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The CXC chemokine receptors (CXCRs) are members of the seven transmembrane (7-TM) G-protein-coupled receptor superfamily that involves innate and adaptive immune systems. In this study, CXCR3a and CXCR3b from Nile tilapia (Oreochromis niloticus) were cloned and identified, designated as OnCXCR3a and OnCXCR3b. The open reading frames of OnCXCR3a and OnCXCR3b were 1074 and 1080 bp, encoding the predicted proteins of 357 and 359 amino acids, respectively. Multiple alignment analysis of OnCXCR3a- and OnCXCR3b-deduced protein sequences with the mammalian and bird sequences indicated the presence of typical structural features of chemokine receptors, including a 7-TM domain and conserved motifs. Quantitative real-time PCR analysis revealed that OnCXCR3a and OnCXCR3b were constitutively expressed in a wide range of tissues. When stimulated with Streptococcus agalactiae, Aeromonas hydrophila, polyinosinic:polycytidylic acid and lipopolysaccharide in vivo or in vitro on leukocytes, the mRNA levels of OnCXCR3a and OnCXCR3b were significantly upregulated. Overall, these results indicated that OnCXCR3s might be involved in host immune responses in Nile tilapia.

Plasma cells are terminally differentiated antibody-secreting B lymphocytes that contribute to humoral immunity by producing large numbers of antibodies. Increasing evidence suggests that teleost fish B cells share certain characteristics with mammalian B1 B cells, including antibody-secreting, phagocytic, and antigen-presenting capacities. However, the difference between mature B cells and plasma cells remains unclear. In this study, we found that, based on their light-scattering characteristics, tilapia anterior kidney (AK) leukocytes can be categorized into two IgM+ B-cell subsets: the lymphoid (L) gate and granulocyte–monocyte/macrophage (G-M) subsets. G-M gate cells are more numerous than L-gate cells and have higher mean fluorescence, but lower forward scatter and side scatter. We analyzed the morphological and ultrastructural features of sorted IgM+ cells and found that L-gate IgM+ cells have a high nucleus–cytoplasm ratio and lymphocyte-like morphology, whereas G-M gate IgM+ cells have a small nucleus, more abundant endoplasmic reticulum, and a larger number of mitochondria, and have a plasma cell-like or macrophage-like morphology. To further characterize the cell types, we examined the specific patterns of expression of B-cell- and T-cell-related genes. We found that B-cell-specific genes were expressed by both L-gate and G-M gate IgM+ cells, and that G-M gate IgM+ cells secreted extremely high levels of IgM. However, T-cell-related genes were highly expressed only in L-gate IgM– cells. These results suggest that G-M gate IgM+ cells are similar to plasma-like cells, with high antibody-secreting capacity. Given that G-M gate cells include the granulocyte, monocyte, and macrophage cell types, but not B cells, monocyte/macrophage markers were used to investigate the cell types further. A macrophage receptor with a collagenous structure was frequently observed, and macrophage-expressed gene-1 was highly expressed, in the G-M gate IgM+ cells. Phagocytic capacity, as determined by ingestion of beads or bacteria, was significantly higher in G-M gate IgM+ cells than in L-gate IgM+ cells, as was antigen-processing capacity. Our findings show that tilapia AK leukocytes can be divided into two IgM+ B-cell subsets and that G-M gate IgM+ cells resemble plasma-like cells, having high antibody-secreting, phagocytic, and antigen-presenting capacities. Thus, this study increases our understanding of the functions of teleost fish plasma-like cells.

Plasma cells are terminally differentiated antibody-secreting B lymphocytes that contribute to humoral immunity by producing large numbers of antibodies. Increasing evidence suggests that teleost fish B cells share certain characteristics with mammalian B1 B cells, including antibody-secreting, phagocytic, and antigen-presenting capacities. However, the difference between mature B cells and plasma cells remains unclear. In this study, we found that, based on their light-scattering characteristics, tilapia anterior kidney (AK) leukocytes can be categorized into two IgM

Complement factor H (CFH), a multifunctional soluble complement regulatory protein, can bind to a variety of pathogens and play a crucial role in host innate immune defense. To explore the functional characteristics of CFH (OnCFH) in Nile tilapia (Oreochromis niloticus), we cloned and characterized the open reading frame (ORF) of OnCFH in this study. The full-length of OnCFH ORF is 1359 bp, encoding 452 aa for a 48.85 kDa peptide, and its predicted structure containing six short complement-like repeats (SCRs). The analysis of tissue distribution showed that OnCFH was constitutively expressed in all tested tissues, with the highest in the liver. Upon Streptococcus agalactiae and Aeromonas hydrophila stimuli in vivo and in vitro, OnCFH mRNA transcript was significantly upregulated in head kidney tissue as well as head kidney monocytes/macrophages. Further, the recombinant OnCFH protein ((r)OnCFH) could bind to pathogenic bacteria in a dose-dependent. Moreover, it got involved in the regulation of inflammation as well as phagocytosis of monocytes/macrophages. The knockdown of OnCFH remarkably decreased the amount of bacteria in the head kidney. In summary, our data demonstrated that OnCFH could participate in the immune response of Nile tilapia against bacterial infection.

Chemokines are a class of small molecular weight cytokines of 6–14 kDa, exerting important roles in the regulation of various inflammatory diseases and immune cell migration. In this study, we have identified the CXCL12 gene from Nile tilapia (Oreochromis niloticus), including CXCL12a (OnCXCL12a) and CXCL12b (OnCXCL12b). The open reading frames of OnCXCL12a and OnCXCL12b are 309 and 297 bp, encoding 102 and 98 amino acids, respectively. Multiple alignment showed that OnCXCL12a and OnCXCL12b have characteristics of CXC chemokines and share high identity with CXCL12 amino acid sequences from the known species. Tissue distribution in the healthy fish indicated that OnCXCL12a and OnCXCL12b expressed in all examined tissues, with the highest expression in muscle and anterior kidney, respectively. After challenged by Streptococcus agalactiae, Poly(I:C) and LPS in vivo and in vitro, OnCXCL12 is transcriptionally up-regulated in immune tissues and cells significantly. The recombinant OnCXCL12 proteins, (r)OnCXCL12a and (r)OnCXCL12b, enhance the release of nitric oxide and increase the expression of inflammatory cytokines (TNF-α, IL-6, and IL-10) in anterior kidney leukocytes, as well as exhibit chemotactic activity for leukocytes from anterior kidney. Summarizing, these results indicate that OnCXCL12 is involved in the immune response of Nile tilapia against pathogen infection and may play an important role in mediating inflammatory response.

The gas-phase propylene epoxidation for noble metal system as a green process has confronted a great challenging task in metal utilization and stability. In this work, we realize the enhancement of Au capture efficiency and high-dispersed Au nanoparticles by supports modification and interfacial regulation to construct Au–Fe–TS-1 interface coupling structure. The Au capture efficiency of the optimum catalyst arrived at 69% and it displayed a 7.3% C3H6 conversion with a PO selectivity of 89.9%, giving a H2 efficiency of about 25.7%. Then some typical characterizations systematically elucidate that Fe is not only incorporated into TS-1 framework and captures Au species with high dispersion and stability, but also inhibits the losing of Ti species. Furthermore, Fe-modified catalysts change the property of Au active site and maintain lower apparent activation energy. This report may provide a new strategy to design advanced catalysts for enhancing metal utilization efficiency by the construction of interface coupling structure.

Present investigation was carried out to study toxicological damages of copper exposure and mitigation of its adverse effects with β-glucan administration in IgM

Copper (Cu), as an essential micronutrient in human and animal metabolism, easily spreads and excessively accumulates in rearing water, which make it more susceptible to fish farms and threatens the health of aquatic animals. In this issue, the protective effect of vitamin C against oxidative damage caused by copper exposure was studied in monocytes/macrophages (MO/MФ) and IgM

Teleost fish anterior kidney (AK) is an important hematopoietic organ with multifarious immune cells, which have immune functions comparable to mammalian bone marrow. Myeloid and lymphoid cells locate in the AK, but the lack of useful specific gene markers and antibody-based reagents for the cell subsets makes the identification of the different cell types difficult. Single-cell transcriptome sequencing enables single-cell capture and individual library construction, making the study on the immune cell heterogeneity of teleost fish AK possible. In this study, we examined the transcriptional patterns of 11,388 AK leukocytes using 10× Genomics single-cell RNA sequencing (scRNA-seq). A total of 22 clusters corresponding to five distinct immune cell subsets were identified, which included B cells, T cells, granulocytes, macrophages, and dendritic cells (DCs). However, the subsets of myeloid cells (granulocytes, macrophages, and DCs) were not identified in more detail according to the known specific markers, even though significant differences existed among the clusters. Thereafter, we highlighted the B-cell subsets and identified them as pro/pre B cells, immature/mature B cells, activated B/plasmablasts, or plasma cells based on the different expressions of the transcription factors (TFs) and cytokines. Clustering of the differentially modulated genes by pseudo-temporal trajectory analysis of the B-cell subsets showed the distinct kinetics of the responses of TFs to cell conversion. Moreover, we classified the T cells and discovered that CD3+CD4−CD8−, CD3+CD4+CD8+, CD4+CD8−, and CD4−CD8+ T cells existed in AK, but neither CD4+CD8− nor CD4−CD8+ T cells can be further classified into subsets based on the known TFs and cytokines. Pseudotemporal analysis demonstrated that CD4+CD8− and CD4−CD8+ T cells belonged to different states with various TFs that might control their differentiation. The data obtained above provide a valuable and detailed resource for uncovering the leukocyte subsets in Nile tilapia AK, as well as more potential markers for identifying the myeloid and lymphoid cell types.

CD5 is a type I transmembrane glycoprotein acting as a pleiotropic functional receptor in the mammalian immune response system, mainly presents on the surface of cells associated with the immune system, and is essential for the classification of B cells. In this study, we identify a CD5 homologue in Nile tilapia (Oreochromis niloticus). The open reading frame of OnCD5 is 507 bp, encoding 168 amino acids. The deduced amino acid sequence contains a signal peptide region, a transmembrane region and a conserved portion of the cytoplasmic region. Expression analysis indicates that the OnCD5 exhibits constitutive expression in the tested tissues, with the highest expression in thymus. Analysis of the OnCD5 transcription in the classified IgM+ and IgM− lymphocytes from anterior kidney, spleen and peripheral blood, and IgMlo and IgMhi lymphocytes from peripheral blood, indicates that the OnCD5 is highly expressed in the IgM + lymphocytes, especially in the IgMhi B lymphocytes. Furthermore, the OnCD5 expression is up-regulated significantly in anterior kidney and spleen following challenges of Aeromonas hydrophila and Streptococcus agalactiae in vivo and in vitro, likewise in IgM+ B lymphocytes sorted from peripheral blood upon stimulation with LPS. Further, the recombinant OnCD5 protein has the bacteria-binding activity. Taken together, these results reveal that OnCD5 participates in host's defense during pathogen infection, and may play an important role in tilapia B cells.

Pax5 (Paired Box 5), a nuclear transcription factor expressed in B cell specifically, is a key regulator for B cell activation. In this study, we cloned and identified a Pax5 gene (OnPax5) from Nile tilapia (Oreochromis niloticus), which has an open reading frame of 1278 bp, encoding deduced amino acid sequence of 425 residues. OnPax5 contains a conserved DNA-binding domain encoding the paired box, an octapeptide, a homeobox homology region, a transactivation and a repressor domain. OnPax5 is constitutively expressed in various analyzed tissues of tilapia, with a relatively high expression in lymphoid organs, including spleen (SPL), anterior kidney (AK), and thymus. What's more, OnPax5 is highly expressed in leukocytes especially in IgM+ lymphocytes sorted from peripheral blood (PBL), SPL and AK. When stimulated with lipopolysaccharide (LPS) in vivo, OnPax5 expression was significantly up-regulated in PBL, SPL and AK. Upon stimulation with LPS, pokeweed mitogen and mouse anti-OnIgM monoclonal antibody in vitro, the expression of OnPax5 was also significantly up-regulated in leukocytes from SPL and AK. Taken together, Pax5, the B cell lineage specific activator factor, might get involved in B cell activation in Nile tilapia.

CXC chemokine receptor 4 (CXCR4), a member of seven-transmembrane (7-TM) G-protein-coupled receptor superfamily, is the receptor of the CXC chemokine ligand 12 (CXCL12), and plays important roles in host defense and inflammation. In the current study, we cloned and identified a homolog of CXCR4 from Nile tilapia (Oreochromis niloticus), designated as OnCXCR4. The open reading frame of OnCXCR4 is 1149 bp encoding a peptide of 382 amino acids, and the predicted molecular weight is 42.65 kDa OnCXCR4 shares common features of CXCR4 family, including a 7-TM domain and a characteristic CXC motif (containing CYC). Expression analysis showed that OnCXCR4 constitutively expresses in various tested tissues of Nile tilapia, with the highest level in the anterior kidney. When stimulated with Streptococcus agalactiae, Aeromonas hydrophila, Poly(I:C), or LPS in vivo and in vitro, the expression of OnCXCR4 was significantly regulated. AMD3100, a CXCR4 antagonist, could not only inhibit the chemotactic activity of the recombinant OnCXCL12 protein on the leukocytes from anterior kidney, but also reduce the expression of OnCXCR4 significantly. Taken together, these results of our study above indicate that OnCXCR4 may play important roles in host defense against bacterial infectionin in Nile tilapia, and being a receptor of OnCXCL12 to exert functions.

Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1), a kind of protein tyrosine phosphatases (PTPs), is a critical regulator of antigen receptor signal transduction. Signal transduction of BCR is regulated by phosphatases in teleost as in mammals. In this study, SHP1 from Nile tilapia (Oreochromis niloticus) (OnSHP1) was identified and characterized, including the expression pattern against bacterial infection and regulation function in BCR signaling pathway. The open reading frame of OnSHP1 contains 1749 bp of nucleotide sequence, encoding a protein of 582 amino acids. The OnSHP1 protein was highly conversed compared to that of other species, including two amino-terminal SH2 domains at the N terminus and a PTP catalytic domain. Transcriptional expression analysis revealed that OnSHP1 was detected in all examined tissues and highly expressed in spleen. The up-regulated OnSHP1 expression was observed in peripheral blood, spleen and anterior kidney following challenge with Streptococcus agalactiae or lipopolysaccharide (LPS) in vivo, as well as that displayed in leukocytes stimulated with S. agalactiae or LPS in vitro. Further, after induction with mouse anti-tilapia IgM monoclonal antibody in vitro, OnSHP1 was significantly up-regulated in leukocytes. When spleen leukocytes treated with PTP Inhibitor II in vitro, the phosphorylation level of OnSHP1 at the phosphorylation sites (Y535 and Y557) and the cytoplasmic free Ca2+ concentration were up-regulated significantly. Overall, the findings of this study indicate that SHP1 gets involved in host defense against bacterial infection and BCR signaling pathway in Nile tilapia.

B lymphocyte-induced maturation protein 1 (Blimp-1), a transcription factor containing zinc finger, is required and sufficient to trigger terminal differentiation of B cells in mammals. The Blimp-1 (OnBlimp-1) from Nile tilapia (Oreochromis niloticus) was identified and characterized its expression pattern during B cell activation and maturation. The cDNA of OnBlimp-1 open reading frame is 2547 bp encoding a protein of 848 amino acids and the predicted molecular weight is 93.36 kDa. OnBlimp-1 contains a SET domain and five ZnF_C2H2 domains, which shares high homology with that of other species. OnBlimp-1 transcription was detected in all examined tissues with high expression in the spleen (SPL). Analysis of sorted lymphocyte populations, including IgM+ and IgM− cells from peripheral blood (PBL), SPL and anterior kidney (AK), indicated that the OnBlimp-1 transcription was highly expressed in the IgM+ B cells. Upon LPS stimulation, OnBlimp-1 expression was up-regulated in tissues of PBL, SPL and AK significantly. The expression of OnBlimp-1, as well as the secreted IgM, was significantly up-regulated in the SPL and AK leukocytes stimulated with anti-OnIgM monoclonal antibody and LPS in vitro, respectively. Above results suggest that OnBlimp-1, a cytokine regulating the terminal differentiation of activated B cells to antibody-secreting cells, is likely to play important roles in B cell activation and maturation in Nile tilapia.

It is well reported that ultraviolet-C (UV-C) treatment enhances the storage or shelf life of many fruits and vegetables. However, biochemical changes associated with this treatment are largely unknown in leaf vegetables. The object of the present study is to evaluate the effects of UV-C treatment on the postharvest quality and biochemistry of three kinds of leaf vegetables (i.e. spinach, leek and cabbage). These vegetables were respectively exposed to single and multiple UV-C irradiations at the same energy level of 2.46 kJ/m 2 , and subsequently stored at 4 °C for 5 days. Total soluble protein, vitamin C and chlorophyll a were determined every day. As a result, the multiple UV-C treatment maintained significantly better quality parameters of postharvest leaf vegetables over the experiment, compared to the single treatment ( p

Complement component 1q (C1q), together with C1r and C1s to form C1, recognize and bind immune complex to initiate the classical complement pathway. In this study, C1q subunit molecules (XlC1qA, XlC1qB, XlC1qC) were cloned and analyzed from Xenopus laevis (X. laevis). The open reading frame (ORF) of XlC1qA is 819 bp of nucleotide sequence encoding 272 amino acids, the ORF of XlC1qB is 711 bp encoding 236 aa, and the XlC1qC is consists of 732 bp encoding 243 aa. The deduced amino acid sequences contain a collagen-like region (CLR), Gly-X-Y repeats in the N-terminus and a C1q family domain at the C-terminus. Phylogenetic analysis revealed that the XlC1qs are clustered with the amphibian clade. Expression analysis indicated that the XlC1qs exhibited constitutive expression in all examined tissues, with the highest expression in liver. Additionally, XlC1q could interact with heat-aggregated mouse IgG and IgM, Xenopus IgM and Nile tilapia IgM, respectively, indicating the functional conservation of XlC1q binding to immunoglobulins. Further, XlC1qs can inhibit C1q-dependent hemolysis of sensitized sheep red blood cells with concentration-dependent manner. These data collectively suggest that the function of C1qs in X. laevis may be conserved in interaction with immunoglobulins, as that of mammals and teleosts.

CD79, composed of two distinct chains called CD79a and CD79b, is a transmembrane protein that forms a B cell antigen receptor with membrane immunoglobulin, and generates a signal following antigen recognition by the B cell receptor. In this study, the CD79a (OnCD79a) and CD79b (OnCD79b) were cloned and identified from Nile tilapia (Oreochromis niloticus). The cDNA of ORF for OnCD79a and OnCD79b are 669 and 627 bp, coding 222 and 208 amino acids, respectively. The deduced protein analysis showed that both CD79a andCD79b contain an immunoreceptor tyrosine-based activation motif in their intracellular tails that used to propagate a signal in a B cell. Expression analysis revealed that both CD79a and CD79b expressed at high levels in immune tissues, such as anterior kidney and spleen, and in IgM+ B cells. Upon Streptococcus agalactiae (S. agalactiae) infection, the expressions of OnCD79a and OnCD79b were significantly up-regulated in anterior kidney and spleen. The significant up-regulations of OnCD79a and OnCD79b were also detected in leukocytes after in vitro challenge with S. agalactiae. Further, stimulations of LPS and anti-OnIgM monoclonal antibody induced significant up-regulations of OnCD79a and OnCD79b in leukocytes. Taken together, the results of this study indicated that CD79 molecule, playing roles in BCR signaling, was likely to get involved in host defense against bacterial infection in Nile tilapia.

The entomopathogenic nematode Heterorhabditis spp. is considered a promising agent in the biocontrol of injurious insects of agriculture. However, different symbiotic bacteria associated with the nematode usually have different specificity and virulence toward their own host. In this study, two symbiotic bacteria, LY2W and NK, were isolated from the intestinal canals of two entomopathogenic nematode Heterorhabditis megidis 90 (PDSj1 and PDSj2) from Galleria mellonela, separately. To determine their species classification, we carried out some investigations on morphology, culture, biochemistry, especially 16S rDNA sequence analyses. As a result, both of them belong to Enterobacter spp., showing the closest relatedness with Enterobacter gergoviae (LY2W) and Enterobacter cloacae (NK), respectively. Moreover, the toxicity to Galleria mellonella was examined using both the metabolites and washed cells (primary and secondary) of these two strains. The results indicated both metabolites and cells of the primary-type bacteria could cause high mortalities (up to 97%) to Galleria mellonella, while those of the primary-type bacteria only killed 20%. These findings would provide new symbiotic bacteria and further references for biological control of the agricultural pest.