不同环境条件下无菌和自然带菌波吉卵囊藻生长的比较

比较不同温度、光照及海水相对密度条件下无菌与自然带菌波吉卵囊藻(Oocystis borgei)生长的差异。结果表明:在20℃时,无菌藻的比增长率(μ)和叶绿素a(Chla)含量分别为0.398d-1和0.117mg/L,均显著高于带菌藻(P<0.05),在28和35℃时,无菌藻和带菌藻生长无显著性差异(P>0.05);在照度为16μmol·m-2·s-1时,无菌藻平均比增长率μ和Chla含量分别为0.345d-1和0.123mg/L,均显著高于带菌藻(P<0.05),在照度为39和88μmol·m-2·s-1条件下,无菌藻和带菌藻生长无显著性差异(P>0.05);在海水密度为1.007、1.020和1.030g/mL时,无菌藻和带菌藻的生长均无显著性差异(P>0.05),μ和Chla含量分别为0.325～0.374d-1和0.085～0.133mg/L。     

波吉卵囊藻对抗生素的敏感性及其无菌化培养

研究了青霉素、链霉素、庆大霉素和卡那霉素对波吉卵囊藻生长的影响,采用4种方法添加抗生素获得无菌藻株,比较波吉卵囊藻除菌前后的生长特点。结果表明:青霉素和链霉素浓度为1000IU/mL时,对波吉卵囊藻生长无明显影响;庆大霉素和卡那霉素浓度达500IU/mL时,对波吉卵囊藻生长无明显影响。微藻培养液中细菌对抗生素敏感性实验表明,除青霉素外,链霉素、庆大霉素和卡那霉素均对藻液中细菌有明显抑制作用。采用4种方法添加抗生素,均可获得无菌藻株。与未除菌时相比,无菌的波吉卵囊藻不易老化。     

虾池微藻定向培育及其对养殖环境因子的影响

采用微藻三级扩大培养技术,将在对虾养殖池选育的波吉卵囊藻Oocysits borgei引入凡纳滨对虾Litopenaeusvannamei高位养殖池,并检测养殖环境中微藻群落结构、水质因子、对虾抗病力相关因子、对虾生长情况,研究了以微藻生态调控为主的对虾防病技术。结果表明:养殖前、中、后期,波吉卵囊藻平均生物量占浮游植物总生物量分别为98.02%、78.89%和45.12%,成为虾池中的绝对优势种;作为优势种的持续时间长达77 d。波吉卵囊藻为主的微藻群落控制的水质较稳定,实验池氨氮和亚硝酸盐氮浓度较对照池低。对虾的血细胞数、溶菌(LSZ)活力、抗菌活力、超氧化物歧化酶(SOD)和酚氧化酶(PO)均显著高于对照池(P<0.05);血清蛋白含量差异不显著(P>0.05)。实验池对虾生长速度显著高于对照池(P<0.05)。可见,通过微藻的定向培育方法来优化虾池微藻群落结构,可改善养殖环境。     

Mg2+、Mn2+对波吉卵囊藻生长的影响

【目的】研究Mg²⁺、Mn²⁺对波吉卵囊藻生长和多糖代谢的影响。【方法】取蒸馏水培养数日后的波吉卵囊藻液接入以f/2培养液的配方为基础的新鲜培养液中,Mg²⁺浓度梯度分别设置为0、1、2、4、8 mg/L,Mn²⁺浓度梯度分别设置为0、0.005、0.050、0.500、5.000 mg/L,实验周期为10 d。【结果与结论】不同浓度Mg²⁺、Mn²⁺对波吉卵囊藻的生长有显著性影响,当Mg²⁺质量浓度为2 mg/L时,波吉卵囊藻的相对增长率、色素蛋白含量均出现最大值,显著高于Mg²⁺添加量为0、4和8 mg/L的组(P<0.05),当Mn²⁺质量浓度为0.005 mg/L时,波吉卵囊藻相对增长率、色素蛋白含量均出现最大值,显著高于Mn²⁺添加量0、0.050、0.500和5.000 mg/L组(P<0.05)。不同浓度Mg²⁺、Mn²⁺对波吉卵囊藻胞外多糖含量有显著影响(P<0.05),当Mg²⁺质量浓度>2 mg/L、Mn²⁺质量浓度>0.005 mg/L时,波吉卵囊藻生长均受到胁迫,其胞外多糖合成量明显增加,以保护藻体不受危害;当Mg²⁺质量浓度为8 mg/L、Mn²⁺质量浓度为5.000 mg/L时,波吉卵囊藻胞外多糖含量分别为对照组的2.43和3.36倍。     

硝酸钠对波吉卵囊藻生长、生化组分及沉降的影响

【目的】研究硝酸钠对波吉卵囊藻(Oocystis borgei)生长、生化组分及沉降的影响。【方法】采用分光光度计法、乙醇法、尼罗红染色法、苯酚硫酸法和考马斯亮蓝法,分别测定波吉卵囊藻在不同硝酸钠浓度下的生物量、叶绿素、脂质、总糖和蛋白质含量,分析波吉卵囊藻沉降与生长及生化组分的关系。【结果】1)硝酸钠对波吉卵囊藻沉降率有显著影响(P 0.05),当硝酸钠74.8 mg/L时,沉降6 h的沉降率达到75%,沉降效果最佳。2)硝酸钠对波吉卵囊藻生物量和叶绿素的影响显著(P 0.05),培养9 d时,硝酸钠37.4～748.0 mg/L组卵囊藻生物量和叶绿素含量较高,生长状态好,沉降率较高;经回归分析,沉降率与生物量呈正相关关系(R~2=0.824 3),与叶绿素含量呈先增加后减少的抛物线关系(R~2=0.819 8)。3)硝酸钠对波吉卵囊藻脂质和总糖含量的影响显著(P0.05),硝酸钠质量浓度为37.4～2 992.0 mg/L时,总糖和脂质含量少,沉降率较高;经一元回归分析,沉降率与脂质和总糖含量呈负相关关系,R~2分别为0.770 5和0.609 4。4)沉降率与生物量和叶绿素有显著正相关性,皮尔逊系数r分别为0.785和0.404;与脂质和总糖有显著负相关性,皮尔逊系数r分别为―0.788和―0.731。【结论】硝酸钠通过影响波吉卵囊藻的生长和生化组分来改变藻细胞的沉降率;波吉卵囊藻的沉降率与生长状态呈正相关关系,与脂质、总糖呈负相关关系,脂质和总糖含量越少,沉降效果越好,与蛋白质无显著相关关系。     

波吉卵囊藻遗传转化体系选择标记的筛选

【目的】确定波吉卵囊藻(Oocystis borgei)遗传转化体系中的选择筛选标记。【方法】使用分光光度计法测定藻体生物量、叶绿素含量及观察藻液颜色变化,综合评定波吉卵囊藻对6种抗生素的敏感性。【结果】1)波吉卵囊藻对博来霉素非常敏感,25μg/m L的博来霉素即能完全抑制藻体生长,叶绿素含量极显著低于对照组(P0.01),藻体颜色发黄变白;2)红霉素和氯霉素各浓度的处理都会使藻体生物量和叶绿素含量极显著降低(P0.01),但是溶解红霉素和氯霉素的乙醇溶剂会对藻体生长产生抑制,不适用作筛选试剂;3)氨苄青霉素、遗传霉素和庆大霉素对波吉卵囊藻生长抑制作用不明显,甚至会促进叶绿素含量的增加。【结论】质量浓度为25μg/m L的博来霉素可以作为波吉卵囊藻遗传转化中的筛选试剂。     

氮浓度、温度和照度对波吉卵囊藻繁殖模式的影响

【目的】探索波吉卵囊藻(Oocystisborgei)的繁殖模式,调控其生殖过程以实现藻生物质稳定增长。【方法】在荧光显微镜下观察波吉卵囊藻似亲孢子形成过程,探究温度、照度、氮浓度对其繁殖模式的影响。【结果】波吉卵囊藻以2、4和8似亲孢子型模式繁殖,在藻细胞第二轮分裂过程中因分裂不同步,有时也形成3个似亲孢子。氮限制和弱光显著影响波吉卵囊藻繁殖模式(P 0.05)。通常情况下,波吉卵囊藻以4似亲孢子型繁殖模式繁殖,但在缺氮和弱光下2和3似亲孢子型模式的频率上升。在15～35℃温度范围内,波吉卵囊藻繁殖模式变化不大。【结论】波吉卵囊藻有4种繁殖模式。在不良环境中,2和3似亲孢子型模式频率上升。     

不同条件下波吉卵囊藻对氨氮的吸收速率

在实验室条件下模拟多种氮源共存的虾池水环境,利用稳定同位素示踪剂,研究温度、光照、盐度、pH、藻浓度对波吉卵囊藻氨氮吸收速率的影响。结果表明:温度、光照和藻浓度对波吉卵囊藻氨氮吸收速率影响极显著(P<0.01)。当温度为20℃,照度为4 500 lx,藻浓度为5.5×105/mL,波吉卵囊藻对氨氮均有最大的吸收速率,分别为1.114、1.400、1.482μg g-1 h-1。单因素方差分析得出波吉卵囊藻吸收氨氮的最优组合为:温度20℃,照度4 500lx,盐度15,pH 7.5,藻浓度5.5×105/mL。可通过对环境因子的调控,提高波吉卵囊藻对水体中氨氮的吸收速率。     

波吉卵囊藻蛋白质提取方法比较及提取条件优化

【目的】探究基于考马斯亮蓝染色法的波吉卵囊藻(Oocystis borgei)蛋白质提取方法,以快速测定波吉卵囊藻的蛋白质含量。【方法】以凯氏定氮法为基准,以考马斯亮蓝法测定经反复冻融法、超声波破碎法和碱提法提取的波吉卵囊藻(鲜品)蛋白含量,比较各提取方法的提取效果;通过单因素和响应面实验,考察碱提法中提取温度、NaOH浓度和提取时间对波吉卵囊藻蛋白提取率的影响。【结果】碱提法显著优于其他提取方法(P 0.01);碱提法中,各因素对波吉卵囊藻蛋白提取率的影响显著性由大到小依次为提取温度、NaOH浓度、提取时间;最佳提取条件:提取温度74.5℃、NaOH浓度0.35mol/L、提取时间69min,此时蛋白得率为1.726%,与模型预测值1.731%接近,以凯氏定氮法测定的藻体蛋白质含量为基准,蛋白提取率高达99.20%。【结论】碱提法是波吉卵囊藻(鲜品)蛋白的最佳提取方法,采用响应面法建立的模型可较好地预测提取温度、NaOH浓度和提取时间对波吉卵囊藻蛋白提取率的影响。     

波吉卵囊藻的固定化培养及利用

以褐藻酸钙为固定化载体,探讨了胶球直径、不同接种量、CaCl2溶液浓度、褐藻酸钠浓度等条件对波吉卵囊藻(Oocystisborgei)固定化培养及对养殖水体水质的影响。结果表明:波吉卵囊藻固定化培养的最好条件是微藻接种量为1×106 mL-1,CaCl2溶液的质量浓度为30g/L,褐藻酸钠溶液的质量浓度为20g/L。在此条件下制备的固定化藻珠,波吉卵囊藻的生物量从1 83×10-2 mg/粒增加到17. 46×10-2 mg/粒,增加了近10倍,生长率相对较高,证明了它的生理活性不会因固定化而受干扰。引入固定化的波吉卵囊藻不但可以降低养殖水体中氨氮、亚硝酸氮等有害因子的浓度,还能提高水中溶解氧含量,使水体环境长时间处于良好的动态平衡状态。     

大亚湾网箱养殖水体弧菌种类组成及变化

研究了海水网箱养殖海域弧菌数量变化、种类组成及其相关因素,探讨弧菌数量的变化与鱼病的关系。结果表明:网箱内水体弧菌数量为390~230 mL-1,平均为20.6×102mL-1,网箱外水体为130~1450 mL-1,平均为530 mL-1,非养殖对照海区为10~390 mL-1,平均为190 mL-1。养殖海区弧菌数量呈现明显的季节性变化,尤其是网箱水体,对照海区只在9月数量较高,其他时间变化不大。平均数量网箱内大于网箱外,网箱外大于对照海区。水体中出现的弧菌种类有河流弧菌Vibrio fluvialis,创伤弧菌Vibrio vulnifgicus,霍乱弧菌Vibrio cholerae,副溶血弧菌Vibrio parahaemolyticus,溶藻弧菌Vibrio alginolyticus,美人鱼弧菌Vibrio damsela,拟态弧菌Vibriomimicus和好利斯弧菌Vibrio hollisae,美人鱼弧菌、溶藻弧菌和霍乱弧菌占有较大优势。弧种数量与海水温度、盐度及营养盐有相关性,但其相关性因不同种和不同点而不同。检测期间,网箱养殖鱼类无明显流行病发生,但弧菌的高峰期明显与报道的鱼病高峰期相吻合。     

Cloning, expressing, and hemolysis of tdh, trh and tlh genes of Vibrio parahaemolyticus

Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea,Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors.They are thermostable direct hemolysin (TDH),TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR).We ligated the three genes into prokaryotic expression vector pET-28a (+),and transformed the recombinant plasmids into Es-cherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins,TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis.The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes’ functions under a single factor level, but also provide evidence for VP vaccine engineering.     

溶藻弧菌双组分调控系统PhoR/PhoB的基因克隆及生物信息学分析

双组分调控系统（Two-component Regulatory System）在致病菌的生长及毒力调控中起重要作用.克隆溶藻弧菌（Vibrio alginolyticus）HY9901 株组氨酸激酶PhoR 和反应调控因子PhoB 的全长基因,并对其进行生物信息学分析.序列分析结果显示,phoR（GenBank 登录号：KJ958404）全长1 299 bp,共编码432 个氨基酸;phoB（GenBank 登录号：KJ863646）全长690 bp,编码229 个氨基酸.构建PhoR/PhoB 的系统进化树,结果显示,溶藻弧菌PhoR/PhoB 与副溶血弧菌（Vibrio parahaemolyticus）、哈氏弧菌（Vibrio harveyi）有较近亲缘关系.利用SWISS-MODEL 软件,对PhoR/PhoB 中两个相对保守的功能域HATPase_c 和REC 进行同源建模,发现HATPase_c具有1 个ATP 结合位点,REC 具有4 个天冬氨酸活性位点.     

Impact of Vibrio parahaemolyticus and white spot syndrome virus (WSSV) co-infection on survival of penaeid shrimp Litopenaeus vannamei

White spot syndrome virus(WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect L itopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus(WSSV+Vp), or we fi rst infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection(Vp+WSSV). The effect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of V ibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction(PCR) method. L v My D88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the different mortality rates. Our results show that(1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection;(2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed signifi cant increases in the numbers of Vibrio( P 0.05);(3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10 5 to 10 7 /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.     

Diversity of bacteria isolated from crustacea larvae and their rearing water

1　Introduction　Blacktigershrimp (Penaeusmonodon)isthemajorshrimpspeciesculturedinIndonesia .Itsculturehasbecomemoreintensivebecauseofitshighdemandandeconomicvalue .Infact,shrimpexporthasyieldedthehighestIndonesianincomeinfisherysector .Itwasestimatedthattheproductionofculturedshrimpwas 80 0 0 0metrictonnesin 1995 .Consequently ,theneedforthefryforstockingpondculturesteadilyin creased .Thetotalnumberoffryrequiredwasabout 6millionperyear (FAO ,1995 )and 90 %oftherequiredanimalswereobtained…     

Effects of Dietary Vitamin C on the Immune Function of Shrimps, Penaeus chinensis

Prepared in this experiment were six groups of diets, i.e. VC0, VC1, VC2, VC3, VC4 and VC5 with the contents of vitamin C (VCmg(100g)-1 diet) of 0, 100, 200, 400, 800, and 1200 respectively. It was found that vitamin C increased the concentrations of immunoglobulin-like (IgG-like, IgA-like and IgM-like) substances in the serum of Penaeus chinensis after a feeding period of 3 weeks. The differences among groups were significant (P＜ 0.01), but there was no difference in the contents of complement3-1ike and complement4-1ike substances in the serum (P ＞0.05). Phenoloxidase (PO) activity in the serum of VC3 group shrimps was higher than that of VC0 and other groups, but no significant difference was observed between VC0 group and other groups. Furthermore, bactericidal activity of the serum to Vibrio parahaemolyticus in shrimps fed with the VC1 diet was higher than that in the other groups (P＜ 0.01), while no difference was demonstrated among all groups for the bactericidal activity to Vibrio alginolyticus (P ＞0.05). It is, therefore, suggested that vitamin C (100-400 mg(100 g) -1 diets) could be used as an immunostimulant of P. chinensis.     

溶藻弧菌对凡纳滨对虾血清中一氧化氮及氧自由基的影响

对凡纳滨对虾注射浓度为1×105、1×106、1×107mL-1的溶藻弧菌悬液,在注射后4、12、24、48、72和120 h于腹血窦取血,测定其血清中一氧化氮(NO)、丙二醛(MDA)含量,以及一氧化氮合酶(NOS)、超氧化物岐化酶(SOD)活力。结果表明,注射溶藻弧菌后,凡纳滨对虾血清中NO含量及NOS活力显著高于对照组,且NOS活力最大值出现时间较NO早;而SOD活力先升高后下降,MDA含量在SOD活力开始下降后逐渐升高,说明一氧化氮系统对凡纳滨对虾感染的溶藻弧菌有清除作用,并能在一定程度上对损伤过程中生成的氧自由基产生作用。     

A Fast and Indirect Fluorescent Antibody Assay for the Vibrio in Large Yellow Croaker Pseudosciaena crocea (Richardson)

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