麻痹性贝毒毒素在中华哲水蚤体内的累积及排出

本文研究了中华哲水蚤对可产生麻痹性贝毒毒素的塔玛亚历山大藻的摄食情况,以及麻痹性贝毒毒素在中华哲水蚤体内的累积和排出过程。实验发现,中华哲水蚤可以摄食有毒塔玛亚历山大藻,并在体内累积麻痹性贝毒毒素;麻痹性贝毒毒素各成分在中华哲水蚤体内发生相互转化的现象,同时中华哲水蚤又可通过自身代谢作用降解掉大部分毒素;中华哲水蚤可通过其排泄物在空间上转移麻痹性贝毒毒素。     

麻痹性贝毒PSP在紫贻贝体内的累积、转化与排出

于1998年9月在青岛鲁迅公园附近礁石区采集紫贻贝（Mytilus edulis)，采用实验室培养的方法，初步研究了塔玛亚历山大藻（Alexandrium tamarense,ATHK)产生的麻痹性贝毒（Paralytic Shellfish Poison,PSP)在其体内累积、转化与排出的规律。结果表明，在累积实验阶段，紫贻贝内脏的和肌肉中的PSP毒素含量均随实验时间的延长而逐渐增加，累积实验结束时，平均每只贝体内的PSP毒素含量为13.40nmol，毒性水平为12.24ugSTXEq/100g，紫贻贝内脏中的毒素含量远远高于肌肉，内脏中PSP毒素占贝体内PSP毒素总量的97.5%。在8天的排出实验阶段，贝体内的PSP毒素总量呈下降趋势，实验结束时，PSP毒素共排除了约50%，每天排除率约为9%。     

塔玛亚历山大藻的麻痹性贝毒研究

对暨南大学水生生物研究所于199芹年10月从香港海域底泥分离后于实验室人工培养的塔玛亚历山大藻运用高效液相色谱分析麻痹性贝毒的组成;按照美国分析化学家协会的小白鼠生物检定标准方法测试其毒性。结果表明,所含毒素成分主要是膝沟藻毒素-2（GTX2）,含量为94.13×10－12g／cell;次要成分是膝沟藻毒素-4的N-磺基氨基甲酸衍生物C4,含量为15．67×10-12g／cell,测得其毒性为（3．23－4.11）×10－6MU／Cell。研究表明,所用的微藻麻痹性贝毒的提取方法和高效液相色谱分析方法都比较容易和有效。     

微小亚历山大藻投喂量与菲律宾蛤仔对麻痹性贝毒累积"转化和排出的动态

选用高毒性微小亚历山大藻台湾株和菲律宾蛤仔为研究对象,研究了不同毒藻投喂量对贝类累积和排出麻痹性贝毒(PSP)的影响.实验分为累积和排出2个部分,累积实验设计了4个投喂梯度(A组:2.0×107cell/d、B组:7.0×107cell/d、C组:1.1×108cell/d和D组:2.0×108cell/d),在排出毒素阶段,每实验组又分成投喂无毒饵料藻组和饥饿组并进行比较.结果显示不同投喂梯度组间PSP毒素累积存在明显差异.随着毒藻投喂量的增加,PSP毒性增加,但毒素累积率下降,这说明在一定范围内毒藻投喂量的大小对蛤仔的毒素累积有影响.A组、B组、C组的最高累积量依次为6.5,82.9,113.9μgSTXeq/100g,D组达到本实验的最高累积量154.1μgSTXeq/(100g),但累积率却随着投喂量的增加而递减,分别为1.79%,0.92%,0.42%和0.39%.停止投喂毒藻后蛤仔体内的PSP毒性迅速下降,在排出阶段,对投喂饵料组和饥饿组的比较结果用单因素方差分析(ANOVA)得出P＜0.05,结果差异显著,饥饿组的毒性均高于投喂饵料藻组,说明投喂无毒饵料有助于菲律宾蛤仔体内的毒素排出.其中D饵料组和饥饿组间排出速率差异最明显,饵料组的排出速率为13.2 μgSTXeq/(100g·d),饥饿组的仅为8.3μgSTXeq/(100g·d),因此将菲律宾蛤仔归属于快速排毒者.     

麻痹性贝毒在毛蚶体内的转化过程研究

麻痹性贝毒能够在贝类体内累积,威胁海产品消费者健康。在以往调查中,多次在毛蚶(Scapharca subcrenata)体内发现高含量的麻痹性贝毒,但对于毛蚶体内麻痹性贝毒的转化过程及其食品安全风险还缺乏认识。通过室内模拟实验,选择太平洋亚历山大藻(Alexandrium pacificum)和链状裸甲藻(Gymnodinium catenatum)作为产毒藻种,研究了两种有毒藻种所产麻痹性贝毒在毛蚶体内的转化过程。结果表明,毛蚶体内主要出现了三种麻痹性贝毒转化过程,一是R1位羟基的还原反应,二是N-磺酰氨甲酰基类毒素R4位磺酸基团的水解反应,三是含羟基苯甲酸(hydroxybenzoate)基团的链状裸甲藻毒素在R4位的水解反应。毛蚶体内麻痹性贝毒的生物转化过程复杂,对毛蚶毒性的影响具有一定的不确定性,未来仍需要进一步深化毛蚶体内毒素累积、代谢、转化过程的研究,同时加强对毛蚶体内毒素含量的全面监测,防范毛蚶可能导致的麻痹性贝毒中毒风险。     

海洋细菌对赤潮藻生长及其产毒量的影响

采用麻痹性贝类毒素小白鼠生物测定法 ,研究了在可控生态条件下两株海洋细菌S1 0 、P42 对塔玛亚历山大藻生长及其产毒量的影响。结果表明 ,菌株S1 0 在较高浓度下对藻细胞的生长有明显的抑制作用 ,在较低浓度下抑藻生长作用较弱 ,不同浓度的菌株S1 0 均能有效地抑制藻细胞内麻痹性贝类毒素的产生 ,且在较低浓度下效果较好 ;菌株P42 对该藻的作用恰好与S1 0 相反 ,在较低浓度下明显抑制藻细胞的生长 ,不同浓度的菌株P42 也能有效地抑藻产毒 ,且在较高浓度下作用较明显。实验用的藻株毒力约为 ( 0 .95— 1 2 .1 4)× 1 0 - 6MU/cell,属于低毒藻株 ,该藻株在培养第 1 4天达到毒性最高峰 ,峰值为 1 2 .1 4× 1 0 - 6MU/cell,之后逐渐下降。讨论了海洋细菌在赤潮生物防治中的应用前景     

微小亚历山大藻投喂量与菲律宾蛤仔对麻痹性贝毒累积、转化和排出的动态关系

选用高毒性微小亚历山大藻台湾株和菲律宾蛤仔为研究对象,研究了不同毒藻投喂量对贝类累积和排出麻痹性贝毒(PSP)的影响。实验分为累积和排出2个部分,累积实验设计了4个投喂梯度(A组:2.0×107cell/d、B组:7.0×107cell/d、C组:1.1×108cell/d和D组:2.0×108cell/d),在排出毒素阶段,每实验组又分成投喂无毒饵料藻组和饥饿组并进行比较。结果显示不同投喂梯度组间PSP毒素累积存在明显差异。随着毒藻投喂量的增加,PSP毒性增加,但毒素累积率下降,这说明在一定范围内毒藻投喂量的大小对蛤仔的毒素累积有影响。A组、B组、C组的最高累积量依次为6.5,82.9,113.9μgSTXeq/100g,D组达到本实验的最高累积量154.1μgSTXeq/(100g),但累积率却随着投喂量的增加而递减,分别为1.79%,0.92%,0.42%和0.39%。停止投喂毒藻后蛤仔体内的PSP毒性迅速下降,在排出阶段,对投喂饵料组和饥饿组的比较结果用单因素方差分析(ANOVA)得出P0.05,结果差异显著,饥饿组的毒性均高于投喂饵料藻组,说明投喂无毒饵料有助于菲律宾蛤仔体内的毒素排出。其中D饵料组和饥饿组间排出速率差异最明显,饵料组的排出速率为13.2μgSTXeq/(100g.d),饥饿组的仅为8.3μgSTXeq/(100g.d),因此将菲律宾蛤仔归属于快速排毒者。     

栉孔扇贝体内麻痹性贝毒的累积与排出过程研究

通过室内摄食实验,研究了栉孔扇贝(Chlamys farreri)对麻痹性贝毒(PSP)产毒藻的敏感性及其累积和排出毒素的特征.有毒微小亚历山大藻(Alexandrium minutum)在短时间内可对栉孔扇贝的摄食有一定的抑制作用,且投喂毒藻的浓度越大,对摄食作用的抑制越明显.栉孔扇贝对PSP贝毒的累积能力很强,实验中,48 h后内脏累积毒素就高达5000μg STXeq100g-1,但其毒素排出速率较慢,在24d解毒阶段后内脏中的毒素还没有下降至国际贝毒食用安全标准水平的80μg STXeq 100g-1.各毒素组成比在整个累积与排除过程中有较大变化,GTX1,GTX4在累积阶段比例下降,同时GTX2,GTX3比例上升;内脏中GTX2:GTX3的比值逐渐增加.毒藻中PSP贝毒毒素组成和贝体中PSP贝毒毒素组成之间的主要差别是GTX4含量的减少和GTX1含量的升高;而扇贝粪便中的毒素组成却与对数生长期的毒藻极为接近.     

赤潮多发区塔玛亚历山大藻的麻痹性贝毒预警值研究

以厦门西海域养殖的翡翠贻贝为实验对象,在实验室内对塔玛亚历山大藻(Alexandrium tamarense(Lebour)Balech)采用单种培养技术,运用“麻痹性贝毒小白鼠生物检测法”进行毒性实验,研究了塔玛亚历山大藻对翡翠贻贝的麻痹性贝毒的毒力,藻密度为1×105个/dm3时,白鼠未死亡;藻密度为1×106个/dm3时,白鼠发生死亡.厦门西海域近年来暴发的赤潮一般起始于局部海区无害硅藻类浮游植物,在生态环境恶化下,硅藻类赤潮在生存竞争中消退,最终暴发有害赤潮.以试验结果为依据,根据厦门海域实际情况,参考各国贝毒临界值行动标准,提出适合南方赤潮多发区塔玛亚历山大藻的麻痹性贝毒藻密度预警值为1×105个/dm3.     

渤海裸甲藻和链状亚历山大藻的麻痹性贝毒毒素分析

目的:分析渤海裸甲藻(Gymnodinium sp.)和链状亚历山大藻(Alexandrium catenella)的麻痹性贝毒毒素,为渤海天津海域的赤潮研究积累基础数据。方法:通过实验室培养裸甲藻和链状亚历山大藻,选取对数生长期、平台生长期的裸甲藻以及平台生长期的链状亚历山大藻,利用高效液相色谱法(HPLC)对这两种微藻进行麻痹性贝毒(PSP)毒素分析。结果:裸甲藻细胞内不含有麻痹性贝毒(PSP);链状亚历山大藻细胞内含有C毒素和GTX1-4毒素,该微藻每个细胞毒素含量约为10.81 fmol/cell。结论:裸甲藻细胞内虽不含有麻痹性贝毒(PSP),但不能排除其含有其它毒素的可能。链状亚历山大藻细胞内含有麻痹性贝毒(PSP),属于有毒微藻,需要对其进行密切监测。     

江苏海域文蛤周年性腺发育的初步研究

江苏文蛤(Meretrixmeretrix)属于较为独立的地理种群。为提高江苏文蛤苗种生产能力,于2003年12月到2004年11月在江苏沿海海域的东凌、新港、吕四、大丰等采样点,定期取样,计算文蛤各采样时期的丰满度,并及时测量水温,对性腺进行直接和显微切片观察,以研究其性腺发育特点。观察表明,其发育可分为5个期:形成期、生长期、成熟期、排放期及耗尽期。形成期在3~4月,水温10.5~16.5℃;生长期在4~5月,水温18~20.5℃;成熟期在5~6月,水温24~25℃;放散期在6~8月,水温24~32℃;耗尽期在9月以后,直到11月份,水温从24℃左右下降到10℃左右。水温在10℃以下时,文蛤的生长发育处于停滞状态,从11月持续到第二年的3月初。江苏文蛤性腺发育的时间比辽宁文蛤早1个月左右,比广西文蛤迟1个月左右。     

文蛤一种球形病毒的分离纯化及细胞病理变化观察

为研究一种球形病毒引起的文蛤(Meretrix meretrix)细胞病理变化及该病毒的分类地位,作者运用透射电镜对异常死亡文蛤的细胞病理变化及感染的病毒粒子进行了超微结构观察,并通过差速离心、酒石酸钾超速离心和氯化铯密度梯度离心结合等方法对首次对该病毒粒子进行了分离纯化。结果表明:患病个体的鳃、消化盲囊、消化道和外套膜均大量存在一种病毒颗粒,该病毒呈球形,无囊膜,直径45~55 nm,核心与核衣壳间无明显界限,未发现病毒包涵体,该病毒存在于细胞间质及被破坏的细胞质中。该病毒粒子的感染引起的细胞病理变化主要表现为:细胞核染色质集聚,溶酶体增多,线粒体肿涨,粗面内质网膨胀,肌原纤维排列混乱,细胞结构变得无规则直至解体。病毒粗提液于4℃150 000 g氯化铯密度梯度离心20 h可该病毒粒子提纯,经纯化后负染显示,该病毒粒子呈球形,二十面体对称,无囊膜,直径在60~75 nm,表面结构粗糙,壳粒结构较明显,该病毒粒子在氯化铯中的浮密度为1.554~1.582 g/m L。本文结果为文蛤死亡病原的研究提供了基础资料。     

Effects of tributyltin at environmental levels on monooxygenase system of digestive gland in hard clam Meretrix meretrix

The effects on ethoxyresorufin O-deethylase （EROD）, NADPH-cytochrome c reductase and NADH cytochrorne b5 reductase activities of digestive gland in Meretrix meretrix exposed to tributyltin （TBT） at environmental levels （0.1,1.0,10.0 ng/dm as stannum concentration）,in experimental condition, were evaluated. The EROD, NADH cytochrorne b5 reductase activities were significantly inhibited after exposure to 10.0 ng/dm^3 TBT for 8 and 20 d, the NADPH cytochrorne c reductase activities were significantly inhibited after exposure to 0.1,1.0 and 10.0 ng/dm TBT for 8 d and to 1.0 and 10.0 ng/dm for 20 d, as compared with the matched control, while NADH cytochrorne b5 reductases and NADPH cytochrome c reductase activities were induced after exposure to 10.0 ng/dm^3 TBT for 2 d. The EROD activity in the 10 ng/dm^3 group,and the NADH cytochrome b5 reductases activities in 1.0 and 10.0 ng/dm groups, were significantly induced when transferred to clean recovery tanks for 7 d. The three enzymic activities in the clams exposed to TBT were recovered to the level corresponding to that of the control group after transfer to clean recovery tanks for 20 d. NADPH cytochrome c reductase activity in Meretrix meretrix seems to be more sensitive to exposure of TBT than that of the EROD and NADH cytochrome b5 reductases. The results suggest that induction and inhibition by TBT to the monooxygenase system enzymic activity in Meretrix meretrix would simultaneously exist. The enzymic activities were inhibited by exposure for a long time. The results suggest that inhibition of the monooxygenase system should be an indication of the exposure to environmentally relevant concentrations of TBT for a long time.     

Growth performance of the clam, Meretrix meretrix, breedingselection populations cultured in different conditions

A breeding-selection program for the clam,Meretrix meretrix,was conducted since 2004.Two of the selection populations were generated with the shell color pattern as an additional selection criterion and named as SP(purple stripes)population and SB(black dots)population.The third-generation SP and SB populations(08G3SP and 08G3SB,respectively)were cultured at two commercial clam farms and a nursery pond and their shell lengths were compared.08G3SB clams had significantly larger sizes than 08G3SP clams at commercial clam farms(p<0.05),yet 08G3SB individuals were significantly smaller than 08G3SP individuals at the nursery pond(p<0.05).Then,we examined the growth of the fourth-generation SP and SB populations(10G4SP and 10G4SB,respectively)at a commercial farm,and found that the shell lengths of the 10G4SB clams increased at a significantly higher growth rate than the 10G4SP clams(p<0.05)from May to September,when the water temperature was between 24.2–27.5 C,while 10G4SB lost the growth advantage in the other months.These results indicated that SP and SB populations responded differently to environmental factors,so it is beneficial for the clam farmers to select a suitable population according to the culture environment.Furthermore,a diallel mating of the SB and SP populations was designed to investigate whether their hybrid population would show heterosis.However,the heterosis was not shown in this study,which might result from the slight genetic divergence between SB and SP populations.     

氨氮对文蛤存活及能量收支的影响

在实验室条件下,研究了氨氮(NH3-N)对文蛤(Meretrix meretrix)的急性毒性作用以及对其能量收支的影响。实验结果表明:在水温(24.5±0.5)℃,盐度21.5,pH8.00的条件下,文蛤(壳长3.85～4.35cm)死亡率随着氨氮质量浓度、胁迫时间的增加而增加(P0.05)。96 h文蛤死亡率与添加氨氮(NH3-Nt)质量浓度对数的回归方程为Y=2.3787x+0.3246(R2=0.978 9),文蛤对总氨氮(NH3-Nt)或分子氨(NH3-Nm)的安全浓度(SC96h)为9.237 mg/L或0.418 mg/L;随着环境中添加氨氮(NH3-Nt)质量浓度的不断上升(0,0.5,1,2,4,9,18,36 mg/L),文蛤摄食能不断下降,呼吸能和排泄能则呈现出先升高后降低的趋势;当总氨氮(NH3-Nt)在0～2 mg/L(或NH3-Nm≤0.090 mg/L)时,文蛤的生长余力没有显著变化(P﹥0.05);当总氨氮(NH3-Nt)≥4 mg/L(或NH3-Nm≥0.181mg/L)时,文蛤的生长余力显著降低(P0.05)。因此,文蛤养殖环境中的NH3-Nt或NH3-Nm应控制在2mg/L或0.090 mg/L以下水平。     

黄渤海海域贝类麻痹性贝毒的检测与分析

采用小白鼠生物测试法和高效液相色谱法对2002~2005年我国黄渤海海域采集的贝类样品进行了麻痹性贝毒毒性检测,结果显示大连海域的虾夷扇贝含有麻痹性贝毒,有毒样品均出现在5月和6月,部分虾夷扇贝样品的毒素含量已经超过食用安全标准。通过高效液相色谱法分析了有毒虾夷扇贝体内的毒素成分,共检出了6种麻痹性贝毒组分,主要以毒性较低的C1和C2毒素为主,GTX3和GTX2次之,STX和neoSTX含量很低。通过高效液相色谱法分析得到的各毒素组分毒性总和与小白鼠生物测试法毒性测试结果基本相当。     

Comparative study on in vitro transformation of paralytic shellfish poisoning (PSP) toxins in different shellfish tissues

Dissected tissues of three shellfish species,the Chinese scallop,Chlamys farreri,Manila clam,Ruditapes philippinarum,and Razor shell,Solen strictu were evaluated for in vitro transformation of paralytic shellfish poisoning(PSP) toxins. Tissue homogenates were incubated with extraction from toxic algae Alexandrium minutum to determine toxin conversion. The effects of heating and addition of a natural reductant(glutathione) on toxin conversion were also assessed. The toxin profile was investigated through high performance liquid chromatography with fluorescence detection(HPLC-FLD) . The evident variations in the toxin content were observed only in Chinese scallop viscera homogenates. The concentration of GTX4 was reduced by 45%(approximately 0.8 μmol/dm 3 ) and 25%(approximately 1 μmol/dm 3 ) for GTX1,while GTX2 and GTX3 increased by six times(approximately 1 μmol/dm 3 ) and 3 times(approximately 0.3 μmol/dm 3 ) respectively. Simultaneously,the total toxicity decreased by 38% during the 48 h incubation period,the final toxicity was 20.4 nmol STXeq/g. Furthermore,heated Chinese scallop viscera homogenates samples were compared with non-heated samples. The concentration of the GTX4 and GTX1 was clearly 28%(approximately 0.53 μmol/dm 3 ) and 17%(approximately 0.69 μmol/dm 3 ) higher in heated samples,GTX2 and GTX3 were four times(0.66 μmol/dm 3 ) and two times(0.187 μmol/dm 3 ) lower respectively. GSH(+) Chinese scallop viscera homogenates samples were compared with GSH(-) samples,the concentration in the GTX4 and GTX1 was 9%(approximately 0.12 μmol/dm 3 ) and 11%(approximately 0.36 μmol/dm 3 ) lower respectively,GTX2 and GTX3 was 17%(approximately 0.14 μmol/dm 3 ) and 19%(approximately 0.006 μmol/dm 3 ) higher respectively. In contrast,there was a little change in the concentration of PSP toxins of Manila clam and Razor shell tissue homogenates. These observations on three shellfish tissues confirmed that there were species-specific differences in PSP toxins transformation. PSP toxins transformation was more pronounced in viscera tissue than in muscle tissue. PSP toxins was possibly interfered by some carbamoylase enzyme,and the activity in Chinese scallop viscera tissue is more remarkable than in the other two species.     

三个文蛤家系双列杂交设计在两种环境下的配合力及杂种优势分析

A 3×3 complete diallel cross comprising three families of the clam Meretrix meretrix(P1, P2 and P3) was used to determine the combining ability of parental families and heterosis of F1 under indoor and openair environments for growth traits. Analysis of variance for shell length and whole body weight indicated highly significant cross effects, environment effects and the interaction of cross by environment. General combining ability(GCA) and specific combing ability exhibited great variation among crosses and between two environments. Pooled over environments, P2 was the top combiner among the three parental families for both traits studied. The cross of P1 and P3 had the highest SCA. Additionally, significant reciprocal effects were observed. For individual environment, about half of the crossbred combinations showed favorable Mid-parent heterosis(MPH)(1%) for the shell length and whole body weight. Our data has shown that non-additive genetic and reciprocal effects constituted the major sources of genetic variation for both shell length and whole body weight, which indicates that crossbreeding among selective families could further explore the heterotic effects.     

文蛤C型凝集素基因(Mm-Lec1)的克隆与表达分析

文蛤(Meretrix meretrix)是我国重要的滩涂养殖贝类,病害严重影响文蛤增养殖业,研究文蛤的免疫机制有助于解决文蛤的病害问题。C型凝集素(C-type lectin)参与先天免疫,在识别病原相关分子模式和激活体液免疫因子等方面发挥重要作用。本研究检索已构建的文蛤全长cDNA 文库,经过Blast比对得到了文蛤C型凝集素1(Mm-Lec1)基因的全长cDNA序列。Mm-Lec1序列全长586 bp,5'和3'非翻译区(UTR)的长度分别为21 bp和79 bp,开放阅读框长度为486 bp,编码161个氨基酸,分子量为18.65 kD,理论等电点为4.98。预测的氨基酸序列中含有信号肽(Met1-Ser19)、糖识别结构域(CRD)和糖结合位点(QPN)。Mm-Lec1的三级结构是紧凑型,含有β片层结构。同源性分析结果表明,Mm-Lec1与其他物种C型凝集素相似度在20%~32%;邻接法(Neighbor-Joining, NJ)进化树分析结果表明,Mm-Lec1与紫贻贝CTL 6和栉孔扇贝CTL A聚为一支。实时荧光定量分析结果显示,Mm-Lec1在文蛤鳃、肝胰腺、闭壳肌、外套膜、性腺和血细胞中均表达,其中鳃表达量最高,血细胞次之,性腺中表达量最少;在鳗弧菌(Vibrio anguillarum)刺激实验中,6 h时Mm-Lec1在血细胞中的表达量最低,48 h表达量最高,暗示Mm-Lec1参与文蛤抵御细菌入侵的免疫过程。