Scott Anne M, Li Ke, Li Weiming
Department of Fisheries and Wildlife, Michigan State University.
Department of Fisheries and Wildlife, Michigan State University;
J Vis Exp. 2018 Jul 17(137):58059. doi: 10.3791/58059.
Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.
生物测定导向的分级分离是一种迭代方法，它利用生理和行为生物测定的结果来指导活性信息素化合物的分离和鉴定。这种方法已成功鉴定出在多种动物物种中起信息素作用的化学信号。海七鳃鳗依靠嗅觉来检测介导行为或生理反应的信息素。我们利用鱼类生物学的这些知识来推测假定信息素的功能，并指导活性信息素成分的分离和鉴定。色谱法用于从条件水中提取、浓缩和分离化合物。进行电嗅图（EOG）记录以确定哪些级分能引发嗅觉反应。然后使用双选迷宫行为测定来确定任何有气味的级分是否也具有行为活性并引起偏好。光谱和波谱方法提供分子量和结构信息，以协助结构解析。通过EOG和行为测定来确认纯化合物的生物活性。在迷宫中观察到的行为反应最终应在野外环境中得到验证，以确认它们在自然溪流环境中的功能。这些生物测定起到双重作用：1）指导分级分离过程；2）确认并进一步确定分离成分的生物活性。在此，我们报告海七鳃鳗信息素鉴定的代表性结果，以例证生物测定导向分级分离方法的效用。海七鳃鳗信息素的鉴定尤为重要，因为调节其信息素通讯系统是控制劳伦琴五大湖入侵海七鳃鳗所考虑的选项之一。这种方法可以很容易地适用于表征广泛类群中的化学通讯，并揭示水生化学生态学。
