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CD4 and CD8 molecules play an important role in the identification of T lymphocytes, and diverse among fish species. In this study, CD4-1 and CD8α gene of spotted sea bass (Lateolabrax maculatus) were cloned, polyclonal antibodies against CD4-1 (CD4-1 pAbs) and CD8α (CD8α pAbs) were produced, respectively. And the variations in CD4-1

A specialized lymphoepithelial tissue termed the interbranchial lymphoid tissue (ILT) is recently identified in several fish species. However, the structural variation and mucosal immune functions of the ILT remain largely unknown. In this study, the anti-Zap-70 MAb was firstly determined to specifically recognize ZAP-70 protein, and CD4-1

Interleukin-2 receptor (IL-2R), as the specific ligand of interleukin-2 (IL-2), binds to IL-2 and transmits signals and then can induce the proliferation of T lymphocytes in mammals. In this paper, the subunit of IL-2R in flounder (Paralichthys olivaceus), interleukin-2 receptor subunit gamma (IL-2Rγ) was cloned, and polyclonal antibodies (Abs) against its extracellular region were produced, then the expression of flounder IL-2Rγ (fIL-2Rγ) at transcriptional and cellular levels were characterized. Moreover, the interaction of flounder IL-2 (fIL-2) with fIL-2Rγ was investigated, and the variations on CD4+/IL-2Rγ+ cells in flounder after treatment with recombinant IL-2 (rIL-2), anti-IL-2Rγ Abs were detected, respectively. The results showed that fIL-2Rγ protein had a typical fibronectin type III (FN3) domain. The Abs could specifically recognize native fIL-2Rγ molecules at 39.9 kDa. FIL-2Rγ was localized on both T and B lymphocytes, and the percentages of CD4+/IL-2Rγ+ and IgM+/IL-2Rγ+ lymphocytes were high in spleen. In addition, pBiFC-VN173-IL-2Rγ plasmids could bind to pBiFC-VC155-IL-2 plasmids. The percentage of CD4+/IL-2Rγ+ lymphocytes was significantly decreased after blocking with anti-IL-2Rγ Abs both in vivo and in vitro. In the meantime, four T cell markers genes and six IL-2-IL-2R pathway genes were down-regulated in anti-IL-2Rγ Abs group. These results first demonstrated that fIL-2Rγ molecules were expressed on both T and B lymphocytes in flounder, and the bond between fIL-2Rγ and fIL-2 activated the CD4 T lymphocytes. This study gave a new sight into the exploration of IL-2R function on T lymphocytes proliferation in fish.

Hirame novirhabdovirus (HIRRV), as a highly pathogenic fish virus, is frequently prevalent in a variety of aquaculture fish in recent years, which seriously threatens the healthy development of aquaculture industry. Epidemiological studies show that the outbreak of HIRRV is obviously temperature dependent. Virus proliferation experiments in vitro and in vivo at different temperatures indicate the antiviral response of flounder is a main reason affect the replication of HIRRV. The RNA-Seq was used to analyze the different antiviral response in flounder which infected with HIRRV at different temperatures, the experiment set two temperatures of 10 °C and 20 °C. The flounder infected with HIRRV by artificial injection, and the spleens were collected at 24 h after infection. Meanwhile, the fish injected with EPC supernatant at different temperatures were set as control groups. It can obtain four pairwise comparison groups if determine a single variable, and the differentially expressed genes were further selected. The results showed that after infection with HIRRV at 10 °C and 20 °C, the differentially expressed genes in spleen were significantly enriched in inflammatory and immune-related pathways like Arachidonic acid metabolism, Cytokine-cytokine receptor interaction, Toll-like receptor (TLR) signaling pathway, RIG-I-like receptor (RLRs) signaling pathway, NOD-like receptor (NLR) signaling pathway and Cytosolic DNA-sensing pathway etc. In addition, the expression of phagocytes, lysosomes, endocytosis related genes were significantly upregulated at high temperature whether HIRRV positive or not. But compared to the infected flounder at 10 °C, some genes of RLRs signaling pathway were significantly upregulated at 20 °C, it can be speculated that RLRs pathway may be related to the anti-HIRRV response of flounder. Therefore, key genes of RLRs signaling pathway including mda5, lgp2, mita, mavs, irf3, irf7, ifn I-3 and ifn-γ were selected, and the temporal expression patterns of these genes in infected flounder at different temperatures were further detected by qRT-PCR. The results showed that HIRRV infection can significantly stimulate and activate the RLRs pathway of flounder, and the response level of this pathway was significantly higher at 20 °C than 10 °C. In general, this study provides important data for the further study about the pathogenesis of HIRRV infection in flounder.

In our previous study, a DNA plasmid encoding the VAA gene of Vibrio anguillarum was constructed and demonstrated to confer moderated protection against V. anguillarum challenge. Here, a bicistronic DNA vaccine (pVAA-IRES-IL2), co-expressing the VAA gene of V. anguillarum and Interleukin-2 (IL2) gene of flounder, was constructed to increase the protective efficacy of VAA DNA vaccine. The potential of pVAA-IRES-IL2 to express both VAA and IL2 in transfected HINAE cell lines was confirmed by immunofluorescence assay. Further, the variation of sIgM+, CD4-1+, CD4-2+ lymphocytes and production of VAA-specific antibodies in flounder, which was intramuscularly immunized with three DNA plasmids (pIRES, pVAA-IRES, pVAA-IRES-IL2), were investigated, respectively. The bacterial burden and relative percentage survival (RPS) of flounder exposed to V. anguillarum infection were both analyzed to evaluate the efficacy of bicistronic DNA plasmid. Our results revealed that the percentages of sIgM+, CD4-1+, CD4-2+ lymphocytes and antibodies specific to VAA were remarkably increased in pVAA-IRES or pVAA-IRES-IL2 immunized fish. Moreover, the co-expression of IL2 enhanced the immune response in response to VAA DNA vaccination, as shown by the higher percentages of sIgM+, CD4-1+, CD4-2+ lymphocytes and production of specific antibody. Importantly, the RPS in pVAA-IRES-IL2 and pVAA-IRES groups reached 64.1% and 51.3%, respectively, when compared with the 97.5% cumulative mortality in pIRES group. Furthermore, the number of V. anguillarum in liver, spleen and kidney of pVAA-IRES or pVAA-IRES-IL2 immunized flounder after V. anguillarum challenge was significantly reduced, as compared to that in pIRES group. These suggest that the bicistronic DNA vaccine can be an effective immunization strategy in inducing immune response against V. anguillarum infection and IL2 has the potential as the adjuvant for VAA DNA vaccine.

B cells have been found to have phagocytic activity in recent years, but the studies exploring the regulation mechanisms are still lacking to date. In the present study, the recombinant interleukin-10 (rIL-10) was obtained to study the function of IL-10 on phagocytosis of flounder (Paralichthys olivaceus) mIgM+ B lymphocytes. Flow cytometric analysis showed that IL-10 significantly enhanced the phagocytosis of Edwardsiella tarda but not Lactococcus lactis by mIgM+ B lymphocytes. Moreover, significantly higher intracellular ROS levels were detected in mIgM+ B lymphocytes following rIL-10 stimulation. The qRT-PCR analysis showed that rIL-10 could upregulate the expressions of IL-10Rb and Stat3 in mIgM+ B lymphocytes, suggesting that IL-10 might modulate the phagocytosis of mIgM+ B lymphocytes by activating IL-10R and Stat3. In addition, we also found that the enhancing effect of IL-10 on phagocytosis and intracellular ROS levels of mIgM+ B lymphocytes were suppressed by the administration of niclosamide. These results collectively demonstrated that IL-10 enhanced mIgM+ B lymphocyte-mediated phagocytosis of E. tarda and intracellular bactericidal ability, and IL-10R and Stat3 might play a curial role in the regulation of IL-10-stimulated phagocytosis, which would deepen our understanding of regulation mechanism of B cell phagocytosis.

Outer membrane protein K (OmpK) is an immunogenic protein that could act as subunit vaccine candidate for Vibrio anguillarum. In this study, a DNA vaccine encoding the OmpK gene of V. anguillarum was constructed and confirmed to express OmpK in vitro and in vivo. To evaluate the potential of pcDNA3.1-OmpK (pOmpK) as vaccine candidate, the humoral and cellular immune responses, and protective effects were analyzed in flounder model. The results showed that the transcription and translation of OmpK gene occurred in both transfected hirame natural embryo (HINAE) cells and injected fish muscles, indicating the functionality of pOmpK to express OmpK. Fish immunized with pOmpK showed significant increase of surface IgM positive (sIgM+), CD4-1+, CD4-2+ lymphocytes and production of specific anti-V. anguillarum or anti-rOmpK antibodies, which indicate the activation of humoral and cellular immune responses after vaccination. Moreover, a relative percent survival (RPS) rate of 50.00% against V. anguillarum infection was obtained for flounder immunized with pOmpK. In conclusion, this study indicates that pOmpK is able to induce humoral and cellular immune responses and can be used as a DNA vaccine candidate.

In order to elucidate the influence of T lymphocytes subpopulations on B lymphocytes immune response, in this paper, CD4-1+, CD4-2+, CD8+ T lymphocytes and B lymphocytes responses to thymus-independent (TI) or thymus-dependent (TD) antigen plus immunosuppressant were investigated in flounder (Paralichthys olivaceus). The results showed that in LPS-immunized group, the percentages of CD4-1+, CD4-2+, CD8β+ T (PCD4-1+ T, PCD4-2+ T and PCD8β+ T) lymphocytes in peripheral blood leucocytes (PBLs) had no significant variations, the percentages of IgM+ B (PIgM+ B) lymphocytes and LPS-specific antibodies (LA) significantly increased and peaked at 3rd or 4th week post-injection; CsA had no inhibition on both T/B lymphocytes and LA; RaPa only suppressed the PIgM+ B lymphocytes and LA, and the inhibition maximum (Imax) were about 35% and 20%, respectively. In KLH-immunized group, the PCD4-1+, PCD4-2+ and PCD8β+ T lymphocytes significantly increased and peaked at 3rd or 5th day, successively the PIgM+ B lymphocytes and KLH-specific antibodies (KA) significantly increased to the peak at 5th week; the PCD4-1+, PCD4-2+ T and PIgM+ B lymphocytes and LA were inhibited significantly by both CsA and RaPa, and the Imax on them were 13%–33%, 11%–25%, 19%–34%, 22%–26%, respectively, while the PCD8β+ T lymphocytes showed no significant suppression. The results indicated that the suppression of PIgM+ B lymphocytes in KLH + CsA group was not directly derived from CsA, but due to the suppression of T lymphocytes, especially CD4+ T lymphocytes subpopulations. The results showed for the first time that, similar to higher vertebrates, T lymphocytes didn't respond to TI antigen, moreover, T lymphocyte subpopulations had a regulation on the immune response of B lymphocyte for TD antigen in flounder.

Previous studies have demonstrated that protein tyrosine phosphorylation plays an important role in WSSV infection. In the present work, in order to further elucidate the potential role of protein tyrosine phosphorylation in white spot syndrome virus (WSSV) infection. The expression variation of tyrosine phosphorylated proteins in hemocytes of shrimp (Litopenaeus vannamei) after WSSV infection were examined by flow cytometric immunofluorescence assay (FCIFA) and enzyme linked immunosorbent assay (ELISA), and results showed that the level of protein tyrosine phosphorylation in hemocytes fluctuated significantly after WSSV infection and exhibited two peaks at 6 and 24 h post infection (hpi). Meanwhile, tyrosine phosphorylated proteins in hemocytes after WSSV infection were also detected by cell immunofluorescence, and results showed that the fluorescence intensity in hemocytes was altered with the course of WSSV infection and showed stronger fluorescent signals at 6 and 24 hpi compared to other time points. Furthermore, two dimensional gel electrophoresis (2-DE) and 2-DE western blotting were applied to identify the differentially expressed tyrosine phosphorylated proteins in hemocytes before and after WSSV infection. The result of 2-DE western blotting showed that there were nine tyrosine phosphorylated proteins in the hemocytes of healthy shrimp, whereas twenty-one tyrosine phosphorylated proteins were detected in the hemocytes of shrimp at 6hpi. Then, the differential tyrosine phosphorylated proteins were analyzed by Mass Spectrometry (MS), and eight of them were identified to be sodium/potassium-transporting ATPase subunit alpha, ubiquitin/ribosomal L40 fusion protein, actin-D, phosphopyruvate hydratase, beta-actin, ATP synthase subunit beta, receptor for activated protein kinase c1 and protein disulfide-isomerase. Moreover, the expression levels of sodium/potassium-transporting ATPase subunit alpha, ubiquitin/ribosomal L40 fusion protein, phosphopyruvate hydratase, ATP synthase subunit beta, receptor for activated protein kinase c1 and protein disulfide-isomerase were examined to be up-regulated post WSSV infection by quantitative real-time RT-PCR. Taken together, these results demonstrated that protein tyrosine phosphorylation was involved in the process of WSSV infection, which might play an important role in the immune response to WSSV infection in shrimp.

Vibrio anguillarum and Edwardsiella tarda are severe aquaculture pathogens shared similar epidemiological characteristics and susceptible to flounder (Paralichthys olivaceus). In our previous studies, recombinant(r) protein heat shock protein 33 (rHsp33) from V. anguillarum and outer membrane protein C (rOmpC) from E. tarda were proved to have protection against V. anguillarum and E. tarda, respectively. In this paper, the cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum, and the protection of divalent vaccine candidate (rHsp33 + rOmpC, rHC) against both V. anguillarum and E. tarda were evaluated. RHC, rHsp33, and rOmpC were vaccinated to flounder, respectively, and the percentages of surface immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBLs), serum IgM, specific antibodies against V. anguillarum or E. tarda, specific antibodies against rHsp33, rOmpC or rHC, the expression of immune-related genes and relative percent survival (RPS) against V. anguillarum or E. tarda were measured. The results showed that: RHC could induced the enhancement of sIg + cells and high levels of specific antibodies against both V. anguillarm and E. tarda; Also a significant increase of specific antibodies against rHsp33, rOmpC or rHC, and up-regulation of gene expression of CD3, CD4-1, CD4-2, CD8α, CD8β and IgM in spleen, head-kidney, and hindgut, RPS of 70 ± 3.45% against V. anguillarum and 60 ± 1.48% against E. tarda, respectively. In addition, rHsp33 induced specific antibodies against E. tarda and rOmpC, and had a RPS of 43.3 ± 3.73% against E. tarda; rOmpC could evoke specific antibodies against V. anguillarum and rHsp33, and had a RPS of 44 ± 1.27% against V. anguillarm; The results demonstrated that there was cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum, rHC as a divalent vaccine can induce significant immune response and efficient protection against both E. tarda and V. anguillarum in flounder.

In our previous work, granulocytes and hyalinocytes were successfully separated by immunomagnetic bead (IMB) method using monoclonal antibodies (mAbs) against granulocytes of shrimp (Fenneropenaeus chinensis). In order to elucidate the proteomic differentiation between granulocytes and hyalinocytes, in this paper, the differentially expressed proteins were analyzed between non-fixed/un-permeabilized (NFP) haemocytes and fixed/permeabilized (FP) haemocytes using two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry (MS). Then the FP haemocytes were separated into two haemocyte subpopulations using IMB method, and the comparative proteome between granulocytes and hyalinocytes was investigated. The results showed that 10 differentially expressed protein spots were detected and identified as 4 proteins in the NFP haemocytes. Twenty one differentially expressed proteins were successfully identified between granulocytes and hyalinocytes, which include 4 unique expressed proteins in granulocytes, 4 significantly highly expressed proteins in granulocytes, and 13 significantly high expressed proteins in hyalinocytes. According to Gene Ontology annotation, the identified proteins between granulocytes and hyalinocytes were classified into six categories, including binding proteins, proteins involved in catalytic activity, enzyme regulator activity, structural molecule activity, translation regulator activity, and ungrouped proteins. Furthermore, quantitative PCR confirmed that the trend of transcription levels of three selected genes were consistent with the proteomic data from 2-DE. The results may lead to better understanding of the functions of haemocyte subpopulations.

The CD3 complex is an important cell surface marker of T lymphocytes and essential for T lymphocytes activation in higher vertebrates. In the present work, the CD3e of Japanese flounder (Paralichthys olivaceus) was recombinantly expressed in E. coli BL21 (DE3) and used as an immunogen to produce mouse anti-rCD3e polyclonal antibodies, which could specifically recognize a 20 kDa protein in the membrane proteins of peripheral blood lymphocytes (PBL) of Japanese flounder by co-immunoprecipitation assay. Mass spectrometric analysis showed the 20 kDa protein was the native CD3e of Japanese flounder. Both the flow cytometric analysis and double immunofluorescence assay (DIFA) showed that the CD3+ T lymphocytes could be identified specifically by the mouse anti-rCD3e polyclonal antibodies, which didn't cross-react with the sIgM+ lymphocytes. Immunohistochemistry showed that CD3+ T lymphocytes could be detected in gill, skin, stomach, intestine, spleen, liver, head-kidney and mid-kidney. Flow cytometric analysis showed the percentages of CD3+ T lymphocytes in the PBL, spleen lymphocytes (SL) and head-kidney lymphocytes (HKL) of Japanese flounder increased rapidly after immunization with formalin-inactivated Edwardsiella tarda, and reached their peak levels at 5th day with 12.6%, 9.7% and 8.7%, respectively, and then decreased gradually. These results suggested that CD3+ T lymphocytes play important roles in mucosal and cell-mediated immunity, and the results would deepen our understanding on the roles of teleost T lymphocytes in the immune response.

In our previous work, two monoclonal antibodies (Mabs) against granulocytes of shrimp (Fenneropenaeus chinensis) had been produced, in this paper, haemocyte subpopulations were analyzed by flow cytometry (FCM) using the Mabs. Then immunomagnetic bead (IMB) method was applied for separation hyalinocytes and granulocytes using the Mabs. The separated hyalinocytes and granulocytes were analyzed by FCM, indirect immunofluorescence assay, Giemsa staining and transmission electron microscopy, respectively. The results showed the proportion of hyalinocytes in haemolymph of F. chinensis was 15.14 ± 1.22%, and that of granulocytes was 75.43 ± 2.31%. After two times separation by IMB, the purity rate of hyalinocytes and granulocytes was 96.27 ± 1.06% and 98.13 ± 0.86%, respectively. The hyalinocytes possessed 0.60-0.85 in nucleus/cytoplasm (N/C) ratio and had few granule in cytoplasm, whereas the separated granulocytes with N/C ratio of 0.12-0.36 and high electronic density of double membrane granules. The results reported the separation of haemocyte subpopulations using Mabs in shrimp for the first time, and the hyalinocytes and granulocytes isolated by IMB could be used for their differential protein analysis.

Our previous study has demonstrated that an integrin β subunit of Chinese shrimp (Fenneropenaeus chinensis) (FcβInt) involved in WSSV infection. In order to further elucidate the potential role of the FcβInt in the WSSV infection, expression response of FcβInt to WSSV infection in shrimp hemocytes was investigated after intra-muscular injection with the virus. Following time-course hemocytes sampling, the expression variation of FcβInt in hemocytes was examined by flow cytometric immunofluorescence assay (FCIFA) and enzyme linked immunosorbent assay (ELISA) using the monoclonal antibody (Mab) 2C5 against FcβInt, which was successfully produced with recombinant partial FcβInt and exhibited binding to a 120 kDa hemocyte protein. Meanwhile, the dynamic state of FcβInt mRNA level and WSSV copies in hemocytes were determined by quantitative real-time PCR. The result of FCIFA showed that FcβInt was mainly expressed on the semi-granular and granular cells, which was down-regulated at 6 h post infection (p.i.), and significantly increased to the peak level at 12 h p.i., then decreased to the control level by 24 h. However, FcβInt on the hyaline cells was lowly expressed and didn't show active response to the viral infection. The variation of FcβInt concentrations in total hemocytes determined by ELISA was roughly in accordance with the changing tendency of FcβInt expressed on the semi-granular and granular cells. FcβInt mRNA level in total hemocytes was significantly up-regulated to the peak level at 12 h p.i. Moreover, the number of WSSV copies in hemocytes began to exhibit a significant increase at 24 h p.i. The present study demonstrated that WSSV infection would induce a differential regulation of FcβInt expression in different type hemocytes, which provided valuable evidences for the close correlation between FcβInt and WSSV infection.

Phenoloxidase (PO) was purified from hemocytes of the scallop Chlamys farreri using native-PAGE and gel permeation column chromatography, and then substrate specificity and antibacterial activity generated from reaction products of purified PO were analyzed. The results showed purified PO had a molecular mass of 576 kDa in native-PAGE and 53 kDa in denatured PAGE, and could catalyze the substrates L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine, catechol and hydroquinone suggesting it is a type of p-diphenoloxidase. Using dopamine as a substrate, PO reaction products significantly inhibited the growth of Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas salmonicida. No significant inhibition was found in Streptococcus dysgalactiae, Streptococcus iniae, Micrococcus lysodeikticus and Edwardsiella tarda. When L-DOPA was used as a substrate, significant inhibition occurred in A. salmonicida only.

Using flow cytometric analysis, the dynamics of surface immunoglobulin positive (sIg+) cells in lymphoid organs of Japanese flounder (Paralichthys olivaceus) reared at 9, 15, 21 and 26 °C, was investigated following intraperitoneal injection with inactivated lymphocystis disease virus (LCDV). The results showed that the percentages of sIg+ cells were suppressed in peripheral blood leucocytes (PBL), spleen leucocytes (SL) and head kidney leucocytes (HKL) from 9 °C to 15 °C immunized groups, and arrived at their peaks (9 °C: 26.12% in PBL, 18.84% in SL, 17.53% in HKL; 15 °C: 38.82% in PBL, 25.38% in SL, 23.95% in HKL) at 9th and 7th week after immunization, respectively. While the proportions of sIg+ cells in PBL, SL and HKL increased most prominent in the 21 °C group and reached the peaks (54.16% in PBL, 30.32% in SL, 30.23% in HKL) at 5th week. The responses of sIg+ cells from 26 °C group were similar to that from 21 °C group and reached the peaks (35.3% in PBL, 26.24% in SL, 21.83% in HKL) at 5th week. Simultaneously, the kinetics of the specific antibody titer against LCDV in sera was determined. It was shown that the antibody response in the 21 °C group was most prominent and reached the peak earliest. These results indicated inactivated LCDV elicited the most powerful immune response when Japanese flounder maintained at the optimal temperature (21 °C) and obtained the most effective immunization, while the response were suppressed at 9 °C, 15 °C or 26 °C.

Haemocyte types of the scallop (Chlamys farreri) were identified by Giemsa stain and flow cytometry (FCM). Additionally, the activities of peroxidase (POD), phenoloxidase (PO) and alkaline phosphatase (ALP) in haemocytes were analysed by immunocytochemical and biochemical methods. The results indicate that there were two types of haemocytes in the scallop, hyalinocytes and granulocytes, and that POD, PO and ALP were more abundant and more active in granulocytes than in hyalinocytes.

Eight monoclonal antibodies produced against haemocytes of shrimp (Litopenaeus vannamei) were used to research the antigenic cross-reactivity of crustacean haemocytes. 2C3 cross-reacted with the haemocytes of all the experimental animals, while 1H8 and 2C11 did not cross-react with the experimental animals. The other five monoclonal antibodies cross-reacted with some of the experimental animals.

Toll-like receptor (TLR) 3 is an important member of the TLR family, recognizes invading pathogens specifically double-stranded RNA viruses. In this paper, turbot (Scophthalmus maximus) TLR3 gene (smTLR3) was cloned and its expression profiles were analyzed with RT-PCR and In situ hybridization. Then its expression was investigated in cultured peripheral blood leukocytes (PBLs) simulated with poly I:C, peptidoglycan (PGN), or lipopolysaccharides (LPS); in liver, spleen, gill and kidney injected with Streptococcus iniae, Edwardsiella tarda hirame rhabdovirus virus (HRV), poly I:C, PGN, or LPS using real-time PCR. The recombinant protein of smTLR3 (rp-smTLR3) and the polyclonal antibody(Pab) was produced, then the reactions of Pab to tissues was studied by Western-blotting, the binding of rp-smTLR3 to all the stimulants was detected using ELISA. The results showed the full length of smTLR3 cDNA consists of about 3100bp, which has the typical construction of the TLR family with 2883 bp of the open reading frame encoding 961 amino acids. It was highly expressed in spleen, liver, gill, skin, intestine, and PBLs, mainly distributed in epithelial cells. SmTLR3 expression was significantly up-regulated by PolyI: C both in vitro and in vivo, no significant change in PGN and LPS groups; SmTLR3 significantly increased in liver and spleen after S. iniae infection with the maximum of 3.6 times and 3.3 times; in liver and kidney after E. tarda infection with the maximum of 3.4 times and 4.1 times; and then in gills and kidney after HRV infection by 3.5 and 4.7 times. The reaction binds of Pab in liver, spleen, gills and kidney was 120kDa, 90kDa and 40kDa. The rp-smTLR3 could bind to all the ligands in vitro. The data suggested the expression of smTLR3 in the turbot and could recognize the ligands more than dsRNA.