Molecular Characterization and Expression Analysis of SKIV Infection of Interferon-Induced Protein with Tetratricopeptide Repeats 1(IFIT1) in Epinephelus lanceolatus

Interferon-induced protein with tetratricopeptide repeats 1(IFIT1), also known as interferon-induced protein 56(IFI56) or Interferon-stimulated protein 56(ISG56), was originally identified as a protein induced upon treatment with interferon and inhibited by viral replication and translational initiation. In this study, Epinephelus lanceolatus IFIT1(ELIFIT1) gene was cloned for the first time. The complete cDNA of El IFIT1 gene includes 2921 nucleotides, and encodes a 437-amino acid(AA) protein. The putative ELIFIT1 protein has 9 TRP domains and is highly similar with IFIT1 proteins in other teleosts. In healthy fish, ELIFIT1 gene was highly expressed in the blood, which indicate its specific function in the peripheral immune system. Its expression was also observed in various immunity-related tissues including spleen, intestine, and kidney, Inducted with spotted knifejaw iridovirus(SKIV), ELIFIT1 gene expression was upregulated in the spleen, kidney, and liver 24 h after induction and reached its peak at 72 h, indicating that ELIFIT1 may play an important role in antivirus. These findings contribute to the understanding of the antiviral regulation of ELIFIT1 gene in teleost.     

Isolation of Prawn(Exopalaemon carinicauda) Lipopolysaccharide and β-1,3-Glucan Binding Protein Gene and Its Expression in Responding to Bacterial and Viral Infections

The pattern recognition proteins(PRPs) play a major role in immune response of crustacean to resist pathogens.In the present study,as one of PRPs,lipopolysaccharide and β-1,3-glucan binding protein(LGBP) gene in the ridge tail white prawn(Exopalaemon carinicauda)(Ec LGBP) was isolated.The full-length c DNA of Ec LGBP was 1338 bp,encoding a polypeptide of 366 amino acid residules.The deduced amino acid sequence of Ec LGBP shared high similarities with LGBP and BGBP from other crustaceans.Some conservative domains were predicted in Ec LGBP sequence.Ec LGBP constitutively expressed in most tissues at different levels,and the highest expression was observed in hepatopancreas.With infection time,the cumulative mortality increased gradually followed by the proliferation of Vibrio parahaemolyticus and white spot syndrome virus(WSSV).The expression of Ec LGBP in response to V.parahaemolyticus infection was up-regulated in hemocytes and hepatopancreas,and the up-regulation in hepatopancreas was earlier than that in hemocytes.Ec LGBP expression after WSSV infection increased at 3 h,then significantly decreased in both hemocytes and hepatopancreas.The results indicated that Ec LGBP was involved in the immune defense against bacterial and viral infections.     

Characterization,Expression and Function Analysis of DAX1 Gene of Scallop （Chlamys farreri Jones and Preston 1904） During Its Gametogenesis

DAX 1,a member of nuclear receptor superfamily,has a function in the sex determination and gonadal differentiation of several vertebrate species.However,little information about DAX1 of invertebrates is available.Here we cloned a homolog of scallop （Chlamys farreri Jones and Preston 1904） dax1,Cf-dax1,and determined its expression characteristics at mRNA and protein levels.The cDNA sequence of Cf-dax1 was 2093 bp in length,including 1404 bp open reading frame （ORF） encoding 467 amino acids.Unlike those of vertebrates,no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1.Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes.Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues,with the highest expression level found in adductor muscle,moderate level in mantle,gill and testis,and low level in kidney,ovary and hepatopancreas.The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher （P＜0.05） in testis than in ovary at the same stage,showing a sex-dimorphic expression pattern.Furthermore,immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary,implying that DAX1 may involve in gametogenesis of bivalves.     

Molecular cloning,tissue expression of gene Muc2 in blunt snout bream Megalobrama amblycephala and regulation after re-feeding

Mucins are important components of mucus,which form a natural,physical,biochemical and semipermeable mucosal layer on the epidermis of fish gills,skin,and the gastrointestinal tract. As the first step towards characterizing the function of Muc2,we cloned a partial Megalobrama amblycephala Muc2 cDNA of 2 175 bp,and analyzed its tissue-specific expression pattern by quantitative real-time PCR(qPCR). The obtained sequence comprised 41 bp 5′-untranslated region(5′-UTR),2 134 bp open reading frame encoding a protein of 711 amino acids. BLAST searching and phylogenetic analysis showed that the predicted protein contained several common secreted mucin-module domains(VWD-C8-TIL-VWD-C8) and had high homology with mucins from other vertebrates. Among four candidate reference genes(β- Actin,RPI13α,RPII,18S) for the qPCR,R PII was chosen as an appropriate reference gene because of its lowest variation in different tissues. M. amblycephala Muc2 was mainly expressed in the intestine,in the order(highest to lowest) middle-intestine fore-intestine hind-intestine. Muc2 was expressed relatively poorly in other organs(brain,liver,kidney,spleen,skin and gill). Furthermore,after 20-days of starvation,M. amblycephala Muc2 expressions after refeeding for 0h,3 h,16 h,3 d,and 10 d were significantly decreased in the three intestinal segments(P 0.05) at 16 h,and were then upregulated to near the initial level at 10 d.     

Histological change and heat shock protein 70 expression in different tissues of Japanese flounder Paralichthys olivaceus in response to elevated temperature

High temperature influences the homeostasis of fish.We investigated the effects of elevated temperature on tissues of Japanese flounder {Paralichthys olivaceus) by analyzing the histology and heat shock protein 70(hsp70) expression of fish reared in warm conditions.In this study,temperature was increased at l±0.5℃/day starting at 24±0.5℃,and was kept at that temperature for 5 days before the next rise.After raising temperature at the rate up to 32±0.5℃,tissue samples from midgut,spleen,stomach,liver,muscle,gill,heart,trunk kidney and brain were collected for histological analysis and mRNA assay.Almost all the tissues showed changes in morphological structure and hsp70 level at 32±0.5℃.Histological assessment of the tissues indicated that the gill had the most serious damage,including highly severe epithelial lifting and edema,curved tips and hyperemia at the ending of the lamellars,desquamation and necrosis.The next most severe damage was found in liver and kidney.The hsp70 levels in all the tissues first increased and then decreased.The gut,stomach,muscle,heart,and brain had the highest expressions in 6 h,whereas the spleen,liver,gill and kidney had the highest expressions in 2 h.Therefore,tissues with the most significant lesions(especially gill and liver) responded much earlier(2 h) in hsp70 expression than other tissues,and these tissues demonstrated the most marked histological disruption and elevated mRNA levels,making them ideal candidates for further studies on the thermal physiology of this species.     

Ontogeny of the Lymphoid Organs of Japanese Flounder, Paralichthys olivaceus

Histological study on the ontogeny of the lymphoid organs, kidney, thymus and spleen of Japanese flounder, Paralichthys olivaceus, from hatching to 40d was carried out. The pronephric kidney duct appeared early in hatching although the primordial haemopoietic stem cells wereobserved within a week after hatching. The spleen was first seen after 8d of hatching. The thymus appeared after 15d, situated near the pronephric kidney. Small lymphoid cells appeared during the later phase of the post-larval stage in the sequence of thymus, kidney and spleen. During the 40d of observations, there were no distinct inner or outer zones in thymus and no red or white pulp in spleen. These results suggest that the nonspecific defense immune system plays a very important role in the early larval stage of Japanese flounder.     

Pharmacokinetics and tissue behavior of enrofloxacin and its metabolite ciprofloxacin in turbot Scophthalmus maximus at two water temperatures

Turbot Scophthalmus maximus, an important aquaculture species in China, currently suffers from epizootic diseases because of high density aquaculture. Enrofloxacin has been used to treat various systemic bacterial fish infections. However, studies concerning the pharmacokinetics of enrofloxacin in turbot are limited. In this study, the pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin, were investigated in the turbot following intravenous and oral administration at 10 mg enrofloxacin/kg body weight, at 16°C and 10°C water temperatures. The concentrations of enrofloxacin and ciprofloxacin in the main tissues (plasma, muscle, liver and kidney) were detected by HPLC. The results show that the plasma concentration-time data for enrofloxacin were best described as a two-compartment open model after intravenous and oral administration. Three pharmacokinetic equations were established between the concentrations and temperatures. The kinetic profile of enrofloxacin was temperature dependent. The absorption half-life of enrofloxacin was 1.99 h and 2.17 h after oral administration, whereas the elimination half-life of the drug was 98.63 h and 136.59 h at 16°C and 10°C, respectively. The peak concentration of enrofloxacin in plasma and tissues was higher at 16°C than that at 10°C, and the peak plasma concentration time in the liver was the shortest at both temperatures among those of other tissues. The plasma C max /MIC ratio varied between 11.08 and 5 540.00 at 16°C; and between 7.92 and 3 960.00 at 10°C. The AUC/MIC ratio was 467.82-280 690.00 at 16°C, and 359.48-215 690.00 at 10°C. These ratios indicate that it is possible to obtain therapeutic efficacy. Very low levels of ciprofloxacin were detected. The AUC ratios of ciprofloxacin and enrofloxacin in plasma suggest that plasma ciprofloxacin might play a minor role in enrofloxacin treatment for turbot.     

Interaction of temperature and salinity on the expression of immunity factors in different tissues of juvenile turbot Scophthalmus maximus based on response surface methodology

Central Composite Design(CCD) and response surface methodology were used in the experiment to examine the combined effect of temperature(16-28℃) and salinity(18-42) on Hsp70 and IgM genes expression levels in turbot(Scophthalmus maximus) liver and kidney.The results showed that the coefficients of determination(R~2=0.965 2 for liver Hsp70,0.972 9 for kidney Hsp70,0.921 for liver IgM and 0.962 1 for kidney IgM) and probability values(P0.01) were significant for the regression model.The interactive effect between temperature and salinity on liver Hsp70,kidney Hsp70 and liver IgM were not significant(P0.05),while the interactive effect between temperature and salinity on kidney IgM was significant(P0.01).The model equation could be used in practice for forecasting Hsp70 and IgM genes expression levels in the liver and kidney of juvenile turbot via applying statistical optimization of the response of interest,at which the maximum liver Hsp70,kidney Hsp70,liver IgM and kidney IgM of1.48,1.49,2.48,and 1.38,respectively,were reached.The present model may be valuable in assessing the feasibility of turbot farming at different geographic locations and,furthermore,could be a useful reference for scientists studying the immunity of turbot.     

Molecular Cloning,Expression and Characterization of Peroxisome Proliferators-Activated Receptors Gamma in the Sea Urchin(Strongylocentrotus intermedius)

Peroxisome proliferators-activated receptor gamma(PPARγ) plays important regulatory roles in adipocyte differentiation. In this study, we cloned the full-length sequence of the PPARγ gene and analyzed its expression profile in different developmental stages and tissues of Strongylocentrotus intermedius. The full-length cDNA of PPARγ contains 2286 base pairs(bp) with a putative open reading frame of 1755 bp, and the gene encodes encoding a polypeptide of 584 amino acid residues. The predicted molecular mass of the protein is 67.27 kDa, and its theoretical isoelectric point(pI) is 10.07. The protein contains conserved motifs, including an RRM(RNA recognition motif) domain. PPARγ expression with the highest level was observed in the gonad, and the lowest was observed in the tube feet of S. intermedius. Time-course expression measurements at different developmental stages showed that the highest expression level of PPARγ is in the eggs and its weakest expression level is in the 32-cells stage. Knock-down of PPARγ by specific siRNA revealed that UCP2 expression is significantly decreased in the gonads and intestines 48 h post-transfection, indicating that the UCP2 is a downstream target gene of PPARγ.This finding suggests that PPARγ and UCP2 have positive regulatory effects in regulating adipocyte differentiation. Changes in fatty acid levels in the gonads before and after PPARγ interference were assessed, and decreased C18:2(trans, n-6) and C20:3(n-6) levels were observed 48 h after siRNA transfection. The results showed the function of PPARγ in fatty acid anabolism, The data are helpful to improve the current understanding of the fatty acid synthesis pathways and regulatory mechanisms in S. intermedius. They also provide an experimental basis for improving fatty acid synthesis in sea urchins, which is important for cultivating sea urchins with high nutritional value.     

Effects of Environmental Cadmium on Cadmium Accumulation,Oxidative Response,and Microelements Regulation in the Liver and Kidney of Hexagrammos otakii

The effects of cadmium(Cd)on metal accumulation,microelements contents,and antioxidant responses in Hexagram-mos otakii were studied.The fish were exposed to 0.2,2.5,or 10μg L?1 Cd for 12 or 24 days.Then,the concentration of Cd and mi-croelements(Ca,Fe,Zn,and Se)were determined in the liver and kidney.Moreover,the activities of antioxidant enzymes including superoxide dismutase(SOD),catalase(CAT),and glutathione-S-transferase(GST),and the content of malondialdehyde(MDA)in the liver and kidney were also measured.A continuous accumulation of Cd was observed throughout the experimental period.Cd accumulation was higher in liver than that in the kidney,while Ca and Fe contents were lower in liver than those in the kidney.Cd exposure resulted in a decrease of Ca and Fe concentrations in the kidney,while there was no effect on the liver.Zn and Se remained unaffected with exposure to Cd.Cd exposure induced severe oxidative stress in H.otakii,as indicated by significant induction of the activities of SOD,CAT,and GST,and a simultaneous increase of MDA content.These data show that antioxidant enzymes and mi-croelements contents can be used as potential biomarkers to monitor environmental health in fish.     

Cloning and expression analysis of a ubiquitin gene (Ub L40 ) in the haemocytes of Crassostrea hongkongensis under bacterial challenge

Ubiquitin, a highly conserved stress-related protein, is assigned multiple functions, such as DNA processing, protein degradation, and ribosome synthesis. The Crassostrea hongkongensis ubiquitin gene (designated ChUbL40) was cloned by a combination of suppressive subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The full-length cDNA of ChUbL40 is 496 bp in length, consisting of a 5’ untranslated region (UTR) of 34 bp, a 3’-UTR of 75 bp and an open reading frame of 387 bp encoding a ubiquitin fusion protein of 128 amino acids. Analysis of the amino acid sequence of ChUbL40 reveals that UbL40 is highly conservative during evolution. The expression patterns of ChUbL40 gene in various tissues were examined by real-time PCR. The expression level of ChUbL40 in haemocytes is down-regulated at 4 h and gradually returned to its original level from 6 h to 24 h after Vibrio alginolyticus challenge. Our results suggest that ChUbL40 is ubiquitously expressed and plays an important role in immune defense against bacterial challenge.     

Cloning and Characterization of a New κ-Carrageenase Gene from Marine Bacterium Pseudoalteromonas sp. QY203

κ-carrageenan oligosaccharides exhibit various biological activities. Enzymatic degradation by κ-carrageenase is safe and controllable. Therefore, κ-carrageenases have captured more and more attentions. In this study, a κ-carrageenase encoding gene, cgk X, was cloned from Pseudoalteromonas sp. QY203 with degenerate and inverse PCR. It comprised an ORF of 1194 bp in length, encoding a protein with 397 amino acid residues. Cgk X is a new member of glycoside hydrolase family 16. The deduced amino acid sequence shared a high similarity with Cgk X of Pseudoalteromonas κ-carrageenase; however, the recombinant Cgk X showed different biochemical characteristics. The recombinant enzyme was most active at p H 7.0 and 55℃ in the presence of 300 mmol L~(~(-1))Na Cl. It was stable in a broad range of acidity ranging from p H 3.0 to p H 10.0 when temperature was below 40℃. More than 80% of its activity was maintained after being incubated at p H 3.6–10.0 and 4℃ for 24 h. Cgk X retained more than 90% of activity after being incubated at 40℃ for 1 h. EDTA and SDS(1 mmol L~(-1)) did not inhibit its activity. Cgk X hydrolyzed κ-carrageenan into disaccharide and tetrasaccharide as an endo-cleaver. All these characteristics demonstrated that Cgk X is applicable to both κ-carrageenan oligosaccharide production and κ-carrageenase structure-function research.