Koch F, Kang Y, Villareal T A, Anderson D M, Gobler C J
Stony Brook University, School of Marine and Atmospheric Sciences, Southampton, New York, USA.
The University of Texas at Austin, Marine Science Institute, Port Aransas, Texas, USA.
Appl Environ Microbiol. 2014 Aug;80(16):4947-57. doi: 10.1128/AEM.00888-14. Epub 2014 Jun 6.
During the past 3 decades, brown tides caused by the pelagophytes Aureococcus anophagefferens and Aureoumbra lagunensis have caused ecological and economic damage to coastal ecosystems across the globe. While blooms of A. lagunensis had previously been confined to Texas, in 2012, an expansive brown tide occurred on Florida's East Coast, causing widespread disruption within the Indian River and Mosquito Lagoons and generating renewed interest in this organism. A major impediment to detailed investigations of A. lagunensis in an ecosystem setting has been the absence of a rapid and reliable method for cell quantification. The combination of their small size (3 to 5 μm) and nondescript extracellular features makes identification and enumeration of these cells with conventional methods a challenge. Here we report the development of an immunological-based flow cytometry method that uses a fluorescently labeled antibody developed against A. lagunensis. This method is species specific, sensitive (detection limit of 1.5 × 10(3) cells ml(-1)), precise (1% relative standard deviation of replicated samples), and accurate (108% ± 8% recovery of spiked samples) over a wide range of cell concentrations. Furthermore, this method effectively quantifies A. lagunensis in both glutaraldehyde- and formalin-preserved samples, yields a high throughput of samples (∼35 samples h(-1)), and is cost-effective, making it an ideal tool for managers and scientists. This method successfully documented the recurrence of a brown tide bloom in Florida in 2013. Bloom densities were highest in June (>2.0 × 10(6) cells ml(-1)) and spanned >60 km from the Ponce de Leon inlet in the northern Mosquito Lagoon south to Titusville in the Indian River Lagoon. Low levels of A. lagunensis cells were found >250 km south of this region. This method also quickly and accurately identified A. lagunensis as the causative agent of a 2013 brown tide bloom in Guantanamo Bay, Cuba, and thus should prove useful for both quantifying the dynamics of ongoing blooms of A. lagunensis as well as documenting new outbreaks of this harmful alga.
在过去30年里，由浮游植物噬藻金球藻（Aureococcus anophagefferens）和拉古纳奥氏藻（Aureoumbra lagunensis）引发的褐潮已对全球沿海生态系统造成了生态和经济破坏。虽然拉古纳奥氏藻的藻华此前一直局限于得克萨斯州，但在2012年，佛罗里达州东海岸出现了一次大规模褐潮，对印第安河和蚊子湖造成了广泛破坏，并引发了人们对这种生物的新关注。在生态系统环境中对拉古纳奥氏藻进行详细研究的一个主要障碍是缺乏一种快速可靠的细胞定量方法。它们体积小（3至5微米）且细胞外特征不明显，这使得用传统方法识别和计数这些细胞成为一项挑战。在此，我们报告了一种基于免疫的流式细胞术方法的开发，该方法使用针对拉古纳奥氏藻开发的荧光标记抗体。该方法具有物种特异性，在很宽的细胞浓度范围内灵敏（检测限为1.5×10³个细胞/毫升）、精确（重复样品的相对标准偏差为1%）且准确（加标样品的回收率为108%±8%）。此外，该方法能有效定量戊二醛和福尔马林保存样品中的拉古纳奥氏藻，样品通量高（约35个样品/小时）且成本效益高，使其成为管理人员和科学家的理想工具。该方法成功记录了2013年佛罗里达州褐潮藻华的再次出现。藻华密度在6月最高（>2.0×10⁶个细胞/毫升），从蚊子湖北部的庞塞·德莱昂河口向南延伸至印第安河泻湖的蒂托维尔，跨度超过60公里。在该区域以南250多公里处发现了少量的拉古纳奥氏藻细胞。该方法还快速准确地确定拉古纳奥氏藻是2013年古巴关塔那摩湾褐潮藻华的致病因子，因此对于量化拉古纳奥氏藻持续藻华的动态以及记录这种有害藻类的新爆发都应是有用的。
