Toxin expression and function by an estuarine model species in a dynamic seasonal community

刺胞动物、蝎子和蛇等有毒动物利用它们的毒素进行多种功能，包括防御捕食者和捕获猎物。 因此，毒液在产生毒液的动物的生态学中起着关键和核心的作用。关于塑造这些分子的多样性和表达的生态因素，有几个主要的假设。 一个中心假设是，这些毒素迅速演变，因为它们参与了生物体与其潜在的捕食者和猎物之间的进化“军备竞赛”。此外，毒素的生产在能量上是昂贵的，因此也假设它们的生产受到严格的调控（“毒液优化假说”），其中个体可以根据饮食的变化和环境因素如温度的变化来调节毒液的量和组成。 目前，缺乏数据来区分这些假设。这项研究将提供一个综合的方法来确定的机制，确定的数量和组成的毒液的摄食生态学的一个模型刺胞动物专门在河口栖息地，容易受到持续的气候变化的变化必不可少的变化。该项目将与Yehu Moran博士（以色列耶路撒冷希伯来大学）进行协同国际合作，将环境变化与毒液蛋白的表达和效力联系起来。本研究将为夏洛特的北卡罗来纳州大学的博士后研究员、研究生和本科生以及北卡罗来纳州斯泰茨维尔的米切尔社区学院的本科生提供指导和培训。额外的研究和教育机会将提供给使用PRODUCE的代表性不足的学生，由NSF通过NC Louis Stokes少数民族参与联盟资助，以招募，保留和研究生STEM教育。 PI将继续通过个人培训，夏洛特夏洛特的推广计划和与奥林匹克高中生物技术学校的巴勒斯惠康科学丰富计划，让高中学生参与研究。以及参与调节毒素蛋白质表达的分子因子对于理解和预测有毒物种将如何对生物群落组成的变化作出反应至关重要。 研究小组将通过研究星状海葵Nematostella vectensis来严格测试毒素肽表达的假设，Nematostella vectensis是一种具有生态重要性的捕食者，生活在北美东部海岸沿着的河口。 N. vectensis已成为生态基因组学的模式刺胞动物，由于基因组的可用性，其在河口食物网中的中心位置，以及易于现场收集和实验室操作;一个组合很少用于有毒动物。 在野外采集的海葵中，毒液基因的表达将与使用显微镜和DNA条形码鉴定的摄食猎物相关。 这一分析将在三个不同的季节进行比较，以确定非生物变量的季节变化以及猎物的可用性的影响。 这些毒素基因在该领域的表达差异可能与调谐的毒素组合物的猎物，捕食者的存在下，和/或由非生物胁迫的程度确定的个人的生理条件。 研究小组将通过受控的实验室实验来区分这些潜在的因果因素，在实验中，海葵暴露于两种常见的压力源（温度，紫外线），个别猎物和捕食者。为了更好地了解这些毒素对特定猎物的选择性，当暴露于不同的动物猎物群体时，将比较每种遗传变体的行为和体外试验毒性，以确定相对效力。 最后，将使用序列分析来研究猎物中毒素的分子靶标，以确定这些靶标是否可能适应限于不同地理位置的毒素等位基因。这些数据将有助于确定有毒动物及其猎物的毒素进化与毒素及其目标是否参与“军备竞赛”之间的潜在因果关系。 该项目预计将对理解毒素在自然环境中的生态相互作用的背景下的演变产生重大影响，并有助于我们对毒素药理学的知识，为未来设计新的杀虫剂。

Venomous animals such as cnidarians, scorpions, and snakes utilize their toxins for a diversity of functions including defense from predators and for capturing prey. Thus, venom plays a pivotal and central role in the ecology of the animal producing it. There are several major hypotheses regarding the ecological factors that shape the diversity and expression of these molecules. A central hypothesis is that these toxins evolve rapidly due to their involvement in an evolutionary "arms race" between the organism and its potential predators and prey. Moreover, toxins are energetically expensive to produce so it is also hypothesized that their production is tightly regulated ("venom optimization hypothesis"), where individuals can module the amount and composition of their venom dependent on changes in diet and shifts in environmental factors such as temperature. Currently, there is a paucity of data to discern between these hypotheses. This research will provide an integrative approach to identify mechanisms that determine shifts in the quantity and composition of the venom essential for the feeding ecology of a model cnidarian specializing in estuarine habitats that are vulnerable to the ongoing climate change. This project will provide a synergistic international collaboration with Dr. Yehu Moran (The Hebrew University of Jerusalem, Israel) to connect environmental variation with the expression and potency of venom proteins. This research will provide mentoring and training for a postdoctoral fellow, graduate and undergraduate students at the University of North Carolina at Charlotte, and undergraduate students at Mitchell Community College in Statesville, NC. Additional research and educational opportunities will be provided for underrepresented students using PRODUCE, funded by the NSF through the NC Louis Stokes Alliance for Minority Participation to recruit, retain, and graduate students in STEM education. The PI will continue to involve high-school students in research through individual training, UNC Charlotte's outreach programs and the Burroughs Wellcome Science Enrichment program with the Olympic High School Biotechnology School.Knowledge of the environmental, genetic, and molecular factors involved in modulating expression of toxin proteins is essential to understand and predict how venomous species will respond to changes in the composition of biological communities. The research team will rigorously test hypotheses in the expression of toxin peptides by studying the starlet sea anemone, Nematostella vectensis, a predator of ecological importance that lives in estuaries along the eastern coast of North America. N. vectensis has emerged as a model cnidarian for ecological genomics due to the availability of a genome, its central position in estuarine food webs, and the ease of field collection and laboratory manipulation; a combination rarely available for venomous animals. Expression of venom genes in field collected anemones will be correlated with ingested prey identified using microscopy and DNA barcodes. This analysis will be compared in three different seasons to identify impacts of seasonal changes in abiotic variables as well as prey availability. Differences in expression of these toxin genes in the field could be related to tuning of the toxin composition to the prey available, presence of predators, and/or the physiological condition of the individual determined by degree of abiotic stress. The research team will differentiate between these potential causal factors through controlled laboratory experiments where anemones are exposed to two common stressors (temperature, UV), individual prey items, and a predator. In order to better understand the selectivity of these toxins to particular prey, behavioral and in vitro assays test toxicity of each genetic variant will be compared when exposed to distinct animal prey groups to determine the relative potency. Finally, sequence analyses will be used to investigate the molecular targets of the toxins in the prey animals to determine if these targets are potentially adapted to toxin alleles restricted to different geographic locations. These data will help determine a potential causative link between toxin evolution of a venomous animal and its prey and whether toxins and their targets are involved in an "arms race". This project is expected to have a significant impact on the understanding of the evolution of toxins in the context of ecological interactions in natural environments and contribute to our knowledge about toxin pharmacology for future design of novel insecticides.