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

像蛇类、蝎子和蛇这样的有毒动物利用它们的毒素具有多种功能，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中扮演着关键和核心的角色。关于塑造这些分子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，这些毒素之所以进化迅速，是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，毒素的生产耗费能量，因此还假设它们的生产受到严格控制(“毒液优化假说”)，即个人可以根据饮食的变化和温度等环境因素的变化来调整其毒液的数量和组成。目前，在这些假说之间缺乏能够辨别的数据。这项研究将提供一种综合方法，以确定决定毒液数量和组成变化的机制，这些机制对于专门研究易受持续气候变化影响的河口生境的模范刺齿动物的摄食生态是必不可少的。该项目将与耶胡·莫兰博士(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。这项研究将为北卡罗来纳大学夏洛特分校的博士后研究员、研究生和本科生以及北卡罗来纳大学米切尔社区学院的本科生提供指导和培训。将利用由国家科学基金会通过北卡罗来纳州路易斯·斯托克斯少数群体参与联盟资助的Products，为未被充分代表的学生提供更多的研究和教育机会，以招收、留住和研究生STEM教育。PI将通过个人培训、北卡罗来纳大学夏洛特分校的外展计划以及与奥林匹克高中生物技术学校合作的Burroughs Wellcome科学丰富计划，继续让高中生参与研究。了解参与调节毒素蛋白质表达的环境、遗传和分子因素对于了解和预测有毒物种将如何应对生物群落组成的变化至关重要。研究小组将通过研究小海葵维氏线虫来严格测试毒素多肽的表达假设。海葵是一种生活在北美东海岸河口的具有生态重要性的捕食者。由于基因组的可获得性、其在河口食物网中的中心位置以及野外采集和实验室操作的简便性，媒介线虫已成为生态基因组学的模范线虫；对于有毒动物来说，这种组合是罕见的。野外采集的海葵毒液基因的表达将与通过显微镜和DNA条形码识别的被摄取的猎物相关。这项分析将在三个不同的季节进行比较，以确定非生物变量和猎物可获得性的季节性变化的影响。这些毒素基因在野外表达的差异可能与毒素成分对可用猎物的调节、捕食者的存在和/或非生物胁迫程度决定的个体的生理状况有关。研究小组将通过受控的实验室实验区分这些潜在的原因，在实验室实验中，海葵暴露在两个常见的应激源(温度、紫外线)、单个猎物和捕食者。为了更好地了解这些毒素对特定猎物的选择性，行为测试和体外测试将比较每个基因变体在接触不同动物猎物组时的毒性，以确定相对效力。最后，序列分析将用于研究被捕食动物体内毒素的分子靶标，以确定这些靶标是否潜在地适应限制在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之间的潜在因果联系，以及毒素及其目标是否参与了一场“军备竞赛”。这个项目有望对在自然环境中生态相互作用的背景下理解毒素的进化产生重大影响，并为未来新型杀虫剂的设计贡献我们对毒素药理学的知识。

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.