Xuejun Li

Toll-like receptor recognition of bacteria in fish: ligand specificity and signal pathways.

Pattern recognition receptors (PRRs) recognize the conserved molecular structure of pathogens and trigger the signaling pathways that activate immune cells in response to pathogen infection. Toll-like receptors (TLRs) are the first and best characterized innate immune receptors. To date, at least 20 TLR types (TLR1, 2, 3, 4, 5M, 5S, 7, 8, 9, 13, 14, 18, 19, 20, 21, 22, 23, 24, 25, and 26) have been found in more than a dozen of fish species. However, of the TLRs identified in fish, direct evidence of ligand specificity has only been shown for TLR2, TLR3, TLR5M, TLR5S, TLR9, TLR21, and TLR22. Some studies have suggested that TLR2, TLR5M, TLR5S, TLR9, and TLR21 could specifically recognize PAMPs from bacteria. In addition, other TLRs including TLR1, TLR4, TLR14, TLR18, and TLR25 may also be sensors of bacteria. TLR signaling pathways in fish exhibit some particular features different from that in mammals. In this review, the ligand specificity and signal pathways of TLRs that recognize bacteria in fish are summarized. References for further studies on the specificity for recognizing bacteria using TLRs and the following reactions triggered are discussed. In-depth studies should be continuously performed to identify the ligand specificity of all TLRs in fish, particularly non-mammalian TLRs, and their signaling pathways. The discovery of TLRs and their functions will contribute to the understanding of disease resistance mechanisms in fish and provide new insights for drug intervention to manipulate immune responses.

Responses of acid/alkaline phosphatase, lysozyme, and catalase activities and lipid peroxidation to mercury exposure during the embryonic development of goldfish Carassius auratus.

This study assessed the impact of mercury exposure on goldfish (Carassius auratus) embryos based on the dynamic characteristics of chemical parameters. Day-old embryos were exposed to different Hg(2+) concentrations (0, 0.2, 1, 5, and 10 μg/L). Subsequently, the embryos were sampled every 24 h during embryonic development to measure acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LSZ), and catalase (CAT) activities, as well as malondialdehyde (MDA) content. The results revealed that the responses of ACP and AKP to mercury exposure presented in dose-dependent and time-dependent manners. The enzyme activities were significantly induced with increased concentrations and extended exposure (at 5 μg/L after 72 h and 10 μg/L after 48 h; p<0.05 or p<0.01). LSZ was not sensitive to lower Hg(2+) concentrations, whereas LSZ significantly increased at higher concentrations and longer exposure (at 5 μg/L at 120 h and 10 μg/L after 72 h; p<0.05 or p<0.01). CAT activities were significantly inhibited at different periods of embryonic development, particularly at 5 and 10 μg/L (p<0.05 or p<0.01). Reduced CAT activities were observed at 72, 96, and 120 h at 1 μg/L (p<0.05 or p<0.01), whereas a decline at 0.2 μg/L was evident at 96 h (p<0.01). MDA content significantly increased at various stages of embryonic development, particularly at 10 μg/L (p<0.05 or p<0.01), and increased further at 72, 96, and 120 h at 5 μg/L (p<0.05 or p<0.01). At 96 h, MDA content was only increased by exposure to 0.2 and 1 μg/L (p<0.01). The activities of ACP, AKP, and LSZ remarkably increased at 120 h in contrast to 96 h (p<0.05 or p<0.01). Therefore, 96 h is an important shifting period of embryonic development because the activity of enzyme has been enhanced at this time. Thus, the increased ACP, AKP, and LSZ activities revealed an enhanced ability of the embryo to synthesize more enzymes and attenuate mercury damage. CAT activity negatively correlates with MDA accumulation. The enhanced enzyme activities after specific embryonic stages are used to strengthen the ability to cope with mercury stress and attenuate mercury damage. The biochemical parameters, except LSZ, exhibited sensitivity to mercury, suggesting that they may act as potential biomarkers in assessing the environmental mercury risk on C. auratus embryos.

Effects of copper exposure on the hatching status and antioxidant defense at different developmental stages of embryos and larvae of goldfish Carassius auratus.

This study aims to assess the effects of copper exposure on hatching status and antioxidant defense at different stages of embryos and larvae of goldfish Carassius auratus. In this study, day-old embryos were randomly grouped after fertilization and then exposed to copper concentrations of 0, 0.1, 0.4, 0.7, and 1.0mgL(-1). Copper-exposed fish embryos were sampled every 24h to determine superoxide dismutase (SOD), and catalase (CAT) activities, as well as malondialdehyde (MDA) content. In addition, cumulative mortality and larval deformity were also investigated. The findings showed that cumulative mortality and larval deformity rate increased gradually with copper concentration increase. SOD and CAT activities were inhibited at higher copper concentrations. At a lower concentration (0.1mgL(-1)), SOD activity increased in larvae, whereas CAT activity showed no significant change (p>0.05). MDA, as the lipid peroxidation product, gradually accumulated in embryos and larvae with increasing copper concentration and the extension of exposure time. At 0.4mgL(-1) and more, copper toxicity was shown in embryos and larvae. In conclusion, copper-exposed effects on hatching status and antioxidant defense in C. auratus embryos and larvae showed concentration- and time-dependent patterns. The biochemical parameters in this study can be used as effective indicators for evaluating the responses of copper-exposed fish embryos. In addition, this study demonstrates that 0.4mgL(-1) copper (corresponding to 1mgL(-1) copper sulfate), used to kill parasites in aquaculture, is not safe concentration, because it can result in toxicity to larvae. Therefore, the copper concentration to kill pathogen should be less than 0.4mgL(-1) for C. auratus.

Expression patterns of Toll-like receptors in natural triploid Carassius auratus after infection with Aeromonas hydrophila.

Toll-like receptors (TLRs) play the important roles in the innate immune system. In the present study, we have cloned fragments of 13 members of TLR family in natural triploid Qihe crucian carp (C. auratus) and investigated the expression profiles of these TLRs in spleen and head kidney at different time points after A. hydrophila infection. The results showed that the expressions of certain TLRs including TLR4, TLR5, and TLR22 were significantly up-regulated after infection, and the expression levels of TLR5 and TLR22 were the highest in spleen with 52.56-fold and 28.14-fold increase, respectively, whereas the other TLRs were down-regulated or no significant changes were observed compared with the control at most time points. These findings suggested that three TLRs (TLR4, TLR5, and TLR22) may play important roles in the immune responses of C. auratus to A. hydrophila infection. Additionally, the expression of most TLRs was significantly up-regulated at 6h post infection, which implied that the immune response of C. auratus reached the highest level at this time point. This work will facilitate our comprehensive understanding for the functions of TLRs in the process of bacterial infection in C. auratus and provide new insights for developing preventive and therapeutic measures against A. hydrophila infection.

Immune effects of the vaccine of live attenuated Aeromonas hydrophila screened by rifampicin on common carp (Cyprinus carpio L)

Aeromonas hydrophila, as a strong Gram-negative bacterium, can infect a wide range of freshwater fish, including common carp Cyprinus carpio, and cause the huge economic loss. To create the effective vaccine is the best way to control the outbreak of the disease caused by A. hydrophila. In this study, a live attenuated A. hydrophila strain, XX1LA, was screened from the pathogenic A. hydrophila strain XX1 cultured on medium containing the antibiotic rifampicin, which was used as a live attenuated vaccine candidate. The immune protection of XX1LA against the pathogen A. hydrophila in common carp was evaluated by the relative percent survival (RPS), the specific IgM antibody titers, serum lysozyme activity and the expression profiles of multiple immune-related genes at the different time points following immunization. The results showed that the variable up-regulations of the immune-related genes, such as the pro-inflammatory cytokine IL-1β, the chemokine IL-10 and IgM, were observed in spleen and liver of common carp injected in the vaccines with the formalin-killed A. hydrophila (FKA) and the live attenuated XX1LA. Specific antibody to A. hydrophila was found to gradually increase during 28 days post-vaccination (dpv), and the RPS (83.7%) in fish vaccinated with XX1LA, was significant higher than that (37.2%) in fish vaccinated with FKA (P<0.05) on Day 28 after challenged by pathogen. It was demonstrated that the remarkable immune protection presented in the group vaccinated with XX1LA. During the late stage of 4-week immunization phase, compared with FKA and the control, specific IgM antibody titers significantly increased (P<0.05) in the XX1LA group. The activity of the lysozyme in serum indicated no significant change among three groups. In summary, the live attenuated bacterial vaccine XX1LA, screened in this study, indicates the better protect effect on common carp against A. hydrophila, which can be applied in aquaculture of common carp to prevent from the disease outbreak in the future.

Tissue distribution of olive flounder VDAC2 and its expression in fish cell lines

AbstractVoltage-dependent anion channel (VDAC) is located in the mitochondrial outer membrane, which plays a crucial role in regulating cell life and death. In this study, the tissue distribution of olive flounder Paralichthys olivaceus VDAC2 (PoVDAC2) was detected by quantitative real-time PCR and Western blot analysis. The qRT-PCR results showed that the expression level of PoVDAC2 was abundant in heart, muscle and gill tissues. Western blot analysis revealed a protein of 32xa0kDa detected in all six tissues. Furthermore, a recombinant eukaryotic expression plasmid pEGFP-N3-PoVDAC2 was successfully constructed and transiently expressed the fusion protein in fish cell lines. Subcellular localization indicated that PoVDAC2-GFP was distributed in a punctate mitochondria-like pattern throughout the cytoplasm in flounder embryonic cells (FEC). The distribution of native VDAC2 in untransfected fish cells was also investigated by immunofluorescence microscopy. The punctate VDAC2 fluorescence signals of both FEC and EPC cells were identically observed in the cytoplasm but not in the nucleus. These results laid a foundation for investigating the functional relevance of VDAC response to pathogens in flounder.n

H2A and Ca-L-hipposin gene: Characteristic analysis and expression responses to Aeromonas hydrophila infection in Carassius aurutus

Abstract Antimicrobial peptide is an important component of the host innate immune system and thus serves a crucial function in host defense against microbial invasion. In this study, H2A and derived antimicrobial peptide Ca‐L‐hipposin were cloned and characterized in Carassius aurutus. The gene H2A full‐length cDNA is 908 bp and includes a 5′‐terminal untranslated region (UTR) of 55 bp and a 3′‐terminal UTR of 466 bp with a canonical polyadenylation signal sequence AATAA, as well as an open reading frame (ORF) of 387 bp encoding a polypeptide of 128 amino acids, with a molecular weight of 13.7 kDa, an isoelectric point of 10.7, and 94% homology with Danio rerio H2A. The secondary structure of H2A includes the &agr;‐spiral with 51 amino acids with a composition ratio of 39.8%, as well as a &bgr;‐corner with 15 amino acids in a composition ratio of 11.7%. The online software ExPaSy predicted that a peptide sequence with 51 amino acids from the 2nd to 52nd amino acids in histone H2A can be produced through hydrolization by protease chymotrypsin, which indicates a difference of only three amino acids, compared with the antimicrobial peptide hipposin in Hippoglossus hippoglossus with a homology of 94%. Ca‐L‐hipposin includes 51 amino acids with a molecular weight of 5.4 kDa and an isoelectric point of 12.0, the secondary structure of which contains an &agr;‐helix of 17 amino acids accounting for 33.3% and a &bgr;‐corner of 8 amino acids accounting for 15.7%. H2A was extensively expressed in the mRNA levels of various tissues, with higher expression levels in kidney and spleen. After C. aurutus was challenged with Aeromonas hydrophila, the mRNA expression levels of H2A were upregulated in the kidney, spleen, and liver. H2A serves an important function in the defense against the invasion of A. hydrophila. In addition, sequence characteristics reveal that Ca‐L‐hipposin could be a potential antimicrobial peptide for use in killing pathogenic bacteria in aquaculture. HighlightsThe complete H2A cDNA was 908 bp with 387 bp ORF in Carassius aurutus.H2A‐derived Ca‐L‐hipposin is a cationic antimicrobial peptide with 51 amino acids.H2A mRNA was higher expressed in kidney and spleen of C. aurutus.H2A were upregulated in fish kidney, spleen and liver after 9 h bacterial challenge.

Molecular cloning, sequence analysis and structure prediction of the related to b 0,+ amino acid transporter (rBAT) in Cyprinus carpio L.

We investigated the long-term responses of tobacco tissues to salt stress, with a particular interest forxa0growth parameters, proline (Pro) accumulation, and carbohydrate metabolism. Exposure of 17-day-old tobacco plants to 0.2 M NaCl was followed by a higher decrease in dry matter in roots than shoots withxa0a decrease of both Chlorophyll a and b. Analysis of potassium, sodium and chloral accumulationxa0showed dramatic effect to K + accumulation especially in shoots, whereas Na + and Cl - accumulationxa0were strongly increased in shoots with NaCl treatment, but with minor effects in the roots. Salinityxa0stress conditions induced a strong increase in sugar free content in the shoot and root and Pro and 2- oxoglutarate biosynthesis in tobacco tissues. However, salt-stress resulted in significant increase in thexa0activity of NADPH and NADH malate dehydrogenase dependency, particularly in the roots, and thisxa0effect was reversed with NADP + and NAD + -dependent malate dehydrogenase, but the activity of thesexa0enzymes was also maintained significantly higher in the absence of NaCl. Significant decreases inxa0NADPH-depend isocitrate dehydrogenase was also observed after long-term salt treatment in tobaccoxa0plants. In addition, a gradual increase was observed in the in vitro aminating GDH activity, but the inxa0vitro deaminating activity remained unchanged under salinity stress. These results confirmed the hypothesis that the salinity-generated MDH and GDH induction act as anti-stress enzymes in ammoniaxa0detoxification and production of Glu for Pro synthesis. Keywords: Tobacco, carbohydrate metabolism, salinity stress