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Social interactions are generally regulated by pheromones that convey information about the identity, physiological state, and location of an individual. The fruit fly, Drosophila melanogaster, offers a powerful model system to study the mechanisms through which pheromones modulate social interactions. Most of the fruit fly’s social behavior is demonstrably modulated by pheromones, and many of the chemical compounds composing its pheromonal profile have been characterized. This chapter describes several behavioral bioassays that can be used to determine the function of contact and short-range volatile pheromones in D. melanogaster’s social behavior. The chapter first provides instructions on how to rear flies for pheromonal experimentation and how to generate flies that cannot produce cuticular hydrocarbons. Afterward, protocols on how to determine the function of pheromones in courtship behavior and mate choice are provided, followed by protocols to determine whether pheromones function as volatile or contact cues during oviposition site selection. Finally, the last section of the chapter gives general advice on how to work with pheromones in the laboratory.

Sociability – an individual’s propensity to engage in group activities – is a conserved trait throughout most of life. Sociability can be considered as a personality type in humans when showing correlated sociability measures across different assays. Our knowledge of the mechanisms that drive sociability and explain its inter-individual variation is still limited. To enhance our mechanistic understanding, the genetically tractable Drosophila melanogaster is increasingly being used as a model for research on social behaviour. High and low sociability phenotypes, indicative of a variation of sociability levels among the population have been documented in this species. However, sociability traits have been measured through unidimensional assays, which do not allow us to assess whether sociability is a personality type in fruit flies. Here, we present a multidimensional approach to assess sociability, using a combination of three behavioural assays that each captures a different functional feature of social interactions in D.melanogaster. We applied our approach to the Drosophila melanogaster Genetic Reference Panel (DGRP) lines and revealed continuous variation in the strength of response to others. We found that sociability levels varied between genetically distinct lines, indicative of a genetic component of sociability.The three sociability traits were, however, not correlated, making it questionable whether sociability can be considered a personality type in D. melanogaster.

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

We follow an adult Drosophila melanogaster female through the major reproductive decisions she makes during her lifetime, including habitat selection, precopulatory mate choice, postcopulatory physiological changes, polyandry, and egg-laying site selection. In the process, we review the molecular and neuronal mechanisms allowing females to integrate signals from both environmental and social sources to produce those behavioral outputs. We pay attention to how an understanding of D. melanogaster female reproductive behaviors contributes to a wider understanding of evolutionary processes such as pre- and postcopulatory sexual selection as well as sexual conflict. Within each section, we attempt to connect the theories that pertain to the evolution of female reproductive behaviors with the molecular and neurobiological data that support these theories. We draw attention to the fact that the evolutionary and mechanistic basis of female reproductive behaviors, even in a species as extensively studied as D. melanogaster, remains poorly understood.

Adaptation to different food resources is a major mechanism shaping local adaptation and speciation. Numerous cases of food-mediated speciation are seen in plant-feeding insects. In this thesis, we first formulate seven key questions using insect literature to explore the processes of food-mediated speciation. We then experimentally investigate two critical questions regarding food-mediated population differentiation and assortative mating using the model organism Drosophila melanogaster and its essential food resource – yeast. We quantified how different fly strains respond to different yeast species, across multiple yeast-dependent life history traits. We found that fly strains respond differently to different yeast species and different life-history traits are maximized on different yeast species. This suggests that the inconsistency of responses between life-history traits forms a possible limitation to population differentiation. Additionally, we built a system for automated tracking of the mating location of flies in environments containing heterogeneous food patches to dissect food-mediated assortative mating. We discovered that flies generally mate on patches containing yeast and fly pairs exert preferences for mating on certain yeast species over others. Notably, the preference of mating on yeast depends on variables including the presence of multiple yeast species and timing of (re)mating with respect to light cycle. This suggests that some level of assortative mating may result from the preference for mating on food resources, but the strength of such assortative mating may vary with environmental conditions. Together, we present the seven-question framework for understanding food-mediated speciation and demonstrate the constraints of food-mediated population differentiation and assortative mating.

Group-living facilitates cooperation between individuals, but also creates competition for limited resources. Individuals should thus modulate their reproductive output in response to the presence of others. However, mechanisms that lead to such social context-dependent modulation of reproduction are poorly understood. In the fruit fly Drosophila melanogaster, females actively attract conspecifics to lay eggs on the same resources, generating groups in which individuals may cooperate or compete. The genetic tractability of this species gave me an opportunity to dissect the mechanisms by which females adjust their behaviour and physiology to their social environment. I discovered social facilitation of oogenesis and egg-laying in Drosophila melanogaster and described the neuro-hormonal mechanisms underlying this modulation. I demonstrated that social environment affects ovarian activity of females in a density-dependent manner. I showed that these responses to the presence of others are mediated by vision through the motion detection pathway and are the result of the stimulation of the juvenile hormone pathway. While social modulation of reproduction is considered a sign of sociality and is mostly expressed in social hymenopterans, my findings thus demonstrate that such phenomenon can already be found in rather solitary species such as D. melanogaster, making the concept of solitary vs social species a bit artificial. I moreover proposed a new approach to explore the variation in sociability among D. melanogaster through the quantification of multiple sociability traits. I found significant variation between the flies in the strength of responses to others, demonstrating the existence of a sociability spectrum in this species.

The fruit fly Drosophila suzukii infests intact, ripening soft fruits using the serrated ovipositor. Originally from Asia, this fly has successfully invaded several parts of America and Europe in the last decade, resulting in direct economic losses in the horticultural sector. Microbes are important in a pest’s life history. This thesis focused on investigating the kinds of microbial associations in D. suzukii in the Netherlands. Overall, this thesis provided us with the fundamental information of D. suzukii’s microbial ecology in the Netherlands.

We humans experience the influence of our circadian clock every day. This clock mechanism causes, for example, a jet lag during transatlantic air travel. We now believe that almost all organisms have developed a circadian clock mechanism.In this thesis I describe the analysis techniques we developed and the newly discovered molecular components of a circadian mechanism in Saccharomyces cerevisiae and Bacillus subtilis. To identify these molecular components, I applied structured zeitgebers, i.e. light and temperature cycling, to yeast and bacillus cultures. All this in conjunction with bioinformatic in-silico approachesIn Bacillus biofilm populations, we found a free-running rhythm of ytvA and KinC activity of nearly 24 hours after entrainment and release to constant dark and temperature conditions. The free-running oscillations are temperature compensated. This is one of the most important features of a circadian clock mechanism, making it very likely that such a system exists in B. subtilis.We found in yeasts that temperature appears to mainly regulate metabolic processes. Light appears to act more indirectly via photo-oxidation of mitochondrial cytochromes.Finally, I present a hypothetical model for an integrated circadian clock mechanism in unicellular microbes with an emphasis on S. cerevisiae. This mechanism involves several metabolic pathways and the main regulator is the stress sensitive transcriptional activator Msn2p. The model shows that in the circadian clock mechanism in yeast, energy metabolism appears to be an important theme. Other processes that are relevant: metabolic process of nitrogen compounds, oxidation-reduction process and fatty acid metabolism. All could serve as a starting point for further research on the circadian clock in yeast.

Sex determination (SD) is an essential part of an individual's development, but the mechanisms controlling SD are incredibly variable and subject to rapid evolutionary change. When new SD genes evolve, this can lead to the formation of sex chromosomes. Sex chromosomes evolve from regular autosomes into a pair of highly-differentiated chromosomes via sex-specific adaptation and genetic degeneration. The evolutionary causes and consequences of SD evolution have been intensively studied using theoretical approaches, but many questions remain. Existing models of SD evolution predict that multiple SD genes generally cannot coexist, while some such systems occur in amongst others insects and amphibians. Here, several models are developed that may help explain why complex SD systems may persist over evolutionary time scales. This is done by assuming that the genes controlling SD do not act in isolation, but instead that SD as a process is integrated into and affected by the biology and ecology of the organism. Early sex chromosome evolution is thought to occur by sex-specific adaptation and the evolution of recombination suppression. Testing this theory has proven difficult as in absence of recombination, sex chromosomes are prone to genetic decay and mutation accumulation. Here, the complex SD system of the housefly Musca domestica is exploited to generate new sex chromosomes, which circumvent the issue of chromosomal decay and may be used to study the early stages of sex chromosome evolution. Further development of the housefly as a model system for evolutionary studies will be needed to bring this potential to full fruition. Altogether, these findings show that SD, and the mechanisms controlling it, is more complex then commonly considered.

The work presented here demonstrates that temperature is a fundamental component of many fundamental aspect of a fly's life. Starting with early development, the temperature at which a fly grows will determine how well it can cope with the climate challenges in its later adult life. Once in adulthood, an intricate system of peripheral and brain thermosensors coordinates how flies respond to dynamic temperature changes. This response is not just a predictable reaction; it is a complex process that can be affected by other internal and external features of the fly, such as its own sex and the sex of surrounding flies. Considering the relevance of Drosophila as a model organism, it is fundamental to continue exploring how temperature interacts with the other features of fly's existence, as it will help us predict how small ectotherms might be affected by climate change, while also answering basic neuroscience questions, such as how a brain integrates temperature information.

Reproductie vergt samenwerking tussen een man en een vrouw om nakomelingen te produceren. Mannetjes initiëren reproductie en verleiden het vrouwtje. Vrouwtjes besluiten of ze hierop ingaan. Vrouwelijke receptiviteit, de ontvankelijkheid tot paring, bepaald dus of een paring plaatsvindt. In dit proefschrift is onderzocht welke interne en externe factoren vrouwelijke receptiviteit beïnvloeden. Wat maakt dat een vrouwtje de verleiding van het mannetje accepteert en welke genen van het vrouwtje zijn belangrijk voor het bepalen van de receptiviteit? In dit proefschrift is beschreven hoe de aanwezigheid van voedsel en sociale context vrouwelijke receptiviteit beïnvloeden en hoe deze worden waargenomen. In omgevingen met grote aanwezigheid van voedsel en andere individuen verhogen vrouwtjes de receptiviteit na paring, maar niet als maagd. Van de genen die vrouwelijke receptiviteit bepalen zijn twee gerelateerde geurreceptoren direct van belang: eentje is alleen belangrijk voor de vrouwelijke receptiviteit na paring en de ander lijkt betrokken bij receptiviteit zowel voor als na paring. Daarnaast laten variatie in receptiviteit en de daarbij behorende genen zien dat de genetische architectuur voor receptiviteit voor en na paring verschillend zijn. Genen betrokken bij variatie in receptiviteit na paring lijken vaker betrokken bij processen voor leren en geheugen, terwijl genen voor maagdelijke receptiviteit betrokken zijn bij geurwaarneming. Samen laten deze resultaten zien dat receptiviteit voor en na paring verschillende mechanismen hebben. De receptiviteit na paring lijkt een complexer gedrag door de betrokkenheid van leren en geheugen en gevoeligheid voor omgevingsfactoren, wellicht om de investering in reproductie overeen te laten komen met de aanwezige bronnen.

Offspring production commences with mate-choice and culminates in the generation of genetically similar individuals. Males and females play distinct roles in this process: males compete to gain access to females, and females exhibit choice. When a female mates more than once, these pre-copulatory interactions extend to the female reproductive tract as sperm from multiple males compete to fertilize her eggs. To investigate the possibility that females bias sperm use after copulation, I employed the fruit fly, Drosophila melanogaster. By reviewing the literature, I found that our understanding of female post-copulatory behaviours is relatively limited and set out to investigate the mechanisms and functions of two female post-copulatory behaviours: sperm storage (movement of sperm into storage organs) and sperm ejection (removal of ejaculate). I found that females alter their attractiveness and chances of remating via ejection of sperm and anti-aphrodisiac pheromones. I also found that females who remate in quick succession produce different patterns of paternity compared to females with slower remating rates indicating an influence of the female on whose sperm is used to fertilize her eggs. To explore how this choice occurs, I artificially modified the activity of various populations of neurons and determined that sperm storage and ejection are regulated by neuronal circuits in the female central nervous system. Overall, I conclude that females’ active control over reproduction extends past pre-copulatory mate choice to include an influence on whose sperm fertilizes her eggs to produce offspring.