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Development of optogenetically controlled gene expression tools for the characterization of neuronal circuits involved in insect reproduction

Reference	BB/N021827/1
Principal Investigator / Supervisor	Dr Matthias Soller
Co-Investigators / Co-Supervisors
Institution	University of Birmingham
Department	Sch of Biosciences
Funding type	Research
Value (£)	150,676
Status	Completed
Type	Research Grant
Start date	01/01/2017
End date	30/06/2019
Duration	30 months

Abstract

Most genes are expressed in complex patterns, which are often repetitive along the body axis. Although many regulatory sequences have been characterized, generally these expression domains can't be further dissected through the control regions to learn about gene function in localized cells. To implement spatial control into existing gene expression patterns we will adapt light-inducible transcription factors derived from bacteria and plants already used in mammalian cell culture to Drosophila to characterize the neuronal circuits involved in reproduction. Reproductive behaviors are excellent models to learn how specific behaviors are implemented into the brain, because they are largely hard-wired. In most insects, female behavior and physiology are fundamentally altered by male substances transferred during mating. In Drosophila, the key-molecule is sex-peptide (SP), which induces refusal to remate and egg-laying. This very robust post-mating response (PMR) to SP allows for identifying the neuronal circuits and the genes involved in mating choice and regulation of egg-laying. Our recent research identified distinct neuronal populations, which can induce refusal to remate and egg laying by expression of membrane-tethered SP. Intriguingly, these two responses can also be separated, but we currently do not know where in the fly these neurons are located. Candidate neurons for SP induced PMRs include sensory neurons in the genital tract and in the legs, but also neurons in the abdominal ganglion and the central brain. To solve the genetically challenging task of subdividing gene expression patterns, it is essential to develop and optimize light controlled gene expression tools to precisely localize neurons able to induce PMRs. These novel optogenetic tools will be widely applicable to solve many biological questions, including novel insights into the control of egg laying, which will instruct its exploitation for crop protection and control of insect born disease.

Summary

Reproductive behaviors and their regulation are most fundamental to all animals, but have been exploited little for population control in insects. Since they are hard-wired into the brain we can learn how this behavioral program is encoded in the brain and shaped by perception and decision-making processes. Understanding how behavior is encoded in the brain is one of the big challenges in biology and requires a behaviorally and genetically tractable model organism, but also tools to manipulate localized neurons. One of the key tools to achieve this aim are light-manipulateable molecules, such ion channels, where light can be used to control neuronal activity in space and time. Due to the small size of insects, however, this technology has its limitation. Here, we want to adapt light-inducible transcription factors derived from bacteria and plants already used in mammalian cell culture to Drosophila to characterize the neuronal circuits involved in reproduction. For this analysis we will capitalize on gene expression regulatory sequences known to characterize neuronal populations involved in reproduction, but these gene expression patterns are complex. To be able to assign functions to localized neurons therefore requires spatial dissection of these gene expression patterns, which can be achieved by light-controlled transcription factors. To develop such light-controlable tools to manipulate gene expression, we will capitalize on the robust post-mating responses (PMRs) of the fruit fly Drosophila melanogaster. Here, male-derived sex-peptide (SP) transferred during mating is the key molecule leading to refusal to remate and induction of egg laying. The very robust behavioral response of Drosophila females to sex-peptide provides the essential prerequisites to map SP responsive neurons and eventually learn how complex behaviors such as mating choice and control of egg laying are encoded in the brain. Our recent studies showed that there are several distinct neuronalpopulations that can via exposure to SP induce refusal to remate and egg laying. We currently do not know where in the fly these neurons are located, however, we could show that these two post-mating responses can be separated. Candidate neurons for SP induced post-mating responses include sensory neurons in the genital tract and in the legs, but also neurons in the abdominal ganglion and the central brain. To identify the neuronal circuitry underlying the sex-peptide response, we will use light induced gene expression directed to neurons in specific parts of the female fly body to express membrane-tethered SP. Such optogentic manipulation of gene expression has the advantage to be fully controllable in space and time. With these experiments we will test the hypothesis that the response to SP is comprised of a modular assembly of individual elements, e.g. refusal to remate or induction of egg laying. Compared to the previous model arguing for central induction of all PMRs, a modular assembly of individual PMRs holds evolutionary flexibility during speciation and adaptation to diverse habitats, but can maintain basic regulatory principles such as the control of egg laying. We therefore anticipate that the knowledge obtained from our studies will be applicable to a wide range of pest insects pinpointing towards novel strategies for pest management to protect crop and control insect born diseases by interfering with egg laying. In particular, our findings are directly transferable to the close relative Drosophila suzukii, one of the few species able to lay eggs into fruits, which is currently invading Europe including the UK and causing damage worth billions of pounds to fruit production.

Impact Summary

Basic research in the genetic model organism Drosophila has contributed fundamental discoveries in neurosciences and reproductive biology. Likewise, the optogenetic gene expression system we will develop is a further addition to the toolbox to allow for further fundamental discoveries. Our laboratory is pioneering in linking these discoveries to applications in pest management. Pest insects cause damage worth billions of pounds in yearly food production and spread deadly diseases. In addition, globalization has brought up numerous species invading novel habitats on different continents further aggravating problems of uncontrolled spreading. Although various chemical and biological methods are used for pest management, problems of resistance and/or introducing profound changes in eco-systems e.g. by eradication of native species requires development of new and/or refined methods for sustainable control. Our research in a Drosophila model builds on the unique feature of most insects to mate only once and to rely on male substances transferred during mating to control use of stored sperm, regulation of egg laying and host seeking behavior, and oviposition, to develop novel routes for pest management. Very little is known about the molecular and cellular mechanism, which govern these essential processes required to guarantee reproductive success and survival of the species. The fundamental nature of these reproductive aspects suggests that key regulatory principles are likely evolutionary conserved over whole taxonomic categories and are accessible to chemical interference. To date, however, such routes for pest management, e.g. by interfering with egg laying, have only been explored little due to the very limited genetic tools available in pest insects. Given the recent advances in transgenic insect technology, it is now feasible and timely, to unravel fundamental molecular and cellular principles of reproduction in the genetic model organism Drosophila melanogaster and subsequently validate the findings in pest insects. Accordingly, the following proposal is making use of the sophisticated genetic tools available in Drosophila melanogaster to pioneer the first step in this direction. MS has made contact with FERA (Food and Environment Research Agency) to implement this vision to current approaches in pest management and presented recent work at Entomological and applied agricultural meetings. Our studies will also provide a better understanding of the reproductive biology of Drosophila and related fly species to help improve the success of SIT (Sterile Insect Technique) and RIDL (Release of Insects carrying a Dominant Lethal) approaches for population control. This is of particular relevance to the invasion of Europe, including the UK, and the US with D. suzukii originating from East Asia. This is one of the few Drosophila species, which posses a serrated ovipositor able to penetrate fruit for egg laying and causes tremendous damage to the soft fruit industry (berries, grapes, etc), up to complete loss in parts of Northern Italy in 2014 (Curr Biol, 2013, 23; R8). Accordingly, our data will be directly transferable to develop novel population management tools for D. suzukii in its native range. Furthermore, MS has the most experience and knowledge in the SP field and our published work had major impacts in the field of insect reproduction. Through disseminating our knowledge in high impact journals and at international meetings we will further foster discussions for novel approaches to insect population control. Through engagement with the general public we will enhance understanding that use of transgenic insects is an essential aspect of innovative management strategies for the control of pest and disease vector insect populations.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	Neuroscience and Behaviour, Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	Tools and Resources Development Fund (TRDF) [2006-2015]
Funding Scheme	X – not Funded via a specific Funding Scheme

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