Cusick Kathleen, Duran Gabriel
Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD 21250, USA.
Microorganisms. 2021 May 23;9(6):1128. doi: 10.3390/microorganisms9061128.
Saxitoxin (STX) is a secondary metabolite and potent neurotoxin produced by several genera of harmful algal bloom (HAB) marine dinoflagellates. The basis for variability in STX production within natural bloom populations is undefined as both toxic and non-toxic strains (of the same species) have been isolated from the same geographic locations. is a STX-producing bioluminescent dinoflagellate that blooms along the east coast of Florida as well as the bioluminescent bays in Puerto Rico (PR), though no toxicity reports exist for PR populations. The core genes in the dinoflagellate STX biosynthetic pathway have been identified, and the gene is essential for toxin production. Using as a molecular proxy for the genetic capacity of STX production, we examined + and - genotype frequency at the single cell level in populations from different locations in the Indian River Lagoon (IRL), FL, and Mosquito Bay (MB), a bioluminescent bay in PR. Multiplex PCR was performed on individual cells with -specific primers targeting the 18S rRNA gene and . The results reveal that within discrete natural populations of , both + and - genotypes occur, and the + genotype dominates. In the IRL, the frequency of the + genotype ranged from ca. 80-100%. In MB, + genotype frequency ranged from ca 40-66%. To assess the extent of variation within individual cells, amplicons from single cells representative of the different sampling sites were cloned and sequenced. Overall, two variants were consistently obtained, one of which is likely a pseudogene based on alignment with cDNA sequences. These are the first data demonstrating the existence of both genotypes in natural sub-populations, as well as presence in from PR. These results provide insights on underlying genetic factors influencing the potential for toxin variability among natural sub-populations of HAB species and highlight the need to study the genetic diversity within HAB sub-populations at a fine level in order to identify the molecular mechanisms driving HAB evolution.
石房蛤毒素（STX）是由几属有害藻华（HAB）海洋甲藻产生的次生代谢产物和强效神经毒素。自然藻华种群中STX产量变异性的基础尚不清楚，因为（同一物种的）有毒和无毒菌株都已从相同地理位置分离出来。是一种产生STX的发光甲藻，在佛罗里达州东海岸以及波多黎各（PR）的发光海湾大量繁殖，不过尚无关于PR种群毒性的报告。甲藻STX生物合成途径中的核心基因已被鉴定，并且该基因对于毒素产生至关重要。利用作为STX产生遗传能力的分子代理，我们在佛罗里达州印度河泻湖（IRL）和PR的一个发光海湾蚊子湾（MB）不同地点的种群中，在单细胞水平上检测了+和-基因型频率。使用针对18S rRNA基因和的特异性引物对单个细胞进行多重PCR。结果表明，在离散的自然种群中，+和-基因型均存在，且+基因型占主导。在IRL中，+基因型频率范围约为80 - 100%。在MB中，+基因型频率范围约为40 - 66%。为了评估单个细胞内的变异程度，对代表不同采样地点的单细胞的扩增子进行了克隆和测序。总体而言，一致获得了两个变体，其中一个基于与cDNA序列的比对可能是假基因。这些是首次证明自然亚种群中两种基因型存在以及PR种群中存在的数据。这些结果为影响HAB物种自然亚种群中毒素变异性潜力的潜在遗传因素提供了见解，并强调需要在精细水平上研究HAB亚种群内的遗传多样性，以确定驱动HAB进化的分子机制。
