硫化物胁迫对凡纳滨对虾血细胞的毒性影响

硫化物是水产养殖过程中常见的水体污染物之一。为探讨水体硫化物胁迫对虾类血细胞的毒性影响,以不同浓度(0.5 mg/L,2.0 mg/L)的硫化物对凡纳滨对虾(Litopenaeus vannamei)进行胁迫,于胁迫后6、12、24和48 h取血淋巴,应用流式细胞术测定血细胞的总数(THC)、活性氧(ROS)含量、一氧化氮(NO)含量以及凋亡率。结果显示,经0.5 mg/L硫化物胁迫48 h后,对虾THC显著下降至11.78×10~6个/mL,为对照组的72.7%(P0.05),ROS含量为对照组的225.2%(P0.05),血细胞凋亡率显著上升至7.42%(P0.05);经2.0 mg/L硫化物胁迫6 h开始,对虾THC呈现显著的下降(P0.05),血细胞ROS含量和凋亡率显著提高(P0.05);表明硫化物胁迫刺激对虾血细胞产生大量ROS,诱导血细胞凋亡,从而导致THC下降,这一过程可能是硫化物导致虾类细胞免疫下降的重要机制;随着硫化物浓度的升高,其细胞毒性作用明显提高。血细胞NO含量在2.0 mg/L硫化物胁迫12和48 h时显著升高(P0.05),推测NO可能在硫化物胁迫防御调控中起着信号因子的作用。     

不同类群九孔鲍免疫防御机能的比较

对不同类群的九孔鲍细胞与体液免疫机能进行比较,结果表明,不同类群的九孔鲍免疫机能具有显著的差异性,复壮鲍(养殖10个月,个体大小为51.8～59.2mm)细胞与体液免疫机能都明显高于退化鲍(养殖20个月,个体大小为37.4～43.1mm)(P<0.01),与野生九孔鲍(个体大小为67.7～76.7mm)相比,复壮鲍细胞免疫机能略高于野生鲍,体液免疫机能则明显低于野生鲍。九孔鲍血细胞在吞噬活动中具有呼吸爆发产生活性氧的功能与特点,活性氧的产生与九孔鲍自身生理、环境温度以及异物数量等因素有显著的相关性(P<0.01)。环境温度由25℃下降至18℃,九孔鲍的细胞与体液免疫机能都有明显的下降。退化鲍在25℃和18℃条件下血淋巴液的抗菌活力水平差异性不明显。酚氧化酶存在于九孔鲍血淋巴液中,但其活性水平不高,在各类九孔鲍之间的差异性也不明显,不能确认为九孔鲍的主要体液免疫因子。     

魁蚶HIF-1α基因结构特征及对低氧胁迫的响应

低氧诱导因子(hypoxia-induciblefactor,HIF)是低氧信号传导途径中的关键因子,在动物低氧应答反应中发挥重要作用。为探讨魁蚶(Scapharca broughtonii) HIF-1α基因结构特征及在低氧胁迫下的应答规律,本研究以魁蚶转录组数据库中的部分序列为基础,通过cDNA末端快速扩增(rapid amplication of cDNA ends, RACE)技术克隆获得了魁蚶HIF-1α基因cDNA全长序列(命名为SbHIF-1α),并检测了其mRNA的组织分布和低氧胁迫下的表达规律。序列和结构分析显示, SbHIF-1α基因cDNA全长为2741 bp,其中包括2136 bp的ORF,编码711个氨基酸,含有HIF保守的HLH、PAS-A、PAS-B和PAC结构域,预测蛋白分子量为80.8kDa,理论等电点为5.57;SbHIF-1α与所选其他物种HIF-1α的序列相似度为56%～95%;系统进化分析显示SbHIF-1α与软体动物的遗传距离最近。qRT-PCR结果显示,在魁蚶的血淋巴、鳃、外套膜、斧足、闭壳肌和肝胰腺6个组织内均能检测到SbHIF-1α基因,在血淋巴中表达量最高,鳃次之,与其他4个组织都存在显著差异(P0.05)。海水溶解氧(DO)为0.5 mg/L、2.5 mg/L、4.5mg/L的低氧胁迫下,每个组织中SbHIF-1α都积极响应,其中血淋巴和鳃比其他四个组织的响应程度大;血淋巴中, DO为0.5 mg/L胁迫4 h后, SbHIF-1α的表达量较对照组即达到极显著差异(P0.01),胁迫64 h后,表达量达到最高,是对照组的519.43倍;每个组织对不同浓度DO处理响应结果表明,3个处理浓度中0.5mg/L处理对SbHIF-1α激活程度相对较大。本研究明确了SbHIF-1α的基因结构特征、时空表达特征及对低氧胁迫的响应规律,丰富了海洋贝类HIF基因的研究资料。     

盐度和胁迫时间对九孔鲍α-淀粉酶基因表达量的联合效应分析

采用中心复合设计和响应曲面法研究了盐度和胁迫时间对九孔鲍(Haliotis diversicolor supertexta)?-淀粉酶基因表达量影响的联合效应,旨在为九孔鲍盐度驯化及海区推广提供指导。实验设定盐度范围为22~40,时间范围为0~72 h,建立了盐度、胁迫时间与?-淀粉酶基因表达量之间关系的定量模型,并通过分析明确了盐度和胁迫时间的最优组合。结果表明,盐度对九孔鲍?-淀粉酶基因表达量影响的一次效应不显著(P0.05),而二次效应极显著(P0.01),说明盐度与九孔鲍?-淀粉酶基因表达量之间为非线性关系。随盐度升高,淀粉酶基因表达量呈先上升后下降的变化趋势,当盐度为31时淀粉酶基因表达量最大,此时九孔鲍消化能力最强。胁迫时间对淀粉酶基因表达量影响的一次效应显著(P0.05),二次效应不显著(P0.05),表明胁迫时间与九孔鲍?-淀粉酶基因表达量之间呈线性关系,即随着胁迫时间的延长,淀粉酶基因从开始低水平表达逐渐升高并恢复至正常表达水平。盐度和胁迫时间之间不存在交互作用。对实验结果建立模型可知,?-淀粉酶基因表达回归模型达极显著水平(P0.05),且失拟项不显著(P0.05),表明实验拟合出的方程有效。回归方程的决定系数为80.05%,校正系数65.80%,预测系数50.18%,表明方程拟合度较好,模型选择较为恰当,可以为不同盐度和胁迫时间诱导下的淀粉酶基因表达量变化提供参考依据。     

应用流式细胞术检测虾类血细胞总数方法的建立

通过对流式细胞术(FCM)检测虾类血细胞总数(THC)的条件和方法进行优化,为虾类血细胞学研究提供快捷、准确的测定方法。2012年7-9月,应用SYBR Green I作为荧光染料标记完整血细胞,设置3个不同染料浓度(1×、10×和100×),测定不同孵育时间下染色细胞比例的变化。结果显示,染料终浓度为10×时染色效果最佳,其最佳孵育时间为60 min;应用建立的FCM方法和显微计数方法测定10尾凡纳滨对虾(Litopenaeus vannamei)的THC,平均值分别为(16.68±1.57)×106个/m L和(15.09±1.76)×106个/m L,2种方法测定结果的相关性极显著(R2=0.8064,P<0.01)。凡纳滨对虾经不同浓度(0.5 mg/L和5.0 mg/L)的Cd2+胁迫,利用建立的FCM方法测定Cd2+胁迫下对虾THC的变化。结果显示,0.5 mg/L和5.0 mg/L Cd2+胁迫下,对虾THC随着胁迫时间的延长不断下降,胁迫48 h时分别下降至对照组的78.7%和64.7%,可见Cd2+胁迫对虾类血细胞产生毒性,抑制了血细胞活性,表明该方法适用于虾类的血细胞学研究。     

中华绒螯蟹分子育种群体与传统育种群体的耐低氧性能

本研究通过低氧-复氧处理,分析了中华绒螯蟹(Eriocheir sinensis)的分子育种群体(M选育系)与传统育种群体(JH选育系)的低氧耐受性能差异。结果显示,M选育系与JH选育系在低氧状态下的半致死浓度(LC₅₀)分别为(0.952±0.058)mg/L和(1.000±0.053)mg/L。在低氧处理12h时,两系的血细胞总数(THC)均达最高峰,M选育系(26.58×10⁶个/mL)高于JH选育系(23.88×10⁶个/mL),但差异不显著(P>0.05);而在低氧处理24h时,M选育系的THC显著高于JH选育系(P<0.05)。在低氧处理3 h和24 h时, M选育系的血蓝蛋白含量均高于JH选育系(P>0.05)。在低氧处理3h时,M选育系与JH选育系的血淋巴中葡萄糖含量分别下降9.55%与14.64%。在低氧处理3h和12 h时, M选育系的肝胰腺中糖原含量显著高于JH选育系(P<0.05)。在整个低氧期间, M选育系的肝胰腺中可溶性蛋白含量均高于JH选育系,而乳酸含量均低于JH选育...     

外源因子介导调控ROS对中国鲎血淋巴细胞参数的影响

以鲎血为主要原料制备鲎试剂是我国鲎资源开发的关键产业链。我国鲎种群数量逐年下降,为实现鲎资源的保护和可持续利用,开展对中国鲎造血作用机理的相关研究刻不容缓。本研究向中国鲎体内注射N-乙酰半胱氨酸(NAC)和灭活鳗弧菌(V),比较注射后0, 6, 12, 24, 48小时(h)中国鲎的血淋巴细胞总数、活性氧含量及非特异性免疫酶活性变化。结果表明,与对照组相比,注射NAC后血淋巴细胞总数(THC)、活性氧(ROS)、丙二醛(MDA)活性均有下降趋势,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、总抗氧化酶(T-AOC)、溶菌酶(LZM)、碱性磷酸酶(AKP)活性均有升高趋势。NAC与不同浓度V共同刺激下,THC、ROS、MDA含量相对仅注射NAC的下降有所减缓,其它酶活性有所升高。而血蓝蛋白(HC)在整个实验中无明显变化。6-48h,NAC组的THC、ROS与V组、NAC和V共刺激组相比呈降低趋势,共同刺激下SOD活性显著高于其它组。48h,NAC、V及共刺激下CAT、T-AOC组间无显著差异,但均高于对照组。NAC+10⁶V的MDA含量在48h最低,AKP活性在12-48h呈升高趋势,而NAC组的LZM活性在48h最高。注射NAC可降低THC、ROS,共刺激可缓和下降,V组THC、ROS升高,但其余血淋巴参数均提高,由此推测NAC和V均能刺激中国鲎血淋巴细胞的免疫机能;机体内的ROS含量对中国鲎血淋巴细胞增殖及再生起着重要作用。     

许氏平鲉低氧耐受能力及血液学和鳃组织学变化

为探明许氏平鲉低氧耐受能力及低氧胁迫过程中其血液生理生化指标和鳃组织形态结构变化,本研究分析了许氏平鲉临界氧分压(Pcrit)和失去平衡点(LOE)时溶解氧含量,观察了低氧胁迫和恢复溶解氧过程中呼吸行为和呼吸频率的变化,分析了血液生理[红细胞数目(RBC),白细胞数目(WBC),血红蛋白(Hb),红细胞积压(HCT)]、生化[葡萄糖、皮质醇]指标以及鳃组织形态学和鳃组织气体交换率(PAGE)变化。结果显示,在温度18 ℃、盐度30,pH值7.85的条件下,许氏平鲉[(88.01±5.34) g] Pcrit和LOE溶解氧含量分别为(3.96±0.11)和(2.60±0.21) mg/L。随着水体中溶解氧含量下降,血浆皮质醇和葡萄糖分别在LOE和Pcrit达到最高。许氏平鲉血液RBC、Hb和HCT在LOE达到最高。低氧过程中,许氏平鲉呼吸频率和PAGE分别于Pcrit和LOE达到最高,且低氧导致许氏平鲉鳃小片长度(SLL)、间距(ID)、周长显著增加;鳃小片宽度(SLW)则随水中溶解氧含量下降而变窄,在LOE处达到最小值。许氏平鲉鳃小片末端膨大、基质肥大、增生比例在低氧过程中显著增加。恢复正常溶解氧24 h后,上述各项检测指标恢复正常,与对照组无显著差异。实验中对照组各项指标无显著变化。研究表明,许氏平鲉对水体低溶解氧变化反应敏感,低氧应激导致其血液生理生化相关指标和鳃小片形态发生显著改变,恢复正常溶解氧24 h后可以显著缓解低氧胁迫应激。相关结果为研究许氏平鲉低氧耐受生理调控机制和高效健康养殖提供了理论依据和技术支撑。     

不同溶解氧水平对青海湖裸鲤肝组织结构及抗氧化酶活性的影响

为研究不同溶解氧水平对青海湖裸鲤(Gymnocypris przewalskii)肝组织结构、线粒体超微结构及抗氧化酶活性的影响,选取120尾平均体重为(97.68±0.12) g的青海湖裸鲤,随机分为三组:中度低氧组(3.0±0.1) mg/L、重度低氧组(0.7±0.1) mg/L和常氧组(8.4±0.1) mg/L,于低氧胁迫8 h和24 h,观察肝组织结构并检测线粒体膜电位及抗氧化酶活性。结果显示:中度和重度低氧胁迫使青海湖裸鲤肝血窦扩张,血窦内红细胞数量增多,中度低氧对肝组织显微和线粒体超微结构未造成损伤,线粒体排列整齐,形态正常,但重度低氧使肝细胞出现空泡化,且随低氧胁迫时间延长肝细胞空泡变性更严重,重度低氧24 h时线粒体数量减少,部分线粒体出现空泡化。肝组织线粒体膜电位在中度低氧胁迫后显著升高,重度低氧胁迫后显著降低。中度和重度低氧胁迫使肝组织过氧化氢(H_2O_2)含量、总抗氧化能力(T-AOC)和谷胱甘肽过氧化物酶(GPX)活性显著升高,但对超氧化物歧化酶(SOD)活性无影响;中度低氧对丙二醛(MDA)含量无影响,但重度低氧使MDA含量显著增加。结果表明,低氧胁迫对青海湖裸鲤肝组织造成氧化应激,但中度低氧对肝组织结构和线粒体未造成损伤,重度低氧导致肝组织结构及线粒体损伤,且低氧胁迫后肝组织通过调整相关抗氧化酶活性提高其低氧适应能力。     

低氧和酸化胁迫对大黄鱼早期发育阶段消化生理的影响

为探究大黄鱼(Larimichthys crocea)早期发育阶段低氧和酸化胁迫对其消化生理的影响,实验设置4个处理组,分别为对照组[溶解氧(DO)=7.0 mg·L~(-1),pH=8.1]、低氧组[溶解氧(DO)=3.5 mg·L~(-1),pH=8.1]、酸化组[溶解氧(DO)=7.0 mg·L~(-1),pH=7.3]和低氧-酸化组[溶解氧(DO)=3.5 mg·L~(-1),pH=7.3],每个处理组4重复,每个重复放置大黄鱼受精卵4.0×10~4粒,并在实验开始后(记录为0 d)和受精卵孵化后1 d、3 d、5 d、10 d、15 d、20 d、27 d分别测定其淀粉酶、胰蛋白酶和脂肪酶活性。实验结果表明:低氧胁迫对大黄鱼早期发育阶段消化酶活性的影响均较为明显;其中对淀粉酶活性的影响主要发生在10 d以后;对胰蛋白酶活性的影响更为明显,其酶活性在前10 d各时间点均显著高于对照组。低氧胁迫和酸化胁迫对脂肪酶活性的影响均主要发生在5～10 d仔鱼刚开口阶段,此阶段低氧胁迫和酸化胁迫均导致脂肪酶活性相比同期对照组显著降低。酸化胁迫对胰蛋白酶的影响则主要体现在5 d之前,导致此阶段胰蛋白酶活性明显较高。低氧-酸化双重胁迫对大黄鱼早期发育阶段消化酶活性的影响相比单一因子胁迫有一定的加剧作用。     

杂色鲍与九孔鲍种质资源研究进展

杂色鲍和九孔鲍是我国南方鲍养殖业的主要对象,九孔鲍与杂色鲍的分类关系,学术界一直存在着不同看法。文章综述了我国杂色鲍和九孔鲍在形态学、染色体核型、生化遗传学及分子遗传学方面种质研究的主要进展,得出二者间差异属于种群差异,台湾的九孔鲍和大陆的杂色鲍的种质差异达不到亚种水平的结论。提出了自己的观点,并提出了合理引种及保护原种的建议。     

Comparative analysis of the predominant carotenoids and chemical components in the common and orange‐muscle mutant of Haliotis gigantea

The Xishi abalone (Haliotis gigantea) is an economically significant aquaculture species in southern China. We identified a novel orange‐muscle mutation present in less than 2% of the cultured population. High‐performance liquid chromatography and electrospray ionization mass spectrometry identified zeaxanthin and β‐carotene as the two main pigments present in the orange‐muscle abalone. The concentrations of zeaxanthin and β‐carotene detected in the orange‐muscle abalones were significantly higher than those detected in the common abalones (p < 0.01). Notably, the concentration of zeaxanthin was approximately 16.5 times higher in orange‐muscle abalones than in common abalones, which was also the species with highest carotenoid content reported in shellfish to date. Furthermore, the contents of total amino acids and essential amino acids in the orange‐muscle abalone were higher than that in the common abalone, though not significantly, while the content of non‐essential amino acids was significantly higher in the orange‐muscle abalone than that in the common abalone (p < 0.05). The saturated fatty acid content in the orange‐muscle abalones was slightly lower than that in the common abalones, and the unsaturated fatty acid content was higher in the orange‐muscle abalones than that in the common abalones. These results could provide a basis for characterizing the mechanism by which carotenoids accumulate in abalones and further guide the breeding of abalone with orange‐muscle mutants in the future.     

盐度对九孔鲍摄食_生长及存活的影响

九孔鲍摄食人工饲料的盐度范围为２０￣３８，其中较适宜的盐度范围为２５￣３５，在此盐度范围内，小规格鲍（平均壳长１．３０ｃｍ，平均体重０．３７ｇ）的壳长日增长为９１．５￣１０２．３μｍ，日增重为８．６０￣１２．５５ｍｇ；大规格鲍（平均壳长２．７ｃｍ，平均体重２．３ｇ）的壳长日增长为５６．３０￣８１．８０μｍ，日增重为１２．６８￣２２．４８ｍｇ，其最适生长的盐度为３０￣３５，存活率为９７％￣１００％。     

患病九孔鲍苗中异养菌产胞外酶的分析

为探寻南方九孔鲍(Haliotis diversicolor supersicolor supertexta)苗大规模掉板死亡病因,从鲍鱼场患病掉板九孔鲍苗中分离到28株异养菌,并对其分泌胞外酶的能力进行分析.结果表明,分离得到的异养菌中有46.4%能分泌(酪)蛋白酶,60.7%能分泌明胶酶,3.57%分泌卵磷脂酶,17.86%分泌溶血素,但均不能分泌脂肪酶和淀粉酶.推测胞外蛋白酶为鲍苗患病的主要致病因子之一.综合考虑每一种胞外酶的潜在致病作用,以及菌株分泌多种胞外酶的能力,初步确认有5株异养菌为鲍苗掉板的潜在致病菌.     

Low‐density culture of red abalone juveniles,Haliotis rufescens Swainson 1822, recirculating aquaculture system and flow‐through system

Commercial abalone culture is carried out using flow‐through systems with a high water volume exchange in Baja California, Mexico. The objective of this work was to compare the growth rate and survival of red abalone cultured in two systems. Flow through (daily water exchange rate of 800%) and recirculating systems consisted of a 250 L fibreglass tank and constant aeration, but biofiltration in the recirculating system was provided with a 28 L (1 ft³) bubble‐washed bead filter. Water variables were measured either daily (dissolved oxygen, temperature, pH and salinity) or three times a week (total ammonia nitrogen, nitrate‐nitrogen, nitrite‐nitrogen and alkalinity). Shell length was measured every 2 weeks for 18 weeks. Only the alkalinity and pH were significantly different due to the addition of sodium bicarbonate to the recirculating system. Abalone growth rate was 26.1 ± 15.96 μm day^?1 in the recirculating systems and 22.21 ± 18.69 μm day^–1 in the flow‐through systems. The final survival was 78.74% in the recirculating systems and 71.82% in the flow‐through systems. Significant differences in the final size and survival of the abalones were found between systems (P<0.05). Therefore, recirculating aquaculture systems is a feasible alternative for juvenile red abalone culture.     

不同单胞藻饵料培养九孔鲍早期稚贝的研究

以单种培养的6种底栖硅藻和具有底栖习性的亚心形扁藻(Platymoras sp.)为饵料,培养九孔鲍(Haliotis diversi-color supertexta)受精后12 d的早期稚贝并观察其生长和存活,培养期为28 d,目的是筛选能维持早期稚贝高存活率和生长率的藻种。实验结果表明,培养稚贝成活率最高的3个藻种由大到小依次是爪哇曲壳藻亚缢变种(Achnanthes javanica var.)、流水双眉藻(Amphora fluninensis)和亚历山大菱形藻(Nitzschia alexandrina),使稚贝生长最快的3个藻种由大到小依次是流水双眉藻、爪哇曲壳藻亚缢变种和亚历山大菱形藻。研究结果揭示了适宜的底栖硅藻种类对鲍早期稚贝阶段生长和存活的重要性。个体小或者胞外产物量大的藻类很可能对九孔鲍早期稚贝的培养是有利的。     

Determination of genome size of Haliotis discus hannai and H.?diversicolor aquatilis (Haliotidae) and phylogenetic examination of this family

Genome size (C value) is an important index for phylogenetic studies. Haliotidae (abalones) includes many species widely distributed throughout the world??s oceans, which makes this family interesting for phylogenetic studies. To examine Haliotidae phylogeny, we determined the C value and adenine and thymine base pair content (AT?%) of Haliotis discus hannai and H.?diversicolor aquatilis by using flow cytometry. The C values of H.?discus hannai and H.?diversicolor aquatilis were 1.84 and 1.45?pg, with AT?% of 62.3 and 66.3?%, respectively. These data represent the first report of abalones classified as Pacific Northwest (H.?discus hannai) and Indo-Pacific (H.?diversicolor aquatilis) groups, and provides new validation for previous theories related to Haliotidae phylogeny.     

Effect of Combined Non‐ionized Ammonia and Dissolved Oxygen Levels on the Survival of Juvenile Dourado,Salminus brasiliensis (Cuvier)

The aim of this study was to assess the mean lethal concentration (LC₅₀) of dissolved oxygen in high ammonia concentration and also the LC₅₀ of ammonia under hypoxic conditions for juveniles of dourado, Salminus brasiliensis. In the first experiment, the non‐ionized ammonia (NH₃) concentrations were: 0.026, 0.447, 0.612, 0.909, and 1.334 mg/L, and the dissolved oxygen concentration was maintained at approximately 1.65 mg/L. In the second experiment, the dissolved oxygen concentrations were: 1.64, 1.99, 3.33, 5.10, and 7.77 mg/L, and the non‐ionized ammonia concentration was kept at approximately 0.927 mg/L. The mean lethal concentrations of non‐ionized ammonia varied from 0.584 to 0.577 mg/L, indicating that LC₅₀ values were almost unaffected by exposure time. The estimated LC₅₀ of dissolved oxygen varied from 4.02 to 5.02 mg/L, indicating a slight increase in the mean lethal concentrations as the exposure time increased. Results from this study indicate that interaction between these two parameters increases mortality and also suggest that dourado is susceptible to the combination of high ammonia with low dissolved oxygen concentrations.     

Colonization and probiotic effects of lactic acid bacteria in the gut of the abalone Haliotis gigantea

Strains of lactic acid bacteria (LAB) were isolated from several different sources and evaluated in vitro for potential probiotic effects in abalones. Two isolates (Lactobacillus sp. strain a3 and Enterococcus sp. strain s6) were highly resistant to bile salt and/or gastric juice and inhibited the growth of three abalone pathogens (Listonella anguillarum, Vibrio harveyi, and V. carchariae). Each of the LAB isolates was used to supplement diet of the abalone Haliotis gigantea for a period of 3 weeks. One group of animals received Lactobacillus sp. strain a3 added to commercial dry feed, one group received Enterococcus sp. strain s6 added to the feed, and a control group received only standard commercial feed. Culturable LAB counts of gut homogenates indicate the a3 colonized in the gut of abalones. Digestive enzyme activities and the concentrations of a number of volatile short-chain fatty acids (VSCFA) were elevated in the gut of abalones receiving feed supplemented with the two LAB strains. These results indicate that dietary supplementation can enable LAB colonization or persistence in the gut of abalone species and can potentially enhance probiotic effects.