Evolution of Neurotoxin Resistance in Pufferfishes and Relatives: A Comparative Genomic Approach

已知的最致命的神经毒素之一是河豚毒素(TTX)。TTX与肌肉、心脏和神经中的钠通道紧密结合并阻断，导致瘫痪和死亡。TTX是由生物起源的，由各种惊人的动物产生，如蓝环章鱼、幽灵蟹、加州纽特和河豚。在一些物种中，TTX被用来捕获猎物，在另一些物种中，TTX被用来防御捕食。由于TTX在制造TTX的动物体内自由循环，这些动物必须对自己的毒素进化为不敏感。某些种类的河豚比其他种类的河豚毒性更强，而毒性程度较高的河豚显然与这些物种的组织对毒素的敏感性较低相匹配。一种假设是，这些河豚的组织对TTX的不同敏感性可能存在于与钠通道相关的蛋白质的氨基酸序列的差异。本项目通过对钠通道蛋白进化的机械研究，重点研究河豚对TTX敏感性的进化。为了重建钠通道基因的进化历史，将从河豚基因组数据库中检测这些基因的序列。接下来，将从表现出不同程度TTX敏感性的各种相关物种中克隆和测序六个钠通道中的三个的基因。河豚之间的系统发育关系是已知的。此外，还将检查一些对TTX不同程度敏感的相关鱼类，以及一些对TTX非常敏感的非相关鱼类。对TTX的敏感性将通过测量与大脑、肌肉和心脏组织样本结合的TTX的量来确定。对这些钠通道基因的序列进行比较，以识别钠通道蛋白中的特定氨基酸，这些氨基酸在对毒素高度不敏感的物种中与那些对毒素不敏感的物种中不同。从这些数据中，可以推断出在这些物种的进化史上，这3个基因中的特定突变是如何累积的。因此，在目前思考毒素如何与钠通道孔隙中的氨基酸相互作用的背景下，我们将获得对这些基因突变如何影响TTX结合的理解。这项关于河豚对自身TTX不敏感的进化工作具有重要的意义，原因有很多。首先，TTX被归类为武器级毒素。了解动物如何保护自己免受它的侵袭，可能有助于设计防御策略。其次，TTX及其相关化合物由海藻释放，引起对渔业和海洋环境造成严重影响的“赤潮”。这个项目将有助于了解一些动物如何保护自己免受这种破坏。第三，因为河豚的基因组很小，所以它们的基因组已经被克隆和测序，所以有关于这个物种的丰富的分子数据。本研究将利用这一信息。最后，从理论角度来看，这是一个耐人寻味的问题。众所周知，鱼类有六个不同的钠通道基因，因此TTX敏感性肯定是在六个基因中或多或少同时进化的。了解河豚对毒素的不敏感性是如何进化的，将成为动物如何在分子水平上对环境挑战做出适应性反应的模型。很少有研究发展出分子变异、不同的生物体表现和相对适合度之间的机制联系。本研究具有建立这些重要联系的巨大潜力。

One of the deadliest neurotoxins known is tetrodotoxin (TTX). TTX binds tightly to and blocks sodium channels in muscles, heart, and nerve causing paralysis and death. TTX is of biological origin and is produced by a striking variety of animals such as the blue-ringed octopus, the ghost crab, the California newt, and the pufferfish. In some species TTX is used to capture prey, in others for defense against predation. Because TTX circulates freely in the body of the animals that make it, these animals must evolve insensitivity to their own toxin. Some species of pufferfishes are more toxic than others, and that higher degree of toxicity is matched, obviously, with lower sensitivity of the tissues of those species to the toxin. One hypothesis is that the varying sensitivity of tissues to TTX among these species of pufferfishes likely resides in variation in the amino acid sequences of the proteins associated with the sodium channels. This project focuses on evolution of TTX sensitivity among pufferfishes by way of a mechanistic examination of the evolution of sodium channel proteins. In order to reconstruct the evolutionary history of the sodium channel genes, the sequences of these genes will be examined from the pufferfish genome database. Next, the genes for three of the six sodium channels will be cloned and sequenced from a variety of related species exhibiting varying degrees of TTX sensitivity. Phylogenetic relationships among the pufferfishes are known. In addition, some related fish with varying degrees of TTX sensitivity, and some unrelated fish that are very sensitive to TTX will be examined. Sensitivity to TTX will be determined by measuring the amount of TTX that binds to tissue samples of brain, muscle and heart. A comparison of the sequences of these sodium channel genes will be made to identify particular amino acids in the sodium channel proteins that are different in those species that are highly insensitive to the toxin versus those that are not. From these data inferences may be drawn to reconstruct how particular mutations accumulated in the 3 genes during the evolutionary history of these species. Thus an understanding of how these genetic mutations influence TTX binding will be gained in the context of current thinking about how the toxin interacts with amino acids in the pore of the sodium channel.This work on the evolution of pufferfish insensitivity to their own TTX is important for a number of reasons. First, TTX is classified as a weapons-grade toxin. Understanding how animals protect themselves against it may help in designing defense strategies against it. Second, TTX and related compounds are released by marine algae and cause the "red tide" which has a serious impact on fisheries industries and the marine environment. This project will help gain understanding about how some animals can protect themselves against this devastation. Third, because they have small genomes, the genome of the Pufferfish has been cloned and sequenced so there is a wealth of molecular data on this species. The present study will take advantage of that information. Finally, from a theoretical point of view, this is an intriguing question. Fish are known to have six different genes for sodium channels, so TTX sensitivity must have evolved more or less simultaneously in six genes. Understanding how toxin insensitivity evolved in pufferfish will be a model for how animals respond adaptively on a molecular level to environmental challenges. Very few studies have developed the mechanistic links between molecular variation, differential organismal performance, and relative fitness. The present study has great potential to forge those important links.