凡纳滨对虾C型凝集素（LvLc1）的特征和活性分析

C型凝集素是一种依赖于Ca~(2+)而发挥功能的糖蛋白,在一线的固有免疫防御过程中发挥着重要作用。围绕对虾C型凝集素开展深入研究,不仅可以丰富无脊椎动物固有免疫学内容,还有望将其开发为具有免疫增强效果的活性饵料,应用于对虾的健康养殖。本实验根据实验室前期转录组信息提示克隆获得了凡纳滨对虾一种新的C型凝集素基因(LvLc1,Gen Bank注册号:KY937940)。生物信息学分析显示LvLc1基因的开放阅读框全长891 bp,编码296个氨基酸,该基因编码的蛋白质含有一个保守的糖识别结构域(carbohydrate recognition domain,CRD),该结构域中具有潜在的半乳糖结合位点(QPD motif),进化发生分析显示LvLc1与来自节肢动物的甘露糖结合凝集素家族成员聚类在一起。对LvLc1基因的CRD结构域进行了原核重组表达与蛋白活性分析研究,结果显示:重组目的蛋白(rLvLc1)在Ca~(2+)存在的条件下,对多种病原菌(G~+、G~–和真菌)具有凝集作用,其凝集活性可被半乳糖、甘露糖、脂多糖等多种病原相关分子模式所抑制。研究表明,LvLc1作为C-型凝集素家族一个新成员,可能通过重要的模式识别受体作用,参与机体应答病原微生物侵染的防御过程。     

A novel mannose-binding lectin from plasma of Labeo rohita

A new mannose-binding lectin was purified from plasma of freshwater fish, rohu (Labeo rohita) by ammonium sulphate precipitation followed by DEAE-cellulose ion-exchange chromatography. The hemagglutinating activity is strong for neuraminidase-treated rabbit erythrocytes. Mannose, glucose and their derivatives inhibit hemagglutination. The apparent molecular weight was determined to be 210 kDa in native PAGE. Analysed on SDS-PAGE under reducing and non-reducing conditions, the protein migrated as a single band of mol.wt. 40,000. The lectin is acidic in nature showing a pI of 4.5. It is a glycoprotein containing complex glycan part as indicated by carbohydrate analysis using high-pressure anion exchange chromatography with a pulsed amperometric detector. The N-terminal sequence (WLNGIGTQIPMKITT) shows no significant homology with known proteins. It appears to be a C-type lectin as Ca²⁺ is essential for carbohydrate-binding activity. Methyl -α- D-mannopyranoside was found to be the most potent inhibitor among the monosaccharides and disaccharides tested. Purified lectin was found to induce intracellular superoxide production following opsonization of E. coli by its own macrophages. This revised version was published online in August 2006 with corrections to the Cover Date.     

中国明对虾C 型凝集素基因(Fclectin)的重组表达及活性分析

研究拟通过分析对虾C型凝集素的活性特点,探讨其在对虾先天免疫应答过程中的潜在功能以及在养殖生产实践中的应用。实验利用原核表达系统对中国明对虾C-型凝集素基因的两个串联的糖识别结构域(carbohydrate recognition domain,CRD)进行了重组表达,并通过纯化复性获得了重组目的蛋白(rFclectin-CRD1和rFclectin-CRD2)。活性分析结果显示,重组目的蛋白对多种病原菌有凝集和抑制生长的作用,并且具有Ca2+依赖活性;其凝集活性可被半乳糖、肽聚糖、脂多糖等多种病原相关分子模式所抑制,研究结果证实,Fclectin是一种典型的C-型凝集素,它可能作为中国明对虾先天免疫中重要的模式识别受体,在一定程度上参与了机体应答病原微生物的防御过程。     

日本沼虾C型凝集素结构域家族3的cDNA克隆、原核表达和定位分析

C型凝集素(C-type lectin)是一类能与糖类结合的非抗体的蛋白质或糖蛋白家族,为了研究C型凝集素基因在日本沼虾组织分布、细胞定位和细菌感染过程中的表达情况,本研究应用cDNA末端快速克隆(rapid-amplification of cDNA ends,RACE)技术首次克隆了日本沼虾C型凝集素结构域家族3基因(MnLec3)的全长序列,通过实时荧光定量PCR(qRT-PCR)分析MnLec3基因在不同组织、细菌感染后不同时间的表达水平,Western blot和免疫荧光分别分析蛋白的表达水平和细胞定位。结果显示,MnLec3基因cDNA全长1 357 bp,包括125 bp的5′末端非翻译区(UTR)、1 026 bp的开放阅读框(ORF)和206 bp的3′UTR,其中开放阅读框编码341个氨基酸。氨基酸序列比对显示,日本沼虾MnLec3基因含有保守钙结合点(Met 1-Glu17)和糖识别结构域(CRD)。同源性分析结果显示,MnLec3与罗氏沼虾C型凝集素3相似度较高;邻接法(Neighbor-Joining,NJ)进化树分析结果显示,MnLec3与其他甲壳动物C型凝集素聚为一支。通过构建原核表达载体获得体外重组蛋白rMnLec3,并将纯化重组蛋白免疫大鼠获得抗血清,免疫荧光结果显示,绿色荧光信号主要在肝胰腺细胞核中表达。qRT-PCR结果显示,MnLec3在日本沼虾所检测组织中均表达,其中肝胰腺中表达量最高,血细胞次之;与对照组相比,在嗜水气单胞菌刺激12～48 h时MnLec3表达量显著升高,48 h表达量最高,Western blot分析结果显示,MnLec3蛋白表达丰度与基因表达模式基本相似,提示克隆得到的MnLec3参与日本沼虾抵御细菌入侵的免疫过程。     

Ca2+- and Mg2+-Induced Conformational and Rheological Changes of Actomyosin Extracted from Fresh and Freeze-Thaw Tilapia

ABSTRACT

The conformational changes of natural actomyosins prepared from fresh and freeze-thaw tilapia (Oreochromis niloticus) in the presence of Ca²⁺ or Mg²⁺ were investigated. The Ca²⁺-activated adenosine triphosphatase (Ca²⁺-ATPase) activities of actomyosins extracted from fresh and freeze-thaw fish were comparable (p > 0.05). The denaturation temperatures (T_d) of actomyosins extracted from fresh fish were lower than those from freeze-thaw counterparts (p < 0.05). The addition of Ca²⁺ or Mg²⁺ reduced the T_d of actomyosins. Ca²⁺ and Mg²⁺ enhanced protein aggregation at ≥ 40°C (p < 0.05). Based on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern, the myosin heavy chain (MHC) and actin bands tended to form large aggregates to a greater extent in the presence of 100mM Ca²⁺ or Mg²⁺. Ca²⁺ and Mg²⁺enhanced disulfide linkages and hydrophobic interactions among actomyosin molecules. The onset temperature of elastic modulus (G′) of both actomyosins was shifted to lower temperature as 100mM of Ca²⁺ or Mg²⁺ was added. Mg²⁺ at 20mM increased the breaking force of washed tilapia mince at 40°C. Our results revealed that the intrinsic properties of actomyosins extracted from fresh and frozen fish were distinct, and divalent ions Ca²⁺ or Mg²⁺ affected their gelation differently.     

鱼蛋白酶水解物的钙螯合修饰及其功能活性研究

以低值鱼蛋白为原料通过复合酶水解法和钙修饰法获得了蛋白质酶水解物的修饰产物并对其功能活性进行了初步研究.结果表明,未脱脂的鱼蛋白酶水解物钙螯合修饰的最适宜条件为蛋白质水解度为5%、螯合pH为7.0、螯合温度为20℃、螯合完成时间为15min;无水乙醇分级分离可获得三种螯合组分,水不溶组分(CA)、50%无水乙醇不溶性组分(CB)和80%无水乙醇不溶性组分(CC);红外光谱分析表明,CA的钙紧密地与氨基和羰基基团结合形成螯合修饰物,并以钙为中心形成五元环,而CB和CC组分中钙只与羰基紧密结合形成环状结构;在这些螯合物组分中,CA具有最高的抗氧化活性,且高达α-生育酚的94%,而CC具有最高的抗大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌的活性,并发现抗菌活性大小与组分的水溶性和分子中电子中继系统的电子缓冲能力有关.本文的研究可望对海洋低值鱼蛋白质的高效利用和蛋白质的酶水解物经螯合修饰后作为食品添加剂在食品工业中的应用奠定理论基础.     

A high amylase‐producing bacterial strain exhibiting the phosphorus‐dissolving ability,isolated from freshwater aquaculture pond

Amylase‐producing bacteria could improve water quality contaminated by waste from feed residue and fish metabolism, thereby increasing the efficiency of aquaculture systems. The objective of this research was to screen and optimize fermentation conditions of a high amylase‐producing strain. Four amylase‐producing bacterial strains (named S458‐1, G05, H38 and B09) were isolated from a grass carp pond, and the strain S458‐1 showed the highest amylase‐producing ability, with 19.58 ± 0.38 mm hydrolysis circle diameter. The strain S458‐1 was identified as Bacillus cereus based on morphological identification, biochemical identification and 16S rDNA sequence analysis. The optimal culture medium formula included (in g/L) Ca²⁺ 0.8, Mg²⁺ 0.2, Mn²⁺ 0.4, Fe²⁺ 0.6, Al³⁺ 0.2, 1% soluble starch and 1% peptone. The optimal fermentation conditions were determined as initial pH 9, culture temperature 37°C, fermentation time of 60 hr and 2% inoculum. Under the optimal formula and condition, its enzyme activity increased from 32 U/ml to 173.01 U/ml, a 5.41‐fold increase. Surprisingly, our research found that the strain S458‐1 also had phosphorus degradation capabilities. Its phosphorus‐dissolving ability was both time‐ and concentration‐dependent. Thus, this study will make a contribution to the bacterial amylase based on the fermentation process and provide a theoretical basis for further research of aquatic probiotics.     

仿刺参体腔液的抗菌特性

为了解仿刺参体腔液的抗菌谱以及不同二价金属离子对仿刺参体腔液抗菌活性的影响,实验采用生长曲线测定法分别测定了仿刺参体腔细胞破碎液上清和体腔液上清对哈维氏弧菌、灿烂弧菌、希瓦氏菌、假交替单胞菌、金黄色葡萄球菌、溶壁微球菌、停乳链球菌、拟诺卡式菌生长的影响,并通过该法以溶壁微球菌为受试菌测定了海洋环境中常见的Fe2+、Ca2+、Cd2+、Mg2+、Mn2+、Zn2+对仿刺参体腔液上清抗菌活性的影响。结果显示,仿刺参体腔细胞破碎液上清对受试的8株细菌的生长均无抑制作用,体腔液上清对溶壁微球菌的生长有明显的抑制作用,而对其他7株受试菌的生长无明显影响;Mn2+和Zn2+可明显增强体腔液上清对溶壁微球菌的生长抑制作用,而Fe2+、Ca2+、Cd2+、Mg2+对体腔液上清的抗菌活性无明显影响。研究表明,仿刺参体腔液在体外状态下只具有窄谱抗菌活性,与抗菌相关的免疫因子主要存在于体腔液上清中;体腔液中的抗菌免疫因子对海洋环境中的常见二价金属离子有一定的适应性,而且适当浓度的Mn2+和Zn2+可能具有促进仿刺参抗菌应答能力的作用。     

Branchial calcium uptake: possible mechanisms of control by stanniocalcin

The branchial Ca²⁺ uptake by teleost fish is under inhibitory control by the hormone stanniocalcin (STC) which is generated by the corpuscles of Stannius (CS). Removal of the CS in North American eel, Anguilla rostrata LeSueur, induced a rapid rise in blood calcium levels. Branchial Ca²⁺ influx following the extirpation of the CS (stanniectomy, STX) increased during the first four days and stayed elevated thereafter (in agreement with previous studies). The transepithelial potential (TEP) across the gills did not change after STX and this means that the electrochemical gradient for Ca²⁺ is less favourable for passive influx of Ca²⁺ in STX eel. Therefore, the Ca²⁺ influx in STX eels is a transcellular flux, with Ca²⁺ crossing the apical and basolateral membrane barrier. The kinetics of ATP-driven Ca²⁺-transport across basolateral plasma membranes from eel gills did not change after STX. Thus, the increased Ca²⁺-influx after STX is not correlated with changes in ATP-dependent Ca²⁺-extrusion across the basolateral membrane, suggesting a regulation at the apical membrane. Moreover, STC did not affect ATP-driven Ca²⁺-transport in isolated basolateral membranes (in vitro). STC (0.1 nM) reduced cAMP levels in dispersed eel gill cells. It had no significant effect on the IP₃ levels in these cells. We postulate that STC controls the permeability to Ca²⁺ of the apical membranes of the Ca²⁺ transporting cells of fish gills by controlling second messenger operated Ca²⁺ channels in the apical membrane.

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Résumé L'entrée de calcium au niveau des branchies est sous le controle inhibiteur de la stanniocalcine (STC) qui est synthétisée au niveau des corpuscules de Stannius (CS). L'ablation des CS chez l'anguille d'Amérique du Nord, Anguilla rostrata LeSueur, induit une augmentation rapide des niveaux de calcium dans le sang. Le flux entrant branchial de calcium consécutif à l'ablation des CS (stanniectomie, STX) augmente pendant les 4 premiers jours et reste élevé au-delà (en accord avec des études antérieures). Le potentiel transépithélial (TEP) à travers les branchies ne change pas après STX, ceci indiquant que le gradient électrochimique du Ca²⁺ est moins favorable pour le flux entrant passif du Ca²⁺ chez l'anguille STX. En conséquence, le flux entrant de Ca²⁺ chez l'anguille STX est un flux transcellulaire, avec le Ca²⁺ traversant la barrière membranaire apicale et basolatérale. La cinétique du transport de Ca²⁺ conduit par l'ATP à travers les membranes plasmatiques basolatérales de branches d'anguille n'est pas modifiée après STX. Ainsi, l'augmentation du flux entrant de Ca²⁺ après STX n'est pas corrlée avec des modifications de l'excrétion de Ca²⁺ conduit par l'ATP à travers la membrane basolatérale, suggérant donc une régulation au niveau de la membrane apicale. De plus, la STC ne modifie pas le transport de Ca²⁺ conduit par l'ATP dans des membranes basolatérales isolées (in vitro). La STC (0.1 nM) réduit les niveaux d'AMPc dans des cellules dispersées de branchies d'anguille. Cette hormone n'a pas d'effet significatif sur les niveaux d'IP₃ dans ces cellules. Nous suggérons que la STC régule la perméabilité au Ca²⁺ des membranes apicales des cellules branchiales transporteuses de Ca²⁺ en controlant un second messager agissant sur les canaux calciques de la membrane apicale.

     

解淀粉芽孢杆菌HZ-1510壳聚糖酶基因的克隆、重组表达及其活性

为了研究壳聚糖酶的水解活性,实验进行了解淀粉芽孢杆菌HZ-1510壳聚糖酶基因编码序列的克隆,并构建了谷胱甘肽转移酶(GST)-壳聚糖酶融合蛋白表达质粒,在大肠杆菌中表达后提取、纯化得到重组蛋白,并通过生物信息学对该蛋白的信号肽、三维结构等进行了分析,最后以胶态壳聚糖溶液为底物研究了该重组壳聚糖酶的活性。结果显示,该壳聚糖酶基因的ORF长为837 bp,编码279个氨基酸,分子量为31.45 ku。在其氨基末端具有信号肽,切割点位于36和37位氨基酸之间。氨基酸序列同源性分析表明该壳聚糖酶属于GH46家族糖苷水解酶。酶的水解活性最适温度约为55°C,最适pH为5.5。而金属离子Fe3+、Ag+、Cu~(2+)、Ba~(2+)和K+对其水解活性都起抑制作用,但是Mn~(2+)、Ca~(2+)和Mg~(2+)对其活性起增强作用。0.1 mmol/L的Zn~(2+)对壳聚糖酶的活性起增强作用,2.0 mmol/L的Zn~(2+)对壳聚糖酶的活性起抑制作用。本实验研究了解淀粉芽孢杆菌HZ-1510壳聚糖酶在不同条件下的水解活性,为壳聚糖酶的工业应用奠定了理论基础。     

The effects of different Ca2+ concentration fluctuation on the moulting,growth and energy budget of juvenile Litopenaeus vannamei (Boone)

A 40‐day experiment was conducted to investigate the effects of different Ca²⁺ concentration fluctuation on the moulting, growth and energy budget of juvenile Litopenaeus vannamei with an initial wet body weight of (1.20±0.01) g. The Ca²⁺ concentration of the control group C385 was 385 mg L^?1 throughout the experiment, while the Ca²⁺ concentration of the C591, C803, C1155 and C2380 groups periodically fluctuated from 385 to 591, 803, 1155 and 2380 mg L^?1 respectively. The moulting frequency (MF) of the shrimp in the Ca²⁺ concentration fluctuating groups was significantly higher than those in the control group (P<0.05). The specific growth rates (SGR_d) and feed intake of the shrimp in the C591 and C803 groups were significantly higher than those in other groups (P<0.05), but no significant differences in feed efficiency were found among all groups (P>0.05). The shrimp in C591 and C803 groups spent significantly less energy in respiration, while depositing significantly more energy for growth than those in other three groups (P<0.05). These results showed that proper Ca²⁺ concentration fluctuation could increase the MF and growth rate of the juvenile L. vannamei, and according to the regression formula made using SGR_d and range of Ca²⁺ concentration fluctuation, periodically enhanced Ca²⁺ concentration of 295 mg L^?1 in the seawater was suggested to be used in shrimp culture.     

Calcium balance in embryos and larvae of the freshwater-adapted teleost,Oreochromis mossambicus

Changes in Ca²⁺ content and flux, and the development of skin chloride cells in embryos and larvae of tilapia, Oreochromis mossambicus, were studied. Tilapia embryos hatched within 96h at an ambient temperature of 26–28°C. Total body Ca²⁺ content was maintained at a constant level, about 4–8 nmol per individual, during embryonic development. However, a rapid increase in body Ca²⁺ level was observed after hatching, 12.8 to 575.3 nmol per individual from day 1 to day 10 after hatching. A significant influx and efflux of Ca²⁺ occurred during development, with the average influx rate for Ca²⁺ increasing from 5.9 pmol mg⁻¹ h⁻¹ at 48h postfertilization to 47.8 pmol mg⁻¹ h⁻¹ at 1 day posthatching. The skin was proposed as the main site for Ca²⁺ influx before the development of gills, and the increased Ca²⁺ influx may be ascribed to gradual differentiation of skin surface and chloride cells during embryonic development. Ca²⁺ efflux was 16–56 pmol mg⁻¹ h⁻¹ in 1-day-old larvae. The resulting net influx of Ca²⁺, 10–12 pmol mg⁻¹ h⁻¹, accounted for the increased Ca²⁺ content after hatching. When comparing the measured and estimated ratios of efflux and influx, active transport was suggested to be involved in the uptake of Ca²⁺. Chloride cells, which may be responsible for the active uptake of Ca²⁺, started to differentiate in the skin of embryos 48h after fertilization, and the density of chloride cells increased following the development. A possibility of active transport for Ca²⁺ in early developmental stages of tilapia is suggested.     

Effects of cadmium on the kinetics of calcium uptake in developing tilapia larvae, Oreochromis mossambicus

The toxic effects of Cd²⁺ on Ca²⁺ influx kinetics in developing tilapia (Oreochromis mossambicus) larvae were evaluated. Addition of 20 µg l^-1 of Cd²⁺ to the environment of 0 and 3 day-old larvae competitively inhibited the Ca²⁺ uptake within 4h resulting in a great increase in K_m values for Ca²⁺ influx (19.3 and 17.4 fold, respectively) as compared with their respective controls. Consequently, the actual Ca²⁺ influx of larvae in solutions of 0.2 mM Ca²⁺ are suppressed by 32–45%. Also, 3 day-old larvae were more sensitive to internally accumulated Cd²⁺ than 0 day-old larvae. Although the Ca²⁺ influx in 0 and 3 day-old larvae may be restored to the levels of their respective controls with 24h of being transferred to a 20 µg l^-1 Cd²⁺ solution, total body Ca²⁺ content was significantly reduced in 3 day-old larvae. Increased Ca²⁺ uptake efficiency ensures sufficient Ca²⁺ for normal growth. However, rapid increase in Ca²⁺ influx after hatching also leads to higher Cd²⁺ uptake. Exposure to Cd²⁺ will lead to a drop in body Ca²⁺ content resulting in retardation of larval growth. Therefore, we conclude that if Ca²⁺ uptake is interfered with at this critical stage of development, larvae will not be able to maintain normal levels of body Ca²⁺ and will show signs of Cd²⁺ poisoning.     

Effect of farming patterns on growth,antioxidant activity and nonspecific immunity of loach,Paramisgurnus dabryanus

This study was carried out to compare growth, intestinal enzymes activity, antioxidation and nonspecific immunity of loach between farmed and imitative ecological farming loach, Misgurnus anguillicaudatus. Loach from farmed (average body weight 15.03 ± 0.36 g) and imitative ecological farming (average body weight 12.44 ± 0.62 g) population were used to analyse growth, whole-body composition, intestinal enzymes, antioxidant enzymes activity and resistance to bacteria. The results showed specific growth rate, average daily gain and protease, lipase, amylase and γ-GT activities in intestine and hepatopancreas of farmed loach were significantly higher (p < 0.05) than of imitative ecological farming loach. However, total protein, lysozyme and albumin contents and superoxide dismutase, catalase and glutathione peroxidase activities of imitative ecological farming loach were significantly higher (p < 0.05) than of farmed loach. Moreover, resistance to Aeromonas hydrophila of imitative ecological farming loach was higher than of farmed loach (p < 0.05). Taken together, ecological farming loach possesses higher antioxidant capacity and resistance to bacteria than farmed loach.     

扇贝内脏团中耐镉菌株的分离及其吸附镉机理

为探讨扇贝内脏团中微生态系统与扇贝高能力富集镉(Cd)的关系,从自然环境中采集的栉孔扇贝内脏团中分离、纯化了2株耐Cd细菌(编号为菌株A和B),运用16S rDNA基因序列进行分子鉴定,通过金属吸附实验研究了耐Cd菌株对Cd的吸附能力及其对Cd的吸附特性,并利用扫描电镜、透射电镜相结合的方法研究了Cd胁迫条件下耐镉菌株细胞形态与结构变化,探讨了其对Cd的吸附机理。结果显示,2株菌株(A和B)在固体培养基上能耐受Cd的浓度分别为100和80 mg/L。经16S rDNA测序鉴定菌株A与Nitratireductor sp.亲源关系最近,菌株B与Ruegeria sp.亲源关系最近。2株菌株对Cd的吸附率远高于铜(Cu)、锰(Mn)、锌(Zn)和铅(Pb)等重金属。在50 mg/L Cd浓度的液体培养基中,菌株A、B对Cd富集量分别为48.57和42.14 mg/g,富集系数分别为971.4和842.8。电镜观察结果显示,经过Cd处理后,2菌株数量均有所减少,细胞中出现空泡,菌株A细胞外沉淀增多,菌株B表面变得粗糙且出现凹陷,表明胞外沉积作用可能是耐Cd菌株对Cd富集作用的重要途径。     

Effects of constant Ca2+ concentration in salinity fluctuations on growth and energy budget of juvenile Litopenaeus vannamei

A 50-day experiment was conducted to investigate the effects of constant Ca²⁺ concentration in salinity fluctuations on growth and energy budget of juvenile Litopenaeus vannamei. The salinity and Ca²⁺ concentration of control group S0 were kept 30‰ and 385 mg l⁻¹, respectively throughout the experiment while the ranges of salinity fluctuations of the treatment groups S5 and S5C, S10 and S10C, S15 and S15C, S20 and S20C were 5, 10, 15, and 20‰, respectively. The Ca²⁺ concentration of the treatment groups S5, S10, S15, and S20 (salinity fluctuation groups) changed with salinities, while the Ca²⁺ concentration of the treatment groups S5C, S10C, S15C, and S20C (constant Ca²⁺ concentration groups) was kept 385 mg l⁻¹ during the experiment. The results were as follows: (1) There were no significant differences in SGR of shrimp in salinity fluctuation groups (P > 0.05); But shrimp in the groups S15C and S20C had significantly higher SGR than those in the groups S5C and S0 in constant Ca²⁺ concentration groups (P < 0.05); (2) Results of the t test showed that significant differences in SGR were also found between S15 and S15C (P = 0.012), and between S20 and S20C (P = 0.013); (3) The shrimp in groups S15C and S20C deposited significantly more energy for growth and spent significantly less energy in respiration than those in groups S15 and S20 (P < 0.05), respectively. These results showed that compared with constant salinity and fluctuating salinities, the growth performance of shrimp under constant Ca²⁺ concentration in large ranges of salinity fluctuations could be better.     

The role of rpoS in the regulation of Vibrio alginolyticus virulence and the response to diverse stresses

Vibrio alginolyticus is a leading aquatic pathogen, causing huge losses to aquaculture. rpoS has been proven to play a variety of important roles in stress response and virulence in several bacteria. In our previous study, upon treatment with Cu²⁺, Pb²⁺, Hg²⁺ and low pH, the expression levels of rpoS were downregulated as assessed by RNA‐seq, while impaired adhesion ability was observed, indicating that rpoS might play roles in the regulation of adhesion. In the present study, the RNAi technology was used to knockdown rpoS in V. alginolyticus. In comparison with wild‐type V. alginolyticus, RNAi‐treated bacteria showed significantly impaired abilities of adhesion, growth, haemolytic, biofilm production, movement and virulence. Meanwhile, alterations of temperature, salinity, pH and starvation starkly affected rpoS expression. The present data suggested that rpoS is a critical regulator of virulence in V. alginolyticus; in addition, rpoS regulates bacterial adhesion in response to temperature, pH and nutrient content changes. These are helpful to explore its pathogenic mechanism and provide reference for disease control.     

pH值和Ca2＋浓度对日本沼虾生长和能量收支的影响

本文报道了不同ｐＨ值（６．５、７．５和８．５）和不同Ｃａ＾２＋浓度（３８．８、６１．１和７８．８ｐｐｍ）对日本沼是生长和能量收支的影响，实验结果表明，ｐＨ值对该虾的生长有一定的影响，Ｃａ＾２＋和ｐＨ值在影响其生长的过程中可能有一定的交互作用，ｐＨ值和Ｃａ＾２＋对该虾生长的影响主要是通过影响其能量摄入量实现的，本实验条件下，该虾摄入的能量平均有１５．８％用于生长，１．８％作为粪便排出体外，其余用于呼     

Mechanism of Cd<Superscript>2+</Superscript> on DNA cleavage and Ca<Superscript>2+</Superscript> on DNA repair in liver of silver crucian carp

The subject of acute injury, apoptosis and canceration of animals induced by heavy metal ions has been one of the hotspots studied worldwide. However, the exact molecular mechanism of Cd-induced carcinogenicity remains largely unclear, and how to relieve the toxicity in vivo has rarely been reported. For this paper, we have investigated the mechanism of Cd²⁺ on DNA cleavage and Ca²⁺ on DNA repair in the liver of silver crucian carp (Carassius auratus gibelio) by agarose gel electrophoresis methods and by estimating biochemical indexes. Our results show that Cd²⁺ induces the classical laddering degradation of DNA in vivo and that DNA cleavage is repaired after injection with Ca²⁺ under various Cd²⁺ concentrations. DNA cleavage caused by Cd²⁺ is due to the activation of deoxyribonuclease (DNase) and the accumulation of reactive oxygen species (ROS). Furthermore, Cd²⁺ destroys the antioxidant system, which diminishes the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), causing an increase of the lipid peroxidation (LPO) level, respectively. However, after the liver is injected with Ca²⁺ under various Cd²⁺ concentrations, the DNase activity, the ROS generating rate and the LPO level are obviously reduced, the activities of SOD, CAT, and POD are greatly increased. At the same time, ROS production and removal recoves its balance. The results show that Ca²⁺ can relieve the toxicity of Cd²⁺ in silver crucian carp.     

Evaluation of Tilapia Effluent with Ion Supplementation for Marine Shrimp Production in a Recirculating Aquaculture System

Reuse of fish effluent for the culture of marine shrimp, such as Pacific white shrimp, Litopenaeus vannamei, could provide an opportunity for the US shrimp farming industry to ease constraints (e.g., environmental concerns and high production costs) that have limited them in the past. In this study under laboratory‐scale conditions, the feasibility of culturing L. vannamei in effluents derived from a commercial facility raising tilapia in recirculating aquaculture systems (RAS), supplemented with various salt combinations, was compared to the shrimp’s survival and growth in well water supplemented with 17.6 (control) and 0.6 (freshwater treatment) g/L synthetic sea salt. Three independent trials were conducted in RAS in which survival and growth in the control, the freshwater treatment, and two effluent treatments were compared. Water quality during this study was within safe levels and no differences (P < 0.05) between treatments were observed for dissolved oxygen, nitrite, pH, total ammonia nitrogen, and temperature. However, average nitrate and orthophosphate levels were consistently more than an order of magnitude greater in the effluent treatments compared to the control and the freshwater treatments. Survival and growth of shrimp over 6‐wk periods did not vary significantly between the control and the freshwater treatments; however, shrimp tested in the tilapia effluents often exhibited significant effects (P < 0.05) depending on the salts added. In the low‐salinity waters, correlations (P < 0.05) were observed between Ca²⁺, Mg²⁺, Ca²⁺ and Mg²⁺, K⁺, Na⁺ : K⁺ and Ca²⁺ : K⁺, and shrimp survival and growth. The results of this study revealed that L. vannamei can be raised in tilapia effluent when supplemented with synthetic sea salt (0.6 g/L), CaO (50 mg/L Ca²⁺), and MgSO₄ (30 mg/L Mg²⁺).