Effects of salinity and body mass on oxygen consumption and ammonia excretion of mudskipper Boleophthalmus pectinirostris

We evaluated the effects of salinity andbody mass on the oxygen consumption rate and ammonia excretion rate of mudskipper Boleophthalmus pectinirostris under laboratory conditions.Salinity and body mass had highly significant effects on the oxygen consumption rate(R_O) and ammonia excretion rate(R_N)(P<0.01).The interactive effects between salinity and body mass onR_O and R_N were insignificant(P>0.05)and highly significant(P<0.01),respectively.R_O and R_N of B.pectinirostris decreased significantly as the individual body mass increased.The relationship between R_O and body mass was represented by R_O=aW^b(R^2=0.956,P<0.01).The relationship between R_N and the body mass of B.pectinirostris was represented by R_N=cW^d(R^2=0.966,P<0.01).The R_O/R_N(O:N) ratios increased significantly as the salinity increased from 12 to 27,but decreased as salinity increased from 27 to 32.The atomic O:N ratios were significantly higher at27 than at other salinity levels.The average O:N ratio was 25.25.Lipid and carbohydrate were the primary energy sources and protein was the secondary energy source within the salinity range 12-32.R_O and R_N were significantly higher at 27 than at other salinity levels.Our results suggest that the optimum salinity level for B.pectinirostris is 27.

Cloning and mRNA expression of macrophage migration inhibitory factor (MIF) gene of large yellow croaker (P seudosciaena crocea)

Mammalian macrophage migration inhibitory factor （MIF） plays an important role as an indispensable mediator in the pathogenesis of inflammatory disease like septicemia, but little is known about the role of MIF homologue in fish septicemia. The authors have cloned the MIF homologue in large yellow croaker Pseudosciaena crocea （LycMIF） using RACE approach. The full-length cDNA of LycMIF was 634 bases and contained an ORF of 345 bases encoding a protein of 115 amino acid residues. As demonstrated by RT-PCR and QRT-PCR assay, MIF mRNAs were constitutively expressed in 11 selected tissues and were abundant in brain and liver. Moreover, the LycMIF transcripts in the liver and head kidney were responsive to bacteria infection and could be significantly up-regulated. Our results provide the first direct evidence that fish MIF was implicated in pathogenesis of fish vibrosis and play an important role in response to bacteria infection.

Influence of temperature on glutathione level and glutathione-related enzyme activities of Antarctic ice microalgae Chlamydomonas sp.ICE-L

GSH system plays a role in the control of the redox balance state, anti-oxidation and protecting life from injury of ROS （ reactive oxygen species）. In present paper, the possible GSH system of Chlamydomonas sp. ICE-L has been investigated by evaluating GSH and GSH-related enzymatic responses at different temperatures using spectrophotometer methods. The results showed that the GSH system is correlated positively to low temperature, and other factors but GR are correlated negatively to high temperature. So GSH and GSH-related enzymes play an important role in the adaptation of Antarctic ice microalgae to low temperature.

Inhibitory Activity of Prunus mume,Copitis chinensis and Crataegus pinnatifida on Vibrio harveyi and Its Biofilm

[Objective] This study was conducted to determine the in-vitro inhibitory effects of Prunus mume,Coptis chinensis and Crataegus pinnatifida on Vibrio harveyi and its biofilm. [Method]The inhibitory zone diameters of the three Chinese herbal medicines against V. harveyi were determined by agar diffusion method; the minimal inhibitory concentration( MIC) and minimal bactericidal concentration( MBC) values of the three Chinese herbal medicines against V. harveyi were determined by doubling dilution method; and the effects of the three Chinese herbal medicines on the formation of V. harveyi biofilm were determined by methyl thiazolyl tetrazolium( MTT) method. [Result]The three Chinese herbal medicines all inhibited V. harveyi to different degrees. C. chinensis and C. pinnatifida and P. mume exhibited the inhibitory zone diameters of( 17. 62 ± 0. 04),( 20. 16 ± 0. 08) and( 30. 76 ± 0. 26) mm against V. harveyi,respectively. P. mume and C. pinnatifida had strong inhibitory effects on V. harveyi. The MIC and MBC values of P. mume against V. harveyi were 7. 812 5 mg/ml; the MIC and MBC values of C. pinnatifida against V. harveyi were 31. 25 mg/ml; and the MIC and MBC values of C. chinensis against V. harveyi were 62. 5 mg/ml. P. mume had the strongest antibacterial and bactericidal ability. The MIC values of C. pinnatifida,C. chinensis and P. mume against V. harveyi were 7. 81,7. 81 and 1. 96 mg/ml,respectively,i. e.,P. mume exhibited the lowest MIC. [Conclusion] P. mume,C. pinnatifida and C. chinensis all have inhibitory effects on V. harveyi and its biofilm,and P. mume has the strongest bactericidal ability.

Molecular Cloning and Bioinformatics Analysis of TypeⅢSecretion System Effector Protein Va1686 Gene of Vibrio alginolyticus

In this study, Va 1686 gene was cloned from Vibrio alginolyticus . The total length of the gene is 1 164 bp, and it could encode 387 amino acids. The physicochemical properties, protein structure, genetic evolutionary relationship and antigenic characteristics of the effect protein Va1686 of V. alginolyticus HY9901 type Ⅲ secretion system were studied and analyzed by bioinformatics methods and tools. The results showed that Va1686 is a stable hydrophilic and acidic protein without a transmembrane region and a signal peptide, and secondary structure to α-helix. The evolutionary analysis showed that V. alginolyticus HY9901 and V. harveyi were clustered together, which indicated that the genetic relationship between the two species was the closest. Va1686 contains a Fic superfamily conserved domain associated with cell division. Bioinformatics analysis showed that the B-cell preponderant epitopes of Va1686 might be localized in the regions of 48-49, 82-85, 125-126, 150-153, 185-186, 236-237 and so on. The 3D structure model of Va1686 subunit was simulated by SWISS-MODEL software and it was found that the vopS of V. parahaemolyticus was similar and the similarity was 89.46%. In this study, the feasibility of Va1686 as a common antigen of Vibrio was verified from the perspective of bioinformatics, which laid the foundation for the next step in vaccine development.

Responses to two-way selection on growth in mass-spawned F_1 progeny of Argopecten irradians concentricus(Say)

A bstract In the present study,the effect of one-generation divergent selection on the growth and survival of the bay scallop(Argopecten irradians concentricus)was examined to evaluate the effi cacy of a selection program currently being carried out in Beibu Bay in the South China Sea.A total of 146 adult scallops were randomly selected from the same cultured population of A.i.concentricus,and divided into two groups in shell length(anterior-posterior measurement): large(4.91–6.02 cm,n=74)and small(3.31–4.18 cm,n=72).At the same time,a control group was also randomly sampled(4.21–4.88 cm,n=80).Mass-spawned F 1 progenies from the three size groups were obtained and reared under identical conditions at all growth phases.The effects of two-way(or upward-downward)selection on fertilization rate,hatching rate,survival rate,daily growth in shell length and body weight were assessed in the three size groups.Results show that signifi cant differences(P<0.01)were found in hatching rate,survival rate and daily growth of F 1 progenies,but not in fertilization rate(P>0.05),among the three groups.The hatching rate,survival rate and daily growth of the progeny of large-sized parents were greater than those of the control group(P<0.05),which in turn were larger than those of small-sized group(P<0.05).Responses to selection by shell length and body weight were 0.32±0.04 cm and 2.18±0.05 g,respectively,for the upward selection,and-0.14±0.03 cm and-2.77±0.06 g,respectively,for the downward selection.The realized heritability estimates of shell length and body weight were 0.38±0.06 cm and 0.22±0.07 g for the upward selection,and 0.24±0.06 cm and 0.37±0.09 g for the downward selection,respectively.The change in growth by bidirectional selection suggests that high genetic variation may be present in the cultured bay scallop population in China.

Combined effects of water temperature and copper ion concentration on catalase activity in Crassostrea ariakensis

A central composite experimental design and response surface method were used to investigate the combined effects of water temperature(18–34℃) and copper ion concentration(0.1–1.5 mg/L) on the catalase(CAT) activity in the digestive gland of C rassostrea ariakensis. The results showed that the linear effects of temperature were significant(P <0.01), the quadratic effects of temperature were significant( P <0.05), the linear effects of copper ion concentration were not significant(P >0.05), and the quadratic effects of copper ion concentration were significant(P <0.05). Additionally, the synergistic effects of temperature and copper ion concentration were not significant(P >0.05), and the effect of temperature was greater than that of copper ion concentration. A model equation of CAT enzyme activity in the digestive gland of C. ariakensis toward the two factors of interest was established, with R 2, Adj. R 2 and Pred. R 2 values as high as 0.943 7, 0.887 3 and 0.838 5, respectively. These findings suggested that the goodness of fit to experimental data and predictive capability of the model were satisfactory, and could be practically applied for prediction under the conditions of the study. Overall, the results suggest that the simultaneous variation of temperature and copper ion concentration alters the activity of the antioxidant enzyme CAT by modulating active oxygen species metabolism, which may be utilized as a biomarker to detect the effects of copper pollution.

Practice Instruction of Innovative and Entrepreneurial Training Program for Aquatic Animal Medicine Students

As an important platform for the combination of theory and practice,college students' innovative and entrepreneurial training program has an important impact on improving the comprehensive ability of undergraduates. The major of aquatic animal medicine is a new sea-related major. The continuous development and transformation and upgrading of aquaculture in the new period have put forward newer and higher requirements for the innovative and practical ability and comprehensive quality of aquatic animal medicine graduates. Taking the innovation and entrepreneurship program of college students majoring in aquatic animal medicine as an example,this paper analyzes the significance of the innovation training program for college students,the cultivation of students' scientific research ability,and the problems existing in the application and operation of the program,in order to provide a reference for the innovative education and talent training for students majoring in aquatic animal medicine.

Construction of Live Attenuated Vaccine against Vib- rio alginolyticus and the Evaluation on Its Immuno- protection Effect

Vibrio alginolyticus, a gram-negative bacterium has been described to be one of the most common and economically important aquatic pathogens of fish and shellfish. Vaccine immunization is an effective approach preventing V. alginolyticus infection. Attenuated vaccine stimulates systemic immune response in the host, but few attenuated vaccine against V. alginolyticus is available. The type III secretion system （T3SS）, an important pathogenic factor of V. alginolyticus, is used by bacterial pathogens to inject effector proteins into the cytoplasm of their host cells. The T3SS forms a structure called needle complex with a multi-ring base that spans the bacteria and a needle-like extension that protrudes several nanometers from the bacterial surface, vscO locates at the ＂needle＂ site of T3SS, playing the role of escorting the molecular chaperone and effector proteins into host cells and further inducing the death of host cells. In this paper, an in-frame deletion mu- tant of vscO was constructed using overlap PCR and homologous recombination technology combining with chloramphenicol （Cm） and sucrose screening. The LDs0 changes of ZJO3AvscO mutant strains compared with the strain ZJ03 were e^amined in grouper （ Epinephelus coioides）. The ZJO3AvscO mutant showed about 150 times decrease in virulence in E. coioides compared with wide type ZJ03. After vaccination with ZJO3AvscO in E. coioides through injection and immersion, the spe- cific antibody titers were markedly higher than that in the saline control group （P 〈 0.05 ）. The titers of injection and immersion group on the forth week reached the maximums at 1：2 048 and 1： 128, respectively. The relative percentage survival （RPS） of injection group was 84%, while that in immersion group was 68%. These results indicate that the ZJO3AvscO of V. alginolyticus has a high immunogenicity, and can be used as live attenuated vaccine. In addition, RPS may be affected by vaccination and infection methods. This study can provide technical support for controlling fish diseases caused by V. alginolyticus.

Molecular Cloning and Bioinformatics Analysis of kdpE Gene from Vibrio alginolyticus

The two-component regulatory system plays an important role in the growth and virulence of bacteria. In this study, the phosphate transcriptional regulatory protein （KdpE） gene of Vibrio alginolyticus strain HY9901 was cloned. Sequence analysis revealed that the length ofkdpE gene is 732 bp and encodes a putative protein of 243 amino acids. The predicted molecular weight （MW） of KdpE was 27.66 kD with an estimated pI of 5.01. Using SignalP 4.0 and TM- HMM Server 2.0 software, it was predicted that the KdpE protein was located in cytoplasm. It did not contain a signal peptide or a transmembranous region. A phylogenetic tree was constructed by MEGA 5.0 software, indicating that the KdpE of V. alginolyticus bad high genetic relationship with Vibrio campbellii and Vibrio parahaemolyticus. Using SWISS-MODEL work-space, the three-dimensional structures of conserved domain REC in the KdpE was determined. These results can provide a basis for further studies on the two-component regulatory systems of V. alginolyticus.

Molecular Cloning and Bioinformatics Analysis of araC Gene of Vibrio alginolyticus

[Objective]To clone araC gene of Vibrio alginolyticus HY9901 strain,and analyze bioinformatics.[Methods]the whole genome sequence of Vibrio alginolyticus on GenBank was used to design specific primers.According to the principle of PCR amplification sequence,the target gene araC was amplified,and then the sequence was further analyzed by bioinformatics method to establish the phylogenetic tree of araC gene and its corresponding subunit three-dimensional structure model.[Results]Sequence analysis revealed araC gene is 711 bp and encodes a putative protein of 236 amino acids.The predicted molecular mass of AraC was 26.92 ku.Using Signal P 4.0 and TMHMM Server 2.0 software for analysis,it was predicted that the AraC protein did not contain a signal peptide or a transmembranous region.The AraC protein had two cAMP and cGMP dependent protein kinase phosphorylation site,five protein kinase C phosphorylation sites,three casein kinase II phosphorylation sites,one prenyl group binding site(CAAX box)and five microbodies C-terminal targeting signal.The predicted results of protein subcellular localization showed that AraC was located in the mitochondria,nucleus and cytoplasm.Its protein is unstable and hydrophilic.The AraC protein is a transcriptional regulatory protein which belongs to HTH_18 superfamily.According to the prediction,secondary structure:a-helix(Alpha helix)accounted for 52.12%,random coil(31.78%),extended strand(11.02%),b-fold(Beta turn)accounted for 5.08%.V.alginolyticus,Vibrio parahaemolyticus and Vibrio palustris were clustered together,which implies that the genetic relationship between these three species was the closest.

Molecular Cloning and Bioinformatics Analysis of TypeⅢSecretion System Effector Protein HY 322 Gene of Vibrio alginolyticus

In this study,Hy322 gene was cloned from Vibrio alginolyticus.The total length of its gene was 969 bp,and it could encode 322 amino acids.The physicochemical properties,protein structure,genetic evolutionary relationship and antigenic characteristics of the effector protein Hy322 of V.alginolyticus HY9901 type Ⅲ secretion system were studied and analyzed by bioinformatics methods and tools.The results showed that Hy322 is an unstable hydrophilic and acidic protein without a transmembrane region and a signal peptide,and secondary structure to α-helix.The evolutionary analysis showed that V.alginolyticus HY9901 and V.harveyi were clustered together,which indicated that the genetic relationship between the two species was closest.HY322 contains a FliN super family conserved domain associated with Flagellar motor switch.Bioinformatics analysis showed that the B-cell preponderant epitopes of Hy322 might be localized in the regions of 32-33,100-102,138-140,215-216,235-238 and 246-249.The 3D structure model of Hy322 subunit was simulated by SWISS-MODEL software and itwas found that the yscQ of Yersinia were similar and the similarity was 42.25%.In this study,the feasibility of Hy322 as a common antigen of Vibrio was verified from the perspective of bioinformatics,which laid the foundation for the next step in vaccine development.

Cloning and Bioinformatics Analysis of TpiA Gene of Vibrio alginolyticus HY9901

[Objectives]To clone and analyze the TpiA gene of Vibrio alginolyticus HY9901.[Methods]According to the TpiA gene sequence of V.alginolyticus,a pair of specific primers was designed,and its full length was amplified by PCR.[Results]The full length of TpiA gene is 771 bp,encoding 256 amino acid residues in total,and the NCBI accession number is OM906798.According to the deduced amino acid sequence,its molecular weight was predicted to be about 26.97548 kDa,and its isoelectric point was 4.78.The amino acid sequence of the N-terminal signal peptide structure was predicted,and it was found that there was no obvious signal peptide cleavage site,no signal peptide,and no transmembrane region;the amino acid sequence contained 3 N-glycosylation sites,4 protein kinase C phosphorylation sites,2 casein kinase II phosphorylation sites,6 N-myristoylation sites,7 microbody C-terminal target signal site,and 1 triose phosphate isomerase active site.The prediction results of protein subcellular localization showed that TpiA may be located in mitochondria or cytoplasm,with probability of 39.1%and 34.8%,respectively.The amino acid sequence of the TpiA gene of V.alginolyticus shared 98.83%-99.61%homology with other Vibrio species,and it was clustered into the same subfamily with Vibrio parahaemolyticus and had a close relationship.In the secondary structure prediction,the proportions ofα-helix,random coil and extended chain were 44.53%,41.41%and 14.06%,respectively,and the similarity of its tertiary structure model to template 1aw1.1.A was 85.16%.[Conclusions]This study is intended to provide a basis for further research on the role of TpiA gene in the type III secretion system and related research on antibiotic resistance.