Award details

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

Reference	BB/M00709X/1
Principal Investigator / Supervisor	Professor Geraldine Wright
Co-Investigators / Co-Supervisors	Professor Dianne Ford
Institution	Newcastle University
Department	Institute of Neuroscience
Funding type	Research
Value (£)	502,854
Status	Completed
Type	Research Grant
Start date	02/03/2015
End date	31/08/2018
Duration	42 months

Abstract

Detection of sugars and toxic 'bitter' compounds in animals is accomplished by gustatory receptors (Grs) housed in sensory neurons in taste buds (vertebrates) or gustatory sensilla (insects). The use of model organisms like Drosophila has made it possible to characterize the functional properties of Grs by manipulating gene expression in gustatory neurons and surveying the chemical ligands that active them. Drosophila has 60 Gr genes an unknown number of receptors, but identifying the ligands for all of its Grs has not yet been accomplished. In contrast, the honeybee (Apis mellifera) has only 10 Gr genes. The research proposed here will use a combination of approaches to identify the functional expression of the bee's Grs with the aim of developing the bee as a model for gustatory coding. First, we will express the bee's Gr genes in a Drosophila heterologous expression system in which all Gr sugar-sensing genes have been knocked out. After making the Drosophila lines, we will use calcium imaging to assay the responses of the flies' 'empty' tarsal neurons during stimulation several potential ligands. We will also employ a loss of function approach to knock down each Gr gene in vivo in the bee using small-interfering RNA (siRNA) and then assay the responses of the bee's gustatory neurons using electrophysiology and behaviour with a series of nutrients and toxic compounds including common pesticides found in the nectar and pollen of flowering crops. By doing this we will: 1) identify which Gr genes encode receptors for sugars, toxins or other nutrients; 2) identify the breadth of tuning of each of the bee's Grs towards classes of compounds like sugars or toxins; 3) have insight into the functional organization of the bee's Grs; 4) identify whether the bee's toxin-detecting Grs also detect common pesticides. These experiments will form the basis of future studies using the honeybee as a model to understand the nature of the gustatory code in animals.

Summary

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 Drosophilawith 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.

Impact Summary

The proposed research has the potential to impact industry, government policy making, land managers, and the public. Taste is an important mechanism that guides food selection and human nutrition. Billions of pounds are spent annually combating conditions resulting from obesity and diet-related diseases. Being able to interrogate the taste system of an insect model like the honeybee will also allow us to understand the basic principles of gustation and how taste information influences the brain's reward pathways. This could lead to discoveries that identify what kinds of taste stimuli cause overeating and how taste is modulated by nutritional feedback. In addition to their expression in taste neurons, gustatory receptors are expressed throughout the body, including the brain and the gut. They are likely to play key roles in regulating nutrition, but we know relatively little about how they function in these locations. Our experiments will contribute directly to understanding how these receptors function and will aid those researching nutrient sensation and food regulation. Food production and pharmaceutical industries may also benefit from our understanding of the way that non-metabolisable substances like sugars can activate sugar receptors so that sugar substitutes can be developed that taste sweet but do not have caloric value. Bees provide important pollination services in native and managed habitats. At the moment, their populations are in critical declines due to many factors including poor nutrition and exposure to pesticides. An important impact of our research will provide information about the way that bees detect nutrients in food and whether they can avoid toxins in food like pesticides. This knowledge will aid research on pesticide development, if pesticides used on flowering crops could be produced that bees could detect and avoid. For example, in March 2013, the EU Commission proposed a 2-year moratorium on the use of neonicotinoid pesticides becauseof evidence for risks posed to bees and other pollinators when these pesticides are used on flowering crops. Our research will aid government bodies, policy makers, and land managers because we will know which pesticides bees can detect and therefore identify the relative risk of specific pesticides to bees. In addition, our understanding of the bee's taste system will improve what is known about how it detects and responds to nutrients. This is important because it will improve what is known about bee nutrition. This knowledge will aid beekeepers and scientists developing appropriate food supplements for bees. The public also has a keen interest in bees, and we expect that the information generated by our research will intrigue many and fuel public understanding of bees and their biology.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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