N-glycosylation sites, species-specific O-glycosylation sites, and two cystatin (CY) domains. FETUB is likely to play regulatory roles in acute inflammation, female infertility, fish organogenesis and tumor suppression. In the present study, transcript of turbot FETUB gene was captured, its protein structure and expression patterns in different tissues with emphasis on mucosal barriers following different bacterial infection were characterized. Turbot FETUB gene showed the closest relationship with Takifugu rubripes in phylogenetic analysis. In addition, FETUB was ubiquitously expressed in all examined tissues with the highest expression level in skin. Finally, FETUB gene showed different expression patterns following both bacterial challenge. The rapidly and significantly differential expression patterns of FETUB in mucosal surfaces against bacterial infections might indicate its key roles to prevent pathogen attachment and entry in turbot mucosal immunity. Functional studies should be carried out to further characterize the FETUB and avail utilization of its function to increase the disease resistance of turbot in maintaining the integrity of the mucosal barriers against infections and to facilitate selection of the fine family/varieties of disease resistance in turbot.

Identification, characterization and expression analysis of TLR5 in the mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

TLRs (Toll-like receptors) are very important pathogen pattern recognition receptors, which control the host immune responses against pathogens through recognition of molecular patterns specific to microorganisms. In this regard, investigation of the turbot TLRs could help to understand the immune responses for pathogen recognition. Here, transcripts of two TLR5 (TLR5a and TLR5b) were captured, and their protein structures were also predicted. Meanwhile, we characterized their expression patterns with emphasis on mucosal barriers following different bacterial infection. The phylogenetic analysis revealed the turbot TLR5 genes showed the closest relationship to Paralichthys olivaceus. These two TLR5 genes were ubiquitously expressed in healthy tissues although expression levels varied among the tested tissues. In addition, the two copies of turbot TLR5 showed different expression patterns after bacterial infections. After Vibrio anguillarum infection, TLR5a was generally up-regulated in intestine and skin while down-regulated in gill, while TLR5b showed a general down-regulation in mucosal tissues. After Streptococcus iniae infection, the TLR5a was down-regulated at 2 h while generally up-regulated after 4 h in mucosal tissues. Interestingly, the TLR5b was up-regulated in intestine while down-regulated in skin and gill after Streptococcus iniae infection. These findings suggested a possible irreplaceable role of TLR5 in the immune responses to the infections of a broad range of pathogens that include Gram-negative and Gram-positive bacteria. Future studies should apply the bacteriological and immune-histochemical techniques to study the main sites on the mucosal tissue for bacteria entry and identify the ligand specificity of the turbot TLRs after challenge.

Characterization and expression analysis of chitinase genes (CHIT1, CHIT2 and CHIT3) in turbot (Scophthalmus maximus L.) following bacterial challenge.

Chitinases are hydrolytic enzymes which have been employed to breakdown chitin coats of pathogenic microorganisms, thereby weaken the defense system of several pathogens and insects. In this regard, we identified the chitinase genes of turbot and characterized their expression patterns in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge. In present study, transcripts of three chitinase genes (CHIT1, CHIT2 and CHIT3) were captured, as well as their protein structures and expression patterns following different bacterial infection were also characterized. The chitinases were widely expressed in all tested tissues with the highest expression levels of CHIT1 and CHIT2 in intestine, and CHIT3 in skin. Finally, these three genes showed different expression patterns following bacterial challenge. The significant quick induction of chitinases in mucosal surfaces against infection indicated their key roles to prevent pathogen attachment and entry in mucosal immunity. Functional studies should further characterize the chitinases and avail utilization of their function to increase the disease resistance in maintaining the integrity of the mucosal barriers against infection and facilitating the disease resistant family/strain selection in turbot.

The involvement of cathepsin F gene (CTSF) in turbot (Scophthalmus maximus L.) mucosal immunity.

Cathepsin F (CTSF) is a recently described papain-like cysteine protease and unique among cathepsins due to an elongated N-terminal pro-region, which contains a cystatin domain. CTSF likely plays a regulatory role in processing the invariant chain which is associated with the major histocompatibility complex (MHC) class II. In this regard, we identified the CTSF gene of turbot as well as its protein structure, phylogenetic relationships, and expression patterns in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge. We also determined the expression patterns of CTSF in mucosal tissues after vaccinated with the formalin-inactivated V. vulnificus whole-cell vaccine. Briefly, turbot CTSF gene showed the closest relationship with that of Paralichthys olivaceus in phylogenetic analysis. And CTSF was ubiquitously expressed in all tested tissues with the highest expression level in gill. In addition, CTSF gene showed different expression patterns following different bacterial challenge. The significant quick regulation of CTSF in mucosal surfaces against infection indicated its roles in mucosal immunity. Functional studies should further characterize avail utilization of CTSF function to increase the disease resistance of turbot in maintaining the integrity of the mucosal barriers against infection and to facilitate selection of the disease resistant family/strain in turbot.

Characterization and expression profiling of NOD-like receptor C3 (NLRC3) in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

The mucosal surfaces are important for teleost as they are directly and continuously exposed to pathogen-rich aquatic environments. Scrutinization and recognition of the attached pathogens is the first crucial step of mucosal immunity initiation. Nucleotide oligomerization domain (NOD)-like receptors (NLRs) are a large group of intracellular pathogen recognition receptors (PRRs) which play key roles in pathogen recognition and subsequent immune signaling pathways activation. In this study, we identified two NLRC3 genes (NLRC3a and NLRC3b), a subfamily of NLRs from turbot, and profiled their expression patterns in mucosal tissues following bacterial challenge. NLRC3a transcript contains an open reading frame (ORF) of 3405 bp that encodes a putative peptide of 1134 amino acids. While NLRC3b has an ORF of 3114 bp encoding 1037 amino acids. A caspase recruitment domain (CARD) at N-terminus characterized turbot NLRC3a, while NLRC3b seems to be unique to teleost, containing a fish specific NACHT associated (FISNA) domain and an extra B30.2 (PRY/SPRY) domain at C-terminus. In addition, NLRC3a and NLRC3b were detected in all the examined tissues, with the highest expression levels in kidney and blood, respectively. After bacteria challenge, expression levels of turbot NLRC3 genes were strongly induced in intestine rather than in skin and gill, while NLRC3a had relatively higher expression level than that of NLRC3b. Taken together, NLRC3 genes found in this study were the first NLR members identified in turbot. The different expression signatures of NLRC3a and NLRC3b in mucosal tissues following two bacterial infections indicated they probably have important roles in early response to bacterial infection in the first line of host defense system.