Pitz Kathleen J, Richlen Mindy L, Fachon Evangeline, Smith Tyler B, Parsons Michael L, Anderson Donald M
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.
Harmful Algae. 2021 Jan;101:101914. doi: 10.1016/j.hal.2020.101914. Epub 2021 Jan 6.
Ciguatera poisoning (CP) is a syndrome caused by the bioaccumulation of lipophilic ciguatoxins in coral reef fish and invertebrates, and their subsequent consumption by humans. These phycotoxins are produced by Gambierdiscus spp., tropical epiphytic dinoflagellates that live on a variety of macrophytes, as well as on dead corals and sand. Recent taxonomic studies have identified novel diversity within the Gambierdiscus genus, with at least 18 species and several sub-groups now identified, many of which co-occur and differ significantly in toxicity. The ability to accurately and quickly distinguish Gambierdiscus species in field samples and determine community composition and abundance is central to assessing CP risk, yet most Gambierdiscus species are indistinguishable using light microscopy, and other enumeration methods are semi-quantitative. In order to investigate the spatial and temporal dynamics of Gambierdiscus species and community toxicity, new tools for species identification and enumeration in field samples are needed. Here, fluorescence in situ hybridization (FISH) probes were designed for seven species commonly found in the Caribbean Sea and Pacific Ocean, permitting their enumeration in field samples using epifluorescence microscopy. This technique enables the assessment of community composition and accurate determination of cell abundances of individual species. Molecular probes detecting G. australes, G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, and the G. silvae/G. polynesiensis clade were designed using alignments of large subunit ribosomal RNA (rRNA) sequences. These probes were tested for specificity and cross-reactivity through experiments in which field samples were spiked with known concentrations of Gambierdiscus cultures, and analyzed to confirm that Gambierdiscus can be successfully detected and enumerated by FISH in the presence of detritus and other organisms. These probes were then used to characterize Gambierdiscus community structure in field samples collected from the Florida Keys and Hawai'i, USA. The probes revealed the co-occurrence of multiple species at each location. Time-series FISH analyses of samples collected from the Florida Keys quantified seasonal shifts in community composition as well as fluctuations in overall Gambierdiscus cell abundance. Application of species-specific FISH probes provides a powerful new tool to those seeking to target individual Gambierdiscus species, including significant toxin-producers, in field populations. Moving forward, analysis of Gambierdiscus community composition across multiple environments and over time will also allow species dynamics to be linked to environmental parameters, improving our ability to understand and manage the current and changing risks of CP worldwide.
雪卡毒素中毒（CP）是一种由亲脂性雪卡毒素在珊瑚礁鱼类和无脊椎动物体内生物累积，随后被人类食用而引起的综合征。这些藻毒素由冈比亚藻属产生，冈比亚藻属是生活在各种大型植物、死珊瑚和沙子上的热带附生甲藻。最近的分类学研究已经确定了冈比亚藻属内的新多样性，目前已鉴定出至少18个物种和几个亚群，其中许多物种共存且毒性差异显著。准确、快速地区分野外样本中的冈比亚藻物种，并确定群落组成和丰度的能力是评估雪卡毒素中毒风险的核心，但使用光学显微镜大多数冈比亚藻物种难以区分，而其他计数方法是半定量的。为了研究冈比亚藻物种和群落毒性的时空动态，需要用于野外样本中物种鉴定和计数的新工具。在此，针对在加勒比海和太平洋常见的7个物种设计了荧光原位杂交（FISH）探针，允许使用落射荧光显微镜对野外样本中的这些物种进行计数。这项技术能够评估群落组成，并准确测定单个物种的细胞丰度。利用大亚基核糖体RNA（rRNA）序列比对设计了检测澳大利亚冈比亚藻、伯利兹冈比亚藻、加勒比冈比亚藻、卡罗来纳冈比亚藻、卡彭特冈比亚藻以及西尔瓦冈比亚藻/波利尼西亚冈比亚藻分支的分子探针。通过在野外样本中添加已知浓度的冈比亚藻培养物进行实验，测试了这些探针的特异性和交叉反应性，并进行分析以确认在存在碎屑和其他生物的情况下，能够通过FISH成功检测和计数冈比亚藻。然后使用这些探针来表征从美国佛罗里达群岛和夏威夷收集的野外样本中的冈比亚藻群落结构。这些探针揭示了每个地点多种物种的共存情况。对从佛罗里达群岛收集的样本进行的时间序列FISH分析量化了群落组成的季节性变化以及冈比亚藻细胞总数的波动。物种特异性FISH探针的应用为那些试图在野外种群中靶向单个冈比亚藻物种（包括重要的毒素产生者）的人提供了一个强大的新工具。展望未来，对多个环境中冈比亚藻群落组成随时间的分析也将使物种动态与环境参数联系起来，提高我们理解和管理全球雪卡毒素中毒当前和不断变化风险的能力。
