Neuronal control of suppression, initiation and completion of egg deposition in Drosophila melanogaster

Dissertation presented to obtain the Ph.D degree in Neuroscience Instituto de Tecnologia Química e Biológica António Xavier | Universidade Nova de Lisboa Oeiras, December, 2021 Neuronal control of suppression, initiation and completion of egg deposition in Drosophila melanogaster Cristina Oliveira Ferreira Cristina Oliveira Ferreira Dissertation presented to obtain the Ph.D degree in Neuroscience Instituto de Tecnologia Química e Biológica António Xavier | Universidade Nova de Lisboa Oeiras, December, 2021 Neuronal control of suppression, initiation and completion of egg deposition in Drosophila melanogaster Research work coordinated by: Neuronal control of suppression, initiation and completion of egg deposition in Drosophila melanogaster Cristina Oliveira Ferreira A Dissertation Presented to the Faculty of Universidade Nova de Lisboa in Candidacy for the Degree of Doctor of Philosophy Supervised by: Maria Luisa Vasconcelos International Neuroscience Doctoral Programme Champalimaud Research Lisbon, Portugal 2021 To the future iii Acknowledgments This 7-year journey was definitely not a smooth one with a lot of difficult moments in the course and, if I reach the end of this path, that was only made possible because I am fortunate to be surrounded by people that are good listeners, patient and caring. Thank you to the Innate Behaviour lab (present and alumni members): Luisa Vasconcelos, Cecilia Mezzera, Miguel Gaspar, Eliane Carvalho, Márcia Aranha and Sophie Dias. It has been a pleasure to be part of this group where the willingness to help was always the prime foundation. For all the expertise and valuable suggestions that helped me to grow as a better scientist and to build a better work. In special, a warm thank you to my supervisor, Luisa Vasconcelos, for being an amazing PI: wise, patient, who trod the whole way next to me always looking for a better trail. Also, a big thank you to Miguel Gaspar for his willingness to contribute to my project, even though he was simultaneously working hard to finish his PhD. To my thesis committee, Eugenia Chiappe and Michael Orger, for the wise suggestions that shape this work for better. To the fly room people −a big thank you for being a buffer on the bad days. Working on such a nice and familiar environment made everything worth it. My life would not be the same without the precious −Gabriela Fioreze and Inês Ferreira −who I met during this journey and became very special friends of mine. Friends for every occasion. To Susana Lima −thank you for your care and, not the least, companionship on the dance floor. To my sister for being the best. The creative mind in the family −thanks for making the exercise of thinking about different alternative careers for me. I´ve lost the count and it is reassuring to know that I´ll never be unemployed with all the ideas popping out your mind. iv There are three people which, without their unconditional love and support, I would have never finished this PhD. This thesis is specially dedicated to: my parents, Emilia and Carlos, and to my companion, Ricardo Neto. I would like to thank my parents for being an unreplaceable support in my life. Without their persistence and love, I would not have passed from the first year of PhD. This is a fact. Love you. To Ricardo Neto −I will never be able to put in worlds how much I thank you. Thanks for your wise advices, endless science-related conversations and listening my presentations over and over. But also, thanks for sharing with me the good moments at work and in life. Your smile and caring make happiness. v Título Controlo neuronal da supressão, iniciação e terminação da deposição do ovo em Drosophila melanogaster Resumo O comportamento de oviposição é um aspeto central na biologia reprodutiva dos insetos tendo um impacto profundo na sobrevivência e fitness das espécies. Em semelhança à maioria das espécies de insetos, as fêmeas Drosophila evoluíram para investir a sua energia na produção de muita progenia em detrimento de cuidado maternal. A relação mãe-progenia termina com a seleção de um local apropriado para a deposição dos ovos que deverá alimentar e proteger a progenia de eventos ambientais extremos e predadores. Enquanto grande progresso tem sido feito na compreensão das bases neuronais da seleção de substrato, existe pouco conhecimento sobre a organização e função de circuitos motores que controlem a deposição do ovo. Esta tese, através da combinação de análise comportamental com a caracterização de circuitos neuronais, fornece conhecimento sobre a arquitetura de circuitos motores inferiores que executam e regulam a deposição do ovo. No segundo capítulo, descrevemos as fases e os elementos comportamentais associados com oviposição em fêmeas D. melanogaster originárias de uma estirpe selvagem. Caracterizamos três fases distintas deste comportamento complexo: deposição do ovo, contorções abdominais e exploração. Estabelecemos que a fase de deposição do ovo −quando o ovo é posto −é sempre seguida pela fase de contorções abdominais, enquanto que a fase de exploração −onde a mosca procura por um local ideal de oviposição −é opcional. Cada fase tem um programa comportamental motor específico que pode progredir numa sequência de comportamentos ou não. Todos estes elementos comportamentais são executados para promover a degustação do substrato, a deposição do ovo e a ovulação. Esta descrição detalhada do comportamento de oviposição em moscas selvagens oferece uma estrutura para questionar os substratos neuronais adjacentes. No terceiro capítulo, fornecemos nova evidência sobre um circuito motor no gânglio abdominal −os neurónios OvAbg −que são suficientes e necessários para o comportamento de oviposição. Usando uma abordagem interseccional baseada no perfil de neurotransmissores para obter subgrupos funcionais, devi scobrimos que os neurónios OvAbg têm três populações neuronais com contribuições diferentes para a oviposição. Experiências de ativação optogenética mostram que os subgrupos glutamatérgicos (OvAbg/VGlut) e colinérgicos (OvAbg/Cha) estão envolvidos na iniciação e terminação da fase da deposição do ovo. Além disso, mostramos que o silenciamento genético de um subgrupo distinto de neurónios GABAérgicos (OvAbg/Gad1) não tem efeito no número de ovos depositados, no entanto, a sua ativação optogenética inibe oviposição. Estes resultados fortemente sugerem que, durante oviposição, os neurónios OvAbg/Gad1 estão silenciados e, quando ativos, podem bloquear a execução de oviposição. Este trabalho fornece informações importantes sobre a identidade e arquitetura de substratos motores inferiores que controlam um comportamento complexo através da descoberta do papel importante do circuito OvAbg no controlo de oviposição. Este trabalho serve de base para a descoberta de como diferentes circuitos estão conectados para coordenar comportamento. vii Contents Acknowledgments ............................ iv Título e Resumo ............................. vi Abstract .................................. viii Author Contributions and Financial Support .......... x List of Abbreviations .......................... xi 1 General Introduction 1 1.1 Egg-Laying: Innate and Reproductive Behaviour . . . . . . . . . 2 1.2 Whentolayanegg.......................... 3 1.2.1 Female Post-Mating Response . . . . . . . . . . . . . . . . 3 1.2.2 Activating the Post-Mating Response . . . . . . . . . . . 4 1.2.3 Circadian Modulation . . . . . . . . . . . . . . . . . . . . 4 1.2.4 Egg-laying Site Value . . . . . . . . . . . . . . . . . . . . 5 1.3 Wheretolayanegg ......................... 6 1.3.1 How to Sense the Environment . . . . . . . . . . . . . . . 6 1.4 Howtolayanegg .......................... 10 1.4.1 Female Reproductive Anatomy . . . . . . . . . . . . . . . 10 1.4.2 Behavioural Motor Programmes . . . . . . . . . . . . . . 11 1.5 Egg-laying Circuits . . . . . . . . . . . . . . . . . . . . . . . . . . 12 1.5.1 When and Where: From mating to the decision of laying anegg............................. 12 1.5.2 How: from the decision to the execution of laying an egg . 14 1.6 Analogous Lower Motor Circuits . . . . . . . . . . . . . . . . . . 18 1.6.1 Drosophila Male Sexual and Locust Egg-laying Behaviours 18 2 Egg-laying behaviour in wild type flies 21 2.1 Characterization of egg-laying behaviour . . . . . . . . . . . . . . 22 xiv 3 Motor control of egg-laying behaviour 26 3.1 Activity of OvAbg neurons promotes egg deposition . . . . . . . 27 3.2 Silencing OvAbg neurons disrupts all motor elements associated with egg-laying behaviour . . . . . . . . . . . . . . . . . . . . . . 31 3.3 Activity of GABAergic OvAbg neurons blocks egg-laying . . . . . 35 3.4 Cholinergic OvAbg neurons are necessary and sufficient for egg deposition............................... 37 3.5 Glutamatergic OvAbg neurons contribute to egg deposition initiation................................. 40 4 Supplementary Information 45 4.1 Supplementary Figure 4.1 . . . . . . . . . . . . . . . . . . . . . . 46 4.2 Supplementary Figure 4.2 . . . . . . . . . . . . . . . . . . . . . . 48 4.3 Supplementary Videos . . . . . . . . . . . . . . . . . . . . . . . . 49 4.3.1 Supplementary Video 1 . . . . . . . . . . . . . . . . . . . 49 4.3.2 Supplementary Video 2 . . . . . . . . . . . . . . . . . . . 49 4.3.3 Supplementary Video 3 . . . . . . . . . . . . . . . . . . . 49 4.3.4 Supplementary Video 4 . . . . . . . . . . . . . . . . . . . 49 4.3.5 Supplementary Video 5 . . . . . . . . . . . . . . . . . . . 49 4.3.6 Supplementary Video 6 . . . . . . . . . . . . . . . . . . . 49 4.3.7 Supplementary Video 7 . . . . . . . . . . . . . . . . . . . 49 4.4 Supplementary Table 1. Fly stocks . . . . . . . . . . . . . . . . . 50 4.5 Supplementary Table 2. Full genotypes used in experiments . . . 51 5 Experimental Procedures 52 5.1 Fly stocks and husbandry . . . . . . . . . . . . . . . . . . . . . . 53 5.2 Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . 53 5.3 Preparation of flies to be assayed . . . . . . . . . . . . . . . . . . 54 5.4 Behavioural assays . . . . . . . . . . . . . . . . . . . . . . . . . . 54 5.4.1 24h egg-laying assay . . . . . . . . . . . . . . . . . . . . . 54 5.4.2 Egg-laying arena and substrate . . . . . . . . . . . . . . . 54 5.4.3 Detailed behaviour . . . . . . . . . . . . . . . . . . . . . . 55 5.4.4 Optogenetics ......................... 55 5.4.5 Imagecapture ........................ 56 5.5 Quantification and statistical analysis . . . . . . . . . . . . . . . 56 5.5.1 Data processing . . . . . . . . . . . . . . . . . . . . . . . . 56 xv 5.5.2 Quantification of behaviours . . . . . . . . . . . . . . . . . 56 5.5.3 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . 58 6 General Discussion and Conclusion 60 6.1 Discussion............................... 61 6.2 Egg-laying behaviour in wild-type flies . . . . . . . . . . . . . . . 62 6.3 OvAbg neurons: lower motor control of egg-laying behaviour . . 63 6.4 Execution of Egg Deposition: Glutamatergic and Cholinergic OvAbgcircuits.............................. 64 6.5 Regulation of Egg-laying: GABAergic OvAbg circuit . . . . . . . 66 6.6 Conclusion .............................. 67 References 68 xvi Chapter 1 General Introduction 1 How the Central Nervous System (CNS) generates a sequence of motor actions to produce behaviour, once a decision is signalled by the brain, is a long-standing question in behavioural neuroscience. It is a complex task as this motor output needs to be: (1) tightly coordinated in time so that the appropriate sequence of behavioural elements is accomplished; (2) synchronised with the animal internal state (e.g. mating, feeding status); and (3) flexible enough to cope with unexpected changes in the environment. The fruit fly has a relatively simple nervous system (∼200,000 neurons) that manages to perform complex behaviours. As such, the fly has been a prime model in behavioural neurobiology research for many years. The current available genetic toolkit, combined with minimal culturing requirements and a short life cycle allow flexible and diverse approaches to scientific questions. The study presented here explores the fruit fly’s potential, along with the study of egg-laying behaviour, in order to strengthen our knowledge about the general principles governing animal motor behaviour. 1.1 Egg-Laying: Innate and Reproductive Behaviour Animals are born with a set of pre-defined behavioural programmes −innate behaviours −that are reliably executed in response to specific stimuli (Tinbergen, 1991). These behaviours are supported by genetically hardwired neural circuits and do not rely on learning (Kim et al., 2017). Reproductive behaviours are included in the repertoire of innate behaviours. Fly sexual behaviours have been highly significant for the study of relevant questions such as: differences in the genetic architecture of gender-specific behaviours, molecular and neuronal basis of social interactions, multisensory integration, neuromodulation and the motor execution of behavioural programmes (Aranha and Vasconcelos, 2018; Kim et al., 2017; Laturney and Billeter, 2014). Egg-Laying behaviour is a sexually dimorphic behaviour essential for the survival and fitness of oviparous species, displayed by females in the postcopulatory phase. Its correct implementation requires the coordination of physiological and behavioural changes that occur to meet the metabolic and nutritional demands of egg production and maturation. Given that Drosophila females do not display maternal behaviours, it is crucial that the behavioural changes include decisions regarding the sites where to lay the eggs to ensure 2 the proper larvae nutrition, and protection from environmental threatening factors. Thus, egg-laying is a complex behaviour and its regulation implies the coordination between the peripheric system and the CNS in order to produce the correct output in space and time. Specifically, as postulated by Cury et al. (2019), three variables should be considered: 1. When (mating, circadian regulation and the value of the egg-laying site); 2. Where (egg-laying site selection); 3. How to lay an egg (behavioural motor programmes and underlying neuronal substrates). In the next sections of this introductory chapter, I will review the current knowledge on the neuroethology of egg-laying, following the three aforementioned points. 1.2 When to lay an egg 1.2.1 Female Post-Mating Response After copulation, female flies initiate a series of changes in different components of their behaviour known as the female Post-Mating Response (PMR). The genetic, physiological and neuronal basis of this response in Drosophila melanogaster have been thoroughly studied over the past twenty years (Carvalho et al., 2006; Chen et al., 1988; Heifetz et al., 2014, 2000; Hussain et al., 2016b; Liu and Kubli, 2003; Mack et al., 2006; McGraw et al., 2008; Peng et al., 2005; Rezával et al., 2014; Soller et al., 1999). The PMR is initiated soon after mating (Liu and Kubli, 2003; McGraw et al., 2008; Shao et al., 2019) and it can persist for several days (Garbe et al., 2016; Peng et al., 2005; Ravi Ram and Wolfner, 2005). The three behavioural hallmarks of the PMR are: a decrease in female sexual receptivity to new courting males (Chen et al., 1988; Connolly and Cook, 1973), an increase in the rate of egg laying (Chen et al., 1988; Heifetz et al., 2000) and a change in the feeding habits that correlates with the nutritional demand of egg production, which is characterized by increased preference for yeast, salt and polyamines-enriched food (Barnes et al., 2008; Carvalho et al., 2006; Hussain et al., 2016b; Ribeiro and Dickson, 2010; Walker et al., 2015). 3 1.2.2 Activating the Post-Mating Response The activation of the PMR was shown to be mostly dependent on the action of a male-specific peptide −Sex Peptide (SP) (Garbe et al., 2016; Peng et al., 2005; Ribeiro and Dickson, 2010; Walker et al., 2015). During copulation, SP is transferred together with the male sperm into the female reproductive tract (Chen et al., 1988; Liu and Kubli, 2003; Peng et al., 2005). The neuronal basis of SP-dependent modulation in the female fly is well dissected (Feng et al., 2014; Häsemeyer et al., 2009; Jang et al., 2017; Rezával et al., 2014, 2012; Yang et al., 2009; Yapici et al., 2008). SP binds to Sex Peptide Receptor (SPR), which is expressed in a group of internal SP Sensory Neurons (SPSNs) located in the uterus (Häsemeyer et al., 2009; Yang et al., 2009). SPR activation reduces SPSNs’ excitability, which in turn dampens the activation of their postsynaptic partners located in the fly Ventral Nerve Cord (VNC) −the SP Abdominal Ganglion (SAG) Neurons. SAG neurons project to the brain where they relay the information to initiate the PMR (Feng et al., 2014). The SAG downstream targets in the central brain were recently identified, thereby unveiling more details about the PMR implementation by the CNS (Wang et al., 2020, 2021). Although SP plays a key role in inducing the PMR, evidence is growing on SP-independent PMR mechanisms. In particular, chronic exposure to the sex pheromone 11-cis-Vaccenyl Acetate (cVA) (Lebreton et al., 2015) and the physical act of copulation (Shao et al., 2019) contribute to the female PMR. Most of the post-mating behaviours are the output of neuronal circuits developmentally controlled by two transcription factors, fruitless (fru) and doublesex (dsx) (Demir and Dickson, 2005; Feng et al., 2014; Häsemeyer et al., 2009; Rezával et al., 2014, 2012; Rideout et al., 2010; Walker et al., 2015; Wang et al., 2021; Yang et al., 2009). 1.2.3 Circadian Modulation The timing of laying an egg is also regulated by the circadian rhythm. Monitoring the egg-laying activity of D. melanogaster in a 12h Light:Dark (L:D) cycle shows a 24h periodicity that is characterized by a peak in egg deposition at the onset of the dark phase (Allemand, 1976; Asburner et al., 2005; Sheeba et al., 2001). The egg deposition rhythm is maintained when flies are subjected to different photoperiod and temperature conditions, as well as after changes in the protein diet quality (Howlader et al., 2006; Sheeba et al., 2001). The persis4 tence of the egg-laying rhythm under atypical conditions suggests the existence of a circadian regulator of egg-laying. After copulation, the PMR needs to be integrated with the circadian clock in order to precisely control the timing of egg-laying. How the CNS orchestrates this integration is still unknown. 1.2.4 Egg-laying Site Value The ‘when’ to lay an egg is not only dependent on the post-copulatory responses and the circadian rhythm. The control over the site where the eggs are laid is of major importance for species perpetuation. D. melanogaster does not display maternal behaviours and the larvae show low mobility, thereby it is crucial that females find a good location to lay their eggs. That location should provide protection from predators and pathogenic microorganisms, environmental stressors, and should also be able to sustain larvae feeding. Therefore, in spite of the drive to lay eggs after mating, if the substrate conditions are not ideal for egg-laying, pregnant flies will pause egg deposition and search for a better egg-laying site. Indeed, there is evidence that females withhold eggs in the reproductive system until circumstances favour egg deposition (Allemand, 1976; Asburner et al., 2005; Drummond-Barbosa and Spradling, 2001; Kacsoh et al., 2015; Kurz et al., 2017; Lefèvre et al., 2012; Yang et al., 2008, 2015). Different environmental cues can trigger a pause in egg-laying at distinct stages of the process, which can manifest as: 1) a decrease in the rate of egg production; 2) a delay in the ovulation of mature eggs (passing an egg from the ovaries to the uterus); and 3) apoptosis of egg precursor cells, which can be induced in extreme environmental conditions, thereby halting egg production. Depriving flies of an appropriate substrate for egg-laying (Asburner et al., 2005; Drummond-Barbosa and Spradling, 2001; Yang et al., 2008, 2015), as well as extreme temperatures (Asburner et al., 2005) are sufficient to reduce egg deposition in mated females. Drummond-Barbosa et al. (2001) observed that mated females maintained in a protein-poor diet reduce the egg production rate. Exposure to predators or to pathogenic bacteria have also been shown to decrease egg deposition and induce egg retention in the ovaries (Kacsoh et al., 2015; Kurz et al., 2017; Lefèvre et al., 2012). A study conducted by Kacsoh et al. (2015) found that apoptosis of the egg precursor cells is observed when mated Drosophila are exposed to female parasitoid wasps. Interestingly though, a different strategy is observed when flies are exposed to pathogenic bacteria (ex. Escherichia coli) characterized by 5 the accumulation of mature eggs in the ovaries (delay in ovulation) (Kurz et al., 2017). 1.3 Where to lay an egg 1.3.1 How to Sense the Environment As already mentioned, fruit flies are very selective about the site where they lay the eggs and, consequently, they may spend a variable amount of time searching for a proper egg-laying spot (Cury and Axel, 2021; Vijayan et al., 2021; Yang et al., 2008, 2015). It is widely accepted that D. melanogaster has a strong preference for laying eggs in rotting fruits, which provide a soft substrate. Flies use multiple sensory modalities to search for possible substrates: olfaction, gustation, vision and mechanosensation are all important to provide accurate information about the substrate qualities. The sensory cues that stimulate or inhibit egg-laying in D. melanogaster are well-known (reviewed in (Cury et al., 2019) and (Aranha and Vasconcelos, 2018) (Fig. 1.1). Figure 1.1: Summary of the sensory systems and some of the attractive (⊕) and aversive (⊖) egg-laying cues involved in egg-laying site selection. Female D. melanogaster figure adapted from the collection of Drosophila species images available in Nicolas Gompel´s laboratory website (http://gompel.org/images-2). For most of the sensory cues, the first-order sensory neurons are identified. However, much less is known about how these peripheric sensory circuits com6 municate with higher-brain centers to accomplish egg-laying site selection. In the next sections, I will review some of the sensory cues identified to positively (attractive) or negatively (aversive) modulate egg-laying. Olfactory cues Flies smell odours from a distance or in site by using the antennae and the maxillary palps. The inventory of olfactory cues evaluated by D. melanogaster during the choice of an egg-laying site is vast. Egg-laying is stimulated by volatile limonene and valencene terpenes present in Citrus fruits, both detected by Olfactory Sensory Neurons (OSNs) expressing the receptor Or19a located in the antennae. The attraction to Citrus fruits protects the larvae from parasitoid wasps, which are repelled by the smell of valencene (Dweck et al., 2013). Ethanol, one of the main metabolites of fermentation present in rotting fruits, is simultaneously an attractive odour for egg-laying (Azanchi et al., 2013) and a potential toxic compound for the larvae (Schumann et al., 2021). Consequently, ethanol preference is dose and contextdependent. Female flies prefer to deposit eggs in food substrates containing the most ecologically beneficial concentrations of ethanol (3-5%) (Azanchi et al., 2013). Yet, they lay eggs in high-ethanol concentration substrates (10-15%) when facing predators, as a way to protect the larvae from infection (Kacsoh et al., 2013; Milan et al., 2012). Evidence suggests that both the olfactory and gustatory systems can contribute for ethanol discrimination (Azanchi et al., 2013). Ethylphenols derived from fruit antioxidants promote egg-laying, and its detection in adult flies is done by maxillary palp neurons expressing the receptor Or71a (Dweck et al., 2015). Egg-laying site selection is also modulated socially through olfactory cues released by other females and by males. The pheromone 9-tricosene deposited by males in food sites was shown to work as an aggregation and an egg-laying attractive cue for females. It is detected by Or7a-expressing neurons in the antennae, the same olfactory sensory neurons that also detect the leaf volatile E2-hexenal, another egg-laying enticing compound (Lin et al., 2015). Mated females also provide information about high-quality egg-laying sites to other females by marking the food spots with a combination of female and male chemicals, including species-specific cuticular hydrocarbons (CHC) and the pheromone cVA (present in the male sperm) (Duménil et al., 2016). 7 input. Silencing aDNs abolishes the preference of mated females to lay eggs in substrates enriched in male-pheromones without affecting the number of eggs laid, suggesting a role for aDNs in egg-laying site selection rather than on the motor execution of this behaviour (Nojima et al., 2021). These findings established a circuit model of how the brain coordinates mating and egg-laying. In the next section, I will expand these circuits to their downstream partners relaying motor commands to the VNC. Figure 1.4: Diagram of neuronal circuits involved in egg-laying and their modulation by mating and site selection cues. Interactions between different neurons involved in the PMR (red), egg-laying site selection (yellow) and execution (light green). In the brain, mating status is processed by pC1-oviIN neurons and substrate cues are integrated by oviEN and aDN neurons. In mated females, pC1-oviIN pathway is silent and, thus, the activity of oviENs and aDNs promotes egg-laying when a suitable substrate is encountered. oviIN connections depicted by dashed lines represent the absence of inhibitory input onto oviEN, oviDN and aDN neurons in mated females. Colour scheme in accordance with Fig. 1.3. Adapted from (Nojima et al., 2021) and (Wang et al., 2020). 1.5.2 How: from the decision to the execution of laying an egg Descending Outputs The decision of laying an egg is executed due to the activity of Descending Neurons (DNs) that relay information from the brain to the VNC. The VNC, the functional analogous of the vertebrate´s spinal cord, is connected with the body thoracic appendages (legs and wings) and abdominal segments/organs to regulate physiology and execute motor programmes. It receives descending signals from the brain to initiate motor actions, but also sends ascending signals carrying motor and sensory feedback (Fig. 1.5b) (Court et al., 2020; Hsu and Bhandawat, 2016). The VNC is located posteriorly to the brain and comprises 14 by three pairs of thoracic units (T1, T2 and T3) fused with a region called the Abdominal ganglion (Abg) (Fig. 1.5a) (Court et al., 2020). Up to now, three DNs −pMN2,DNp42 and oviDNs −were found to be involved in egg-laying behaviour, even though, only DNp42 and oviDNs were characterized in a more integrative way within egg-laying circuits (Huoviala et al., 2018; Kimura et al., 2015; Vijayan et al., 2021; Wang et al., 2020). Huoviala et al. identified that the geosmin avoidance LHAV1a1 neurons output to the descending neuron DNp42 (Fig. 1.4). Silencing DNp42 abolishes geosmin avoidance during egg-laying, as observed upon LHAV1a1 inactivation. The female-specific dsx+ pMN2 was the first DN described to elicit the motor elements linked with egg deposition (Fig. 1.4) (Kimura et al., 2015). More recently, a set of dimorphic fru+ oviposition descending neurons (oviDNs) were characterized as necessary and sufficient for egg-laying. Silencing oviDNs abolishes egg-laying in mated females, whereas their activation triggers the motor elements for egg deposition: abdomen bending to contact the substrate, ovipositor extrusion and egg expulsion. Imaging oviDNs activity during spontaneous egg-laying revealed that its signal rises during the search period and peaks during egg deposition. Interestingly, the rate of rise in oviDNs activity during exploration is modulated by substrate quality, which strongly affects how quickly oviDNs hit the threshold to initiate the motor programme for egg expulsion (Vijayan et al., 2021). The activity of oviDNs is not affected by mating, as abdomen bending and ovipositor extrusion are similarly triggered in virgin and mated females, which suggests a mating-dependent modulation in brain circuits upstream of OviDNs (Wang et al., 2020). Importantly, oviDNs are integrated in the egg-laying circuit composed by oviIN, oviEN and aDN neurons (Fig. 1.4) (Wang et al., 2020). Further studies are required to characterize the downstream partners of oviDN neurons in order to draw a more complete picture of a sensory-motor pathway dedicated to egg-laying. Motor Executors How is egg-laying execution, downstream of DNs, implemented? The motor circuits involved in the execution of the egg-laying behavioural programme (search, egg deposition and clean and rest) are poorly explored. The neural substrates that integrate commands from the brain to generate motor outputs are located in the VNC and are referred to as lower motor 15 Figure 1.5: Logic of motor control by the Drosophila CNS. (a) Structural and functional organization of the VNC. (b) In order to generate motor behaviour, the nervous system integrates a variety of stimuli; those are first detected by the peripheric sensory systems (e.g. probosics, antennae, legs, ovipositor) and associated sensory circuits, which relay the sensory information to the brain. In the brain, the information is integrated in higher brain circuits involved in motivation, learning, choice and internal state, which select a behavioural outcome. The behaviour comes to action via brain descending neurons that activate lower motor circuits in the VNC involved in the execution and coordination of motor behaviour. Mechanosensory, proprioceptive and chemical feedback from the periphery into the VNC, as well as ascending inputs into the brain, are an important source of sensory feedback to the system; such feedback allows to fine tune behaviour accordingly with the external conditions and, also, ensures a coordinated sequence of motor events. 16 circuits. These circuits comprise local neurons and motor neurons. While motor neurons generate motor output by directly innervating muscle fibers, local neurons act on downstream motor neurons to control behaviour (Fig. 1.5b) (Purves, 2001; Venkatasubramanian and Mann, 2019). The motor execution of egg-laying implies the coordination among behaviours that promote ovulation, fertilization, search and, ultimately, egg deposition into the substrate. There is supporting evidence that dsx-expressing neurons are key in the control of egg-laying behaviour. Silencing either the activity of all dsx+ neurons, or a restricted subset of ∼9 VNC dsx+/octopaminergic neurons, is sufficient to reduce, or even abolish egg-laying in mated females; in turn, their activation triggers an increase in the number of eggs laid by virgin females (Rezával et al., 2014; Rideout et al., 2010). Yet, how and which sub-populations of the dsx+ VNC network control the egg-laying motor programme is still to be uncovered. The mechanisms by which the VNC controls the oviduct muscles to promote ovulation are fairly understood. The female reproductive tract is a muscular system which distends when the egg passes through, being innervated by motor and sensory processes. There is mounting evidence that ovulation in Drosophila is modulated by glutamate and OA (Lee et al., 2003; Lim et al., 2014; Rodríguez-Valentín et al., 2006). While OA promotes oviduct relaxation by binding to OAMB and Octβ2R receptors in the oviduct wall, glutamate induces oviduct contractions (Lee et al., 2003; Lim et al., 2014; Rodríguez-Valentín et al., 2006). Impairing octopaminergic signalling results in sterile females with enlarged ovaries full of mature eggs that are not ovulated and, therefore, not laid (Lee et al., 2003; Li et al., 2015; Lim et al., 2014; Monastirioti, 2003; Monastirioti and Linn, 1996). Interestingly, the re-establishment of OA production in a population of Abg neurons innervating the ovaries and oviducts is sufficient to rescue the sterile phenotype (Monastirioti, 2003). Moreover, stimulating a cluster of Abg glutamatergic neurons innervating the oviduct triggers reproductive tract contractions (Gou et al., 2014) while its silencing causes eggs jammed in the oviducts (Castellanos et al., 2013; Gou et al., 2014; Yang et al., 2008). 17 1.6 Analogous Lower Motor Circuits 1.6.1 Drosophila Male Sexual and Locust Egg-laying Behaviours Insects are prime models to study the neuronal basis of motor behaviours. Works on D. melanogaster and locust species (Locusta migratoria migratorioides and Schistocerca americana) have been fundamental to our comprehension of the identity and function of lower motor circuits in a wide range of behaviours, from locomotion to reproduction (Ayali and Lange, 2010; Feng et al., 2020; Howard et al., 2019). Here, I will briefly review two studies, which provide general principles of how motor circuits are organized to coordinate reproduction in insects. I will first introduce a circuit in Drosophila males involved in the successful execution of genital coupling during courtship (Pavlou et al., 2016). Next, I will review work on circuits controlling different behaviours during egg deposition in locust females (Ayali and Lange, 2010; Lange, 2009a,b). The study undertaken by Pavlou et al. showed that the initiation and termination of genital coupling in males is coordinated by an Abg circuit composed by two dsx+ populations: a glutamatergic subset (dsx+/VGlut+) that innervates the male genitalia, and a local GABAergic subset (dsx+/Gad1+). The dsx+/VGlut+ neurons initiate copulation by allowing genital coupling with the female, whereas dsx+/Gad1+ neurons mediate genital uncoupling to terminate copulation, likely by gating the activity of the dsx+/VGlut+ population. This circuit is modulated by sensory feedback from dsx+ mechanosensory neurons located in the genitalia, and is poised to connect with local dopaminergic neurons to modulate copulation persistence (Fig. 1.6a). The work developed over the past years in locust has been instrumental to understand how VNC circuits coordinate a wide range of behaviours. Here, I will focus on the motor control of egg-laying behaviour. The knowledge presented here is based on three exhaustive reviews on the field (Ayali and Lange, 2010; Lange, 2009a,b). The locust reproductive system has identical anatomical structure to the one in Drosophila: two ovaries linked by lateral oviducts converging on a central oviduct connected with the genital chamber or uterus. There is one spermatheca to store sperm and a pair of accessory glands. During egg-laying, females dig a deep hole in the substrate with the ovipositor valves to burrow the eggs. While digging the substrate, females retain the ovulated 18 eggs in the lateral oviducts and, only once the digging process is completed, the eggs descend to the uterus for fertilization and expulsion. Therefore, the coordination between digging, egg retention and fertilization behaviours is crucial for successful egg-laying. How is this coordination accomplished to produce an orderly sequence of behaviours? Three motor circuits located in the abdominal ganglion were shown to control digging, egg retention and fertilization (Fig. 1.6b). The digging circuit innervates the ovipositor valves to induce contractions for substrate digging. An egg-retention circuit whose activity stimulates oviduct contractions ensures that eggs are not expelled during the digging process. The coordinated activity of digging and egg-retention neurons ensures that eggs are only expelled once the hole is completed. The digging circuit receives sensory feedback from mechanosensory neurons in the ovipositor valves to maintain or stop digging behaviour. The egg then descends to the uterus where fertilization must occur before expulsion. A fertilization circuit promotes contractions of the spermatheca for sperm release and fertilization. The fertilization module likely receives feedback from sensory neurons sensing the egg in the uterus to ensure fertilization at the appropriate timing (Ayali and Lange, 2010; Lange, 2009a,b). Taken together, these studies allow us to speculate that the same circuit logic may be applied in the control of egg-laying behaviour in D. melanogaster. In my thesis work, I drew inspiration from the work outlined in the previous sections, and used D. melanogaster egg-laying behaviour as a paradigm to further our understanding of the mechanisms underlying motor behaviour. 19 Figure 1.6: VNC motor circuits controlling reproductive behaviours in insects. (a) Model of the tripartite circuit controlling genital coupling in Drosophila male. Adapted from (Pavlou et al., 2016). (b) The three motor circuits located in the Abg that control egg-retention, digging and fertilization behaviours in locust. These circuits are located in the VII and VIIIth segments of the Abg. Egg-laying is accomplished by the elongation of the abdomen into the substrate, while digging a hole by using the ovipositor valves. Adapted from (Lange, 2009a). 20 Chapter 2 Egg-laying behaviour in wild type flies 21 2.1 Characterization of egg-laying behaviour In order to understand how the execution of the egg-laying motor programme is coordinated, we analysed wild-type egg-laying behaviour in detail. Fig. 2.1a shows a schematic representation of the behavioural setup. Single mated females were placed in an arena lined with 1% agarose on three walls. Videos were recorded for 45 minutes. We first analysed the temporal structure of egg expulsion (Fig. 2.1b). For the duration of the video, each female laid from 4 to 17 eggs, with a median of 11.5 (Fig. 2.1c). All eggs were laid on the agarose walls and 94% of eggs were buried in the agarose. The median inter-egg expulsion interval calculated for each female ranges from 2 to 3 minutes with the exception of one fly (fly#7) that showed a median of 5 minutes (Fig. 2.1d). These data indicate a low inter-individual variability when rearing conditions and environment are controlled. To obtain a detailed description of egg-laying behaviour, we analysed egg-laying motor elements which we associated with different egg-laying phases. Egg-laying behaviour involves: 1) an exploration phase where the fly presumably searches for appropriate egg-laying sites, 2) the egg deposition phase in which the ovipositor motor programme is activated to lay an egg, and 3) the rest phase. We chose to call the rest phase, ‘abdominal contortions’, because we and others (Bräcker et al., 2019; Cury and Axel, 2021) observed that this phase is accompanied by strong and prolonged abdominal contortions, as described below. Fig. 2.1e shows videoframes capturing each of the different egg-laying motor elements. Some behavioural denominations were based on other descriptions of egg-laying (Bräcker et al., 2019; Cury and Axel, 2021). Egg deposition motor elements all progress in a sequence culminating in egg expulsion and ending with grooming of the terminalia. We show more than one videoframe for egg pushing as it has two different postures. Abdominal contortions, as mentioned earlier, are the single motor element of its phase. We defined exploration motor elements as elements where females probe the substrate either with the ovipositor or the proboscis without progressing in continuous sequence to egg expulsion. To understand the temporal sequence of the different behavioural elements, we plotted the probability of each motor element around the moment of egg expulsion (Fig. 2.2a-d). During egg deposition phase, the motor elements leading to egg expulsion are performed every time an egg is deposited (Fig. 2.2a). After an egg is expelled, the female curls the abdomen while walking away, which can 22 Figure 2.1: Egg-laying behaviour in wild type flies. (a) Schematic of setup and arenas used to record egg-laying behaviour. Each arena is composed by two chambers allowing to record two flies simultaneously. The egg-laying substrate used in this study was 1% agarose. Infrared (IR) LEDs were used for illumination and IR cameras were used to record fly behaviour. (b) Raster plot showing the temporal profile of egg expulsion bouts. Each black dot marks the moment of egg expulsion. n = 10 flies. (c) Number of eggs laid by mated Canton S (CS) flies. Median = 11.5; total number of flies = 10; total number of eggs = 112. (d) Time intervals between egg expulsion bouts per fly. Each dot represents the interval between consecutive egg deposition bouts. Median values of the distribution for each fly are shown on the right side. n = 10 flies. (e) Video snapshots illustrating the different egg-laying motor patterns analysed within each egg-laying phase. Open triangles between snapshots denote sequential behaviours. Ovipositor contact (marked by the black arrowhead) is defined by abdomen bending accompanied by extrusion of the ovipositor and contact with underlying substrate. It can culminate in egg expulsion or not. Burrowing is characterized by scratching the surface eventually leading to digging the substrate with the ovipositor. Egg pushing is characterized by a rigid posture that initiates at the end of burrowing behaviour and accompanies egg expulsion. Note that this behaviour can be displayed in two different postures: 1. erect body posture (fly head is elevated in relation to the abdomen, or 2. leaning body posture (fly leans towards the substrate). Abdomen curling is characterized by curling the abdomen followed by walking forward, either lifting the curled abdomen, or by dragging it through the substrate. Grooming terminalia is self-explanatory. Abdominal contortions are undulated abdominal movements (represented by the shape of the black line) accompanied by extrusion of the ovipositor. Proboscis extension is characterized by the proboscis being extended to contact the substrate (marked by the black arrow). 23 Figure 3.3: Chronic silencing of OvAbg neurons blocks egg-laying and promotes egg-jamming. (a) Number of eggs laid per female in the 24h after mating during inhibition of OvAbg neurons. n = 45 (control) and 55 (OvAbg) females. Mean ±s.d. is shown on top the scatter plot. Mann-Whitney test, ****p < 0.0001. (b) Percentage of females with eggs jammed in the lateral oviducts during inhibition of OvAbg neurons. n = 32 (control) and 36 (OvAbg) females. Fisher´s exact test, ****p < 0.0001. (c) Image representing an OvAbg female reproductive system (right) with two eggs jammed (white arrows) in the lateral oviducts and a control (left) reproductive system with no eggs in the oviducts. Note that, besides the egg arresting, OvAbg silenced females also have enlarged ovaries containing more mature eggs when compared with control ovaries. This result together with the observation that flies survive and appear healthy with constitutive silencing of OvAbg neurons indicates that they are specifically involved in egg-laying. To ascertain that egg-laying defect does not result from a defect in egg production, we dissected the reproductive system. We observed eggs jammed in the lateral oviducts in all test flies together with an excess of mature eggs in the ovaries (Fig. 3.3b-c), indicating that egg production is not affected. In this series of experiments, we found a group of neurons that are part of the egg-laying motor circuits as they are directly involved in the execution of egg pushing and egg expulsion, and that are necessary for egg-laying. These Abg neurons provide a great entry point to address how different circuits coordinate to execute egg deposition. 30 3.2 Silencing OvAbg neurons disrupts all motor elements associated with egg-laying behaviour We have shown that upon activation of OvAbg neurons a single egg deposition motor element - egg pushing - is induced and that OvAbg silenced females do not lay eggs. How do OvAbg silenced females behave? Do they perform all the behaviour elements with the exception of egg pushing, or are other motor elements are affected? To answer these questions we used the anion channelrhodopsin GtACR1 (Mohammad et al., 2017) for acute optogenetic silencing of OvAbg neurons. We analysed 15 minutes of light stimulation as well as 10 minutes preand 5 minutes post-stimulation (Fig. 3.4a). Acute silencing of OvAbg neurons blocked egg-laying; the number of eggs laid during the stimulation was severely reduced and partially recovered in the post-stimulation period (Fig. 3.4a and b). Figure 3.4: Acute silencing of OvAbg neurons blocks egg-laying (a) (top) Neuronal GtACR1 silencing protocol scheme. Green shaded area represents the stimulation period. (bottom) Raster plot shows the egg expulsion events. n = 19 (OvAbg) and 8 (control) flies. (b) Quantification of the number of eggs laid per fly by OvAbg and control females during 5 min periods. n = 19 (OvAbg) and 8 (control) flies. Pre: Mann-Whitney test, ns p≥0.05; Silencing: Mann-Whitney test, ****p < 0.0001; Post: Mann-Whitney test, *p < 0.05. Analysis of the behavioural elements showed that, with the exception of grooming, all egg deposition motor elements are abolished during stimulation and partially recovered post-stimulation (Fig. 3.5a-e). The expulsion of four eggs during the silencing period was done without using most of the egg deposition motor programme (Supplementary Video 4). The number of terminalia grooming bouts does not differ from control during silencing (Fig. 3.5e). 31 However, the time the female spent grooming the terminalia is much larger in the test condition during silencing (Fig. 3.5f). Interestingly, both measures of grooming are reduced compared to control in the post-stimulation period, suggesting a rebound effect on circuits modulating grooming behaviour. The data, so far, shows a wide effect of silencing OvAbg neurons in all the egg deposition elements. Figure 3.5: Silencing OvAbg neurons disrupts all motor elements associated with egg deposition phase. Quantification of the number of behaviour bouts during 5 min periods. n = 19 (OvAbg) and 8 (control) flies. Pre: Mann-Whitney test in (a),(c) and t-test in (b),(d),(e) ns ≥0.05; Silencing: Mann-Whitney test in (a),(e), ****p < 0.0001 and ns ≥0.05 and t-test in (b),(c),(d), **p < 0.01; Post: Mann-Whitney test in (a),(b),(c), (d),(e), *p < 0.05. (f) Quantification of the mean duration of grooming terminalia bouts. n = 19 (OvAbg) and 8 (control) flies. Pre: Mann-Whitney test, ns p≥0.05; Silencing: t-test, ****p < 0.0001; Post: Mann-Whitney test, **p < 0.01. We next analysed the other egg-laying phases. Abdominal contortions are reduced compared to control, both in number of bouts (Fig. 3.6a) and behaviour duration (Fig. 3.6b). Additionally, the intensity of the contortions and the extent of the ovipositor extrusion are reduced in test flies compared to controls, as exemplified in Fig. 3.6c. The behaviour elements of the exploration motor programme are reduced during silencing (Fig. 3.6d-f). Interestingly, both the 32 exploration and abdominal motor programmes during the post-stimulation period are not different from the respective controls (Fig. 3.6a-b, d-f), indicating that the regulation of these phases is simpler than that of the egg deposition programme where the inhibitory effects of GtACR1 stimulation persist. Figure 3.6: Silencing OvAbg neurons disrupts all motor elements associated with abdominal contortions and exploration phases (a) and (b) Quantification of the number of abdominal contortions bouts during 5 min periods and the corresponding bout mean duration. n = 19 (OvAbg) and 8 (control) flies. Pre: t-test in (a) and Mann-Whitney test in (b), ns p ≥0.05; Silencing: MannWhitney test in (a) and (b) *p < 0.05; Post: Mann-Whitney test in (a) and t-test in (b), ns p ≥0.05. (c) Video snapshots of test (bottom) and control (top) flies displaying abdominal contortions during the silencing period. Silenced flies also display less extended ovipositor extrusions during abdominal contortions (arrowheads). (d),(e) and (f) Exploration phase-associated motor elements and corresponding quantification of the number of behaviour bouts during 5 min periods. n = 19 (OvAbg) and 8 (control) flies. Pre: t-test in (d) and Mann-Whitney test in (e) and (f), ns p ≥0.05; Silencing: Mann-Whitney test in (d),(e), and (f), **p < 0.01, ***p < 0.001, ****p < 0.0001; Post: Mann-Whitney test in (d),(e), and (f), ns p ≥0.05. Our findings show that silencing OvAbg neurons affects all phases of egglaying behaviour. This dramatic result could reflect a direct involvement of OvAbg neurons in all egg-laying phases. Alternatively, they could reflect an 33 arrest on the egg-laying cycle (Fig. 2.2e) induced by the loss of egg pushing behaviour and inability to complete egg deposition. 34 3.3 Activity of GABAergic OvAbg neurons blocks egg-laying To address how different neurons within the OvAbg population contribute to the execution of egg-laying, we used an intersectional approach to obtain functional subgroups (Diao et al., 2015). The GABAergic OvAbg (OvAbg/Gad1) neurons will be discussed here while cholinergic and glutamatergic OvAbg neurons will be discussed in the ensuing sections. OvAbg/Gad1 neurons (∼96 neurons, n=7 flies) are local interneurons with sparse and faint projections to other VNC ganglia (Fig. 3.7a). No projections of OvAbg/Gad1 neurons were observed in the brain (Fig. 3.7b) or the reproductive system (Fig. 3.7c). Figure 3.7: GABAergic OvAbg anatomy. (a),(b), and (c) Confocal images of female VNC (a), brain (b) and reproductive system (c) of OvAbg/Gad1 neurons and corresponding innervations stained with antiGFP (green) to reveal the anatomy and nc82 for neuropil. Anti-F-actin was used in (c) to visualize the muscle fibers. ovar: ovary; ov: oviducts; sr: seminal receptacle; ut: uterus. Anti-GFP is targeting the fluorescent protein Venus from OvAbg/Gad1-LexA > CsChrimson-mVenus expressing flies. Scale bars a), b) 50 µm and c) 200 µm. Silencing OvAbg/Gad1 had no effect on the number of eggs laid in 24h (Fig. 3.8a) and its optogenetic activation did not elicit any behaviour associated with the egg-laying motor programme (data not shown). Therefore, if OvAbg/Gad1 neurons contribute to egg-laying, they may do so by inhibiting egg-laying. To test this, we activated OvAbg/Gad1 neurons overnight (16h activation) and measured the number of eggs laid. We observed that activation of OvAbg/Gad1 abolishes egg-laying (Fig. 3.8b) and, the dissection of the ovaries at the end of the experiment, revealed that the eggs are jammed at the lateral oviduct (Fig. 3.8c-d). In summary, the results show that, during egg-laying, OvAbg/Gad1 neurons are silent, and that activity in OvAbg/Gad1 neurons prevents egg-laying. This subset of OvAbg neurons contributes to opposing outcomes compared to the 35 general line and, thus, have the potential to gate egg-laying execution by other neurons in the OvAbg population. Figure 3.8: Activation of OvAbg GABAergic neurons is sufficient to block egg-laying. (a) Number of eggs laid per female in the 24h after mating during inhibition of OvAbg/Gad1 neurons. n = 46 (control) and 49 (OvAbg/Gad1) females. Mean ±s.d. is shown on top of the scatter plot. Mann-Whitney test, ns p ≥0.05. (b) Number of eggs laid per female during the 16h photoactivation with CsChrimson of OvAbg/Gad1 neurons. n = 29 (control) and 40 (OvAbg/Gad1) females. Mean ±s.d. is shown on top of the scatter plot. Mann-Whitney test, ****p < 0.0001. (c) Percentage of females with eggs jammed in the lateral oviducts after 16h photoactivation of OvAbg/Gad1 neurons. n = 25 (control) and 35 (OvAbg/Gad1) females. Fisher´s exact test, ****p < 0.0001. (d) (bottom) Image representing a reproductive system of an OvAbg/Gad1 female after 16h photoactivation with one egg jammed (yellow arrow) in the lateral oviduct and a control (top) reproductive system with clear oviducts. Note that OvAbg/Gad1 activated females also have enlarged ovaries containing more mature eggs when compared with control ovaries (similar to OvAbg silencing egg jamming phenotype, see figure 3.3c). 36 3.4 Cholinergic OvAbg neurons are necessary and sufficient for egg deposition Cholinergic neurons (OvAbg/Cha) are a large fraction of OvAbg neurons (Fig. 3.9a) that include projections to the brain (Fig. 3.9b) and the reproductive system (Fig. 3.9c). Silencing OvAbg/Cha neurons leads to a severe reduction in the number of eggs laid (Fig. 3.9d) and a large fraction of the females display egg jamming in the lateral oviduct (Fig. 3.9e). These results show a very similar phenotype to that observed when silencing all OvAbg neurons (Fig. 3.3a-b). Figure 3.9: Silencing Cholinergic OvAbg neurons blocks egg-laying. (a),(b), and (c) Confocal images of female VNC (a), brain (b) and reproductive system (c) showing OvAbg/Cha neurons and corresponding innervations stained with anti-GFP (green) to reveal the membranes and nc82 for synapses. Anti-F-actin was used in (c) to visualize the muscle fibers. ovar: ovary; ov: oviduct; ut: uterus. AntiGFP is targeting the fluorescent protein Venus from OvAbg/Cha-LexA > CsChrimsonmVenus expressing flies. Scale bars a), b) 50 µm and c) 200 µm. (d) Number of eggs laid per female in the 24h after mating during inhibition of OvAbg/Cha neurons. n = 45 (control) and 25 (OvAbg/Cha) females. Mean ±s.d. is shown on top of the scatter plot. Mann-Whitney test, ****p < 0.0001. (e) Percentage of females with eggs jammed in the lateral oviducts during inhibition of OvAbg/Cha neurons. n = 28 (control) and 19 (OvAbg/Cha) females. Fisher´s exact test, ****p < 0.0001. Likewise, activation of OvAbg/Cha neurons using the protocol shown in Fig. 3.2a leads to both virgin and mated females assuming an egg pushing 37 posture each time the light is ON (Fig. 3.10a-b, Supplementary Video 5), as observed when all OvAbg neurons are activated (Fig. 3.2b-c). Interestingly, quantification of the number of females laying eggs during the stimulation protocol revealed that all OvAbg/Cha females laid one egg (Fig. 3.10c), in contrast to less than half of OvAbg females (Fig. 3.2f). Figure 3.10: OvAbg Cholinergic neurons are involved in egg pushing and expulsion. (a) Percentage of stimulation events in which OvAbg/Cha flies displayed an egg pushing-like posture during photoactivation with CsChrimson. n = 150 (control), 144 (M, OvAbg/Cha) and 72 (V, OvAbg/Cha) stimulations. (b) Video snapshot (lateral view) of an OvAbg/Cha female displaying an egg pushing-like posture in response to the stimulation with CsChrimson. (c) Percentage of OvAbg/Cha females that lay eggs during photoactivation with CsChrimson. n = 25 (control) and 24 (OvAbg/Cha) females. Fisher´s exact test, ****p < 0.0001. (d) Percentage of eggs laid by OvAbg/Cha females during stimulations and ISI. n = 24 eggs. (e) Latency (seconds) to egg expulsion (period of time to egg expulsion during stimulation) of OvAbg and OvAbg/Cha photoactivated females. n = 9 (OvAbg) and 24 (OvAbg/Cha) females. Mean ±s.d. is shown on top of the scatter plot. Mann-Whitney test, ****p < 0.0001. Additionally, all eggs laid during the stimulation protocol by OvAbg/Cha females were laid during the first stimulus (Fig. 3.10d), whereas egg-laying timing by OvAbg females during the stimulation protocol was variable, with females 38 laying eggs in the third and fifth stimulus as well as in the interstimulus intervals (Fig. 3.2g). The results show that, upon activation, if and when an egg is laid is variable for OvAbg, but not for OvAbg/Cha females. We also quantified, within the 10 second stimulation bout, when the females expelled the egg. We found a striking difference between OvAbg and OvAbg/Cha females (Fig. 3.10e), with OvAbg females taking a longer time to expel the egg. The increased variability regarding when the egg is expelled during the stimulation protocol, and the increased latency to lay an egg upon light ON of the OvAbg females compared to the OvAbg/Cha females, likely results from inhibition by the GABAergic neurons in the OvAbg population. Activation of OvAbg neurons encompasses simultaneous activation of inhibitory OvAbg/Gad together with egg-laying promoting OvAbg/Cha, which results in conflicting information leading to variability and delay of the behavioural execution. Overall, the results indicate that OvAbg/Cha neurons underlie the execution of egg pushing leading to egg expulsion. Unlike egg-laying behaviour of wildtype flies, eggs expelled by optogenetically activated OvAbg/Cha females were never buried and were equally distributed between the acrylic and the agarose surfaces, highlighting the importance of other behavioural components for egglaying site selection and egg burial. 39 4.1 Supplementary Figure 4.1 46 Supplementary Figure 4.1 (a) and (b) Confocal images of female abdominal muscles in the nsyb-Gal4 (a) and OvAbg line (b). Neuronal innervations are stained with anti-GFP (green) and muscle fibers with anti-F-actin (magenta). A7 and A8 indicate the position of the abdominal segments. nsyb-Gal4 line expression is shown for comparison with the OvAbg line expression. Anti-GFP is targeting the fluorescent protein GFP and Venus from nsybGal4 > mCD8::GFP and OvAbg > CsChrimson-mVenus flies. Scale bars a) and b) 50 µm. (c-k) Confocal images of OvAbg neuronal polarity in the female VNC (c-e), brain (f-h) and reproductive system (i-k). Dendrites (inputs) are labelled using the somatodendritic marker, DenMark, and axons (outputs) are labelled using the synaptic vesicle marker, Synaptotagmin. Anti-GFP is targeting EGFP-tagged Synaptotagmin and anti-DsRed is targeting mCherry-tagged DenMark. Scale bars c-k) 50 µm. (l-n) Confocal images of male brain (l), as well as male (m) and female (n) dorsal view of VNC showing OvAbg neurons and corresponding innervations stained with anti-GFP (green) to reveal the anatomy and nc82 for synapses. Anti-GFP is targeting the fluorescent protein Venus from OvAbg > CsChrimson-mVenus expressing flies. Scale bars l), m) and n) 50 µm. (o) Percentage of stimulation events in which male OvAbg flies displayed abdomen curling and aedeagus extrusion behaviours during photoactivation with CsChrimson. n = 48 (control) and 48 (OvAbg) stimulations. (p) Video snapshot (lateral view) of OvAbg male displaying abdomen curling and aedeagus extrusion (asterisk) behaviours in response to the stimulation with CsChrimson (top). A snapshot of the same male during a light off period (below) is also shown for comparison. (q) Percentage of receptive females during inhibition of OvAbg neurons. n = 60 (control) and 62 (OvAbg) females. Fisher´s exact test, ns p ≥0.05. 47 4.2 Supplementary Figure 4.2 aand bProbabilities of grooming terminalia behaviour during a 1-min time window around egg expulsion for (a) control and (b) OvAbg/VGlut silenced flies. Time = 0 minutes marks the moment of egg expulsion (represented by the grey vertical line). n = 126 (control) and n = 105 (OvAbg/VGlut) egg expulsions. 48 4.3 Supplementary Videos Videos can be accessed trough this link: www.biorxiv.org/content/10.1101/2021. 08.23.457359v1.supplementary-material 4.3.1 Supplementary Video 1 Motor elements displayed by Canton S mated females during the exploration and egg deposition phases. 4.3.2 Supplementary Video 2 Abominal contortions displayed by Canton S mated females during the abdominal contortions phase. 4.3.3 Supplementary Video 3 CsChrimson optogenetic stimnulation of OvAbg neurons in mated females. The first part of the video shows egg pushing accompained by egg expulsion. The second part of the video shows egg pushing without egg expulsion. The red dot on the top right side of the video marks the stimulation period. 4.3.4 Supplementary Video 4 Egg deposition bout during GtACR1 inhibition of OvAbg neurons in mated females. The green dot on the top right side of the video marks the stimulation period. 4.3.5 Supplementary Video 5 CsChrimson optogenetic stimnulation of OvAbg/Cha neurons in mated females. The first part of the video shows egg pushing accompained by egg expulsion. The second part of the video shows egg pushing without egg expulsion. The red dot on the top right side of the video marks the stimulation period. 4.3.6 Supplementary Video 6 CsChrimson optogenetic stimnulation of OvAbg/VGlut neurons in mated females eliciting ovipositor contact behaviour. The red dot on the top right side of the video marks the stimulation period. 4.3.7 Supplementary Video 7 Egg deposition bout during Kir2.1 inhibition of OvAbg/VGlut neurons in mated females. 49 4.4 Supplementary Table 1. Fly stocks 50 4.5 Supplementary Table 2. Full genotypes used in experiments 51 Chapter 5 Experimental Procedures 52 5.1 Fly stocks and husbandry See Supplementary Table 1 and 2 for genotypes of Drosophila used in this study. Fruit flies D. melanogaster were raised in standard cornmeal-agar medium, using Vienna food recipe (in 1 Liter of water: 80 g molasses-barley malt, 22 g beet syrup, 80 g corn flour, 18 g granulated yeast, 10 g soy flour, 8 g agar-agar, 8 mL propionic acid, 12 mL 15% nipagin, 35 mL Bavistin), at 25ºC and 70% relative humidity in a 12h dark:12h light cycle. Detailed information on fly housing and age for each experiment are indicated in the relevant section. 5.2 Immunohistochemistry Adult brains, VNCs, reproductive systems and abdomen cuticles were dissected in cold Phosphate-Buffered Saline (PBS) and immediately transferred to cold Paraformaldehyde (PFA) 4% in PBL (PBS with 0.12 M Lysine) and fixed for 30 min at Room Temperature (RT), washed three times for 5 min in PBT (PBS with 0.5% Triton X-100) and blocked for 30 min at RT in 10% normal goat serum in PBT (Sigma, cat# G9023). Samples were incubated with the primary antibodies in blocking solution, for 72h at 4°C. The following primary antibodies were used: rabbit anti-GFP 1:1000 (Molecular Probes, cat#A11122), chicken anti-GFP 1:1000 (abcam, ab13970), mouse anti-nc82 1:10 (Developmental Studies Hybridoma Bank, cat# AB2314866), rabbit anti-DsRed 1:1000 (Takara, cat# 632496). Samples were washed three times for 5 min in PBT and incubated in Alexa Fluor 488 or 594 secondary antibodies 1:500 (Invitrogen) for 72h at 4ºC. To counterstain the female reproductive system, Alexa 594-conjugated phalloidin (Molecular Probes, cat# A12381) was used. Samples were washed three times for 5 min in PBT and mounted in VectaShield medium (Vector Laboratories, cat#H-1000). Images were acquired on a Zeiss LSM 710 confocal microscope using a 25X immersion objective (Zeiss) for the brains/VNCs and a 10X objective (Zeiss) for the reproductive systems and abdomen cuticles. After acquisition, colour levels were adjusted using Fiji (Schindelin et al., 2012) for optimal display. 53 5.3 Preparation of flies to be assayed Low fly density crosses (10-15 virgin females x 5 males per bottle) were used in all experiments for rearing flies with the appropriate genotype. In order to maximize the occurrence of egg deposition events during behavioural experiments, we followed the egg-laying deprivation protocol described by Yang et al. (2015) in all experiments, except in the 24h egg-laying assays. Briefly, groups of 5-7 virgin females per vial of the appropriate genotypes and 2-3 Canton S males (for mating) were collected into normal food vials with the exception of optogenetic experiments in which flies were housed in normal food containing all-trans-Retinal (Sigma, R2500) (all-trans-Retinal concentrations used: 0.2 mM for CsChrimson activation and 0.4 mM for GtACR1 silencing). In contrast with the original protocol (Yang et al., 2015), wet yeast paste was not supplied to the food. Flies were left in the vials for 4 to 7 days at 25ºC and 70% relative humidity. Behavioural assays were performed within that 4–7 days’ time window. 5.4 Behavioural assays 5.4.1 24h egg-laying assay Single virgin females were gently aspirated and transferred to 35 mm Petri dishes of 10 mm of height (Thermo Fisher Scientific) coated with apple agar (750 mL water, 250 mL apple juice, 19,5 g agar, 20 g sugar, 10 mL 10% nipagin) and incubated with a naive CS male for 2h under constant observation to check for mating occurrence. Plates where mating did not happen were discarded. Flies were kept in the plate for 24h before eggs were counted. After egg counting, the female´s reproductive system was dissected to measure egg jamming. 5.4.2 Egg-laying arena and substrate Custom made small rectangular-shaped arenas with 2 chambers were designed to allow recording of 2 flies simultaneously. Each chamber measures 1.8 (H) x 1.2 (L) x 0.3 (D) cm. During behavioural assays, the chambers were partially filled with the egg-laying substrate, which in this study was always 1% agarose (SeaKam®LE Agarose, cat# 50004) diluted in distilled water (MilliQ®Water Purification Systems Merk). Flies had a free walking space of 1.5 x 54 0.7 x 0.3 cm in the chamber for egg-laying. The base and the lid of the arena were made of white opaque and transparent acrylic, respectively. 5.4.3 Detailed behaviour To analyse the motor elements associated with egg-laying behaviour, females were collected soon after eclosion and housed in groups following the egg-laying deprivation protocol described above. Aged 4-7 days females were tested. Flies were gently aspirated into the egg-laying arena and behaviour was recorded at 20 frames per second during 45 min for CS (Fig. 2.1 and 2.2) and during 15 min for OvAbg/VGlut silenced females (Fig. 3.11f, 3.12, 3.13). The same fly handling procedure was performed for optogenetic experiments in which egglaying motor elements were analysed (for more detailed information, see the optogenetic stimulation section). 5.4.4 Optogenetics For all experiments using CsChrimson, except in the 16h egg-laying assay (Fig. 3.8b-d), the stimulation protocol included 1 min baseline period followed by 6 repetitions of 10 s red-light stimuli with a power of 4.40 mW/cm2and 20 s interval between stimuli. Fly behaviour was recorded at 20 frames per second, except in the OvAbg line activation experiments (Fig. 3.2b-g) in which we used 15 frames per second. In the OvAbg headless females’ photoactivation (Fig. 3.2d), the head was gently cut using dissection forceps (Dumont #55 Forceps, 11295-51) under CO2 anaesthesia. Flies were transferred to the egglaying arena and allowed to recover from this procedure for 5-10 min before photoactivation. In the 16h photoactivation egg-laying assay (Fig. 3.8b-d), mated females were transferred to the apple agar plates (described in the 24h egg-laying assay section). The stimulation protocol included constant red-light with a power ranging 4.19-4.85 mW/cm2during 16h. At the end of this period, the eggs were counted and the reproductive system was dissected to measure egg jamming. For the silencing experiment using GtACR1 (Fig. 3.4-3.6), the stimulation protocol included a pre-stimulation period that lasted for 10 min, followed by constant green-light stimulation with a power of 5-6.23 mW/cm2 during 15 min and a post-stimulation period of 5 min. Videos were recorded at 20 frames per second. 55 6.2 Egg-laying behaviour in wild-type flies Complementary to Yang et al. (2008), we showed that egg-laying behaviour is structured in three phases - egg deposition, abdominal contortions and exploration. Each phase includes a specific and stereotyped repertoire of behavioural elements conserved across Drosophila species (Bräcker et al., 2019) and executed to promote substrate probing, egg deposition and ovulation. Different groups simultaneously working on this topic reached complementary results that were recently published (Cury and Axel, 2021; Vijayan et al., 2021), highlighting the need in the field for a more comprehensive characterization of this complex behaviour. We show that the egg deposition phase follows a reliable behavioural sequence in which grooming terminalia is the only optional behaviour, as also shown by Cury and Axel (2021). We established that egg deposition always transits to abdominal contortions, the sole behaviour of this phase, which is in line with Vijayan et al. showing that abdominal contortions represent the ovulation period and, thus, are critical for the positioning of a new egg to be laid (Vijayan et al., 2021). The exploratory phase shares common behavioural elements with the egg-deposition phase but, in this case, they are used by flies to sample the substrate and do not culminate in egg expulsion. The exploratory behaviours are not performed in a behavioural sequence and can be displayed with different timings, in contrast with the egg deposition motor programme. The variability in the sequence and timing of exploratory behaviours, which was also reported by Cury and Axel (2021), may offer to the organism a behavioural flexibility to cope with changes in the environment during egg-laying site selection. On this analysis, we did not distinguish proboscis extension bouts performed for feeding or exploration in the context of egg-laying. However, since the substrate we used lacks nutritive cues and the flies tested are well fed, we hypothesize that most of the proboscis extension bouts observed in our experiments are specific for egg-laying behaviour. Interestingly, females show reduced exploration in our behavioural assays and this phase is optional. Two factors may explain these observations: 1) there may be exploration that does not include the motor elements we considered and, instead, flies use mechanosensory and chemosensory information from the legs and antennae; 2) we use very small arenas with a restricted space for exploration and without complex sensory cues. This feature may allow a quick spatial and sensory recognition of the environment making 62 exploration less frequent. Future work on the features of exploration and site selection should use more complex arenas and environments. In this work, moments of distinct behaviours were annotated manually, which represents an important investment of time and limits the use of this behavioural assay in high-throughput experiments. The implementation of automated annotation systems (e.g. DeepLabCut, JAABA) to track fly kinematics and classify egg-laying motor elements will certainly offer an advantage for future studies (Kabra et al., 2013; Mathis et al., 2018). Overall, this detailed description of egg-laying behaviour provided a framework to interrogate the underlying neuronal substrates. 6.3 OvAbg neurons: lower motor control of egglaying behaviour This study offers unprecedented insights into the lower motor control of egglaying through the characterization of a novel population located in the Abg −the OvAbg neurons. The analysis of their anatomy and function showed that OvAbg neurons are critical for egg-laying behaviour and belong to the egg-laying motor circuits. We show that OvAbg is a dimorphic population involved in the control of reproductive behaviours, as shown by the distinct CNS anatomy and function between females and males. The differential regulation of dsx expression supports sex-specific behaviours (Kohl et al., 2013; Nojima et al., 2021; Pavlou et al., 2016; Rideout et al., 2010). We assume that we are targeting part of the dsx+ population at the Abg based on our intersectional approach using splitGal4 lines controlled by dsx regulatory regions, and given the anatomy reported for dsx+ neurons in the VNC (Pavlou et al., 2016; Rideout et al., 2010). The Abg egg-laying circuit is poised to receive commands from the brain for the execution of egg deposition. Which are the upstream targets of OvAbg neurons? OviDNs innervations at the VNC are anatomically poised to connect with OvAbg neurons (Vijayan et al., 2021; Wang et al., 2020). Tools to study connectivity between neurons, such as GRASP (Feinberg et al., 2008) and optogenetic stimulation integrated with calcium imaging, await the development of new genetic drivers labelling OviDNs and OvAbg populations, respectively. 63 The OvAbg neurons provided a great entry point to address how different circuits coordinate egg deposition. A detailed discussion of the three abdominal ganglion populations characterized in this study follows below. 6.4 Execution of Egg Deposition: Glutamatergic and Cholinergic OvAbg circuits We characterized two sub-populations within the OvAbg circuit −a glutamatergic and a cholinergic −that implement the egg deposition motor programme. The OvAbg/VGlut circuit represents a restricted fraction of the dsx+ glutamatergic-expressing neurons in the Abg of Drosophila females. Given the total number of dsx+ neurons expressing glutamate at the female Abg (∼100 neurons) reported by Pavlou et al (2006), we reasoned that OvAbg/VGlut population represents only 10-18% of all Abg glutamatergic dsx+ neurons. The neuronal manipulations of OvAbg/VGlut population uncovered a specific role in the initiation of the egg deposition motor sequence through the execution of ovipositor contact and burrowing behaviours. Interestingly, these motor elements of egg deposition, that are also displayed during the exploration phase, were not affected in the exploration phase when these neurons were silenced, suggesting a different control for the same motor elements during different phases. Curiously, the fact that OvAbg/VGlut neurons are involved in the initiation of the egg deposition sequence led us to speculate that they could represent the analogous neurons on the female Abg to the male-specific glutamatergic neurons controlling the initiation of genital coupling during male courtship (Pavlou et al., 2016). Additionally, the OvAbg/VGlut population could be the equivalent circuit in the fly to the digging motor circuit found in locust females (see introduction section 1.6). Burrowing behaviour is one of the motor elements elicited by OvAbg/VGlut activation and, complementary to our work, this behaviour was recently associated with digging the substrate to allow subterraneous egg deposition (Cury and Axel, 2021). Based on the architecture of the locust egg deposition circuits, we hypothesized that the OvAbg/VGlut population could locally interact with an egg-retention circuit to block egg expulsion while digging is still occurring. 64 Egg-laying is mostly performed by mated females, although virgin females may deposit unfertilized eggs residually. Activation of OvAbg/VGlut neurons leads to fewer events of egg deposition initiation in virgin females when compared to mated females, thus suggesting that mating status modulation of egg-laying is occurring locally at the Abg, in addition to the modulation in the brain (Feng et al., 2014; Shao et al., 2019; Wang et al., 2020). Local modulation may result from direct octopaminergic modulation or in downstream targets. The OvAbg/Cha circuit represents a broad population that is necessary and sufficient for egg deposition. Optogenetic activation of OvAbg/Cha neurons triggers the completion of egg deposition through the execution of egg pushing and egg expulsion, while their silencing abolishes egg-laying and promotes eggjamming. The egg-jamming phenotype suggests a role for OvAbg/Cha neurons in oviduct contractions. Such modulation is most likely indirect, because we did not find any projections between this population and the lateral oviducts. As discussed in the introductory section, a role for OA in the oviduct activity during egg-laying has been intensely studied in insects. Do OvAbg/Chat neurons belong to the octopaminergic network involved in this process? We propose that the OvAbg population is unlikely to be octopaminergic, given the differences in the Abg cell body anatomy between OvAbg and the octopaminergic representative dTdc2-GAL4 line (data not shown). Nonetheless, cholinergic OvAbg neurons may still modulate reproductive function by responding to octopaminergic signalling (e.g. through the expression OAMB and/or Octβ2R receptors). An RNAi screening targeted to octopaminergic receptors and functional connectivity experiments should help clarify at the role of OA on the OvAbg circuit function. Our findings hint at a possible interaction between the OvAbg glutamatergic and cholinergic populations in the execution of egg deposition. How is this interaction set so that flies perform a sequential order of behavioural elements leading to egg expulsion? Are OvAbg/VGlut presynaptic to OvAbg/Cha neurons? If glutamatergic OvAbg were upstream partners of cholinergic OvAbg neurons in a linear sequential motor circuit, we would expect that activation of OvAbg/VGlut population elicited egg pushing and egg expulsion, which are never observed upon this manipulation. However, we cannot rule out the possibility for such a sequential pathway because OvAbg/VGlut neurons may work 65 together with other excitatory descending input to activate OvAbg/Cha population (see model schematic, Fig. 3.13e). It is also interesting to speculate that both populations may receive sensory feedback from the reproductive system to shape the egg deposition motor programme. For example, Posterior Uterine (PU) sensory neurons located in the uterus (Cury and Axel, 2021) could relay feedback to OvAbg/VGlut neurons about the presence of the egg in the ovipositor during burrowing behaviour and, therefore, modulate the activity of this motor circuit to fine tune the progression along the behavioural sequence to complete egg deposition. 6.5 Regulation of Egg-laying: GABAergic OvAbg circuit The GABAergic OvAbg circuit represents about half (∼46%) of all dsx+ GABAergic-expressing neurons in the Abg according with Pavlou et al (2006). While the glutamatergic and cholinergic OvAbg populations represent motor circuits involved in the implementation of the egg deposition motor programme, the OvAbg/Gad1 population seems to play a more regulatory role on egg-laying. This evidence comes from the fact that (1) the number of eggs laid is not affected when OvAbg/Gad1 neurons are silenced and (2) optogenetic activation does not elicit any behavioural element associated with the egg-laying motor programme (data not shown) but, instead, is sufficient to block egg-laying. Taken together, our data indicate that OvAbg/Gad1 activity negatively regulates egg-laying whereby they need to be silent during egg-laying, likely through inhibitory descending input. Local suppression of egg-laying may be required when negative egg-laying cues arise or in virgin females (mating status modulation), which could be mediated by an increase in activity in the GABAergic population. OvAbg/Gad1 neurons could suppress egg-laying by acting on the glutamatergic and cholinergic populations (see model schematic, Fig. 3.13e). We may hypothesize that OvAbg/VGlut and OvAbg/Gad1 populations form a circuit to coordinate egg deposition initiation and suppression with analogous architecture to the one proposed by Pavlou et al. (2016). If OvAbg/Gad1 neurons indeed supress egg-laying in unfavourable environmental conditions, to which negative cues do they respond? Who is relaying the information about the environment to OvAbg/Gad1? We envision 66 that it could be either transmitted from sensory systems (e.g. legs, reproductive system) or from descending input coding negative cues (e.g. DNp42 pathway for geosmin avoidance). As a conclusion, we identified different populations of OvAbg neurons involved in the control of egg-laying motor output. The fundamental questions on the connectivity between these OvAbg populations require further investigation, which is currently limited to the genetic drivers available to label different OvAbg neurons. Future screenings on additional enhancer lines (e.g. enhancer bashing) and different expression systems (e.g. LexA driver lines) would allow to independently label and manipulate each of the relevant clusters. This will open new possibilities of dissecting the circuit. More recently, the EM-based connectivity for the T1, T2 and T3 VNC segments was recently published highlighting the sensory and motor networks controlling limb movement (Phelps et al. 2021). Mapping the structural connectivity of the Abg awaits future work and would certainly contribute for our understanding on how the OvAbg circuit coordinates and regulates egg-laying behaviour. 6.6 Conclusion 1. Our findings provide a detailed description of egg-laying. 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