Development of Novel Toxin Detection Methodologies Applicable to Marine and Fresh Waters

有害藻华（HABs）会对生态、经济和人类健康造成严重影响。至少有32个州报告了人类或哺乳动物接触有害藻华毒素的情况。此外，研究显示，在过去几十年，赤潮事件的频率和强度有所增加，并将在本世纪继续增加。在这些事件的生态驱动因素方面仍然存在知识差距，这阻碍了制定成功的缓解战略。大多数有害藻华由几个属组成，每一种都含有有毒和无毒菌株。这使得使用传统方法理解这些事件的生态变得极其困难。分子技术（如定量PCR）的进步促进了对这些复杂事件的深入理解的第一步，虽然以前的研究一般集中在一个单一的生物体。在这个项目中，研究人员在苏必利尔湖上级州立大学和得克萨斯州A M大学科珀斯克里斯蒂将开发和采用灵活的实时多重定量聚合酶链反应（MQPCR）测定同时定量的有毒有害藻华属。这将促进我们进一步了解这些复杂事件的下一个重要步骤，并将大大改善有害赤潮活动与现有的州和联邦监测和观测方案的结合。本研究的主要目的有四个：（1）开发用于检测产生类毒素-a的蓝藻以及有毒裸藻的MqPCR特异性TaqMan引物和探针，以结合现有的MqPCR方法;（2）设计并现场验证可针对特定HAB事件定制的灵活的MqPCR;（3）利用MqPCR技术分别研究了产微囊藻毒素/节球藻毒素、产柱孢藻毒素、产石房蛤毒素和产类毒素a的蓝藻以及具有潜在毒性的裸藻对环境变量的响应，以及相互之间的关系，在水华的过程中;和（4）确定的作用知之甚少的产毒藻类与美国淡水。 预计这项研究将为今后的赤潮事件建模和预测以及建立早期预警网络提供重要数据，以保护人类免受赤潮毒素的影响。 这项工作将进一步促进我们的理解的环境驱动力迫使在劳伦特五大湖和其他重要的商业，经济和娱乐淡水资源系统在美国的有害生物属的转变。由于该国的不同地区存在不同的毒素问题，即将开发的灵活的MqPCR将使研究人员和水管理人员能够根据需要定制MqPCR检测方法，以适应特定的水华情况。 更一般地说，这项工作应显着推进目前对这些事件的复杂生态的理解，使更好地预测水华毒性，并改善现有的州和联邦监测和观测计划的赤潮活动的整合。 该项目还将提供一名博士后研究员的培训。本项目所依据的原始提案（R 01 ES 021968 -01）已提交给美国国立环境健康科学研究院（NIH/NIEHS）响应资助机会公告RFA-ES-11-013，“海洋、五大湖和人类健康（R 01）"，这是NSF联合赞助的一个机会。 该项目通过NSF和NIEHS的单独奖励合作资助。

Harmful algal blooms (HABs) can have severe ecological, economical, and human health impacts. At least 32 states have reported human or mammal exposures to HAB toxins. Furthermore, studies suggest that the frequency and intensity of HAB events have increased over the past few decades and will continue to increase this century. Knowledge gaps remain regarding the ecological drivers of these events which have hindered the development of successful mitigation strategies. Most HABs are comprised of several genera with each species containing toxic and non-toxic strains. This has made understanding the ecology of these events using traditional methods extremely difficult. Advances in molecular techniques (e.g. quantitative PCR) have facilitated the first steps towards a deeper understanding of these complex events although previous studies have generally focused on a single organism. In this project, researchers at Lake Superior State University and the Texas A & M University at Corpus Christi will develop and employ a flexible real-time multiplex quantitative polymerase chain reaction (MqPCR) assay for the simultaneous quantification of co-occurring toxic HAB genera. This will facilitate the next important step in furthering our understanding of these complex events and will significantly improve the integration of HAB activities into existing state and federal monitoring and observational programs. There are four primary objectives: (1) to develop MqPCR specific TaqMan primers and probes for the detection of anatoxin-a producing cyanobacteria as well as toxic eugleniods to incorporate into the existing MqPCR method; (2) to design and field validate a flexible MqPCR that can be tailored to specific HAB events; (3) to use the MqPCR to investigate the response of microcystin/nodularin-, cylindrospermopsin-, saxitoxin-, and anatoxin-a-producing cyanobacteria as well as potentially toxic euglenoids to environmental variables individually, as well as in relation to each other, over the course of a bloom; and (4) to identify the role of poorly understood toxin-producing algae with U.S. freshwaters. This study is expected to provide important data for the modeling and prediction of future HAB events as well as the development of early warning networks to protect humans from exposure to HAB toxins.Broader Impacts. This work will further our understanding of the environmental drivers forcing shifts in HAB genera in the Laurentian Great Lakes and other important commercial, economical, and recreational freshwater resources systems across the USA. As different regions of the country have problems with different suites of toxins, the flexible MqPCR to be developed will give researchers and water managers the ability to tailor the MqPCR assay to suit specific bloom situations as needed. More generally, this work should significantly advance current understanding of the complex ecology of these events, enable better prediction of bloom toxicity, and improve the integration of HAB activities into existing state and federal monitoring and observational programs. The project would also provide for the training of a postdoctoral researcher.JOINT FUNDING BY NSF AND NIEHS: The original proposal on which this project is based (R01 ES021968-01) was submitted to the National Institutes of Environmental Health Sciences (NIH/NIEHS) in response to Funding Opportunity Announcement RFA-ES-11-013 , "Oceans, Great Lakes and Human Health (R01)", an opportunity jointly sponsored by NSF. This project is cooperatively funded through separate awards from NSF and NIEHS.