塔玛亚历山大藻毒素及其对中国对虾的急性毒性

利用美国分析化学家协会(Association of Official Analytical Chemists, AOAC)所推荐的小鼠生物法(mouse bioassay, MBA), 测定了塔玛亚历山大藻(Alexandrium tamarense)(ATHK藻株)毒素粗提液的麻痹性贝毒(paralytic shellfish poisoning, PSP)毒性;采用浸浴方式, 研究了该藻株对中国对虾的急性毒性; 采用HE染色方法, 分别对有毒藻浸浴处理和毒素粗提液注射处理后96 h中国对虾(Fenneropenaeus chinensis)的鳃和肝胰腺石蜡切片进行观察。结果表明, 该藻株麻痹性贝毒毒性为3.9×10^–5 MU/cell (相当于7.3 pg STX Equal/cell), 在同种藻株中属低毒藻株; 在96 h急性毒性实验中, 塔玛亚历山大藻对中国对虾的半致死浓度(LC₅₀)为1.0×10⁴ cells/mL, 安全浓度(SC)为1.0×10³ cells/mL; 石蜡切片观察发现, 塔玛亚历山大藻与毒素粗提液分别引起了鳃和肝胰腺组织出现细胞肿胀、空泡化等病理变化。以上研究结果提示, 塔玛亚历山大藻对中国对虾有急性致死作用。为了保证中国对虾的健康养殖, 养殖水体中的塔玛亚历山大藻浓度至少应该控制在1.0×10³ cells/mL以下; 鳃的病变直接导致对虾窒息死亡可能是塔玛亚历山大藻暴露后对虾急性致死作用的最重要原因;塔玛亚历山大藻所产的PSP毒素可引起虾体代谢和解毒的主要器官—肝胰腺的病变。     

产麻痹性贝毒塔玛亚历山大藻共培养菌群的物种多样性研究

产毒赤潮藻与其共生菌的互作关系是揭示麻痹性贝毒(PSP)产生机制的关键,而其共培养菌群的分离及其多样性解析是开展此研究的必要前提。塔玛亚历山大藻(Alexandrium tamarense)是典型产PSP赤潮原因甲藻,为揭示其共培养菌群的物种多样性信息,本文通过免培养海洋微生物高通量测序技术,解析了东海塔玛亚历山大藻(at3#)共培养菌群的种类、丰度及多样性信息。结果表明,其共培养菌群共有87个种与已有数据库匹配,此外尚有5%的未知种。其包含57个属,其中优势菌属5个,分别为Rhodobacteraceae、Marinobacter、Methylophaga、Nitratireductor及Phycisphaera。研究表明东海产PSP塔玛亚历山大藻(at3#)含有物种丰富的共培养菌群,且具有发掘海洋特殊生境微生物新种属的良好潜力。     

Development of a quantification assay for the cysts of the toxic dinoflagellate <Emphasis Type="Italic">Alexandrium tamarense</Emphasis> using real-time polymerase chain reaction

ABSTRACT:   The cysts of toxic dinoflagellate Alexandrium tamarense are the seed population for the bloom responsible for paralytic shellfish poisoning (PSP). However, it is impossible to identify the Alexandrium spp. cyst on the basis of morphological features. In this study, we prepared A. tamarense cysts by sexual conjugation in laboratory conditions and developed an efficient DNA extraction method for polymerase chain reaction (PCR) assay. Using the A. tamarense cysts, we established the identification and quantification method showing the species specificity and the high sensistivity for A. tamarense cysts using real-time PCR. This assay was also able to detect and quantify the A. tamarense cysts accurately when mixed with excess cysts of A. catenella (Whedon and Kofoid) Balech prepared by conjugation experiment.     

Development and application of fluorescence in situ hybridization (FISH) method for simple and rapid identification of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella in cultured and natural seawater

ABSTRACT:   The toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and A. catenella (Whedon and Kofoid) Balech produce potent neurotoxins, such as saxitoxin and gonyautoxin and have been mainly responsible for paralytic shellfish poisoning (PSP) in Japan. To prevent a negative effect on the fishery industry, it is necessary to identify these toxic species precisely and rapidly before and during the bloom. In this paper, a rapid and simple protocol of a fluorescence in situ hybridization (FISH) method using ribosomal RNA (rRNA)-targeted probes has been established for identifying the cultured strains and natural cells of A. tamarense and A. catenella . Using the FISH method established in this study, it was possible to identify these toxic species species-specifically and rapidly, within 30 min. The procedure of detection constituted three steps: (i) fixation/dehydration; (ii) hybridization; and (iii) washing; this made the identification simple. Moreover, this method did not require either special techniques or equipment, and the cost for detection was low. The specificity, rapidity, and simplicity of the developed method suggest that it might be useful for routine monitoring of these toxic microalgae.     

Electrophysiological Evaluation of Pacific Oyster (Crassostrea gigas) Sensitivity to Saxitoxin and Tetrodotoxin

Pacific oysters (Crassostrea gigas) may bio-accumulate high levels of paralytic shellfish toxins (PST) during harmful algal blooms of the genus Alexandrium. These blooms regularly occur in coastal waters, affecting oyster health and marketability. The aim of our study was to analyse the PST-sensitivity of nerves of Pacific oysters in relation with toxin bio-accumulation. The results show that C. gigas nerves have micromolar range of saxitoxin (STX) sensitivity, thus providing intermediate STX sensitivity compared to other bivalve species. However, theses nerves were much less sensitive to tetrodotoxin. The STX-sensitivity of compound nerve action potential (CNAP) recorded from oysters experimentally fed with Alexandrium minutum (toxic-alga-exposed oysters), or Tisochrysis lutea, a non-toxic microalga (control oysters), revealed that oysters could be separated into STX-resistant and STX-sensitive categories, regardless of the diet. Moreover, the percentage of toxin-sensitive nerves was lower, and the STX concentration necessary to inhibit 50% of CNAP higher, in recently toxic-alga-exposed oysters than in control bivalves. However, no obvious correlation was observed between nerve sensitivity to STX and the STX content in oyster digestive glands. None of the nerves isolated from wild and farmed oysters was detected to be sensitive to tetrodotoxin. In conclusion, this study highlights the good potential of cerebrovisceral nerves of Pacific oysters for electrophysiological and pharmacological studies. In addition, this study shows, for the first time, that C. gigas nerves have micromolar range of STX sensitivity. The STX sensitivity decreases, at least temporary, upon recent oyster exposure to dinoflagellates producing PST under natural, but not experimental environment.     

文蛤对四种单胞藻的表观消化状况及同化率

水温22℃下,显微观察了文蛤对塔玛亚历山大藻及海水小球藻,等鞭金藻和青岛大扁藻的表观消化状况,以灰化法测定了文蛤对四种单胞藻的同化率。结果表明,低浓度时文蛤对四种藻类的表观消化状况优于高浓度组;文蛤对海水小球藻、等鞭金藻和青岛大扁藻的同化率随着藻类浓度的升高而降低,其同化率变化范围分别为87.9%~67.4%、83.4%~58.7%和67.5%~37.9%。当塔玛亚历山大藻浓度低于5.0×103cell/mL时,文蛤不会产生假粪,该藻能够被文蛤摄食。藻浓度在1.0×103cell/mL到3.0×103cell/mL时,文蛤对该藻的同化率从81.8%降至52.3%,4.0×103cell/mL组的同化率升高至67.6%,藻浓度升高至5.0×103cell/mL时同化率又降至52.4%,塔玛亚历山大藻3.0×103cell/mL浓度组是一个分界点,同化率最低,大于这一浓度时,同化率又呈上升趋势。     

3种滤食性贝类对塔玛亚历山大藻的摄食研究

20 0 3年 4月在实验室内采用静水法 ,研究了长牡蛎、紫贻贝、栉孔扇贝对塔玛亚历山大藻的清滤率 (CR)和摄食率 (IR) ,实验时间持续 3d ,水温 17℃。实验中每 1种贝都分别投喂 :(1)叉鞭金藻 (对照藻 ) ,(2 )叉鞭金藻 +塔玛亚历山大藻去藻过滤液 1.5L ,(3)叉鞭金藻 +塔玛亚历山大藻细胞。结果表明 ,塔玛亚历山大藻过滤液 ,对 3种贝的清滤率的无明显影响 (P >0 .0 5 ) ;3种贝对塔玛亚历山大藻和叉鞭金藻混合液的清滤率与对照组相比明显降低 (P <0 .0 1) ;而对塔玛亚历山大藻的摄食率和保留率 3d间无显著变化 ;实验期间 ,随时间的变化 ,牡蛎对混合液的清滤率变化最大 ,贻贝次之 ,扇贝则无明显著变化 (P >0 .0 5 ) ;如果以摄食率作为指标 ,贻贝在控制赤潮方面的潜力较大 ,值得今后进一步研究。     

Paralytic shellfish poisoning toxin analysis of the genus <Emphasis Type="Italic">Alexandrium</Emphasis> (Dinophyceae) occurring in Korean coastal waters

ABSTRACT:   Paralytic shellfish poisoning (PSP) toxins produced by Alexandrium isolates from Korea were analyzed by high-pressure liquid chromography. Species designation of the regional isolates was determined by morphological criteria and ribotyping inferred from sequences of the 28S rDNA D1-D2 region. Toxin analysis performed at the exponential growth phase, revealed that the two strains of A. fraterculus were non-toxic, while the strains of A. tamarense and A. catenella were toxic. Toxic isolates DPC7 and DPC8 of A. catenella produced GTX1, 2, 3, 4, 5, dcGTX2, 3, C1, 2, neoSTX and STX with trace or non-detectable levels of C3 and C4, while isolates UL7, KDW981, SJW97043, SJW97046, KJC97111 and KJC97112 of A. tamarense produced GTX1, 2, 3, 4, dcGTX3, C1, 2, neoSTX with trace or non-detectable levels of C3, 4, dcSTX and STX, and no GTX5 and dcGTX2. The major toxins produced by A. catenella were C1 +2, and those of A. tamarense were C1 +2 and GTX4 in most of the isolates. A. tamarense strains other than SJW97046 produced a relatively high proportion of carbamate toxins, reflecting the high toxicity scores of shellfish intoxication in sampled coastal areas. Two representative toxic isolates, A. tamarense SJW97043 and A. catenella DPC7, were cultured for 30 days in batch mode and subjected to toxin analysis at 5-day intervals. Comparison of toxin productivity in terms of total toxin content, toxin components, and their variations with culture age revealed marked differences between the two strains.