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蓄积并随后被人类食用而引起的综合征。这些藻毒素由Gambierdiscus spp产生。，生活在各种大型植物以及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Gambierdiscus属内的新多样性，现在至少确定了 18 个物种和几个亚组，其中许多同时发生并且毒性显着不同。准确、快速地区分田间样品中的甘比尔盘属物种并确定群落组成和丰度的能力是评估 CP 风险的核心，但大多数使用光学显微镜无法区分Gambierdiscus物种，其他计数方法是半定量的。为了研究Gambierdiscus物种和群落毒性的时空动态，需要在田间样本中进行物种鉴定和计数的新工具。在这里，荧光原位杂交 (FISH) 探针是为加勒比海和太平洋常见的七种物种设计的，允许使用落射荧光显微镜在野外样本中进行计数。该技术能够评估群落组成并准确确定单个物种的细胞丰度。检测G. australes、G. belizeanus、G. caribaeus、G. carolinianus、G. carpenteri 的分子探针，和G. silvae/G。波利尼西亚进化枝是使用大亚基核糖体 RNA (rRNA) 序列的比对设计的。这些探针通过实验测试了这些探针的特异性和交叉反应性，在这些实验中，田间样品中加入了已知浓度的Gambierdiscus培养物，并进行分析以确认在存在碎屑和其他生物的情况下，FISH 可以成功检测和计数Gambierdiscus。然后使用这些探针来表征Gambierdiscus从美国佛罗里达群岛和夏威夷采集的现场样本中的群落结构。探针揭示了多个物种在每个位置的共存。从佛罗里达群岛收集的样本的时间序列 FISH 分析量化了群落组成的季节性变化以及整体Gambierdiscus细胞丰度的波动。物种特异性 FISH 探针的应用为那些寻求在野外种群中针对单个Gambierdiscus物种（包括重要的毒素生产者）的人提供了一个强大的新工具。展望未来，对Gambierdiscus 的分析 随着时间的推移，跨多个环境的群落组成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 CP 当前和不断变化的风险的能力。