The Adaptive Significance of an Evolutionary Constraint: from Protein to Organism

研究人员将确定特定蛋白质中氨基酸突变的生物物理特性如何影响不同水平的生物组织，最终影响个体在不同群落中生活的能力，甚至影响区域尺度上的物种相互作用。本研究的模型系统是有毒的太平洋蝾螈（Taricha）和它们的抗性掠食性袜带蛇（Thamnophis）之间的相互作用。蝾螈由河豚毒素（TTX）保护，这是迄今为止发现的最致命的毒素之一。这种神经毒素与电压门控钠通道的外孔结合，钠通道是负责在肌肉和神经中传播电脉冲的蛋白质，从而使肌肉和神经器官瘫痪。尽管如此，多种吊带蛇以蝾螈为食，这在很大程度上是由于肌肉中表达的钠通道蛋白的特定变化。然而，这些适应性变异可能承担很高的成本；对钠通道的生物物理学研究表明，减少TTX与蛋白质结合的氨基酸突变可能会降低钠通道的功能。如果这种权衡适用于生物体，那么它就可以解释为什么抗TTX的吊带蛇在地理上是局部的，而没有席卷整个景观。此外，这种权衡可以解释为什么蛇类中具有抗性的突变只代表了已知减少TTX与蛋白质结合的突变的一小部分。然而，自然发生的蛋白质变异的成本仍未被探索，这种生物物理权衡、组织功能和机体性能之间的关系也未被探索。使用真正的综合方法，研究人员将：1)量化这种分子适应的明显权衡；2)确定对猎物毒素和生物物理权衡的抵抗力如何扩大到组织；3)确定组织阻力和不同器官（肌肉和神经）的表现如何结合起来决定蛇的适应性。这项研究将为理解分子水平的权衡如何影响个体对环境的反应提供一个框架，最终影响整个景观的适应性进化和物种相互作用。该研究提供了一个具有清晰生态背景的易于处理的模型，以帮助确定适应性进化何时和为何可能受到限制，以及这些限制对适应可能产生的下游后果。该项目将培训两名研究生和几名本科生进行高度整合的研究，他们将参与分子数据生成、蛋白质分析、生理分析和整个生物体性能试验，所有这些都是在捕食者-猎物共同进化的背景下进行的。该项目的研究主题将扩展到内华达州里诺市的家庭户外教育项目。该项目将资助5个市中心家庭参加一个周末的户外活动，他们将在一个野外站停留，探索距离他们居住的城市仅30分钟路程的森林生态系统的生物多样性和生态学。申请将向参加联合国大学数学、工程和科学成就项目（MESA）的中小学生开放。MESA致力于吸引低收入的第一代学生，并将成为识别对科学感兴趣但有经济需求的学生的理想渠道，否则这些学生将无法参加像家庭营地这样的项目。PI之所以选择以家庭为基础的模式，是因为他观察到，如果一个孩子的直系亲属也参与进来，那么让他接触这种以自然为基础的体验，会产生更高、更持久的影响。这种模式提供了一套积极的、共享的家庭经历，可以记住和加强，为孩子继续发展和探索科学兴趣创造了一个支持性的环境。这样的教育机会在内华达州这样的EPSCoR州尤其罕见，但将对促进我们社区的生物多样性和基于有机体的研究和教育产生重大影响。

The investigators will determine how the biophysical properties of amino acid mutations in a specific protein influence various levels of biological organization, ultimately affecting the capacity of individuals to live in various communities, and even influencing species interactions at a regional scale. The model system for this research is the interaction between toxic Pacific newts (Taricha) and their resistant predatory garter snakes (Thamnophis). Newts are defended by tetrodotoxin (TTX), one of the most lethal toxins ever discovered. This neurotoxin binds to the outer pore of voltage-gated sodium channels, the proteins responsible for propagating electrical impulses in muscles and nerves, thereby paralyzing muscle and nerve organs. Despite this, multiple garter snake species feast on newts, in large part due to specific changes to the sodium channel protein expressed in muscle. However, these adaptive variants likely bear a high cost; biophysical work on sodium channels suggests that amino acid mutations that reduce TTX binding to the protein are likely to reduce sodium channel function. If this tradeoff scales to the organism, then it may explain why TTX resistant garter snakes are geographically localized, and have not swept across the landscape. Further, this tradeoff may explain why resistance-conferring mutations in snakes represent only a small subset of mutations known to reduce TTX binding to the protein. Nevertheless, the cost of naturally occurring protein variants remains unexplored, as does any relationship between this biophysical tradeoff, tissue function, and organismal performance. Using a truly integrative approach, the investigators will: 1) quantify the apparent tradeoff in this molecular adaptation; 2) determine how resistance to prey toxin and biophysical tradeoffs scale up to the tissue; and 3) determine how tissue resistance and performance in different organs (muscle and nerve) combine to determine the adaptation of the snake. This research will provide a framework for understanding how a tradeoff at the molecular level can influence an individual's response to its environment, ultimately impacting adaptive evolution and species interactions across the landscape. The study provides a tractable model with a clear ecological context to help determine when and why adaptive evolution may be limited, as well as the possible downstream consequences of those constraints on adaptation.The project will lead to training of two graduate students and several undergraduates in a highly integrative investigation where they will participate in molecular data generation, protein analysis, physiological assays, and whole organism performance trials, all in the context of predator-prey coevolution. The research themes of this project will be extended to an outdoor education program for families from Reno, Nevada. This project will sponsor 5 inner-city families to participate in a weekend-long outdoor program where they stay at a field station and explore the biodiversity and ecology of forest ecosystems that exist only 30 minutes from the urban setting in which they live. Applications will be made available to elementary and middle school children engaged in the UNR Mathematics, Engineering, and Science Achievement program (MESA). MESA strives to engage low-income, first-generation students and will be an ideal conduit for identifying students that have interest in science but with financial needs that would otherwise preclude attendance of a program like Family Camp. The PI has chosen a family-based model because he has observed that exposing an individual child to this nature-based experience has a much higher and longer-lasting impact if their immediate family participates as well. This model provides a set of positive, shared family experiences that can be remembered and reinforced, creating a supportive environment as the child continues to develop and explore an interest in science. Such educational opportunities are particularly rare in an EPSCoR state like Nevada but will have significant impact on promoting biodiversity and organismally-based research and education in our community.