2023, Vol. 47
文章信息
- 张昊宇, 吴海燕, 董晨帆, 郑关超, 郭萌萌, 谭志军. 2023.
- ZHANG Hao-yu, WU Hai-yan, DONG Chen-fan, ZHENG Guan-chao, GUO Meng-meng, TAN Zhi-jun. 2023.
- 利玛原甲藻产毒差异及影响因素研究进展
- Research progress on the difference in toxin production and factors influencing production in Prorocentrum lima
- 海洋科学, 47(3): 137-146
- Marine Sciences, 47(3): 137-146.
- http://dx.doi.org/10.11759/hykx20210905002
-
文章历史
- 收稿日期：2021-09-05
- 修回日期：2022-02-28
2. 中国水产科学研究院 黄海水产研究所, 山东 青岛 266071;
3. 青岛海洋科学与技术试点国家实验室, 山东 青岛 266071;
4. 海洋食品精深加工关键技术省部共建协同创新中心 大连工业大学, 辽宁 大连 116034
2. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;
3. Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266071, China;
4. Collaborative Innovation Center for key Technologies of Marine Food intensive and Deep processing, Dalian University of Technology, Dalian 116034, China
利玛原甲藻(Prorocentrum lima, P. lima), 隶属于甲藻门(Pyrrophyta)双鞭甲藻纲(Dinophyceae)纵裂甲藻亚纲(Desmokontae)原甲藻目(Prorocentrales), 广泛分布于河口和沿海海域[1], 由于其对近海生态、食品安全和人体健康造成严重影响, 因而受到国际社会的广泛关注。P. lima是我国近海四大海域广泛分布的典型海洋底栖有毒赤潮藻[2-3], 也是两大国际有害藻华研究计划GEOHAB和GlobalHAB纳入的重点研究对象。P. lima主要产生腹泻性贝毒(Diarrhetic Shellfish Toxins, DSTs), 包括大田软海绵酸毒素(okadaicacid, OA)、鳍藻毒素1-3(dinophysis toxins, DTX1-3), 以及其他衍生物如酰化衍生物(7-O-酰基酯), 硫酸酯衍生物(Sulfated dister)和乙二醇酯衍生物(Diolesters), 如C8∶2OA、C9∶2OA、C10∶2OA、OA二醇酯和35S DTX1[2-3]。DTX-1比OA在C35上多了一个甲基, DTX-2和OA是同分异构体, 均易溶于甲醇、乙醇、氯仿和乙醚等有机溶剂, 这三种组分是目前的主要监管对象。
DSTs通常在贝类等海洋动物体中积累[4-5], 贝类摄食含有DSTs的P. lima不断在消化腺等组织中富集高含量DSTs, 同时, DSTs在贝类体内进一步代谢转化, 并且与贝类组织中脂质成分形成稳定的复合物, 更加难以消除[6-7], 最终通过食物链进入人体[4-5]。OA和DTXs都可以高效、专一性地抑制PP1(蛋白磷酸酶1)和PP2a(蛋白磷酸酶2a)型的蛋白磷酸酶, 引起中毒症状包括腹泻、恶心、呕吐及肠胃绞痛等[8], 甚至促进肿瘤的形成[9]。通常贝类被认为是DSTs对人类健康风险的主要传播媒介[10]。截至目前, 因贝类中DSTs高浓度蓄积已导致全球包括美国、日本、澳大利亚和欧洲国家等产品禁售[11-16]。而贝类中DSTs残留风险与P. lima种类或地理株密切相关, 具有显著的品种-种类相关性[3, 17]。因此, P. lima中DSTs产毒状况是影响贝类产品风险程度的关键因素之一, 除产毒藻自身生理原因外, 外界环境因子也对P. lima产毒能力具有决定性影响[18-20]。
现有研究发现, P. lima的生长和产毒的品种和地域差异性, 取决于不同地域的环境因素[2, 21], 比如环境中的氮磷和微量元素, 同时温度和光照是也影响藻类生长和毒素产生的重要物理因素, 它们对藻类的生长, 细胞分裂的速度和毒素的产生具有不可忽视的影响[22]。如前所述, P. lima种群在我国近海广泛分布, 这些产毒藻的地理分布和产毒情况直接影响区域产品的安全。然而, 现阶段我国对这些P. lima的生长生理和产毒情况知之甚少, 从而影响进一步评价P. lima对我国近海生态、食品安全和消费者健康危害的研究。因此, 本文总结了目前国内外对P. lima的分布、产毒机理研究及产毒影响的研究进展, 以期为开展我国近海P. lima毒性风险评价并进而构建科学的风险评估及防控技术提供科学基础。
1 P. lima的分类学及全球分布概况 1.1 P. lima的基本状况及鉴定方法原甲藻属(Prorocentrum), 其在分类学上地位十分特殊, 既介于动物与植物之间, 又介于真核生物和原核生物之间, 许多学者称之为“介核生物”或“间核生物”[23]。P. lima细胞通常在光学显微镜下呈黄绿色倒卵形[3](图 1a, b), 长33~44 μm, 宽23~ 33 μm, 细胞中央的淀粉核清晰可见, 并且周围散布着颗粒状的淀粉颗粒和叶绿体, 在淀粉核和根尖孔之间可以看到较大的液泡。P. lima藻体由左右两个壳组成, 壳厚且光滑, 在表面上分布有壳孔, 一般62~87个, 边缘的刺胞孔均匀分布在壳板周围(图 1c, d), 边缘的刺胞孔是一个重要特征, 可将利玛原甲藻和其他原甲藻分开[24-25]。通常在研究中也可以通过更详细的形态学研究(使用扫描电子显微镜)和分子分析来正确鉴定该物种。不同P. lima菌株在形态上表现出高度的可变性[26], 包括形状、长度和宽度, 以及瓣膜和边缘孔的数量和形状等细胞特征[27]。P. lima的准确识别对其生态、分布和毒性的研究具有重要意义[28], 最近的一些研究, 揭示了P. lima中遗传距离较远的核型的复杂结构, 通过对P. lima内部转录间隔区(ITS1- 5.8S-ITS2)、18S rDNA和大亚基(LSU) rDNA的分析结果也表明, P. lima发育树分支与P. cf. maculosum和P. caipirignum的分支很接近。这三个物种只有细胞形状或边缘孔隙形状有所不同[29]。多项研究也表明P. lima的不同形态类型是以遗传为基础的[17, 26, 30], 即不同谱系的P. lima生理特征可能不同[31]。
原甲藻属(Prorocentrum)包含约60种已发表和可识别的原甲藻物种, 其中近半为底栖甲藻[33-34]。近些年研究发现P. lima等底栖甲藻正在向逐渐变暖的温带生态系统扩张[32]。P. lima目前被认为在世界各地的水域均有分布[1], P. lima主要分布于热带和亚热带地区, 在营养水平较高的海峡如加勒比海与地中海海域中尤为丰富, 同时随着P. lima的扩张在高纬度也偶有报道(见图 2), 其中部分依赖于质谱技术对其毒素组成和单细胞毒素含量有详细报道(表 1)。自然界中, P. lima主要附生在大型藻类、珊瑚礁、海草及海底沙粒上, 大多被发现在0.5 m到4 m的深度中, 如意大利、比利时、英国、爱尔兰、西班牙、法国和美国和中国的近海海域[11, 14, 35-36]。如在美国西北部的沿海水域, P. lima每年在3月和6月有两个高峰期, 峰值达到5×103 cell/mL细胞以上[37]。在我国东海、南海等海域也均常有发现P. lima, 例如在我国海南省三亚海区珊瑚礁海域的大型海藻上就发现多有P. lima附着, 也是当地重要的赤潮原因种之一。
| 藻株名 | 气候带 | 位置 | 毒素种类及含量/(pg·cell–1) | 参考文献 |
| P. lima(TIO302) | 热带 | 中国台湾 | OA: 10.260; DTX1: 1.810 | [43] |
| P. lima(QD502、WZD12) | 中国南海 | OA: 0.55-10.26; DTX1: 0.91-1.81 | [6] | |
| P.lima(Syn. Exuviaella lima) | 墨西哥 | OA: 5.2 | [44] | |
| P. lima(LM002、003) | 西大西洋 | OA: 9.50-10.6 | [3] | |
| P. lima(UNR-01、09) | 南大西洋 | OA: 15.2-38.5; DTX1: 0.5 | [30] | |
| P.lima(CSIRCSMCRI005) | 北印度洋 | OA: 0.022 | [25] | |
| P. lima(TIO124) | 温带 | 中国北海 | OA: 2.834 | [43] |
| P. lima(a~j) | 日本 | OA: 13.39; DTX1: 11.26 | [2] | |
| P. lima | 英国 | OA: 0.51-1.5; DTX1: 0.35-5.4 | [45] | |
| P. lima(IO66-01) | 葡萄牙 | OA: 0.7-2.6; DTX1: 0.2-1.1 | [46] | |
| P. lima(Ehrenberg) | 意大利 | OA: 6.69-15.80; DTX1: 0.12-0.39 | [47] | |
| P.lima(CCAP1136/11) | 西班牙 | OA: 0.1-1.25 | [48] |
现有研究普遍认为OA可能在P. lima的叶绿体中合成并储存, 是P. lima光合作用的直接产物之一[49], 并且单位细胞中的OA含量与脂质和叶绿素浓度密切相关[50]。Hu等[51]则明确指出DTX-4和其他水溶性硫酸盐衍生物是在叶绿体中合成的, 并且是底栖原甲藻的初始生物合成产物。但也有学者提出不同观点, 认为OA及其衍生物被储存在细胞质周围的液泡中[52]。Pan等[53]认为P. lima通过光介导作用, 水溶性毒素(例如DTX-4和DTX-5)被转移到液泡中, 并进一步转化为乙二醇酯衍生物和游离毒素(例如OA和DTXs)。采用同位素示踪实验发现DSTs分子中的所有碳均来自乙酸盐和乙醇酸[54], 其碳骨架由疑似由PKS合成[55](图 3)。通常聚酮类化合物合成过程需要首先通过丙酮酸合成乙酰辅酶A, 作用基础结构单元[55]。而P. lima中乙酰辅酶A的羧基氧并没有被H218O标记[55], 阻止OA合成中乙酰辅酶A的氧原子被水标记的机制目前还不清楚。有学者提出OA的生物合成中可能存在特殊的氧化机制——Baeyer- Villiger氧化[56]。然而到目前为止, P. lima毒素的产生机理尚不完全清楚。
目前, 部分研究者认为, P. lima产生DSTs可能受到细菌的刺激[57-58]。通过扫描和透射电子显微镜可以观察P. lima胞外和胞内均有大量附着或游离细菌分布[57]。并且多株共附生菌被证明与P. lima赤潮有关[58]。Tarazona-Janampa等[59]发现P. lima附生菌的丰富度与DTX1a-D8浓度呈正相关。Perez等[60]发现P. lima培养液中存在和细菌高度同源的聚酮化合物合酶(PKS)基因, 而在无毒的东海原甲藻(Prorocentrum donghaiense)和米氏凯伦藻(Karenia mikimotoi)中没有发现。与之相反, Morton[61]同时在无菌和细菌环境培养P. lima均可生成DSTs。此外, P. lima进行洗涤或在培养基加入抗生素后, 对其产毒也并无显著影响[59]。中立观点则认为, 细菌可能对P. lima细胞产生毒素具有一定的协同作用, 通常在P. lima的衰老阶段, 毒素的产生水平会增加, 而这些细菌可能在这个阶段参与降解P. lima细胞并加速细胞的衰老, 其裂解作用可能会增加溶解的有机碳的量从而提供更多营养物质以促进毒素的产生[19]。而P. lima及其附生菌共同参与构造了底栖系统中的细菌-甲藻群落, 其中甲藻细胞可以提供的独特且营养丰富的微生境[59], 而细菌能够合成如维生素B12等甲藻生长所必需的营养物质[62]。虽然目前是否有细菌参与及其促毒素生成的机制还不清晰, 但因其共同生存现象的存在, 仍然是未来研究的重点方向之一。
3 营养因素对P. lima的产毒的影响 3.1 氮氮是藻类细胞中蛋白质、脂质和叶绿素等的重要成分, 氮的含量会影响细胞的生长和代谢以及其他生命活动。研究发现高浓度的氮(﹥882 μmol/L)会抑制藻类细胞毒素的合成[19], 而低浓度氮或氮限制培养条件下单细胞产毒量则会升高[47, 63]。Hou等[64]研究就发现氮限制培养P. lima组中单细胞OA含量是对照组的3.54倍, 其他研究也证明氮限制培养中P. lima的细胞毒素水平增加了近1倍[47, 65-66]。Lee等[67]在不添加氮的培养基中(除用于制备培养基的天然海水中残留的硝酸盐和磷酸盐外)培养, 获得藻的单细胞OA浓度是基础氮水平(883 µmol/L)的3.7倍, 而在含氮10倍(8 830 µmol/L)的培养基中, 细胞内OA浓度与基础氮水平相似。除了限制氮的浓度, Mclachlan[22]还通过逐步添加硝酸盐(即30、100、300、1 000和3 000 µmol/L), 并将其连续添加到P.lima生长环境中培养(间隔10 d), 得出富氮会导致藻类细胞持续生长, 但细胞毒素水平保持在相对较低的水平(≤8 pg/cell)。由此可知, 如果局部海域中氮的含量突然增加, 造成海水富营养化, 会显著促进P. lima的生长。而毒素的产生可能是P. lima藻对在低营养条件下的一种补偿性竞争策略, 营养缺乏时毒素可被来维持细胞元素平衡, 并且可以作为对摄食浮游藻类生物的威慑, 成为一种自我防护措施[20]。
3.2 磷磷是藻代谢过程中另一种必需营养物质, 包括光合作用、细胞膜合成、信号转导以及分解代谢等[68]。但不同磷源下P. lima的产毒也有所不同, 磷酸二氢钠(NaH2PO4)作为磷源OA产量最高(35 pg/cell), 其次是ATP(30 pg/cell), 最后是甘油磷酸钠(18 pg/cell)[69]。通常磷缺乏会引起细胞蛋白磷酸化, 从而抑制藻类细胞的增殖[20]和提高毒素产生能力[70]。仲云等[63]的研究表明, 磷浓度对藻细胞OA含量影响显著, 并且在
诸多如微量元素、重金属元素、有机污染物等因素也会影响藻类细胞的生理活性。研究发现0.1 μmol/L的硒诱导P. lima单细胞产毒增加约50%[72]。Gu等[72]观察到提高铜浓度(从40 nmol/L增加到5 039 nmol/L) P. lima的单细胞OA含量增加了1.8倍(27 pg/cell)。Kana[73]用在日本发现的MIO12P菌株培养基中加入金属离子, 得到了极高的DTX1产量(1 265.3 ng/mL)。研究指出环境污染物(例如有机化合物)会导致细胞质壁分离、叶绿素含量降低, 影响其光合作用, 进而也会影响藻类细胞的生长和毒素产生[74]。然而, 很少有研究集中在重金属和有机化合物对P. lima产生毒素特性的影响上。此外有研究发现环境中的抗生素也会刺激赤潮藻类的生长, 海洋生态系统中的抗生素污染物可能对赤潮的爆发起到了积极的作用[75]。高等植物释放的克生物质也会抑制藻类的生长, 贾睿等[76]的研究表明新鲜的缘管浒苔(Enteromorpha linza)组织和干粉对P. lima的生长有很强的抑制作用, 而煮沸的培养物滤液并没有抑制的P. lima生长。据此目前有提出可以利用克生物质来治理赤潮的想法[77], 以望能够达到高效降低毒性的防治目标。
4 物理因素对P. lima的产毒的影响 4.1 温度温度主要是通过影响藻细胞内碱性磷酸酶的活性来影响藻细胞生长和产毒等代谢活动[77]。同时温度变化引起的胁迫也会促进P. lima产生毒素。P. lima具有广温性(5~30 ℃), 通常其最佳生长温度为25 ℃[20]。在4~20 ℃的范围内, 光营养生长速率的Q10值为2.3[78-79]。Aquino-Cruz等[80]研究得出藻细胞毒素含量在15 ℃可达到最高值, 游离态OA和DTX-1分别可达6.04和4.92 pg/cell, 高于30 ℃时的毒素含量。Wang等[71]发现单个P. lima细胞中的OA和DTX1含量在15 ℃和30 ℃下均高于其他温度, 在30 ℃时最大OA和DTX1含量分别为12.731和16.587 pg。但P. lima在15 ℃和30 ℃的生长率均不是最高, 这表明P. lima最佳生长和产毒温度存在差异性。同时温度对
盐度也是影响藻类生长的一个重要因素, 是影响藻细胞生长代谢的关键因子。藻类通常具有最佳的生长盐度, 盐度太高或太低都不利于藻类的生长。盐度增加会抑制藻细胞的分裂, 甚至对藻细胞自身的细胞结构产生严重的破坏[82]。研究表明, P. lima生长的盐度范围为20.0~45.0(pSU), 而且单细胞毒素含量随盐度增加而升高[71]。即盐度对P. lima生长速率和藻毒素产生的影响通常显示出相互矛盾的结果。一般来说, 生长率随着盐度的升高而降低[61]。而单细胞产毒量则与之相反, 当盐度从15增加到45时, OA从≈1.0 pg/cell增加到≈3.0 pg/cell, DTX1从≈4.7 pg/cell增加到≈22.5 pg/cell[71], 单细胞OA和DTX-1的含量在盐度为45.0达到最高。但是P. lima的最适生长盐度为30.0, 这表明藻细胞的生长和产毒的最适盐度条件不同, 也可能与原产地生长环境的长期适应有关。
4.3 光照目前已知不同光照强度和周期以及波长均会影响藻细胞的产毒, 适宜的光照强度可以促进P. lima的光合作用效率, 但是过度的光照强度会抑制藻细胞的光保护作用机制(通过叶绿素荧光参数的变化来评估), 进而影响其代谢和繁殖[83]。研究表明, P. lima在1.23×104~1.38×104 lx光照强度范围内可以达到最大的生长速率, 然而单细胞DSTs含量在低光照强度(约2 000 lx)下达到最高[19]。同时不同的光照周期对毒素也有影响, 一般认为毒素仅在光期内合成, 这表明在P. lima毒素的生成可能是一个光介导的代谢过程。而且单细胞OA和DTX-1含量也随光照周期的延迟(由12~16 h)显著下降, 两者含量在光照周期为12 h∶12 h时分别为1.80 pg/cell和11.0 pg/cell, 是16 h∶8 h时的1.20倍和2.20倍[71]。且在不同的细胞周期阶段, 合成特定毒素衍生物有所不同[53], DTX4水平的上升先于OA和DTX1细胞浓度的增加(延迟3~6 h), DTX4的合成开始于细胞周期的G1期, 并持续到S期, 而OA和DTX1的合成则晚于S期和G2期。有人对此提出假设[84], DTX4和OA合成的顺序模式可能是由于DTX4在细胞间的运输, 而这个过程可能是由光介导的机制调节的。
5 总结P. lima是产生腹泻性贝毒的主要甲藻之一, 其分布也非常广泛, 因为近期P. lima出现的频率、持续时间和数量都在增加, 这不仅直接影响海洋产品的品质和食用安全, 更加威胁了海洋养殖业的健康发展, 对食品安全造成极大威胁, 所以有关P. lima产毒的研究的重要性也日益提升。目前细胞毒素的产生与营养盐、细胞周期、温度等有关, 这些环境因素可能会通过影响细胞周期或细胞分裂速度来影响毒素的产生。其中, 营养盐的影响最为显著。例如氮和磷含量的突然增加导致富营养化, 富营养化和良好的气候条件将促进底栖生物的生物量增加。当营养水平低时, 出于补偿性或竞争策略, P. lima产毒会显著上升。在目前的研究中, 藻毒素标准的商业可获得性很低, 且供应时间长和价格高, 严重限制了我国海洋环境和海产品中相关毒素的日常检测。而通常藻毒素在P. lima中也是以微量存在, 因此从P. lima中收获毒素获得可持续供应目前还具有较大挑战。因此, 通过研究P. lima的产毒进一步开发DSTs相关研究非常必要, 同时跟进对P. lima影响因素的相关研究, 对我国近海生态、食品安全和消费者健康也有深远的意义。
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