Molecular characterization and mRNA transcript profile of vitellogenin in Chinese shrimp, Fenneropenaeus chinensis

A full-length cDNA encoding vitellogenin (Vg) was cloned from Chinese shrimp, Fenneropenaeus chinensis using RACE method. The full-length cDNA consist of 7,942 nucleotides including a 7,761 bp open reading frame, which encodes 2,587 amino acid residues. The deduced amino acid sequence showed high (from 94% to 37%) identity with other known crustacean Vgs. In addition, a consensus cleavage site (R-X-K/R-R) recognized by an endopeptidase and a member of subtilisin family of serine protease were identified in the deduced Vg precursor. RT-PCR analysis shown that Vg mRNA can be detected in both ovary and hepatopancreas of vitellogenic females but not in other experimental tissues including muscle, heart, lymph organ, gill, haemocytes and intestine. These results suggest that the Vg gene may be expressed exclusively in mature females, and both ovary and hepatopancreas are the possible tissues for Vg synthesis in F. chinensis. In addition, Vg gene is detected in genomic DNA of both females and males.     

Synaptonemal complex analysis in spermatocytes of diploid and triploid Chinese shrimp Fenneropenaeus chinensis

A modified surface spreading technique for synaptonemal complex (SC) analysis was tested to assess the process of chromosome synapsis in spermatocytes of diploid and induced triploid Fenneropenaeus chinensis. Spermatocytes of diploid shrimp showed typical morphological characteristics of eukaryote SC, with complete synapsis of bivalents. No recognizable bivalent associated with sex chromosomes was observed in spermatocytes of diploid shrimp. However, differences in morphology of SC, including unsynapsed univalents, bivalents, totally paired trivalents with non-homologous synapsis, partner switches and triple synapsis were identified at early pachytene stage of triploid spermatocytes. Triple synapsis was especially common at late pachytene stage in spermatocytes of triploid shrimp. The observed abnormal synapsis behavior of chromosomes in spermatocytes indicated that triploid male shrimp may find it difficult to develop normal haploid sperm.     

The effect of fucoidan from brown seaweed Sargassum wightii on WSSV resistance and immune activity in shrimp Penaeus monodon (Fab)

The polysaccharide-fucoidan was extracted from brown seaweed Sargassum wightii and characterized through FT-IR and ¹³C & ¹H NMR analysis. The extracted fucoidan was supplemented with pellet diets at three different concentrations (0.1, 0.2 and 0.3%). The fucoidan supplemented diets were fed to Penaeus monodon for 45 days, then challenged with WSSV and the mortality percentage was recorded daily up to 21 days. During the challenge test, the control group showed 100% mortality within 10 days, but in the experimental groups, the mortality percentage (51-72% within 21 days) was decreased considerably (P < 0.05) with respect to the concentrations of fucoidan. The reduction in mortality percentage of experimental groups over control group was ranged from 50.81 to 68.06%. During challenge experiment, the immunological parameters such as THC, prophenoloxidase activity, respiratory burst activity, superoxide dismutase activity and phagocytic activity were measured before injection of WSSV (0 day) and after the injection of WSSV on 10th and 21st days, respectively. All the immunological parameters of experimental groups were significantly (P < 0.05) increased than control group. RT-PCR analysis confirmed the considerable reduction of WSSV DNA copy numbers with respect to the concentration of fucoidan. It was concluded that P. monodon fed with fucoidan of S. wightii supplemented diet had enhanced the innate immunity and increased resistance against WSSV infection.     

Comparison of Gene Expression Profiles of Fenneropenaeus chinensis Challenged with WSSV and Vibrio

Microarray technique was used to analyze the gene expression profiles of shrimp when they were challenged by WSSV and heat-inactivated Vibrio anguillarum, respectively. At 6 h post challenge (HPC), a total of 806 clones showed differential expression profile in WSSV-challenged samples, but not in Vibrio-challenged samples. The genes coding energy metabolism enzyme and structure protein were the most downregulated elements in 6 h post WSSV-challenged (HPC-WSSV) tissues. However, a total of 155 clones showed differential expression in the Vibrio-challenged samples, but not in WSSV-challenged samples. Serine-type endopeptidase and lysosome-related genes were the most upregulated elements in tissues 6 h post Vibrio challenge (HPC-Vibrio). Totally, 188 clones showed differential expression in both 6 and 12 HPC-WSSV and HPC-Vibrio samples. Most of the differentially expressed genes (185/188) were downregulated in the samples of 12 HPC-WSSV, whereas upregulated in the samples at 6 and 12 HPC-Vibrio and 6 HPC-WSSV. The expression profiles of three differentially expressed genes identified in microarray hybridization were analyzed in hemocytes, lymphoid organ, and hepatopancreas of shrimp challenged by WSSV or Vibrio through real-time PCR. The results further confirmed the microarray hybridization results. The data will provide great help for us in understanding the immune mechanism of shrimp responding to WSSV or Vibrio. Wang and Li contributed equally to this work.     

Effect of hypoxic stress on the immune response and the resistance to vibriosis of the shrimpPenaeus stylirostris

The immune response of shrimp exposed to severe hypoxia, 1 mg O₂ml⁻¹for 24 h, was measured in terms total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity (PO) and respiratory burst by NBT reduction. Results showed that hypoxia induced a significant decrease (P<5%) of the THC due to a significant decrease of semi-granular cells (SGC) (P<5%) and hyaline cells (HC) (P<5%). There was also a decrease in the NBT reduction. On the other hand, PO activity increased significantly (P<1%) and seemed to be related to a lower amount of plasma inhibitors regulating the prophenoloxidase system. Experimental infection with a virulentVibrio alginolyticuswas carried out by intramuscular injection of 2×10⁴colony forming units (cfu) per shrimp. The mortality of stressed shrimp was 48% while the mortality of control shrimp was 32%. This difference was significant (P<1%). The variations of immunological parameters, except for the PO activity, seem to be consistent with thein vivoresponse against the pathogenicVibrio. Coyright 1998 Academic Press     

A double WAP domain (DWD)-containing protein with proteinase inhibitory activity in Chinese white shrimp, Fenneropenaeus chinensis

The whey major component, whey acidic protein (WAP), has one or more WAP domains characterized by a four-disulfide core (4-DSC) structure. These kinds of proteins are involved in multiple functions, including proteinase inhibitor activity, antimicrobial activity, ATPase inhibitor activity, and regulatory function in cell proliferation. Recent research indicates that WAP domain-containing proteins play an important role in the innate immunity of crustaceans. In this study, a novel double WAP domain (DWD)-containing protein named Fc-DWD was found for the first time in Chinese white shrimp, Fenneropenaeus chinensis. The open reading frame of Fc-DWD encodes a protein of 117 amino acids, including a signal peptide of 16 amino acids and two WAP domains. The predicted molecular mass of the mature protein is 12.78 kDa with an estimated pI of 8.49. The first WAP domain, named WAP 1, composed of 49 amino acids locates in the amino-terminal of Fc-DWD, and the second WAP domain, named WAP 2, composed of 45 amino acids locates in the carboxy-terminal. Fc-DWD mRNA was upregulated in hemocytes, hepatopancreas, gills, and stomach of bacteria- and virus-challenged shrimp. Results of the binding assay showed that rFc-DWD could bind to both Gram-negative bacteria and Gram-positive bacteria. rWAP 1 could only bind to Gram-positive bacteria, but rWAP 2 could bind to both Gram-negative and positive bacteria. Moreover, rFc-DWD exhibited proteinase inhibitory activity against the secretory proteinase(s) from Bacillus subtilis and Pseudomonas aeruginosa. All of these findings suggest that Fc-DWD may play an important role in enabling the host defense to execute its proteinase inhibitory activity against pathogens.     

Styrax japonica supplementation diet enhances the innate immune response in Epinephelus bruneus against bacterial and protozoan infections

Kelp grouper, Epinephelus bruneus, fed for 30 days with 0% (control), 0.1%, 1.0%, and 2.0% of Styrax japonica supplementation diets, led to reductions in mortality after being challenged with a bacterium (Vibrio harveyi) and a ciliate protozoan (Uronema marinum). The enriched diets significantly increased the survival rate as compared to the controls. The phagocytic and respiratory activities were significantly increased in kelp groupers given 1.0% and 2.0% enriched diets. The complement activity, lysozyme activity, serum bactericidal activity, and total protein level significantly increased with any enriched diet against the pathogens; however antiprotease activity and myeloperoxidase levels significantly increased only with 1.0% and 2.0% enriched diets while the α2-macroglobulin level was significantly enhanced with 1.0% enriched diet. The study suggests that incorporation of S. japonica at 1.0% and 2.0% level in the diet significantly enhances the immune responses in the kelp grouper E. bruneus against V. harveyi and U. marinum.     

High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a approximately 6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use.     

哈氏弧菌dam基因的克隆与分析

利用兼并PCR的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶(dam)基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bpDNA片段皆具有启动子活性,但前者的活性明显高于后二者。     

Effect of copper sulfate on the immune response and susceptibility to Vibrio alginolyticus in the white shrimp Litopenaeus vannamei

White shrimp Litopenaeus vannamei (Boone) held in 35 per thousand seawater were challenged with Vibrio alginolyticus at a dose of 3 x 10(5) colony-forming units (cfu) shrimp(-1), and then placed in water containing concentrations of Cu2+ at 0 (control), 1, 5, 10 and 20 mg l(-1). Mortality of shrimp in 5, 10 and 20 mg l(-1) Cu2+ was significantly higher than those in 1 mg l(-1) Cu2+ and the control solution after 24-96 h. In another experiment, L. vannamei which had been exposed to control, 1, 5, 10 and 20 mg l(-1) Cu2+ for 24, 48 and 96 h were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity and clearance efficiency to V. alginolyticus. Copper concentrations at 1 mg l(-1) or greater for 24h resulted in decreased THC, phenoloxidase activity, phagocytic activity and clearance efficiency, whereas copper concentration at 20 mg l(-1) caused significant increase in respiratory burst of L. vannamei. In conclusion, concentration of Cu2+ at 1 mg l(-1) or greater increased the susceptibility of L. vannamei to V. alginolyticus infection by a depression in immune ability. The release of superoxide anion by L. vannamei exposed to 20 mg l(-1) Cu2+ was considered to be cytotoxic to the host.     

Purification and characterization of hatching enzyme from shrimp Penaeus chinensis

By using Penaeus chorion as a specific substrate, the hatching enzyme (HE) from Penaeus chinensis was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties in this study. It was found that the molecular weight of Penaeus HE is about 43.0 kDa in SDS-PAGE. The Penaeus HE had obvious choriolytic activity, which was optimal at pH 6.0 and temperature of 40 degrees C, respectively. The Km value of the HE for casein was 7.47 mg ml(-1). The HE activity was almost completely inhibited by SBTI, p-APMSF, bestatin, and NEM, greatly inhibited by ovomucoid, TLCK, IAM, chymostatin, and PMSF, and slightly inhibited by pepstatin A, TPCK, LBTI, and leupeptin. These results indicate that the HE is most probably a trypsin-type serine protease. Besides of these, the HE was extremely sensitive to EDTA, Zn2+, Ca2+, Mg2+, and Cu2+. Combined with the results that the EDTA-pretreated HE activity could be perfectly recovered by Zn2+, it is indicated that shrimp HE is most probably a kind of Zn-metalloprotease.     

Primary culture and characteristic morphologies of medulla terminalis neurons in the eyestalks of Chinese shrimp, Fenneropenaeus chinensis

A method for culturing medulla terminalis (MT) neurons in the eyestalk of Chinese shrimp, Fenneropenaeus chinensis, was first established. The neurons showed immediate outgrowth in the culture medium supplemented with glutamine, glucose and antibiotics. The cells grew for about 2-7 days and then sustained for a week or more. At least six types of neurons were distinguished on the basis of size and form of soma and outgrowth pattern of cells.     

The immunostimulatory effects of hot-water extract of Gelidium amansii via immersion, injection and dietary administrations on white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

The total haemocyte count (THC), phenoloxidase activity, and respiratory burst were examined when white shrimp Litopenaeus vannamei were immersed in seawater (34 per thousand) containing hot-water extract of red alga Gelidium amansii at 200, 400 and 600 mg l(-1), injected with hot-water extract at 4 and 6 microg g(-1) shrimp, and fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg(-1). These parameters increased significantly when shrimp were immersed in seawater containing hot-water extract at 400 and 600 mg l(-1) after 1h, when shrimp were injected with hot-water extract at 6 microg g(-1) shrimp after one day, and when shrimp were fed diets containing hot-water extract at 1.0 and 2.0 g kg(-1) after 14 days. Phagocytic activity and clearance efficiency were significantly higher for the shrimp that were fed diets containing hot-water extract at 1.0 and 2.0 g kg(-1) than those of shrimp that were fed diets containing hot-water extract at 0 and 0.5 g kg(-1) after 14 and 28 days. In a separate experiment, L. vannamei which had received hot-water extract via injection, or fed diets containing hot-water extract, were challenged after 3h or 28 days with V. alginolyticus at 2 x 10(6) cfu shrimp(-1) and 1 x 10(6) cfu shrimp(-1), respectively, and then placed in seawater. The survival of shrimp that were injected with hot-water extract at 6 microg g(-1) was significantly higher than that of control shrimp after 1 day, and the survival of shrimp fed diets containing hot-water extract at 0.5, 1.0 and 2.0 g kg(-1) increased significantly after 3 days as well as at the end of the experiment (6 days after the challenge), respectively. It was concluded that L. vannamei that were immersed in hot-water extract at 400 mg l(-1), injected with hot-water extract at 6 microg g(-1) shrimp, and fed hot-water extract of G. amansii at 2.0 g kg(-1) or less showed increased immune ability as well as resistance to V. alginolyticus infection.     

Genetic variation and evolutionary demography of Fenneropenaeus chinensis populations, as revealed by the analysis of mitochondrial control region sequences

Genetic variation and evolutionary demography of the shrimp Fenneropenaeus chinensis were investigated using sequence data of the complete mitochondrial control region (CR). Fragments of 993 bp of the CR were sequenced for 93 individuals from five localities over most of the species' range in the Yellow Sea and the Bohai Sea. There were 84 variable sites defining 68 haplotypes. Haplotype diversity levels were very high (0.95 ± 0.03-0.99 ± 0.02) in F. chinensis populations, whereas those of nucleotide diversity were moderate to low (0.66 ± 0.36%-0.84 ± 0.46%). Analysis of molecular variance and conventional population statistics (F(ST) ) revealed no significant genetic structure throughout the range of F. chinensis. Mismatch distribution, estimates of population parameters and neutrality tests revealed that the significant fluctuations and shallow coalescence of mtDNA genealogies observed were coincident with estimated demographic parameters and neutrality tests, in implying important past-population size fluctuations or range expansion. Isolation with Migration (IM) coalescence results suggest that F. chinensis, distributed along the coasts of northern China and the Korean Peninsula (about 1000 km apart), diverged recently, the estimated time-split being 12,800 (7,400-18,600) years ago.     

Effects of Zn supplementation on the growth,amino acid composition,polysaccharide yields and anti-tumour activity of <Emphasis Type="Italic">Agaricus brasiliensis</Emphasis>

The effects of Zn supplementation on the growth, amino acid composition, polysaccharide yields and anti-tumour activity of Agaricus brasiliensis were studied. An initial Zn concentration within the range of 0–300 mg/l had a significant effect on the cell growth and Zn biosorption. At an initial Zn concentration of 300 mg/l, a maximal extracellular polysaccharide (EPS) yield of 5.08 ± 0.25 g/l was obtained, as well as a maximal intracellular polysaccharide (IPS) of 12.25 ± 0.31 mg/g DW. Amino acid analysis results showed that the total amino acid contents of mycelia and culture filtrate decreased from 1090.08 ± 0.76 (233.62 ± 0.06) to 1077.40 ± 0.77 mg/100 g DW (229.52 ± 0.05 mg/l), respectively, while the total essential amino acid contents of mycelia and culture filtrate markedly increased from 429.51 ± 0.86 (58.84 ± 0.05) to 476.9 ± 0.85 mg/100 g DW (59.99 ± 0.04 mg/l), respectively. The anti-tumour activity of Zn-enriched mycelial powder against sarcoma 180 in mice showed that the tumour inhibition ratio was 61.5% and was enhanced markedly as compared to normal mycelial powder of 30.8. The fundamental information obtained in this study will be useful for efficient production of Zn-enriched foods or drugs.