Molecular basis for the detection of nutrients and toxins by the honeybee

我们的味觉是我们检测食物中营养和毒素的主要手段，它的功能对我们的健康和幸福很重要。哺乳动物和昆虫的味觉原理是相同的，这使得用昆虫作为模式生物来理解化学信息是如何被味觉系统检测和编码的成为可能。当动物摄取食物时，像糖这样的营养物质是由舌头上的味蕾细胞检测到的，而昆虫则是由化学感受器中的神经元检测到的。一般来说，味觉系统的组织结构是这样的：一部分味觉细胞或神经元被糖激发，而另一部分则被有毒（苦）化合物、氨基酸、盐或水激发。在昆虫中，味觉神经元膜上的味觉受体（Grs）选择性地与不同种类的化合物（如糖）结合，它们的激活表明食物中存在哪些化合物。动物可能有几百个Grs，但存在数百万个潜在的配体。我们不知道Gr多样性如何提供化合物的检测和分类。因此，解码味觉，首先需要识别在一个动物物种中激活特定Grs的营养物质或毒素。这可能与它的Gr神经元的反应特性以及它的味觉和敏锐度有关。这里提出的研究将发展蜜蜂作为一个模型系统来理解味觉代码的逻辑。蜜蜂只有10亿个基因，是基因组测序的昆虫中报道的最少的。根据与果蝇的序列同源性以及我们对蜜蜂Gr基因结构的了解，我们预测它的功能Gr少于20个。由于这个原因，鉴定这些基因产生的Grs的化学配体是可能的，目的是利用蜜蜂作为模型来理解味觉编码的原理。与拥有60个基因的果蝇相比，拥有少量Gr基因的蜜蜂是一个易于控制的模型系统。目前只确定了13种果蝇的配体。该提案描述了一个项目，将使用两种方法来识别与蜜蜂的Gr基因相关的受体配体。使用“功能获得”方法，我们将采用一种新开发的转基因果蝇系，其中所有假定的糖受体基因都已被敲除。每个蜜蜂的Gr基因都将在这条线上表达。每个蜂系的果蝇将使用它们跗味觉神经元的钙成像进行分析。通过一系列配体的刺激，我们将能够确定是否由单个Gr基因的表达产生功能性受体，并确定其Gr的配体。基于我们对果蝇糖受体及其蜜蜂同源物的了解，我们还将测试编码糖受体的多个Gr基因的表达是否需要形成功能性的Gr。我们不知道是否必须表达几个Gr基因才能形成检测糖类以外化合物的功能受体。出于这个原因，我们还必须使用“功能丧失”方法，即使用小干扰RNA （siRNA）敲低蜜蜂体内每个Gr基因的表达。利用这种方法，我们将敲除每个Gr基因的表达，并利用电生理学和行为分析蜜蜂的味觉神经元。我们将测试一系列营养物质和有毒化合物，其中包括蜜蜂在开花作物中遇到的常见杀虫剂。尽管蜜蜂只有10个Gr基因，但它们仍然能够检测到一些毒素，并调节它们对Gr检测到的糖和氨基酸等营养物质的摄入。这里提出的实验将揭示蜜蜂的少数Gr基因是如何转化成它能尝到的味道的，并将导致未来的工作，以确定Gr神经元群体如何编码有关食物的化学性质和复杂性的信息。

Our sense of taste is our primary means of detecting nutrients and toxins in food, and its function is important for our health and well-being. The principles of gustation are shared in mammals and insects making it possible to use insects as model organisms to understand how chemical information is detected and encoded by the gustatory system. When animals ingest food, nutrients like sugars are detected by cells in taste buds on the tongue, and in insects by neurons in chemosensory sensilla. In general, the gustatory system is organized such that a subset of gustatory cells or neurons is excited by sugars, whereas others are excited by toxic (bitter) compounds, by amino acids, by salts, or by water. In insects, gustatory receptors (Grs) on the membranes of taste neurons selectively bind to classes of chemical compounds (e.g. sugars) and their activation indicates which compounds are present in food. Animals may have as many as a few hundred Grs, but millions of potential ligands exist. We do not know how Gr diversity affords the detection and classification of chemical compounds. Decoding gustation, therefore, first requires the identification of the nutrients or toxins that activate specific Grs in one animal species. This could then be related to the response properties of its Gr neurons and to its taste perception and acuity. The research proposed here will develop the honeybee as a model system for understanding the logic of the gustatory code. The honeybee has only 10 Gr genes - the least reported from insects with sequenced genomes. Based on sequence homology with Drosophila and what we know about the structure of the bee's Gr genes, we predict it has less than 20 functional Grs. For this reason, it would be possible to identify the chemical ligands for the Grs produced by these genes with the aim of using the bee as a model to understand the principles of gustatory coding. Having few Gr genes makes the bee a tractable model system in contrast to Drosophila with its 60 genes. Ligands have been determined for only 13 Drosophila Grs.This proposal describes a project that will use two approaches to identify the ligands for the receptors associated with the honeybee's Gr genes. Using a 'gain-of-function' approach, we will employ a newly-developed transgenic fruit fly line in which all of the putative genes for sugar receptors have been knocked out. Each of the bee's Gr genes will be expressed in this line. Flies from each bee Gr line will be assayed using calcium imaging of their tarsal gustatory neurons. By stimulating with a series of ligands, we will be able to identify whether functional receptors are produced by the expression of single Gr genes and to identify their Grs' ligands. Based on what we know about fruit fly sugar receptors and their bee homologues, we will also test whether expression of multiple Gr genes that encode sugar receptors is necessary to form functional Grs. We do not know if several Gr genes must be expressed to form functional receptors for the detection of compounds other than sugars. For this reason, we must also use a 'loss-of-function' approach in which we knock down expression of each Gr gene in vivo in the bee using small-interfering RNA (siRNA). Using this method, we will knock down expression of each Gr gene and assay the bee's taste neurons using electrophysiology and behaviour. We will test a suite of nutrients and toxic compounds that includes common pesticides encountered by bees in flowering crops. In spite of the fact that bees have only 10 Gr genes, they are still able to detect some toxins and to regulate their intake of nutrients like sugars and amino acids that are detected by Grs. The experiments proposed here will reveal how the bee's few Gr genes translates into the spectrum of what it can taste and will lead to future work that identifies how populations of Gr neurons encode information about the chemical nature and complexity of food.