The quantitative proteomic analysis of rare minnow, Gobiocypris rarus, infected with virulent and attenuated isolates of grass carp reovirus genotype â ¡.

Grass carp reovirus genotype Ⅱ (GCRV II) causes severe hemorrhagic disease in grass carp and affects the aquaculture industry in China. GCRV Ⅱ isolates have been collected from different epidemic areas in China, and these isolates can lead to different degrees of hemorrhagic symptoms in grass carp. Rare minnow (Gobiocypris rarus) is widely used as a model fish to study the mechanism of hemorrhagic disease because of its high sensitivity to GCRV. In this study, the protein levels in the spleen of rare minnow after infection with GCRV virulent isolate JZ809 and attenuated isolate XT422 were investigated using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics. 109 and 50 differentially expressed proteins (DEPs) in the virulent and attenuated infection groups were obtained, respectively, among which 40 DEPs were identified in both groups. Combining protein expression profiling with gene ontology (GO) annotation, the responses of rare minnow to the two genotypes GCRV Ⅱ in terms of upregulated proteins were similar, focusing on ATP synthesis, in which ATP can serve as a "danger" signal to activate an immunoreaction in eukaryotes. Meanwhile, the virulent genotype JZ809 induced more immunoproteins and increased the levels of ubiquitin-proteasome system members to adapt to virus infection. However, together with a persistent and excessive inflammatory response and declining carbon metabolism, rare minnow presented more severe hemorrhagic disease and mortality after infection with virulent JZ809 than with attenuated XT422. The results provide a valuable information that will increase our understanding of the pathogenesis of viruses with different levels of virulence and the mechanism of interaction between the virus and host. Furthermore, the 6 proteins that were only significantly upregulated in the XT422 infection group all belonged to cluster 2, and 28 of 30 proteins that were only upregulated in JZ809 infection group were clustered into cluster 1. For the downregulated proteins, all DEPs in the XT422 infection group were clustered into cluster 4, and 25 of 39 proteins that were only significantly downregulated in the JZ809 infection group belonged to cluster 3. The results indicated that the DEPs in the attenuated XT422 infection group might be sensitive and their abundance changed more quickly when fish experienced virus infection.

Characterization of reference genes for qRT-PCR normalization in rice-field eel (Monopterus albus) to assess differences in embryonic developmental stages, the early development of immune organs, and cells infected with rhabdovirus.

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) has become a popular technique to assess gene expression. Suitable reference genes are normally identified first to ensure accurate normalization. The aim of the present study was to select the most stable genes in embryonic developmental stages, the early development of immune organs, and cells infected with Chinese rice-field eel rhabdovirus (CrERV) of the rice-field eel (Monopterus albus). Four reference genes, including those encoding 18S ribosomal RNA (18SrRNA), beta actin (ß-actin), elongation factor 1 alpha (EF1ɑ), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were assessed using geNorm, NormFinder, BestKeeper, and RefFinder software. Analyses indicated the stability ranking was 18SrRNA > ß-actin > GAPDH > EF1α in the embryonic stage, with 18SrRNA as the most stable reference gene. For immunity-related organs at different developmental stages, the order in the thymus was ß-actin > GAPDH > EF1α > 18SrRNA, with ß-actin as the most stable gene. In both spleen and kidney tissues, the rank order was EF1ɑ > GAPDH > ß-actin > 18SrRNA, with EF1α as the most stable gene. Furthermore, in rice-field eel kidney (CrE-K) cells infected with CrERV, the ranking was EF1ɑ > ß-actin > GAPDH > 18SrRNA, with EF1α as the most stable gene. The results for cells infected with CrERV were verified by testing signaling pathway genes catenin beta 1 (CTNNB1) and NOTCH1 based on the above four genes after virus infection in CrE-K cells. This study laid the foundation for choosing suitable reference genes for immunity-related gene expression analysis in rice-field eel.

Rag1 and Rag2 Gene Expressions Identify Lymphopoietic Tissues in Larvae of Rice-Field Eel (Monopterus albus).

In immature lymphocytes, recombination activating genes 1 and 2 are necessary for antigen receptor V (D) J recombination, representing immature lymphocyte biomarkers. Herein, we cloned and sequenced rice-field eel rag1 and rag2 genes. Their expressions in the thymus, liver, and kidney were significant from 0 days post hatching (dph) to 45 dph, peaking at 45 dph in these three tissues. In situ hybridization detected high rag1 and rag2 expressions in the liver, kidney, and thymus of rice-field eel from 2 to 45 dph, suggesting that multiple tissues of rice-field eel contain lymphocyte lineage cells and undergo lymphopoiesis. Tissue morphology was used to observe lymphopoiesis development in these three tissues. The thymus primordium began to develop at 2 dph, while the kidney and liver have generated. Our findings verified that the thymus is the primary lymphopoietic tissue and suggested that, in rice-field eel, lymphocyte differentiation also occurs in the liver and kidney.

Azalomycin F5a Eradicates Staphylococcus aureus Biofilm by Rapidly Penetrating and Subsequently Inducing Cell Lysis.

Antimicrobial resistance has emerged as a serious threat to public health. Bacterial biofilm, as a natural lifestyle, is a major contributor to resistance to antimicrobials. Azalomycin F5a, a natural guanidine-containing polyhydroxy macrolide, has remarkable activities against Gram-positive bacteria, including Staphylococcus aureus, a major causative agent of hospital-acquired infections. To further evaluate its potential to be developed as a new antimicrobial agent, its influence on S. aureus biofilm formation was evaluated using the crystal violet method, and then its eradication effect against mature biofilms was determined by confocal laser scanning microscopy, the drop plate method, and regrowth experiments. The results showed that azalomycin F5a could significantly inhibit S. aureus biofilm formation, and such effects were concentration dependent. In addition, it can also eradicate S. aureus mature biofilms with the minimum biofilm eradication concentration of 32.0 µg/mL. As extracellular deoxyribonucleic acid (eDNA) plays important roles in the structural integrity of bacterial biofilm, its influence on the eDNA release in S. aureus biofilm was further analyzed using gel electrophoresis. Combined with our previous works, these results indicate that azalomycin F5a could rapidly penetrate biofilm and causes damages to the cell membrane, leading to an increase in DNase release and eventually eradicating S. aureus biofilm.

Molecular characterization of caspase-8-like and its expression induced by microcystin-LR in grass carp (Ctenopharygodon idella).

Caspase-8, an initiator caspase, plays a vital role in apoptosis. In this study, caspase-8-like (named as Cicaspase-8-like), a homologue of caspase-8, was identified in grass carp (Ctenopharygodon idella). The full-length cDNA sequence of CiCaspase-8-like was 1409 bp and contained a 162 bp 5'-UTR, a 239 bp 3'-UTR and a 1008 bp coding sequence. The putative amino acids sequence was 335 residues long, including a large subunit (P20) and a small subunit (P10), but lacking conserved death effector domains. A histidine active site DHSQMDAFVCCVLSHG and a cysteine active-site motif KPKLFFIQACQG were found in P20. Phylogenetic analysis showed that Cicaspase-8-like clustered with the caspase-8 and caspase-8-like of other fish and grouped closely with Carassius auratus caspase-8-like. Quantitative real-time PCR revealed that the Cicaspase-8-like mRNA were expressed constitutively in all tested tissues from healthy grass carp, with high expression level in the blood, spleen, liver and gill, indicating its role in immune reaction. The expression of Cicaspase-8-like mRNA was decreased significantly in the liver because of the stress caused by microcystin-LR (MC-LR) (75 and 100 µg MC-LR/kg BW) at 24 h and 96 h post injection (P < 0.05), but it was increased significantly in grass carp treated with 25 µg MC-LR/kg BW at 24 h (P < 0.05) post injection. Cleaved fragments of Cicaspase-8-like were observed using western blot analysis, and the expression of Cicaspase-8-like protein was increased after MC-LR treatments. Moreover, the expression of both caspase-9 and caspase-3 mRNA increased significantly after treatment with the three doses of MC-LR. TUNEL assay results showed remarkable changes in apoptosis after the MC-LR treatment. These results suggest that Cicaspase-8-like is an important caspase and plays an essential role in MC-LR-induced apoptosis.

Identification, structural characterization, and expression analysis of toll-like receptors 2 and 3 from gibel carp (Carassius auratus gibelio).

Toll-like receptors (TLRs) are important components of innate immunity. TLRs recognize pathogen-associated molecular patterns (PAMPs) and initiate downstream signaling pathways in response. In present study, we report the identification of two TLRs from gibel carp (Carassius auratus gibelio), TLR2 and TLR3 (designated CagTLR2 and CagTLR3, respectively). We report on the genomic structures and mRNA expression patterns of CagTLR2 and CagTLR3. Five exons and four introns were identified from the genomic DNA sequence of CagTLR3 (4749 bp in total length); this genomic organization is similar to that of TLR3 in zebrafish and human. However, only one intron was identified from the CagTLR2 genomic locus (3166 bp in total length); this unique genomic organization of CagTLR2 is different from that of TLR2 in fish and humans. The cDNAs of CagTLR2 and CagTLR3 encoded 791 and 904 amino acid residues, respectively. CagTLR2 and CagTLR3 contained two distinct structural/functional motifs of the TLR family: a leucine-rich repeat (LRR) domain in the extracellular portion and a toll/interleukin-1 receptor (TIR) domain in the intracellular portion. The positions of critical amino acid residues involed in PAMP recognition and signaling pathway transduction in mammalian TLRs were conserved in CagTLR2 and CagTLR3. Phylogenetic analysis revealed a closer clustering of CagTLR2 and CagTLR3 with TLRs from freshwater fish than with marine fish species. In healthy gibel carp, transcripts of these genes were detected in all examined tissues, and high expression levels of CagTLR2 and CagTLR3 were observed in liver and brain, respectively. Following injection with CyHV-2, expression levels of CagTLR2 and CagTLR3 were significantly upregulated in the spleens of gibel carp after three days, and CagTLR3 transcript levels were rapidly increased in head kidney after 12 h. These results suggest that CagTLR2 and CagTLR3 are functionally involved in the induction of antiviral immune response.

Identification and characterization of IL-1 receptor-associated kinase-4 (IRAK-4) in half-smooth tongue sole Cynoglossus semilaevis.

As a crucial component in TLR/IL-1R signaling pathways, IRAK-4 plays a central role in innate and adaptive immunity. In the present study, the cDNA of IRAK-4 was cloned for the first time from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of csIRAK-4 was 2149 bp and contained a 168 bp 5' UTR, a 580 bp 3' UTR and a 1401 bp CDS. The predicted protein sequence of csIRAK-4 had two typical domains, a death domain (DD) at the N terminus and a serine/threonine/tyrosine protein kinase domain (STYKc) at the C terminus. RT-PCR showed that csIRAK-4 mRNA was detected in all tested tissues, especially in immune-related organs, gonads and brain. After injected with inactivated Vibrio anguillarum, the expressions of csIRAK-4 were up-regulated significantly (P<0.05) in spleen and head kidney. During development, csIRAK-4 was expressed at all selected stages and low-level expression was detected at metamorphosis. Taken together, the present study indicated that csIRAK-4 played a crucial role in immune responses and might be involved in the process of development.

Oral Vaccination of Grass Carp (Ctenopharyngodon idella) with Baculovirus-Expressed Grass Carp Reovirus (GCRV) Proteins Induces Protective Immunity against GCRV Infection.

The grass carp reovirus (GCRV) causes severe hemorrhagic disease with high mortality and leads to serious economic losses in the grass carp (Ctenopharyngodon idella) industry in China. Oral vaccine has been proven to be an effective method to provide protection against fish viruses. In this study, a recombinant baculovirus BmNPV-VP35-VP4 was generated to express VP35 and VP4 proteins from GCRV type Ⅱ via Bac-to-Bac baculovirus expression system. The expression of recombinant VP35-VP4 protein (rVP35-VP4) in Bombyx mori embryo cells (BmE) and silkworm pupae was confirmed by Western blotting and immunofluorescence assay (IFA) after infection with BmNPV-VP35-VP4. To vaccinate the grass carp by oral route, the silkworm pupae expressing the rVP35-VP4 proteins were converted into a powder after freeze-drying, added to artificial feed at 5% and fed to grass carp (18 ± 1.5 g) for six weeks, and the immune response and protective efficacy in grass carp after oral vaccination trial was thoroughly investigated. This included blood cell counting and classification, serum antibody titer detection, immune-related gene expression and the relative percent survival rate in immunized grass carp. The results of blood cell counts show that the number of white blood cells in the peripheral blood of immunized grass carp increased significantly from 14 to 28 days post-immunization (dpi). The differential leukocyte count of neutrophils and monocytes were significantly higher than those in the control group at 14 dpi. Additionally, the number of lymphocytes increased significantly and reached a peak at 28 dpi. The serum antibody levels were significantly increased at Day 14 and continued until 42 days post-vaccination. The mRNA expression levels of immune-related genes (IFN-1, TLR22, IL-1ß, MHC I, Mx and IgM) were significantly upregulated in liver, spleen, kidney and hindgut after immunization. Four weeks post-immunization, fish were challenged with virulent GCRV by intraperitoneal injection. The results of this challenge study show that orally immunized group exhibited a survival rate of 60% and relative percent survival (RPS) of 56%, whereas the control group had a survival rate of 13% and RPS of 4%. Taken together, our results demonstrate that the silkworm pupae powder containing baculovirus-expressed VP35-VP4 proteins could induce both non-specific and specific immune responses and protect grass carp against GCRV infection, suggesting it could be used as an oral vaccine.

Purification and characterization of hatching enzyme from brine shrimp Artemia salina.

By using Artemia chorion as a specific substrate, the hatching enzyme from Artemia salina (AHE) was purified by gel-filtration and ion-exchange chromatography, and characterized biochemically and enzymatically in this study. It was found that the AHE had a molecular weight of 82.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and often contained 73.3 kDa molecules in preparation. The AHE had obvious choriolytic activity, which was optimal at pH 7.0 and a temperature of 408C. The Km value of the AHE for dimethyl casein was 8.20 mg/ml. The AHE activity was almost completely inhibited by soybean trypsin inhibitor and p-amidinophenyl methane sulfonyl fluoride hydrochloride, greatly inhibited by N-tosyl-L-lysyl chloromethyl ketone, phenylmethanesulfonyl fluoride, and lima bean trypsin inhibitor, slightly inhibited by pepstatin, N-tosyl-L-phenylalanyl chloromethyl ketone, leupeptin, N-ethylmaleimide, and iodoacetamide, and not inhibited by chymostatin and bestatin. All these results imply that AHE is most probably a trypsin-type serine protease. Besides of these, AHE was also sensitive to EDTA and Zn21. Combined with the results that the EDTA-pre-treated HE activity could be perfectly recovered by Zn21, it is indicated that AHE might be also a kind of Zn-metalloprotease.

A Novel Subunit Vaccine Based on Outer Capsid Proteins of Grass Carp Reovirus (GCRV) Provides Protective Immunity against GCRV Infection in Rare Minnow (Gobiocypris rarus).

The grass carp hemorrhagic disease, caused by the grass carp reovirus (GCRV), has resulted in severe economic losses in the aquaculture industry in China. VP4 and VP35 are outer capsid proteins of GCRV and can induce an immune response in the host. Here, three recombinant baculoviruses, AcMNPV-VP35, AcMNPV-VP4, and AcMNPV-VP35-VP4, were generated to express recombinant VP4 and VP35 proteins from GCRV type II in insect cells by using the Bac-to-Bac baculovirus expression system to create a novel subunit vaccine. The expression of recombinant VP35, VP4, and VP35-VP4 proteins in Sf-9 cells were confirmed by Western blotting and immunofluorescence. Recombinant VP35, VP4, and VP35-VP4 were purified from baculovirus-infected cell lysates and injected intraperitoneally (3 µg/fish) into the model rare minnow, Gobiocypris rarus. After 21 days, the immunized fish were challenged with virulent GCRV. Liver, spleen, and kidney samples were collected at different time intervals to evaluate the protective efficacy of the subunit vaccines. The mRNA expression levels of some immune-related genes detected by using quantitative real-time PCR (qRT-PCR) were significantly upregulated in the liver, spleen, and kidney, with higher expression levels in the VP35-VP4 group. The nonvaccinated fish group showed 100% mortality, whereas the VP35-VP4, VP4, and VP35 groups exhibited 67%, 60%, and 33% survival, respectively. In conclusion, our results revealed that recombinant VP35 and VP4 can induce immunity and protect against GCRV infection, with their combined use providing the best effect. Therefore, VP35 and VP4 proteins can be used as a novel subunit vaccine against GCRV infection.

Isolation and characterization of Toll-like receptor 9 in half-smooth tongue sole Cynoglossus semilaevis.

Toll-like receptors (TLRs) are considered as key sensors to trigger the host's innate immune system and adaptive immune responses by recognizing various PAMPs and initiating signal transduction. TLR9, as a member of TLR family, mediates the recognition of unmethylated CpG dinucleotide motifs commonly found in both bacterial and viral genomes. In the current study, the TLR9 gene was isolated from one of flatfish species, half-smooth tongue sole (Cynoglossus semilaevis). In the 4588 bp genomic sequence, three exons, two introns, and 5' UTR of 23 bp and 3' UTR of 342 bp were identified. Putative amino acid sequence was 1062 residues long, including a typical conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, 14 leucine-rich repeat (LRR) motifs, with greater than 60% identity to gilthead sea bream Sparus aurata and Japanese flounder Paralichthys olivaceus orthologs. Quantitative RT-PCR analysis indicated a broad expression of csTLR9, especially in spleen and gonads. No statistically significant changes were observed for csTLR9 mRNA levels in spleen and head kidney after inactive Vibrio anguillarum immunisation. In C. semilaevis ontogeny, the expression of csTLR9 appeared to be developmentally regulated. The presence of maternal TLR9 mRNA and the dramatic decrease of TLR9 expression at metamorphic stage indicated TLR9 might be involved in C. semilaevis development. Comparing sequence and expression profile of csTLR9 with mammalian and other piscine TLR9s suggested that the main function of TLR9 might be conserved across vertebrates, although species-specific features were present.

Systematic evaluation of the gut microbiome of swamp eel (Monopterus albus) by 16S rRNA gene sequencing.

BACKGROUND: The swamp eel (Monopterus albus) is a commercially important farmed species in China. The dysbiosis and homeostasis of gut microbiota has been suggested to be associated with the swamp eel's disease pathogenesis and food digestion. Although the contributions of gut microbiome in fish growth and health has been increasingly recognized, little is known about the microbial community in the intestine of the swamp eel (Monopterus albus). METHODS: The intestinal microbiomes of the five distinct gut sections (midgut content and mucosa, hindgut content and mucosa, and stools) of swamp eel were compared using Illumina MiSeq sequencing of the bacterial 16S rRNA gene sequence and statistical analysis. RESULTS: The results showed that the number of observed OTUs in the intestine decreased proximally to distally. Principal coordinate analysis revealed significant separations among samples from different gut sections. There were 54 core OTUs shared by all gut sections and 36 of these core OTUs varied significantly in their abundances. Additionally, we discovered 66 section-specific enriched KEGG pathways. These section-specific enriched microbial taxa (e.g., Bacillus, Lactobacillus) and potential function capacities (e.g., amino acid metabolism, carbohydrate metabolism) might play vital roles in nutrient metabolism, immune modulation and host-microbe interactions of the swamp eel. CONCLUSIONS: Our results showed that microbial diversity, composition and function capacity varied substantially across different gut sections. The gut section-specific enriched core microbial taxa and function capacities may perform important roles in swamp eel's nutrient metabolism, immune modulation, and host-microbe interactions. This study should provide insights into the gut microbiome of the swamp eel.

LI1035, a putative effector secreted by Lawsonia intracellularis, targets the MAPK pathway and regulates actin organizationin yeast and mammalian cells.

Lawsonia intracellularis is an obligate intracellular Gram-negative bacterium that has been identified as the etiological agent of the contagious disease proliferative enteropathy (PE) in a wide range of animals, mainly pigs. The genome sequence of L. intracellularis indicates that this bacterium possess a type III secretion system (T3SS), which may assist the bacterium during cell invasion and host innate immune system evasion and could be a mechanism for inducing cellular proliferation. However, the effectors secreted by the T3SS (T3Es) of L. intracellularis have not been reported. T3Es often target conserved eukaryotic cellular processes, and yeast is an established and robust model system in which to reveal their function. By screening the growth inhibition of an ordered array of Saccharomyces cerevisiae strains expressing the hypothetical genes of L. intracellularis, LI1035 was identified as the first putative effector that inhibits yeast growth. The LI1035-induced growth inhibition was rescued in two of the 14 mitogen-activated protein kinase (MAPK) yeast haploid deletion strains, suggesting that LI1035 interacts with the components of the MAPK pathway in yeast. Phosphorylation assays confirmed that LI1035 inhibits MAPK signaling cascades in yeast and mammalian cells. Actin staining assays revealed that LI1035 regulates actin organization in yeast and mammalian cells. Taken together, these results indicate that LI1035 alters MAPK pathway activity and regulates actin organization in the host. These findings may contribute to the understanding the pathogenesis of L. intracellularis and support the use of yeast as a heterologous system for the functional analysis of pathogen-specific gene products in the laboratory.

Azalomycin F5a, a polyhydroxy macrolide binding to the polar head of phospholipid and targeting to lipoteichoic acid to kill methicillin-resistant Staphylococcus aureus.

Azalomycin F5a was a polyhydroxy macrolide produced by streptomycete strains. Our preliminary researches indicated that it could kill methicillin-resistant Staphylococcus aureus (MRSA) likely by increasing the permeability of cell membrane, and that cell-membrane phospholipids were likely important targets. To confirm this, membrane permeability assay was performed and visualized by fluorescence staining, and then the detailed interactions between azalomycin F5a and model membranes prepared with 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) were determined using attenuated total reflectance fourier transform infrared spectroscopy and 31P nuclear magnetic resonance techniques. The results indicated that there were strong interactions between azalomycin F5a and model membranes, especially between azalomycin F5a and the polar head of phospholipid. For further evidence and details, the molecular dynamics (MD) simulation of the interactions between azalomycin F5a and DPPG or lysyl-DPPG were performed using Amber16 software package. A strong interaction between the lactone ring of azalomycin F5a and the polar head of DPPG or lysyl-DPPG had been clearly observed. Moreover, a larger distribution probability out of phospholipid bilayer had been discovered for the guanidyl side chain of azalomycin F5a, especially when probable anion molecules anchoring on the cytoplasmic membrane occurred. Therefore, lipoteichoic acid (LTA), a vital component of gram-positive bacterial envelope, was investigated for its probable interactions with azalomycin F5a using broth microdilution method. The results showed that azalomycin F5a-induced MRSA lysis could be prevented by LTA. This deduced that there were some interactions between azalomycin F5a, more likely its guanidyl side chain, and LTA. Thereby, azalomycin F5a increasing the cell-membrane permeability of MRSA had likely achieved by the synergy of its lactone ring binding to the polar head of phospholipid and its guanidyl side chain targeting to LTA, and which had eventually led to the autolysis of MRSA cells.

Expression profiling and validation of potential reference genes during Paralichthys olivaceus embryogenesis.

Differential expression of genes is crucial to embryogenesis. The analysis of gene expression requires appropriate references that should be minimally regulated during the embryonic development. To select the most stable genes for gene normalization, the expression profiles of eight commonly used reference genes (ACTB, GAPDH, rpL17, alpha-Tub, EF1-alpha, UbcE, B2M, and 18S rRNA) were examined during Japanese flounder (Paralichthys olivaceus) embryonic development using quantitative real-time polymerase chain reaction. It was found that all seven mRNA genes appeared to be developmentally regulated and exhibited significant variation of expression. However, further analyses revealed the stage-specific expression stability. Hence when normalization using these mRNA genes, the differential and stage-related expression should be considered. 18S rRNA gene, on the other hand, showed the most stable expression and could be recommended as a suitable reference gene during all embryonic developmental stages in P. olivaceus. In summary, our results provided not only the appropriate reference gene for embryonic development research in P. olivaceus, but also possible guidance to reference gene selection for embryonic gene expression analyses in other fish species.

Disinfection effect of povidone-iodine in aquaculture water of swamp eel (Monopterus albus).

The swamp eel (Monopterus albus) is an important commercial farmed fish species in China. However, it is susceptible to Aeromonas hydrophila infections, resulting in high mortality and considerable economic loss. Povidone-iodine (PVP-I) is a widely used chemical disinfectant in aquaculture, which can decrease the occurrence of diseases and improve the survival. However, environmental organic matter could affect the bactericidal effectiveness of PVP-I, and the efficacy of PVP-I in aquaculture water is still unknown. In this paper, disinfection assays were conducted to evaluate the effectiveness of PVP-I against the A. hydrophila in different types of water. We found that the effective germicidal concentration of PVP-I in outdoor aquaculture water was 25 ppm for 12 h. In indoor aquaculture water with 105 CFU/mL bacteria, 10 ppm and 20 ppm of PVP-I could kill 99% and 100% of the bacteria, respectively. The minimal germicidal concentration of PVP-I in Luria-Bertani broth was 4,000 ppm. Available iodine content assay in LB solutions confirmed that the organic substance had negative impact on the effectiveness of PVP-I, which was consistent with the different efficacy of PVP-I in different water samples. Acute toxicity tests showed that the 24 h-LC50 of PVP-I to swamp eel was 173.82 ppm, which was much higher than the germicidal concentrations in outdoor and indoor aquaculture water, indicating its safety and effectivity to control the A. hydrophila. The results indicated PVP-I can be helpful for preventing the transmission of A. hydrophila in swamp eel aquaculture.

[Advances in fish antifreeze protein research].

Antifreeze proteins (AFPs) can highly effectively protect cells and embryos from damages in freezing process by lowering the freezing points of their cytoplasmic matrix and body fluids in a noncolligative manner. Based on their origins and properties, AFPs have been classified into four types, i.e. type I, II, III and IV. Each of them possesses rather distinct characteristics both in structure and composition, although all of them have ability of lowering freezing points of fluids. AFPs' genes have been characterized as members of a multigene family and the levels of their mRNA synthesis vary significantly with seasons. Adsorption-inhibition operating at the ice surface is nowadays a hypothesis widely used to interpret the molecular mechanisms of noncolligative lowering of the freezing point, but the details of the mechanism on how the different types of AFP are adsorbed onto ice remain uncertain. Progresses in research on structures, amino acid compositions, genes, antifreeze mechanisms of the 4 distinct types of AFPs, and the application of the AFPs in cryopreservation of cells and embryos are reviewed here.

Molecular cloning and characterization of interleukin-1ß in half-smooth tongue sole Cynoglossus semilaevis.

As a member of the interleukin (IL)-1 family, IL-1ß is a prototypical proinflammatory cytokine, which plays a crucial role in immune responses. Herein, we reported the full length cDNA of IL-1ß in half-smooth tongue sole (Cynoglossus semilaevis). The csIL-1ß cDNA contained a 130 bp 5' UTR, a 417 bp 3' UTR and a 741 bp coding sequence (CDS) that translated into a polypeptide of 246 amino acids. The protein sequence included a typical IL-1 family signature and lacked an interleukin-converting enzyme (ICE) cut site. RT-PCR analysis indicated a broad expression of csIL-1ß, especially in immune-related organs. After injection with inactive Vibrio anguillarum, the expression of csIL-1ß was induced in the head kidney and spleen and reached the highest level at 8 h post injection. Higher expression of csIL-1ß was observed at gastrula stage, eye-bud stage and hatching stage and lower level of csIL-1ß mRNA was detected at metamorphic stage. The expression of csIL-1ß during development suggested IL-1ß might be involved not only in immunity but also development. Taken together, the present study indicated that csIL-1ß played an important role in immune responses and development like its mammalian counterparts, although species-specific features were present.

Molecular cloning, tissue distribution, and ontogeny of gonadotropin-releasing hormone III gene (GnRH-III) in half-smooth tongue sole (Cynoglossus semilaevis).

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that plays a vital role in hypothalamus-pituitary-gonad (HPG) axis. In the present study, the GnRH-III gene was isolated from half-smooth tongue sole (Cynoglossus semilaevis). In the 1160 bp genomic sequence, four exons, three introns, and 5'-/3'-flanking sequences were identified. The putative peptide was 92 residues long, including a putative signal peptide containing 23 amino acids, the GnRH decapeptide, a proteolytic cleavage site of three amino acids and a GnRH associated peptide of 56 amino acids. The overall amino acid sequence of C. semilaevis GnRH-III (csGnRH-III) was highly conserved with other teleost GnRH-III genes. Phylogenetic analysis showed the evolutionary relationships of csGnRH-III with other known GnRH genes. A 320 bp promoter sequence of the csGnRH-III was also analyzed, and several potential regulatory motifs were identified which were conserved in the GnRH promoters of other teleosts. Quantitative real-time PCR analysis indicated csGnRH-III was expressed only in brain and gonads. In C. semilaevis, the csGnRH-III transcript was maternally deposited and appeared to be developmentally regulated during embryogenesis and early larval development. Comparing sequence and expression patterns of csGnRH-III with other teleosts GnRH-IIIs suggested that the main function of GnRH-III might be conserved in teleosts.

Cloning and characterization of type II interleukin-1 receptor cDNA from Japanese flounder (Paralichthys olivaceus).

The type II interleukin-1 receptor (IL-1RII) cDNA was cloned from Japanese flounder (Paralichthys olivaceus) by mRNA differential display (DD-PCR) and rapid amplification of cDNA ends (RACE). The full-length cDNA is 1793 bp in length, including a 100 bp 5'-terminal untranslated region (UTR), a 430 bp 3'-terminal UTR, and a 1263 bp open reading frame (ORF). The ORF encodes 420 amino acid residues with an estimated molecular mass of 46.65 kDa. The protein possesses signature features of the IL-R family, consisting of one N-terminal signal peptide, one transmembrane (TM) domain, two Ig-like domains in its extracellular region, one short cytoplasmic tail of 17 amino acids and four conserved proline residue sites. The predicted amino acid sequence has 65.3%, 52.5% and 51.6% identity with gilthead seabream, rainbow trout and Atlantic salmon IL-1RII, respectively. Phylogenetic analysis supported a close relation to mammalian IL-1RII. Reverse Transcription Polymerase Chain Reaction (RT-PCR) analysis indicated that the IL-1RII gene expression of Japanese flounder was weakly up-regulated and reached the peak expression in the kidney, spleen, and gill at 6 h after infection with Vibrio anguillarum, at 1.9, 2.0 and 1.5 times relative to the uninfected fish, respectively. These results suggest that IL-1RII has a signal transduction function and possibly plays a minor role in immune regulation against bacterial(s) infection in Japanese flounder.