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

有害藻华(HAB)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HAB毒素的情况。此外，研究表明，过去几十年来，赤潮发生的频率和强度有所增加，本世纪还将继续增加。关于这些事件的生态驱动因素的知识差距仍然存在，这阻碍了成功的缓解战略的制定。大多数赤潮由几个属组成，每个物种都含有有毒和无毒的菌株。这使得用传统方法理解这些事件的生态变得极其困难。分子技术(如定量聚合酶链式反应)的进步促进了深入了解这些复杂事件的第一步，尽管以前的研究一般集中在单个生物体上。在这个项目中，苏必利尔湖州立大学和位于科珀斯克里斯蒂的德克萨斯农工大学的研究人员将开发和使用一种灵活的实时多重定量聚合酶链式反应(MqPCR)分析方法，用于同时定量共存的有毒HAB属。这将有助于下一步进一步加深我们对这些复杂事件的理解，并将显著改善将卫生与公众服务局的活动整合到现有的州和联邦监测和观测计划中。有四个主要目标：(1)开发MqPCR特异的TaqMan引物和探针，用于检测产生类毒素a的蓝藻以及有毒的真球藻，以纳入现有的MqPCR方法；(2)设计并现场验证可针对特定的赤潮事件量身定做的灵活的MqPCR；(3)使用MqPCR来研究在水华发生过程中产生微囊藻/节藻毒素、圆柱状精液蛋白、萨氏毒素和Anatoxin a的蓝藻以及潜在有毒的真球藻对环境变量的响应以及它们之间的关系；以及(4)确定鲜为人知的产毒素藻类在美国淡水中的作用。这项研究有望为未来赤潮事件的建模和预测以及开发早期预警网络以保护人类免受赤潮毒素的影响提供重要数据。这项工作将进一步加深我们对迫使劳伦斯五大湖和美国各地其他重要的商业、经济和娱乐淡水资源系统中HAB属转移的环境驱动因素的理解。由于该国不同地区存在不同毒素套件的问题，将开发的灵活的MqPCR将使研究人员和水资源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。更广泛地说，这项工作应该会显著推进当前对这些事件的复杂生态的理解，使更好地预测水华毒性，并改进将赤潮活动纳入现有的州和联邦监测和观测计划。该项目还将提供一名博士后研究人员的培训。NSF和NIEHS的资助：本项目所依据的原始提案(R01 ES021968-01)已提交给国家环境卫生科学研究所(NIH/NIEHS)，以回应由NSF联合赞助的资助机会公告RFA-ES-11-013“海洋、五大湖与人类健康(R01)”。该项目由NSF和NIEHS通过单独的奖项提供合作资金。

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.