Salidroside inhibits the ferroptosis to alleviate lung ischemia reperfusion injury via the JAK2/STAT3 signalling pathway.

OBJECTIVE: The present study aims to investigate the protective potential of salidroside in both lung ischemia/reperfusion injury (LIRI) mice model and cell hypoxia/reoxygenation (H/R)model and the involvement of ferroptosis and JAK2/STAT3 pathway. MATERIALS AND METHODS: After we established the IR-induced lung injury model in mice, we administered salidroside and the ferroptosis inhibitor, ferrostatin-1, then assessed the lung tissue injury, ferroptosis (levels of reactive oxygen species level, malondialdehyde and glutathione), and inflammation in lung tissues. The levels of ferroptosis-related proteins (glutathione peroxidase 4, fibroblast-specific protein 1, solute carrier family 1 member 5 and glutaminase 2) in the lung tissue were measured with Western blotting. Next, BEAS-2B cells were used to establish an H/R cell model and treated with salidroside or ferrostatin-1 before the cell viability and the levels of lactate dehydrogenase (LDH), inflammatory factor, ferroptosis-related proteins were measured. The activation of the JAK2/STAT3 signaling pathway was measured with Western blotting, then its role was confirmed with STAT3 knockdown. RESULTS: Remarkably, salidroside was found to alleviate ferroptosis, inflammation, and lung injury in LIRI mice and the cell injury in H/R cell model. Severe ferroptosis were observed in LIRI mice models and H/R-induced BEAS-2B cells, which was alleviated by salidroside. Furthermore, salidroside could inhibit JAK2/STAT3 activation induced by LIRI. STAT3 knockdown could enhance the effect of salidroside treatment on H/R-induced cell damage and ferroptosis in vitro. CONCLUSIONS: Salidroside inhibits ferroptosis to alleviate lung ischemia reperfusion injury via the JAK2/STAT3 signaling pathway.

The role of hematological parameters in asymptomatic and non-severe cases of Omicron variant infection.

BACKGROUND: Omicron variants are currently the predominant circulating lineage worldwide and most cases are mild or asymptomatic. The Omicron variant is characterized by high transmissibility and immune evasion. Early identification of Omicron cases in clinical settings is crucial for controlling its spread. Previous studies have indicated that changes in hematological parameters can be used to predict the severity of coronavirus disease 2019 (COVID-19). However, the role of hematological parameters in non-severe and asymptomatic cases remains unknown. This study aimed to investigate the role of hematological parameters in non-severe and asymptomatic Omicron variant infections. METHODS: Hematological parameters and results were analyzed and compared in symptomatic (n = 356) and asymptomatic (n = 171) groups respectively, and between these two groups with positive COVID-19 tests. The utility of hematological parameters for predicting positive COVID-19 tests was analyzed using receiver operating characteristic curves. RESULTS: Individuals with non-severe cases exhibited decreased levels of platelets, lymphocytes, eosinophils, basophils, lymphocytes (%), eosinophils (%), and basophils (%), while exhibiting elevated counts of monocytes, neutrophils (%), monocytes (%), neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio (PLR), and C-reactive protein (CRP) when compared to suspected cases or asymptomatic carriers. In asymptomatic patients, positive carriers had lower leukocyte, neutrophil, and lymphocyte counts but higher monocyte, monocyte (%), PLR, and CRP levels than negative carriers. Basophil counts combined with lymphocytes or the PLR demonstrated a more significant predictive value in screening non-severe cases earlier compared to other parameters. The combined assessment of the monocyte (%) and the PLR had the highest area under the curve for diagnosing asymptomatic carriers. CONCLUSIONS: Circulating basophils, alone or in combination with other hematological parameters, may be used as efficient biomarkers for early screening of non-severe Omicron cases.

TLR21 is involved in the NF-κB and IFN-ß pathways in largemouth bass (Micropterus salmoides) and interacts with TRIF but not with the Myd88 adaptor.

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-ß (IFN-ß) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-ß (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

Molecular characterization, expression and functional analysis of TAK1, TAB1 and TAB2 in Nile tilapia (Oreochromis niloticus).

The MAPK pathway is the common intersection of signal transduction pathways such as inflammation, differentiation and proliferation and plays an important role in the process of antiviral immunity. Streptococcus agalactiae will have a great impact on tilapia aquaculture, so it is necessary to study the immune response mechanism of tilapia to S. agalactiae. In this study, we isolated the cDNA sequences of TAK1, TAB1 and TAB2 from Nile tilapia (Oreochromis niloticus). The TAK1 gene was 3492 bp in length, contained an open reading frame (ORF) of 1809 bp and encoded a polypeptide of 602 amino acids. The cDNA sequence of the TAB1 gene was 4001 bp, and its ORF was 1491 bp, which encoded 497 amino acids. The cDNA sequence of the TAB2 gene was 4792 bp, and its ORF was 2217 bp, encoding 738 amino acids. TAK1 has an S_TKc domain and a coiled coil structure; the TAB1 protein structure contains a PP2C_SIG domain and a conserved PYVDXA/TXF sequence model; and TAB2 contains a CUE domain, a coiled coil domain and a Znf_RBZ domain. Homology analysis showed that TAK1 and TAB1 had the highest homology with Neolamprologus brichardi, and TAB2 had the highest homology with Simochromis diagramma (98.28 %). In the phylogenetic tree, TAK1, TAB1 and TAB2 formed a large branch with other scleractinian fishes. The tissue expression analysis showed that the expression of TAK1, TAB1 and TAB2 was highest in the muscle. The expression of TAK1, TAB1 and TAB2 was significantly induced in most of the tested tissues after stimulation with LPS, Poly I:C and S. agalactiae. The subcellular localization results showed that TAK1 was located in the cytoplasm, and TAB1 and TAB2 had certain distributions in the cytoplasm and nucleus. Coimmunoprecipitation (Co-IP) results showed that TRAF6 did not interact with the TAK1 protein but interacted with TAB2, while TAB1 did not interact with P38γ but interacted with TAK1. There was also an interaction between TAK1 and TAB2.

Nile tilapia TBK1 interacts with STING and TRAF3 and is involved in the IFN-ß pathway in the immune response.

Nile tilapia (Oreochromis niloticus) occupies an important position in the culture of economic fish in China. However, the high mortality caused by streptococcal disease has had a significant impact on the tilapia farming industry. Therefore, it is necessary to clarify the immune mechanism of tilapia in response to Streptococcus agalactiae. As a hub in the natural immune signaling pathway, the junction molecule can help the organism defend against and clear pathogens and is crucial in the signaling pathway. In this study, the cDNA sequence of Nile tilapia TBK1 was cloned, and the expression profile was examined in normal fish and challenged fish. The cDNA sequence of the TBK1 gene was 3378 bp, and its open reading frame (ORF) was 2172 bp, encoding 723 amino acids. The deduced TBK1 protein contained an S_TKc domain, a coiled coil domain and a ubiquitin-like domain (ULD). TBK1 had the highest homology with zebra mbuna (Maylandia zebra) and Lake Malawi cichlid fish (Astatotilapia calliptera), both at 97.59%. In the phylogenetic tree, TBK1 forms a large branch with other scleractinian fish. TBK1 expression was highest in the brain and lowest in the liver. LPS, Poly I:C, and S. agalactiae challenge resulted in significant changes in TBK1 expression in the tissues examined. The subcellular localization showed that TBK1-GFP was distributed in the cytoplasm and could significantly increase IFN-ß activation. Pull-down results showed that there was an interaction between TBK1 and TRAF3 and an interaction between STING protein and TBK1 protein. The above results provide a basis for further investigation into the mechanism of TBK1 involvement in the signaling pathway.

Angiotensin-Converting Enzyme 2 Inhibits Lipopolysaccharide-Caused Lung Fibrosis via Downregulating the Transforming Growth Factor ß-1/Smad2/Smad3 Pathway.

BACKGROUND: In our previous studies, angiotensin-converting enzyme 2 (ACE2) was shown to alleviate the severity of acute lung injury, but its effects on the development of lung injury-caused lung fibrosis have not been studied. METHODS: In the present study, the effects of ACE2 on lipopolysaccharide (LPS)-induced fibrosis in the lung were studied. The role of epithelial-mesenchymal transition (EMT) and that of the transforming growth factor ß-1 (TGF-ß1)/Smad2/Smad3 pathway in LPS-induced fibrosis in the lung were investigated. RESULTS: ACE2 expression in the mouse model of LPS-induced lung fibrosis was significantly increased. ACE2 activator diminazene aceturate (DIZE) significantly reduced pulmonary fibrosis, decreased alpha-smooth muscle actin expression, collagen I, hydroxyproline, and TGF-ß1 in the lung. DIZE significantly decreased TGF-ß1 expression and the activation of Smad2 and Smad3. ACE2 overexpression inhibited the LPS-induced EMT in MLE-12 cells (lung epithelial cells) and small interfering RNA treatment of ACE2 stimulated EMT. ACE2 overexpression also inhibited TGF-ß1 expression and activation of Smad2 and Smad3 in MLE-12 cells. Finally, after MLE-12 cells were treated with both ACE2 and TGF-ß1 plasmid, TGF-ß1 plasmid significantly abolished the effect of ACE2 plasmid on the EMT in MLE-12 cells. CONCLUSION: Combined with the in vivo study, it was revealed that ACE2 can suppress the TGF-ß1/Smad2/Smad3 pathway in lung type II epithelial cells, thus reversing their EMT and lung fibrosis. The present study provides basic research data for the application of ACE2 in lung injury-caused lung fibrosis treatment and clarifies the intervention mechanism of ACE2 in pulmonary fibrosis, which has potential value for clinical application. SIGNIFICANCE STATEMENT: Angiotensin-converting enzyme 2 (ACE2) can inhibit the epithelial-mesenchymal transition (EMT) in lung type II epithelial cells and lung fibrosis. ACE2 can regulate the transforming growth factor ß-1/Smad2/Smad3 pathway in lung type II epithelial cells, which may be the underlying mechanism of ACE2's effect on EMT and lung fibrosis.

Characterization of the core gut microbiota of Nile tilapia (Oreochromis niloticus): indication of a putative novel Cetobacterium species and analysis of its potential function on nutrition.

The genus Cetobacterium has been considered a dominant group of gut bacteria in many freshwater fish, and members of this genus contribute to anaerobic metabolism. Because of its significant place in the gut of freshwater fish, many studies on Cetobacterium were performed. Those studies mostly focused on the temporal and spatial changes of its abundance in fish intestine, which were affected by food or other environmental conditions. However, only a few studies isolated strains from genus Cetobacterium and reported their characteristics. In the present study, we performed 16S rRNA sequencing of the intestinal mucosa of Nile tilapia (Oreochromis niloticus) and found that Cetobacterium sp. existed widely in the foregut, midgut and hindgut mucosa, and a strain of Cetobacterium was successfully isolated from the gut of tilapia. We sequenced its whole genome and predicted it to be a novel candidate species of Cetobacterium sp. and named it NK01. The size of its genome was 3,095,946 bp, with a guanine + cytosine content of 28.8%. Among the identified genes, 2855 were predicted to be coding DNA sequences, 84 were tRNA and 34 were rRNA. We found that NK01 produced amino acids, including leucine, isoleucine, valine, glycine, alanine, phenylalanine and proline. Strain NK01 could use starch, sucrose, maltose, glucose, and mannose and synthesize and utilize glycogen. INV, GPI, malQ, malZ, sacA, scrK, glgC, glgA and glk, which were related to carbohydrate metabolism, were detected. yiaY and adhE, which oxidize ethanol to acetaldehyde and participate in a variety of metabolic pathways, were also present in the genome. No coding genes directly involved in acetate or butyrate production were detected. NK01 could also catabolize a variety of vitamins, and all genes involved in folate synthesis were detected, including folP, folC, folA and eutT, which converted vitamin B12s into vitamin B12 coenzyme. Here, we investigated the draft genome and in vitro function of Cetobacterium isolated from the intestinal tract of Nile tilapia. The results provided a preliminary understanding of the core microbiota of fish gut.

Genome survey of Misgurnus anguillicaudatus to identify genomic information, simple sequence repeat (SSR) markers, and mitochondrial genome.

BACKGROUND: The dojo loach Misgurnus anguillicaudatus is an important economic species in Asia because of its nutritional value and broad environmental adaptability. Despite its economic importance, genomic data for M. anguillicaudatus is currently unavailable. METHODS AND RESULTS: In the present study, we conducted a genome survey of M. anguillicaudatus using next-generation sequencing technology. Its genome size was estimated to be 1105.97 Mb by using K-mer analysis, and its heterozygosity ratio, repeat sequence content, GC content were 1.45%, 58.98%, and 38.03%, respectively. A total of 376,357 microsatellite motifs were identified, and mononucleotides, with a frequency of 42.57%, were the most frequently repeated motifs, followed by 40.83% dinucleotide, 7.49% trinucleotide, 8.09% tetranucleotide, and 0.91% pentanucleotide motifs. The AC/GT, AAT/ATT, and ACAG/CTGT repeats were the most abundant motifs among dinucleotide, trinucleotide, and tetranucleotide motifs, respectively. Besides, the complete mitochondrial genome was sequenced. Based on the Maximum Likelihood and Bayesian inference analyses, M. anguillicaudatus yingde in this study was the "introgressed" mitochondrial type. Seventy microsatellite loci were randomly selected from detected SSR loci to test polymorphic, of which, 20 microsatellite loci were assessed in 30 individuals from a wild population. The number of alleles (Na), observed heterozygosity (Ho), and expected heterozygosity (He) per locus ranged from 7 to 19, 0.400 to 0.933, and 0.752 to 0.938, respectively. All 20 loci were highly informative (PIC > 0.700). Eight loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.05). CONCLUSIONS: This is the first report of genome survey sequencing in M. anguillicaudatus, genome information, mitochondrial genome, and microsatellite markers will be valuable for further studies on population genetic analysis, natural resource conservation, and molecular marker-assisted selective breeding.

Isolation, identification, and pathogenic characteristics of Nocardia seriolae in largemouth bass Micropterus salmoides.

The largemouth bass Micropterus salmoides is an important freshwater aquaculture fish in China. Recently, largemouth bass at a fish farm in Guangdong province experienced an outbreak of a serious ulcer disease. As part of the investigations conducted to identify the aetiology and identify potentially effective control measures, we isolated a pathogenic bacterium (NK-1 strain) from the diseased fish. It was identified as Nocardia seriolae through morphological observation, physiological and biochemical analysis, and molecular identification, and its pathogenicity was verified by experimental infection. Pathological changes in the diseased fish included granulomatous lesions in the liver and spleen, destruction of renal tubules, necrosis of intestinal epithelial cells, infiltration of inflammatory cells in the brain, vacuolation of cells, and swelling and cracking of the mitochondria and endoplasmic reticulum. Bacterial detection using qPCR showed that the spleen and intestine were the main organs targeted by N. seriolae. The mortality of largemouth bass experimentally infected with N. seriolae at 21°C was significantly lower than that in fish infected at higher temperatures between 24 and 33°C; there were no significant differences in the levels of mortality at these higher temperatures. The level of mortality of largemouth bass infected with N. seriolae was lowest at a neutral water pH of 7 but increased significantly at higher and lower pH. Of the tested Chinese herbal medicines, Chinese sumac Galla chinensis and Chinese skullcap Scutellaria baicalensis exhibited the best antibacterial effects. This study lays a foundation for the clinical diagnosis and scientific control of ulcer disease in largemouth bass.

Nile tilapia TRIM39 recruits I3K413 and I3KL45 as adaptors and is involved in the NF-κB pathway.

Tripartite motif (TRIM) proteins play a regulatory function in cancer, cell apoptosis and innate immunity. To understand the role of TRIM39 in Nile tilapia (Oreochromis niloticus), TRIM39 cDNA was isolated. The total length of TRIM39 cDNA was 5025 bp. The deduced OnTRIM39 protein contains 549 amino acids and has conserved domains of the TRIM family, which are the RING, B-box, coiled-coil and PRY-SPRY domains. OnTRIM39 mRNA was widely expressed in various tissues. After challenge with Streptococcus agalactiae and stimulation with polyinosinic polycytidylic acid [poly (I:C)] and lipopolysaccharides (LPS), the amount of OnTRIM39 transcript was changed in various tested tissues. OnTRIM39 overexpression increased NF-κB activity. OnTRIM39 was present in the cytoplasm. Mass spectrometry of proteins pulled down with recombinant OnTRIM39 showed that 250 proteins potentially interact with OnTRIM39. The authors selected I3K4I3 from the 250 candidate proteins to verify its interaction with TRIM39. They also selected I3KL45, a member of the same 14-3-3 protein family, to verify its interaction with TRIM39. The results of pull-down assays showed that OnTRIM39 interacted with both I3K413 and I3KL45. These results contribute to further study of the innate immune mechanism of tilapia.

Transcriptomic response to three osmotic stresses in gills of hybrid tilapia (Oreochromis mossambicus female × O. urolepis hornorum male).

BACKGROUND: Osmotic stress is a widespread phenomenon in aquatic animal. The ability to cope with salinity stress and alkaline stress is quite important for the survival of aquatic species under natural conditions. Tilapia is an important commercial euryhaline fish species. What's more tilapia is a good experimental material for osmotic stress regulation research, but the molecular regulation mechanism underlying different osmotic pressure of tilapia is still unexplored. RESULTS: To elucidate the osmoregulation strategy behind its hyper salinity, alkalinity and salinity-alkalinity stress of tilapia, the transcriptomes of gills in hybrid tilapia (Oreochromis mossambicus ♀ × O. urolepis hornorum ♂) under salinity stress (S: 25‰), alkalinity stress(A: 4‰) and salinity-alkalinity stress (SA: S: 15‰, A: 4‰) were sequenced using deep-sequencing platform Illumina/HiSeq-2000 and differential expression genes (DEGs) were identified. A total of 1958, 1472 and 1315 upregulated and 1824, 1940 and 1735 downregulated genes (P-value < 0.05) were identified in the salt stress, alkali stress and saline-alkali stress groups, respectively, compared with those in the control group. Furthermore, Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted in the significant different expression genes. In all significant DEGs, some of the typical genes involved in osmoregulation, including carbonic anhydrase (CA), calcium/calmodulin-dependent protein kinase (CaM kinase) II (CAMK2), aquaporin-1(AQP1), sodium bicarbonate cotransporter (SLC4A4/NBC1), chloride channel 2(CLCN2), sodium/potassium/chloride transporter (SLC12A2 / NKCC1) and other osmoregulation genes were also identified. RNA-seq results were validated with quantitative real-time PCR (qPCR), the 17 random selected genes showed a consistent direction in both RNA-Seq and qPCR analysis, demonstrated that the results of RNA-seq were reliable. CONCLUSIONS: The present results would be helpful to elucidate the osmoregulation mechanism of aquatic animals adapting to saline-alkali challenge. This study provides a global overview of gene expression patterns and pathways that related to osmoregulation in hybrid tilapia, and could contribute to a better understanding of the molecular regulation mechanism in different osmotic stresses.

Overexpression of protein regulator of cytokinesis 1 facilitates tumor growth and indicates unfavorable prognosis of patients with colon cancer.

BACKGROUND: Protein regulator of cytokinesis 1 (PRC1) has been reported to play important role in the pathogenesis of various cancers. However, its role in colon cancer has not been studied. Here, we aimed to investigate the biological functions and potential mechanism of PRC1 in colon cancer. METHODS: The expression level of PRC1 in colon cancer tissues and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemical (IHC) staining of a tissue microarray (TMA). Furthermore, colon cancer cell lines HCT116 and SW480 were treated with short hairpin RNAs against PRC1. The biological function of PRC1 was determined by MTT proliferation, colony formation assay, cell cycle, and apoptosis assays. Then, an in vivo tumor formation assay was conducted to explore the effects of PRC1 on tumor growth. RESULTS: The mRNA and protein expression levels of PRC1 were highly expressed in colon cancer tissues and cell lines. PRC1 expression was associated with clinicopathological characteristics and overall survival of patients with colon cancer. Knockdown of PRC1 could decrease proliferation and colony forming ability of colon cancer cells, as well as arrested more cells at G2/M phase and promoted cell apoptosis. In cancer cells, the expression pattern of protein regulators included in cell cycle and apoptosis progress were reverted by PRC1 down-regulation. Additionally, PRC1 down-regulation could suppress colon tumor growth and differentiation. CONCLUSIONS: We confirmed that PRC1 was overexpressed in colon cancer and was associated with poor prognosis of colon cancer patients. PRC1 down-regulation could arrest cell cycle at G2/M stage, inhibit proliferation, and elicit apoptosis. These findings showed the potential of PRC1 to be used for therapeutic approaches in colon cancer.

The angiotensin-converting enzyme 2/angiotensin (1-7)/mas axis protects against pyroptosis in LPS-induced lung injury by inhibiting NLRP3 activation.

Previous studies have suggested that pyroptosis may play an important role in LPS-induced acute lung injury (ALI), but the exact mechanism of pyroptosis induction and the role of Angiotensin-converting enzyme 2 (ACE2)/Ang (1-7)/Mas axis in pyroptosis has not been investigated yet. The present study aimed to establish a mice model of ALI and clarify the involvement of pyroptosis and ACE2/Ang (1-7)/Mas axis. The results showed that LPS induced pyroptosis in lung, demonstrated by increased expression of Gasdermin D (GSDMD), cleaved GSDMD, IL-1ß, and Caspase-1. Treatment of Ang (1-7) significantly reduced the severity of ALI and pyroptosis, while AngII significantly exaggerated them. Furthermore, ACE2 activator resorcinolnaphthalein (RES) significantly reduced the severity of ALI and pyroptosis, but ACE2 inhibitor MLN-4760 and Mas inhibitor A779 significantly exaggerated them, suggesting that the ACE2/Ang (1-7)/Mas axis was involved in the pyroptosis in LPS-induced ALI. In addition, Ang (1-7) and RES significantly decreased the levels of NLRP3, which were increased by AngII and A779. NLRP3 knockout significantly reduced the severity of ALI and pyroptosis. In conclusion, pyroptosis played an important role in ALI induced by LPS. The ACE2/Ang (1-7)/Mas axis negatively regulated the pyroptosis and protected mice against LPS-induced ALI through NLRP3 inhibition. The present study expanded our understating of the role of ACE2/Ang (1-7)/Mas axis in ALI by providing a novel explanation that it may regulate the pyroptosis in ALI.

Molecular characterization, expression and functional analysis of IRAK1 and IRAK4 in Nile tilapia (Oreochromis niloticus).

Interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 are critical signalling mediators and play pivotal roles in the innate immune and inflammatory responses mediated by TLR/IL-1R. In the present study, two IRAK family members, OnIRAK1 and OnIRAK4, were identified in the Nile tilapia Oreochromis niloticus with a conserved N-terminal death domain and a protein kinase domain, similar to those of other fishes and mammals. The gene structures of OnIRAK1 and OnIRAK4 are organized into fifteen exons split by fourteen introns and ten exons split by nine introns. OnIRAK1 and OnIRAK4 were broadly expressed in all of the tissues tested, with the highest expression levels being observed in the blood and the lowest expression levels being observed in the liver. Both genes could be detected from 2 d post-fertilization (dpf) to 8 dpf during embryonic development. Moreover, the expression levels of OnIRAK1 and OnIRAK4 were clearly altered in all five tissues after Streptococcus agalactiae infection in vivo and could be induced by LPS, Poly I: C, S. agalactiae WC1535 and △CPS in Nile tilapia macrophages. The overexpression of OnIRAK1 and OnIRAK4 in 293T cells showed that they were both distributed in the cytoplasm and could significantly increase NF-κB activation. Interestingly, after transfection, OnIRAK1 significantly upregulated OnMyd88-induced NF-κB activation, while OnIRAK4 had no effect on OnMyd88-induced NF-κB activation. Co-immunoprecipitation (Co-IP) assays showed that OnMyd88 did not interact with either OnIRAK1 or OnIRAK4 and that OnIRAK1 did not interact with OnIRAK4. Taken together, these findings suggest that OnIRAK1 and OnIRAK4 could play important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

Identification and characterisation of tumour necrosis factor receptor (TNFR) associated factor 3 from Nile tilapia, Oreochromis niloticus.

In this study, we cloned the complementary (c)DNA sequences of tumour necrosis factor receptor (TNFR)-associated factor 3 (traf3) in Nile tilapia, Oreochromis niloticus. The expression patterns of the traf3 gene were investigated and preliminary functional analyses were performed. In healthy fish, traf3 transcript was broadly expressed in all examined tissues, with the highest expression level in the blood and the lowest in the liver. The traf3 gene reached its highest expression at 8 days post-fertilisation (dpf) during embryonic development. Moreover, we found that expression of traf3 was clearly altered following stimulation with Streptococcus agalactiae in vivo and that traf3 could be induced by lipopolysaccharides (LPS), Poly I: C and S. agalactiae WC1535 in Nile tilapia macrophages. Overexpression in 293T cells showed that Traf3 protein was mainly distributed in the cytoplasm and could significantly increase nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Taken together, these results implied that traf3 could play important roles in the immune response to pathogen invasion.

Digital gene expression analysis in the liver of ScpB-vaccinated and Streptococcus agalactiae-challenged Nile tilapia.

In recent years, streptococcal diseases have severely threatened the development of tilapia aquaculture, but effective prevention and control methods have not yet been established. To understand the immune responses of vaccinated Nile tilapia (Oreochromis niloticus), digital gene expression (DGE) technology was applied in this study to detect the gene expression profile of the Nile tilapia (O. niloticus) liver in response to ScpB (Streptococcal C5a peptidase from group B Streptococcus, ScpB) vaccination and a Streptococcus agalactiae-challenge. The control and the ScpB-vaccinated Nile tilapia yielded a total of 25,788,734 and 27,088,598 clean reads, respectively. A total of 1234 significant differentially expressed unigenes were detected (P < 0.05), of which 236 were significantly up-regulated, and 269 were significantly down-regulated (P < 0.05, |fold|>2, FDR<0.05). Of the differentially expressed gene, the identified genes which were enriched using databases of GO and KEGG could be categorized into a total of 67 functional groups and were mapped to 153 signaling pathways including 15 immune-related pathways. The differentially expressed genes (TLR1, TLR2, TLR3, TLR5, TLR9, MyD88, C3, IL-1ß, IL-10) were detected in the expression profiles, and this was subsequently verified via quantitative real-time PCR (qPCR). The results of this study can serve as a basis for future research not only on the molecular mechanism of S. agalactiae invasion, but also on the anti-S. agalactiae mechanism in targeted tissues of Nile tilapia.

Effects of Lactococcus lactis subsp. lactis JCM5805 on colonization dynamics of gut microbiota and regulation of immunity in early ontogenetic stages of tilapia.

The administration of probiotics during early ontogenetic stages can be an effective way to manipulate the gut microbiota of animals. Specifically, the administration of probiotics can enhance gut-colonization success and regulate the immune response. In this study, the effects of early contact with probiotic Lactococcus lactis subsp. lactis JCM5805 on the gut microbial assembly of larvae Nile tilapia were examined. The effects of JCM5805 on IFNα expression through the TLR7 and TLR9-dependent signal transduction pathway as well as larval disease resistance were studied. Three days postfertilization, embryos were randomly allocated into nine 30 L tanks with a concentration of 20 eggs L-1. Triplicate tanks were performed for each treatment. Treatments included a control group (C), a low probiotic concentration group (T1), where JCM5805 was added to the water at 1 × 104 cfu ml-1, and a high probiotic concentration group (T2), where JCM5805 was added to the water at 1 × 108 cfu ml-1. Probiotics were administered continuously for 15 days. qPCR was used to analyze transcript levels of the TLR7, TLR9, MyD88, IRF7 and IFNα genes using RNA extracted from whole embryos on day 5 and 10, and from the intestine of larvae on day 15. Transcription of these genes was also measured in the intestine, liver and spleen of larvae one month after the cessation of probiotic administration. The results showed that MyD88 and IRF7 were significantly elevated on days 5 and 10 in the T2 group. TLR9 and IFNα were also significantly elevated on days 5, 10 and 15 during probiotic application of T2 (P < 0.05). One month after the cessation of probiotics administration, no significant difference was observed in the expression of these genes (P > 0.05). The larvae were fed probiotics for 15 days and were infused with Streptococcus agalactiae strain WC1535 at a final concentration of 1 × 106 cfu ml-1. The survival rate of T2 was significantly higher than that of the C group (P < 0.05). Microbial characterization by Illumina HiSeq sequencing of 16S rRNA gene amplicons showed the significantly higher presence of JCM5805 in the guts of T2 after 15 days of probiotic continuous application. Although JCM5805 was below the detection level after the cessation of probiotic for 5 days, the gut microbiota of the exposed tilapia larvae in T2 remained clearly different from that of the control treatment after the cessation of probiotic administration. These data indicated that a high concentration of the probiotic strain JCM5805 upregulated the expression of IFNα via the TLR7/TLR9-Myd88 pathway and enhanced disease resistance of larvae. JCM5805 was only transiently detected and thus was not included in the stable larval microbiota. The early microbial exposure of tilapia larvae affects the gut microbiota at later life stages. However, whether the upregulation of related genes is related to the presence of JCM5805 strain in the intestine requires further verification.

MHC Class IIB gene polymorphisms associated with resistance/susceptibility to Streptococcus agalactiae in Nile tilapia Oreochromis niloticus.

Genetic variation in the major histocompatibility complex (MHC) Class IIB was tested in Nile tilapia Oreochromis niloticus, and the association between the MHC IIB alleles and disease resistance was also studied. F3 fry offspring (n = 1200) from 12 full-sib families were challenged with Streptococcus agalactiae, which caused significantly different mortalities in different Nile tilapia families (11.00-81.10%). Twenty fry (F1) from each of the 12 families were selected to study the polymorphisms of the MHC Class IIB gene using PCR followed by cloning and sequencing methods. The results showed that the size of the amplified fragment was 770-797 bp. Thirty-seven sequences from 240 individuals revealed 22 different alleles, which belonged to 9 major allele types. Up to 63.58% of nucleotide positions were variable, while the proportion of the amino acid variable positions was up to 68.73%. According to the survival rate of offspring (F3) from 12 full-sib families, we deduced that the alleles Orni-DAB*0107, Orni-DAB*0201 and Orni-DAB*0302 were highly associated with resistance to S. agalactiae, while the allele Orni-DAB*0701 was associated with susceptibility to S. agalactiae. In addition, our previous study found that the allele Orni-DAB*0201 was more frequently distributed in the disease-resistant groups. Therefore, the allele Orni-DAB*0201 could be used as an S. agalactiae resistance-related MHC marker in molecular marker-assisted selective breeding programs for S. agalactiae-resistant Nile tilapia.

Capsular polysaccharide of Streptococcus agalactiae is an essential virulence factor for infection in Nile tilapia (Oreochromis niloticus Linn.).

Streptococcus agalactiae (Group B Streptococcus, GBS) is associated with diverse diseases in aquatic animals. The capsule polysaccharide (CPS) encoded by the cps gene cluster is the major virulence factor of S. agalactiae; however, limited information is available regarding the pathogenic role of the CPS of serotype Ia piscine GBS strains in fish. Here, a non-encapsulated mutant (Δcps) was constructed by insertional mutagenesis of the cps gene cluster. Mutant pathogenicity was evaluated in vitro based on the killing of whole blood from tilapia, in vivo infections, measuring mutant survival in tilapia spleen tissues and pathological analysis. Compared to wild-type (WT) GBS strain, the Δcps mutant had lower resistance to fresh tilapia whole blood in vitro (p < 0.01), and more easily cleared in tilapia spleen tissue, and was highly attenuated in tilapia and zebrafish. Additionally, compared to the Δcps mutant, numerous GBS strains and severe tissue necrosis were observed in the tilapia spleen tissue infected with WT strains. These results indicated that the CPS is essential for GBS pathogenicity and may serve as a target for attenuation in vaccine development. Gaining a better understanding of the role, the GBS pathogenicity in fish will provide insight into related pathogenesis and host-pathogen interactions.

Effects of dietary Lactobacillus rhamnosus JCM1136 and Lactococcus lactis subsp. lactis JCM5805 on the growth, intestinal microbiota, morphology, immune response and disease resistance of juvenile Nile tilapia, Oreochromis niloticus.

The present study aimed to evaluate the individual and combined effects of Lactobacillus rhamnosus (LR) JCM1136 and Lactococcus lactis subsp. lactis (LL) JCM5805 on the growth, intestinal microbiota, intestinal morphology, immune response and disease resistance of juvenile Nile tilapia (Oreochromis niloticus). A total of 720 apparently healthy juvenile Nile tilapia (0.20 ± 0.05 g) were randomly divided into four equal groups. Fish were fed with a basal diet (CK) supplemented with JCM1136 (LR), JCM5805 (LL), and JCM1136 + JCM5805 (LR+LL) at 1 × 108 CFU/g basal diet for 6 weeks, followed by a basal diet for 1 week. After 6 weeks of feeding, the LL treatment significantly increased the growth and feed utilization of Nile tilapia when compared with the CK. Light microscopy and transmission electron microscopy images of the midgut revealed that probiotic supplementation significantly increased gut microvilli length and microvilli density compared to CK. The transcript levels of several key immune-related genes in the mid-intestine and liver of fish were analyzed by means of quantitative polymerase chain reaction (qPCR) at the end of the sixth week. The results showed the following: when compared to CK group, fish in LR had significantly increased transcript levels of IFN-γ, lyzc, hsp70 and IL-1ß in the intestine; LL fish showed significantly increased expressions of TNF-α, IFN-γ, lyzc, hsp70 and IL-1ß in the intestine and liver; and intestine lyzc, hsp70 and IL-1ß and liver TNF-α, IFN-γ, hsp70 and IL-1ß were significantly increased in LR+LL fish. Following a 6-week period of being fed probiotics or a control diet, the tilapia were challenged with an intraperitoneal injection of 20 µl of the pathogenic Streptococcus agalactiae (WC1535) (1 × 105 CFU/ml). The survival rates of the probiotic-fed groups were significantly higher than that of the CK group, and the LL group had the highest survival rate. High-throughput sequencing revealed a significantly higher presence of JCM5805 in the guts of LL fish during the period of probiotic application, but this was no longer detected in all LL samples 1 week post cessation of probiotic administration. Cessation of probiotic administration led to disorders of individual gut microbes within the LR and LL groups. Statistical analysis (LEfSe) demonstrated that three phyla, namely, Bacteroidetes, Fusobacteria and Actinobacteria were enriched in the CK group, while the abundance of Proteobacteria was greater in the probiotic-fed fish. At the genus level, Plesiomonas, which includes potential pathogens of fish, were significantly decreased in the probiotic-fed groups. In contrast, a significant increase of Rhizobium and Achromobacter, which can produce a variety of enzymes with cellulolytic and pectolytic activity, were observed in fish fed with probiotics, indicating that dietary probiotics were helpful in the propagation of some probiotic bacteria. Our data revealed that JCM1136 and JCM5805, as a feed additive at 108 CFU/g feed, could improve intestinal morphology, enhance immune status and disease resistance, and affect the gut microbiota of tilapia; thus, these additives could be used as probiotics for juvenile Nile tilapia. JCM5805 was more effective than JCM1136 or the mixture of the two for promoting the growth, enhancing the immune status and disease resistance of tilapia.