Regulation of PhoB on biofilm formation and hemolysin gene hlyA and ciaR of Streptococcus agalactiae.

PhoB is a response regulator protein that plays a key role in the PhoBR two-component signal transduction system. In this study, we used transcriptome and proteomics techniques to evaluate the detect the gene network regulated by PhoB of Streptococcus agalactiae. The results showed that expression of biofilm formation and virulence-related genes were changed after phoB deficiency. Crystal violet and CLSM assay confirmed that the deletion of the phoB increased the thickness of S. agalactiae biofilm. The results of lacZ reporter and the bacterial one-hybridization method showed that PhoB could directly bind to the promoter regions of hemolysin A and ciaR genes but not to the promoter regions of cylE and hemolysin III. Through the construction of an 18-base pair deoxyribose nucleic acid (DNA) random fragment library and the bacterial one-hybridization system, it was found that the conservative sequence of PhoB binding was TTGGAGAA(G/T). Our research has uncovered the virulence potential of the PhoBR two-component system of S. agalactiae. The findings of this study provide the theoretical foundation for in-depth research on the pathogenic mechanism of S. agalactiae.

An IL-6 gene in humphead snapper (Lutjanus sanguineus): Identification, expression analysis and its adjuvant effects on Vibrio harveyi OmpW DNA vaccine.

Interleukin 6 (IL-6) is a pleiotropic cytokine that plays important role in mediating the innate and adaptive immune responses against pathogen infection. In this study, an IL-6 homolog (Ls-IL6) was identified and characterized from humphead snapper, Lutjanus sanguineus. The full-length cDNA of Ls-IL6 was 1066 bp, containing an open reading frame (ORF) of 639 bp encoding 212 amino acids, 5' untranslated region(UTR) of 63 bp and 3' UTR of 605 bp. The predicted Ls-IL6 protein had typical motif of IL-6 family and shared high identities to teleost IL-6s. Ls-IL6 extensively expressed in various tissues, and the highest expression of Ls-IL6 was detected in head kidney, spleen and thymus. In vivo, the transcript levels of Ls-IL6 were significantly up-regulated in response to Vibrio harveyi infection. Moreover, the DNA plasmid containing the OmpW of V. harveyi together with the gene encoding Ls-IL6 were successfully constructed and administered to fish, the protective efficacy of Ls-IL6 was investigated. Compared with the pcDNA-OmpW group, the level of specific antibodies against V. harveyi increased in pcDNA-IL6-OmpW injected group. After V. harveyi infection, the pcDNA-IL6-OmpW vaccinated fish showed higher relative percent survival (76%) than the relative survival of fish immunized with pcDNA-OmpW (60%). These results indicated that Ls-IL6 was involved in immune response against V. harveyi infection and could be applied as a promising adjuvant for DNA vaccines against V. harveyi.

Identification of Beclin-1 from orange-spotted grouper (Epinephelus coioides) involved in viral infection.

Beclin-1 is an essential autophagic regulator that plays diverse roles in physiology and disease. However, reports about the function of fish Beclin-1 during pathogen infection are still very limited. In this study, a Beclin-1 homolog (EcBeclin-1) from orange-spotted grouper (Epinephelus coioides) was identified and its roles in viral infection were investigated. EcBeclin-1 encoded 447amino acids protein with a BH3 domain, a CCD domain and an ECD domain, which shared high identities (97%-82%) with reported Beclin-1 proteins from mammal to fish. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBeclin-1 was predominantly expressed in brain and muscle of healthy grouper. Using fluorescence microscopy, we found that EcBeclin-1 was co-localized with endoplasmic reticulum (ER) in grouper spleen cells (EAGS). After red-spotted grouper nervous necrosis virus (RGNNV) infection in vitro, EcBeclin-1 transcript was significantly up-regulated, implying that EcBeclin-1 might be involved in viral infection. Furthermore, the in vitro studies of EcBeclin-1 overexpression promoted RGNNV induced autophagy, as well as the expression of coat protein (CP) and RNA-dependent RNA polymerase (RdRp). The overexpression of EcBeclin-1 suppressed the expressions of interferon pathway-related factors, inflammatory-related factors and activities of NF-κB and ISRE. Additionally, EcBeclin-1 could interact with EcBcl-xL in vitro. These data suggest that EcBeclin-1 affect viral replication through modulating IFN and inflammatory responses, as well as virus-induced cell death, which will help us to further explore the immune response of fish during viral infection.

Antibacterial activity of erythrocyte from grass carp (Ctenopharyngodon idella) is associated with phagocytosis and reactive oxygen species generation.

Red blood cells (RBCs) are widely accepted as their primary function in respiration. Recent studies in mammals have revealed a vital role in immune responses of RBCs; however, little is known about immune function of teleost erythrocytes. Here we demonstrated that RBCs from grass carp (Ctenopharyngodon idella) were capable of binding and aggregating the bacteria with apparent morphological alterations. The phagocytosis by teleost RBCs (erythrophagocytosis) was visualized by confocal, scanning and transmission electron microscopy. Hb-FeII of hemoglobin (Hb) could quickly be auto-oxidated to Hb-FeIII (methemoglobin/metHb) in the presence of oxygen (O2), and release superoxide radical (O2-.) which could be spontaneously dismutated into H2O2 that could further oxidize Hb-FeIII to transient HbFeIV-OH (ferryl-Hb). Furthermore, bacterial extracellular proteases and pathogen-associated molecular patterns (PAMPs) binding to Hb could synergistically activate pseudoperoxidase, subsequently facilitated the generation of reactive oxygen species (ROS) which were toxic to the bacteria. Our results indicated that erythrocyte pertains anti-bacterial activity using unique ROS generation pathway via oxidation of hemoglobin and associated with its phagocytosis.

A comprehensive profile of the tilapia (Oreochromis niloticus) circular RNA and circRNA-miRNA network in the pathogenesis of meningoencephalitis of teleosts.

The pathogenesis of tilapia meningoencephalitis is still unclear, where the involvement of circRNA is considered for its active role as a "miRNA sponge". Therefore, we aimed to investigate the profile of circRNA in tilapia meningoencephalitis further by constructing the circRNA-miRNA network for in-depth mechanism exploration. Briefly, a nile tilapia model of meningitis was established by injecting Streptococcus agalactiae (1.0 × 107 cfu per mL) and we evaluated the infected tilapia brain for the expression profile of circRNAs, their potential functions and their correlation with genes involved in inflammatory pathways. A total of 11 263 circRNAs were identified from RNA sequencing (RNA-seq) data in nile tilapia (Oreochromis niloticus), a commercially important fish in China and East Asia. GO and KEGG analyses revealed that the biological functions of genes hosting the circRNAs were enriched in the progression of metabolism and binding. Notably, we found that 99% circRNAs in tilapia had abundant miRNA-binding sites and a total of 2136 of the identified circRNAs had two to six miRNA-binding sites. Six circRNAs were validated by qRT-PCR and the final circRNA-miRNA network was constructed. This is the first report of comprehensive identification of O. niloticus circRNAs being differentially regulated in the brain in normal conditions relating to S. agalactiae infection. This work will shed novel light on gene expression mechanisms under disease conditions and may identify circRNAs as new biomarkers for meningoencephalitis and neurodegenerative disorders.

Bax inhibitor-1 from orange spotted grouper, Epinephelus coioides involved in viral infection.

Bax inhibitor-1 (BI-1) is a conserved anti-apoptotic protein that suppresses endoplasmic reticulum (ER) stress-induced cell death. However, the function of fish BI-1 is not quite clear. In the present study, a bi-1 homolog (Ecbi-1) from orange spotted grouper, Epinephelus coioides was identified and its roles in viral infection were investigated. EcBI-1 encoded 237 amino acids protein, contained six transmembrane regions and a conservative C-terminus motif. Ecbi-1 predominantly expressed in kidney and spleen of healthy grouper. After SGIV stimulation, Ecbi-1 transcript was significantly increased in vitro. Subcellular localization analysis revealed that EcBI-1 was localized throughout the cytoplasm and co-localized with ER. Furthermore, overexpression of EcBI-1 suppressed SGIV infection induced cell death, caspase-3 activity and viral genes transcription. And C-terminus motif was critical for regulation roles of EcBI-1 during SGIV infection. In addition, EcBI-1 could interact with EcBNIP3 in vitro. Together, our data firstly demonstrated that fish BI-1 play important roles in response to viral infection.

AcfA is an essential regulator for pathogenesis of fish pathogen Vibrio alginolyticus.

V. alginolyticus is an important opportunistic pathogen which causes vibriosis in aquatic animals. AcfA, as an accessory colonization factor, is hypothesized to be involved in the pathogenesis of infection. In this study, a mutant strain with an in-frame deletion removed nucleotides 86 to 561 of the acfA gene was constructed to reveal the role of AcfA in the physiology and virulence from V. alginolyticus. An acfA mutant showed a similar growth level, an obvious decrease in swarming motility and the activity of ECPase, a higher LD50 value by intraperitoneal injection of grouper fish compared to that of the wild-type. Furthermore, the deletion of acfA could enhance the level of biofilm formation and suppress the polar flagellum forming. The comparative proteomic analysis demonstrated the deletion mutation of acfA could up-regulate the expression of 4 proteins of p4alcd, deoD, phb and DctP, and down-regulate the expression of 8 proteins of Clp, hpV36980, ABCtp, pepD, arA, aggp, fla and ompA compared to that of the wild-type. The analysis of RT-qPCR showed the mRNA levels of DctP and deoD were significantly induced, and the mRNA levels of pepD, arA, fla and ompA were significantly reduced in acfA mutant compared with the wild-type. The results suggest that acfA may contribute to the overall success in the pathogenesis of V. alginolyticus by regulating the expression of some relevant genes.

Identification and characterization of CD40 from humphead snapper (Lutjanus sanguineus).

CD40 is known as "master switch" in immune response to pathogen infection in mammals. However, limited information of CD40 is known in lower vertebrates. In this study, a novel CD40 homolog (Ls-CD40) was cloned and characterized from humphead snapper, Lutjanus sanguineus. The Ls-CD40 cDNA composed of 2073 bp with a 69 bp of 5'-UTR, a 1020 bp of 3'-UTR and an open reading frame (ORF) of 984 bp, encoding 327 amino acid residues. Sequence analysis showed that Ls-CD40 contained a single peptide, a transmembrane domain and four cysteine-rich domains. The deduced amino acid sequence of Ls-CD40 shared 40%-53% identities with other known fish CD40. The qRT-PCR showed that Ls-CD40 gene expressed in all examined tissues with the most abundant in spleen and lowest level in intestine. After V. harveyi and poly I:C stimulation, the expression of CD40 were significantly induced in spleen. Moreover, Ls-CD40 could interact with Ls-TRAF3 in vitro. These data indicate that Ls-CD40 might play a regulatory role in immune response of L. sanguineus.

Complete genome sequence analysis of the fish pathogen Flavobacterium columnare provides insights into antibiotic resistance and pathogenicity related genes.

We analyzed here the complete genome sequences of a highly virulent Flavobacterium columnare Pf1 strain isolated in our laboratory. The complete genome consists of a 3,171,081 bp circular DNA with 2784 predicted protein-coding genes. Among these, 286 genes were predicted as antibiotic resistance genes, including 32 RND-type efflux pump related genes which were associated with the export of aminoglycosides, indicating inducible aminoglycosides resistances in F. columnare. On the other hand, 328 genes were predicted as pathogenicity related genes which could be classified as virulence factors, gliding motility proteins, adhesins, and many putative secreted proteases. These genes were probably involved in the colonization, invasion and destruction of fish tissues during the infection of F. columnare. Apparently, our obtained complete genome sequences provide the basis for the explanation of the interactions between the F. columnare and the infected fish. The predicted antibiotic resistance and pathogenicity related genes will shed a new light on the development of more efficient preventional strategies against the infection of F. columnare, which is a major worldwide fish pathogen.

Immunogenicity and efficacy of DNA vaccine encoding antigenic AcfA via addition of the molecular adjuvant Myd88 against Vibrio alginolyticus in Epinephelus coioides.

DNA vaccines had been widely used against microbial infection in animals. The use of molecular adjuvants to improve the immunogenicity of DNA vaccines has been increasingly studied in recent years. MyD88 is one of the adapter molecules to activate the signaling cascades and produces inflammatory mediators, and its immunological role and adjuvant potential which had been proved in mammals were rarely reported in fish species. In this study, plasmid pcMyD88 was constructed and the capacity of MyD88 as molecular adjuvant was explored by co-injecting with a DNA vaccine encoding AcfA against Vibrio alginolyticus infection in orange spotted grouper. The results suggested that it needed at least 7 days to transported DNA vaccine pcacfA or molecular adjuvant pcMyD88 from the injected muscle to kidney and spleens and stimulate host's immune system for later protection. The co-injection of pcMyD88 with DNA vaccine pcacfA could increase significantly specific antibody levels and the expression levels of the immune-related genes including MHCIα, MHCIIα, CD4, CD8α, IL-1ß and TNFα. Furthermore, pcMyD88 enhanced the immunoprotection of pcacfA against V. alginolyticus infection, with the significantly higher RPS of 83.3% in pcMyD88 + pcacfA group compared with that of pcacfA alone (73.3%) at challenging test of 10 weeks post vaccination. Together, these results clearly demonstrate that MyD88 is an effective adjuvant for the DNA vaccine pcacfA in orange spotted grouper.

Molecular characterization and expression of interleukin-10 and interleukin-22 in golden pompano (Trachinotus ovatus) in response to Streptococcus agalactiae stimulus.

In the present study, members of the interleukin (IL)-10 family of cytokines, including IL-10 (TOIL-10) and IL-22 (TOIL-22) of golden pompano (Trachinotus ovatus), were cloned for the first time, and their expression patterns and 3D structures analyzed. The full-length cDNA sequences of TOIL-10 and TOIL-22 contained open reading frames of 564 and 567 bp, respectively. TOIL-10 and TOIL-22 shared higher homology (78%-89%) with the corresponding genes from various fish relative to other species (25%-34%) and contained the IL-10 family signature and four cysteine residues that are well conserved in other vertebrate IL-10 members. Phylogenetic tree analysis of our sequences alongside other IL-10 family proteins revealed that TOIL-10 and TOIL-22 cluster together with other teleost IL-10 and IL-22 molecules. Expression of TOIL-10 and TOIL-22 genes was ubiquitous in all tissues examined. The TOIL-10 gene was also highly expressed in skin, heart, gill, spleen, kidney, brain and liver, and lower levels were detected in intestine and muscle. High expression of the TOIL-22 gene was observed in gill, intestine, kidney, spleen, with the lowest levels in liver. TOIL-10 and TOIL-22 were rapidly activated after SAΔphoB immunization and significantly increased to peak levels at 12 h and 4 d in golden pompano kidney and spleen respectively following challenge. Expression in the brain reached peak levels at 4 d and 3 d respectively after post-immunization. Our results collectively indicate that TOIL-10 and TOIL-22 participate in the host immune response to bacterial infection. Moreover, TOIL-22 plays a potentially important role in mucosal immunity.

BNIP3, a cell pro-apoptotic protein, involved in response to viral infection in orange spotted grouper, Epinephelus coioides.

BNIP3 is a kind of BH3-only protein that induces both cell death and autophagy. Here, a BNIP3 gene (EcBNIP3) was identified from orange spotted grouper, Epinephelus coioides. EcBNIP3 possessed 236 amino acids residues, contained a conservative BNIP3 domain and a transmembrane region. Besides, EcBNIP3 expressed at a relative high level in heart and spleen. EcBNIP3 transcript was up-regulated after SGIV infection in vitro. Subcellular localization analysis revealed that EcBNIP3 was predominantly localized in the cytoplasm and co-localized with mitochondria. In addition, overexpression EcBNIP3 accelerated SGIV infection induced cell death but inhibited viral genes transcription. Taken together, these results provided new evidence that fish BNIP3 might involved in response to virus infection.

Characterization of microRNAs in orange-spotted grouper (Epinephelus coioides) fin cells upon red-spotted grouper nervous necrosis virus infection.

Nervous necrosis virus (NNV), one of the most prevalent fish pathogens, has caused fatal disease of viral nervous necrosis (VNN) in many marine and freshwater fishes, and resulted in heavy economic losses in aquaculture industry worldwide. However, the molecular mechanisms underlying the pathogenicity of NNV remain elusive. In this study, the expression profiles of microRNA (miRNA) were investigated in grouper fin (GF-1) cells infected with red-spotted grouper nervous necrosis virus (RGNNV) via deep sequencing technique. The results showed that a total of 220 miRNAs were identified by aligning the small RNA sequences with the miRNA database of zebrafish, and 18 novel miRNAs were predicted using miRDeep2 software. Compared with the non-infected groups, 51 and 16 differentially expressed miRNAs (DE-miRNAs) were identified in the samples infected with RGNNV at 3 and 24 h, respectively. Six DE-miRNAs were randomly selected to validate their expressions using quantitative reverse transcription polymerase chain reaction (qRT-PCR), the results showed that their expression profiles were consistent with those obtained by deep sequencing. The target genes of the DE-miRNAs covered a wide range of functions, such as regulation of transcription, oxidation-reduction process, proteolysis, regulation of apoptotic process, and immune response. In addition, the effects of four DE-miRNAs including miR-1, miR-30b, miR-150, and miR-184 on RGNNV replication were evaluated, and the results showed that over-expression of each of the four miRNAs promoted the replication of RGNNV. These data provide insight into the molecular mechanism of RGNNV infection, and will benefit for the development of effective strategies to control RGNNV infection.

Construction of a Streptococcus agalactiae phoB mutant and evaluation of its potential as an attenuated modified live vaccine in golden pompano, Trachinotus ovatus.

Streptococcus agalactiae is a Gram-positive pathogen that can survive inside professional phagocytes and nonphagocytic cells to cause septicemia and meningoencephalitis in freshwater and marine fish. However, vaccines based on extracellular products (ECP) and formalin-killed whole S. agalactiae cells, as well as subunit vaccine are unable to protect fish from infection by variant serotypes S. agalactiae. The search for live attenuated vaccine with highly conserved and virulent-related genes is essential for producing a vaccine to help understand and control streptococcosis In this study, the phoB gene was cloned from pathogenic S. agalactiae TOS01 strain and the mutant strain SAΔphoB was constructed via allelic exchange mutagenesis. The results showed that the deduced amino acid of S. agalactiae TOS01 shares high similarities with other Streptococcus spp. and has high conserved response regulator receiver domain (REC) and DNA-binding effector domain of two-component system response regulators (Trans_reg_C). Cell adherence and invasion assays, challenge experiments and histopathological changes post-vaccination were performed and observed, the results showed that the mutant strain SAΔphoB has a lower adherence and invasion rate and less virulent than the wild type strain in golden pompano, and it doesn't induce clinical symptoms and obvious pathological changes in golden pompano, thereby indicating that the deletion of phoB affects the virulence and infectious capacity of S. agalactiae. Golden pompano vaccinated via intraperitoneal injection SAΔphoB had the relative percent survival value of 93.1% after challenge with TOS01, demonstrating its high potential as an effective attenuated live vaccine candidate. Real-time PCR assays showed that the SAΔphoB was able to enhance the expression of immune-related genes, including MHC-I, MyD88, IL-22 and IL-10 after vaccination, indicating that the SAΔphoB is able to induce humoral and cell-mediated immune response in golden pompano over a long period of time.

Construction of glutathione peroxidase (GPx) DNA vaccine and its protective efficiency on the orange-spotted grouper (Epinephelus coioides) challenged with Vibrio harveyi.

The main aims of this study were to construct glutathione peroxidase (GPx) DNA vaccine of Vibrio harveyi ZJ0603 and to investigate its immune protective efficiency as a vaccine candidate on the orange-spotted grouper (Epinephelus coioides) treated with V. harveyi. Base on the cloning of ZJ0603 GPx gene, a DNA vaccine, named as pcDNA-GPx, was constructed by inserting GPx gene into pcDNA3.1 (+) plasmid. Orange-spotted groupers were immunized with the pcDNA-GPx plasmid by injection intramuscularly. The relative percent of survival (RPS) of fish vaccinated with the DNA vaccine against pathogenic V. harveyi infection was 77.5%. The expression of DNA vaccine was analyzed in the tissues of orange-spotted grouper by PCR and RT-PCR. The results indicated that pcDNA-GPx distributed and expressed in the head kidney, liver, spleen, gill and injected muscle at 7 and 28 days after vaccination. Significant specific antibody responses were also detected in the vaccinated orange-spotted groupers by indirect ELISA method. In a conclusion, DNA vaccine pcDNA-GPx showed an effective immune protection to the orange-spotted grouper treated with V. harveyi. The GPx can be used as a candidate DNA vaccine for the control of vibriosis.

Integrated analysis neurimmiRs of tilapia (Oreochromis niloticus) involved in immune response to Streptococcus agalactiae, a pathogen causing meningoencephalitis in teleosts.

MicroRNAs (miRNAs) are a class of noncoding RNA molecules and play important roles in a wide spectrum of biological processes, including in immune response. Recent years have witnessed considerable amount of research interest in studies on miRNA-mediated modulation gene function during neuroinflammation. Here, we evaluated Streptococcus agalactiae infected tilapia (Oreochromis niloticus) brain for the expression profile of miRNAs, potential functions and their correlation with genes involved in inflammatory pathways. A total of 1981 miRNAs were identified, including in 486 miRNAs which have homologues in the currently available databases and 1945 novel miRNAs. The expression levels of 547 miRNAs were significantly altered at 6 h-48 h post-bacterial infection, and these miRNAs were therefore classified as differentially expressed tilapia miRNAs. Real-time PCR were implemented for 14 miRNAs co-expressed in five samples, and agreement was confirmed between the high-throughput sequencing and real-time PCR data. For the 486 differentially expressed miRNAs target 41,820 genes. GO and KEGG enrichment analysis revealed that some target genes of miRNAs were grouped mainly into the categories of apoptotic, signal pathwayand immune response. This is the first report of comprehensive identification of teleost miRNAs being differentially regulated in brain in normal conditions relating to bacterial infection.

Transcriptomic analysis of the head kidney of Topmouth culter (Culter alburnus) infected with Flavobacterium columnare with an emphasis on phagosome pathway.

Flavobacterium columnare (FC) has caused worldwide fish columnaris disease with high mortality and great economic losses in cultured fish, including Topmouth culter (Culter alburnus). However, the knowledge about the host factors involved in FC infection is little known. In this study, the transcriptomic profiles of the head kidney from Topmouth culter with or without FC infection were obtained using HiSeq™ 2500 (Illumina). Totally 79,641 unigenes with high quality were obtained. Among them, 4037 differently expressed genes, including 1217 up-regulated and 2820 down-regulated genes, were identified and enriched using databases of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The differently expressed genes were mainly associated with pathways such as immune response, carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Since phagocytosis is a central mechanism of innate immune response by host cells to defense against infectious agents, genes related to the phagosome pathway were scrutinized and 9 differently expressed phagosome-related genes were identified including 3 up-regulated and 6 down-regulated genes. Five of them were further validated by quantitative real-time polymerase chain reaction (qRT-PCR). This transcriptomic analysis of host genes in response to FC infection provides data towards understanding the infection mechanisms and will shed a new light on the prevention of columnaris.

An alpha-2 macroglobulin in the pearl oyster Pinctada fucata: Characterization and function in hemocyte phagocytosis of Vibrio alginolyticus.

Alpha-2 macroglobulin (α2M) is a ubiquitous protease inhibitor and considered to be an evolutionarily conserved constituent of innate host defence system. Here, an α2M gene (designated as Pfα2M) was obtained from the pearl oyster Pinctada fucata by RT-PCR, PCR walking and rapid amplification of cDNA ends (RACE). The Pfα2M cDNA consists of 6394 bp with an open reading frame (ORF) of 5745 bp encoding a protein of 1914 amino acids with a 19 residues signal peptide. Pfα2M sequence contains three putative functional domains, including a bait region, a thiol ester domain and a receptor-binding domain. Phylogenetic analysis revealed that Pfα2M is closely related to the α2Ms from other molluscs. Pfα2M was expressed in all tested tissues including digestive gland, gill, adductor muscle, mantle and foot, while the highest expression was found in hemocytes. Following challenge with Vibrio alginolyticus, Pfα2M expression in hemocytes was significantly up-regulated at 2 h and then returned to the original level at 48 h. Knockdown of Pfα2M by RNA interference significantly reduced the phagocytosis of V. alginolyticus by hemocytes in vivo, and similar results were obtained upon chemical inactivation of the reactive thioester bond in Pfα2M by methylamine treatment. Taken together, it is suggested that Pfα2M is an immune-relevant molecule and involved in phagocytosis of V. alginolyticus by P. fucata hemocytes, and the function of Pfα2M in phagocytosis is dependent on the active thioester bond.

Comprehensive identification and profiling of Nile tilapia (Oreochromis niloticus) microRNAs response to Streptococcus agalactiae infection through high-throughput sequencing.

MicroRNAs are a kind of small non-coding RNAs that participate in various biological processes. Deregulated microRNA expression is associated with several types of diseases. Tilapia (Oreochromis niloticus) is an important commercial fish species in China. To identify miRNAs and investigate immune-related miRNAs of O. niloticus, we applied high-throughput sequencing technology to identify and analyze miRNAs from tilapia infected with Streptococcus agalactiae at a timescale of 72 h divided into six different time points. The results showed that a total of 3009 tilapia miRNAs were identified, including in 1121 miRNAs which have homologues in the currently available databases and 1878 novel miRNAs. The expression levels of 218 tilapia miRNAs were significantly altered at 6 h-72 h post-bacterial infection (pi), and these miRNAs were therefore classified as differentially expressed tilapia miRNAs. For the 1121 differentially expressed tilapia miRNAs target 41961 genes. GO and KEGG enrichment analysis revealed that some target genes of tilapia miRNAs were grouped mainly into the categories of apoptotic process, signal pathway, and immune response. This is the first report of comprehensive identification of O. niloticus miRNAs being differentially regulated in spleen in normal conditions relating to S. agalactiae infection. This work provides an opportunity for further understanding of the molecular mechanisms of miRNA regulation in O. niloticus host-pathogen interactions.

Molecular Cloning and Expression Analysis of IgD in Nile Tilapia (Oreochromis niloticus) in Response to Streptococcus agalactiae Stimulus.

IgD is considered to be a recently-evolved Ig and a puzzling molecule, being previously found in all vertebrate taxa, except for birds. Although IgD likely plays an important role in vertebrate immune responses, the function of IgD in Nile tilapia (Oreochromis niloticus) is virtually unknown. In the present study, a membrane form of IgD (mIgD) heavy chains were cloned from the GIFT strain of Nile tilapia (designated On-mIgD). The On-mIgD heavy chain's cDNA is composed of 3347 bp with a 31 bp of 5'-UTR, 3015 bp open reading frame (ORF) and 301 bp 3'-UTR, encoding a polypeptide of 1004 amino acids (GenBank accession no: KF530821). Phylogenetic analysis revealed that On-mIgD heavy chains showed the highest similarity to Siniperca chuatsi. Quantitative real-time PCR (qRT-PCR) analysis showed that On-mIgD expression occurred predominately in head kidney, thymus, spleen, and kidney. After Streptococcus agalactiae infection, transcripts of On-mIgD increased and reached its peak at 48 h in the head kidney and thymus, and 72 h in the spleen, respectively. Taken together, these results collectively indicated that IgD could possibly have a key role to play in the immune response when bacterial infections in Nile tilapia.