Understanding multi-level impact of male-derived sex peptide on female reproductive behaviours

Abstract
Reproductive behaviors and their regulation are most fundamental to all animals. Since they are largely hard-wired into the brain we can learn how behavior 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. Female reproductive behaviors of the fruit fly Drosophila melanogaster profoundly change after mating leading to refusal to remate and induction of egg laying. Male-derived sex-peptide (SP) is the key molecule inducing these post-mating behaviors, which can last up to one week in the presence of sperm. This 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.
A key tool to find SP-target neurons is membrane-tethered SP (mSP), that will induce a response when the same neuron that expresses mSP also expresses an SP receptor. Our recent studies showed that there are several distinct neuronal populations that can via exposure to mSP induce refusal to remate and egg laying. Importantly, we could also show that these two post-mating responses can be separated by mSP expression either in the trunk or in the head leading to induction of egg laying or reduction of receptivity, respectively. These exciting findings draw a much more complex picture how SP interferes through multiple sites with female reproductive behaviors to coordinate the post-mating response, yet how this works at a brain systems level remains to be elaborated.
We currently have very limited understanding of where SP-target neurons are located in the fly brain. To identify the neuronal circuitry underlying the sex-peptide response, we have used expression of mSP in subsets of neurons of genes involved in the SP-response and the sex-determination pathway. In particular, we subdivided the regulatory region of the broadly expressed SP receptor (SPR) gene into small regulatory fragments expressing only in subsets of neurons. Using this paradigm-shifting approach we have identified small populations of neurons, one that reduces receptivity and induces egg laying, and another one that only induces egg laying upon expression of mSP. Through these experiments we now have worked out, in principle, how to identify all the different populations of SP target neurons. Hence, we now need to identify small regulatory fragments in sex determination genes and SP response pathway genes to identify additional SP target neurons. These experiments will be then be the resource to identify the relevant neurons in the recently become available high-resolution Drosophila brain from the fly connectome project to build the circuitry directing the female post-mating response.
With these experiments we will test the hypothesis that SP-target neurons are present at multiple sites in the brain to direct female reproductive behaviors for a coordinated post-mating response. Compared to a 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 in billions to fruit production.

Technical Summary
Reproductive behaviors are excellent models to learn how specific behaviors are implemented into the brain and shaped by decision-making processes, because they are largely hard-wired. In most insects, female behavior and physiology are altered by 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 now for identifying the neuronal circuits involved in mating choice and regulation of egg-laying.
We have recently shown by expression of membrane-tethered SP (mSP) that several distinct populations of stimulatory or inhibitory neurons can induce refusal to remate and egg laying. Intriguingly, these two responses can be separated, but we currently only have a limited understanding where these neurons are located in the central brain and abdominal ganglion.
Although neuronal gene expression patterns have been identified, which can induce PMRs, these expression patterns are generally complex. To solve the genetically challenging task of subdividing complex gene expression patterns, we used enhancer fragments of a gene in the SP response pathway for intersectional splitGAL4 expression to restrict mSP to very few neurons which induce PMRs and thus are SP targets.
To understand how SP interferes with female reproductive behaviors at multiple sites for coordinated PMRs, we will expand this approach of intersectional gene expression to identify additional SP target neurons. We will identify the architecture of SP-target neurons and map their identity to the Drosophila model brain developed by the fly connectome to reveal how their connectivity and activity contribute to PMRs.
These studies will identify neuronal circuits coding for reproductive behaviors in a model insect. Our research will provide novel insights into the control of egg laying and instruct its exploitation for crop protection and control of insect born diseases.