Divergent evolution of sleep in Drosophila species.

Living organisms synchronize their biological activities with the earth's rotation through the circadian clock, a molecular mechanism that regulates biology and behavior daily. This synchronization factually maximizes positive activities (e.g., social interactions, feeding) during safe periods, and minimizes exposure to dangers (e.g., predation, darkness) typically at night. Beyond basic circadian regulation, some behaviors like sleep have an additional layer of homeostatic control, ensuring those essential activities are fulfilled. While sleep is predominantly governed by the circadian clock, a secondary homeostatic regulator, though not well-understood, ensures adherence to necessary sleep amounts and hints at a fundamental biological function of sleep beyond simple energy conservation and safety. Here we explore sleep regulation across seven Drosophila species with diverse ecological niches, revealing that while circadian-driven sleep aspects are consistent, homeostatic regulation varies significantly. The findings suggest that in Drosophilids, sleep evolved primarily for circadian purposes. The more complex, homeostatically regulated functions of sleep appear to have evolved independently in a species-specific manner, and are not universally conserved. This laboratory model may reproduce and recapitulate primordial sleep evolution.

OptoPi: An open source flexible platform for the analysis of small animal behaviour.

Behaviour is the ultimate output of neural circuit computations, and therefore its analysis is a cornerstone of neuroscience research. However, every animal and experimental paradigm requires different illumination conditions to capture and, in some cases, manipulate specific behavioural features. This means that researchers often develop, from scratch, their own solutions and experimental set-ups. Here, we present OptoPi, an open source, affordable (∼ £600), behavioural arena with accompanying multi-animal tracking software. The system features highly customisable and reproducible visible and infrared illumination and allows for optogenetic stimulation. OptoPi acquires images using a Raspberry Pi camera, features motorised LED-based illumination, Arduino control, as well as irradiance monitoring to fine-tune illumination conditions with real time feedback. Our open-source software (BioImageProcessing) can be used to simultaneously track multiple unmarked animals both in on-line and off-line modes. We demonstrate the functionality of OptoPi by recording and tracking under different illumination conditions the spontaneous behaviour of larval zebrafish as well as adult Drosophila flies and their first instar larvae, an experimental animal that due to its small size and transparency has classically been hard to track. Further, we showcase OptoPi's optogenetic capabilities through a series of experiments using transgenic Drosophila larvae.

Sensory processing during sleep in Drosophila melanogaster.

During sleep, most animal species enter a state of reduced consciousness characterized by a marked sensory disconnect. Yet some processing of the external world must remain intact, given that a sleeping animal can be awoken by intense stimuli (for example, a loud noise or a bright light) or by soft but qualitatively salient stimuli (for example, the sound of a baby cooing or hearing one's own name1-3). How does a sleeping brain retain the ability to process the quality of sensory information? Here we present a paradigm to study the functional underpinnings of sensory discrimination during sleep in Drosophila melanogaster. We show that sleeping vinegar flies, like humans, discern the quality of sensory stimuli and are more likely to wake up in response to salient stimuli. We also show that the salience of a stimulus during sleep can be modulated by internal states. We offer a prototypical blueprint detailing a circuit involved in this process and its modulation as evidence that the system can be used to explore the cellular underpinnings of how a sleeping brain experiences the world.

Sleep in Drosophila and Its Context.

A prominent idea emerging from the study of sleep is that this key behavioural state is regulated in a complex fashion by ecologically and physiologically relevant environmental factors. This concept implies that sleep, as a behaviour, is plastic and can be regulated by external agents and changes in internal state. Drosophila melanogaster constitutes a resourceful model system to study behaviour. In the year 2000, the utility of the fly to study sleep was realised, and has since extensively contributed to this exciting field. At the centre of this review, we will discuss studies showing that temperature, food availability/quality, and interactions with conspecifics can regulate sleep. Indeed the relationship can be reciprocal and sleep perturbation can also affect feeding and social interaction. In particular, different environmental temperatures as well as gradual changes in temperature regulate when, and how much flies sleep. Moreover, the satiation/starvation status of an individual dictates the balance between sleep and foraging. Nutritional composition of diet also has a direct impact on sleep amount and its fragmentation. Likewise, aggression between males, courtship, sexual arousal, mating, and interactions within large groups of animals has an acute and long-lasting effect on sleep amount and quality. Importantly, the genes and neuronal circuits that relay information about the external environment and internal state to sleep centres are starting to be elucidated in the fly and are the focus of this review. In conclusion, sleep, as with most behaviours, needs the full commitment of the individual, preventing participation in other vital activities. A vast array of behaviours that are modulated by external and internal factors compete with the need to sleep and thus have a significant role in regulating it.

Ethoscopes: An open platform for high-throughput ethomics.

Here, we present the use of ethoscopes, which are machines for high-throughput analysis of behavior in Drosophila and other animals. Ethoscopes provide a software and hardware solution that is reproducible and easily scalable. They perform, in real-time, tracking and profiling of behavior by using a supervised machine learning algorithm, are able to deliver behaviorally triggered stimuli to flies in a feedback-loop mode, and are highly customizable and open source. Ethoscopes can be built easily by using 3D printing technology and rely on Raspberry Pi microcomputers and Arduino boards to provide affordable and flexible hardware. All software and construction specifications are available at http://lab.gilest.ro/ethoscope.

Regulation of sleep homeostasis by sexual arousal.

In all animals, sleep pressure is under continuous tight regulation. It is universally accepted that this regulation arises from a two-process model, integrating both a circadian and a homeostatic controller. Here we explore the role of environmental social signals as a third, parallel controller of sleep homeostasis and sleep pressure. We show that, in Drosophila melanogaster males, sleep pressure after sleep deprivation can be counteracted by raising their sexual arousal, either by engaging the flies with prolonged courtship activity or merely by exposing them to female pheromones.

The role of contact chemoreception in the host location process of an egg parasitoid.

Taste allows insects to detect palatable or toxic foods, identify a mate, and select appropriate oviposition sites. The gustatory system strongly contributes to the survival and reproductive success of many species, yet it is rarely studied in insect parasitoids. In order to locate and assess a host in which they will lay their eggs, female wasps actively search for chemical cues using their sensory organs present mainly on the antennae. In this paper, we studied the role of antennal taste sensilla chaetica in the perception of contact semiochemicals in Trissolcus brochymenae (Hymenoptera: Platygastridae), an egg parasitoid of the brassicaceae pest Murgantia histrionica (Heteroptera: Pentatomidae). Methanolic extracts obtained from male and female hosts elicited action potentials in taste neurons housed in antennal sensilla chaetica, indicating that these sensilla are involved in the perception of non volatile host kairomones. In behavioural assays, wasp females displayed an intense searching behaviour in open arenas treated with host extracts, thus confirming that these kairomones are soluble in polar solvents. We further investigated the extracts by Gas Chromatography-Mass Spectrometry (GC-MS) and found that they contain several compounds which are good candidates for these contact kairomones. This study contributes to better understanding contact chemoreception in egg parasitoids and identifying gustatory receptor neurons involved in the host location process.

Drosophila Bitter Taste(s).

Most animals possess taste receptors neurons detecting potentially noxious compounds. In humans, the ligands which activate these neurons define a sensory space called "bitter". By extension, this term has been used in animals and insects to define molecules which induce aversive responses. In this review, based on our observations carried out in Drosophila, we examine how bitter compounds are detected and if bitter-sensitive neurons respond only to molecules bitter to humans. Like most animals, flies detect bitter chemicals through a specific population of taste neurons, distinct from those responding to sugars or to other modalities. Activating bitter-sensitive taste neurons induces aversive reactions and inhibits feeding. Bitter molecules also contribute to the suppression of sugar-neuron responses and can lead to a complete inhibition of the responses to sugar at the periphery. Since some bitter molecules activate bitter-sensitive neurons and some inhibit sugar detection, bitter molecules are represented by two sensory spaces which are only partially congruent. In addition to molecules which impact feeding, we recently discovered that the activation of bitter-sensitive neurons also induces grooming. Bitter-sensitive neurons of the wings and of the legs can sense chemicals from the gram negative bacteria, Escherichia coli, thus adding another biological function to these receptors. Bitter-sensitive neurons of the proboscis also respond to the inhibitory pheromone, 7-tricosene. Activating these neurons by bitter molecules in the context of sexual encounter inhibits courting and sexual reproduction, while activating these neurons with 7-tricosene in a feeding context will inhibit feeding. The picture that emerges from these observations is that the taste system is composed of detectors which monitor different "categories" of ligands, which facilitate or inhibit behaviors depending on the context (feeding, sexual reproduction, hygienic behavior), thus considerably extending the initial definition of "bitter" tasting.

Antennal gustatory perception and behavioural responses in Trissolcus brochymenae females.

Trissolcus brochymenae (Hymenoptera: Platygastridae) is an egg parasitoid that could be used to control stink bugs like Murgantia histrionica (Heteroptera: Pentatomidae), a pest of brassicaceous crops. Before laying their eggs, parasitoid females spend considerable time examining the substrate with their antennae, which are also used during feeding and mating behaviours. This suggests that contact chemoreception plays a prominent role in many aspects of parasitoid ecology. Therefore, we examined the sensitivity of antennal taste sensilla chaetica to several classical tastants including those that are appetitive or aversive. In addition we studied the taste preferences of wasps when presented with these chemicals alone or mixed. The sensilla chaetica of T. brochymenae responded to serial concentrations of sucrose, salts, and quinine, but no concentration-dependent effect was observed when testing sinigrin, a secondary metabolite found in many brassicaceae. However, both sinigrin and quinine inhibited responses to 0.1 M sucrose when mixed with this sugar. Behavioural taste preference assays confirmed that wasps showed a dose dependent preference for sucrose over agarose. In addition, a behavioural avoidance of sucrose solutions containing quinine was observed. This effect was not observed when sinigrin was used as a feeding deterrent. In the two-choice tests the wasp did not discriminate between sucrose solutions mixed with salts and sucrose alone. Further no preference for salts or sinigrin compared to agarose alone was observed. This work represents the first step towards the identification of gustatory receptor neurons implicated in the detection of different types of chemical cues in egg parasitoids.

Dual mechanism for bitter avoidance in Drosophila.

In flies and humans, bitter chemicals are known to inhibit sugar detection, but the adaptive role of this inhibition is often overlooked. At best, this inhibition is described as contributing to the rejection of potentially toxic food, but no studies have addressed the relative importance of the direct pathway that involves activating bitter-sensitive cells versus the indirect pathway represented by the inhibition of sugar detection. Using toxins to selectively ablate or inactivate populations of bitter-sensitive cells, we assessed the behavioral responses of flies to sucrose mixed with strychnine (which activates bitter-sensitive cells and inhibits sugar detection) or with L-canavanine (which only activates bitter-sensitive cells). As expected, flies with ablated bitter-sensitive cells failed to detect L-canavanine mixed with sucrose in three different feeding assays (proboscis extension responses, capillary feeding, and two-choice assays). However, such flies were still able to avoid strychnine mixed with sucrose. By means of electrophysiological recordings, we established that bitter molecules differ in their potency to inhibit sucrose detection and that sugar-sensing inhibition affects taste cells on the proboscis and the legs. The optogenetic response of sugar-sensitive cells was not reduced by strychnine, thus suggesting that this inhibition is linked directly to sugar transduction. We postulate that sugar-sensing inhibition represents a mechanism in insects to prevent ingesting harmful substances occurring within mixtures.

The role of serotonin in feeding and gut contractions in the honeybee.

Serotonin (5-hydroxytryptamine, 5-HT) is involved in the regulation of feeding and digestion in many animals from worms to mammals. In insects, 5-HT functions both as a neurotransmitter and as a systemic hormone. Here we tested its role as a neurotransmitter in feeding and crop contractions and its role as a systemic hormone that affected feeding in adult foraging honeybees. We found 5-HT immunoreactive processes throughout the gut, including on the surface of the oesophagus, crop, proventriculus, and the midgut, as well as in the ventral nerve cord. mRNA transcripts for all four of the known bee 5-HT receptors (Am5-ht1A,2α,2ß,7) were expressed in the crop and the midgut suggesting a functional role for 5-HT in these locations. Application of a cocktail of antagonists with activity against these known receptors to the entire gut in vivo reduced the rate of spontaneous contraction in the crop and proventriculus. Although feeding with sucrose caused a small elevation of endogenous 5-HT levels in the haemolymph, injection of exogenous 5-HT directly into the abdomen of the bee to elevate 5-HT in the haemolymph did not alter food intake. However, when 5-HT was injected into directly into the brain there was a reduction in intake of carbohydrate, amino acid, or toxin-laced food solutions. Our data demonstrate that 5-HT inhibits feeding in the brain and excites muscle contractions in the gut, but general elevation of 5-HT in the bee's haemolymph does not affect food intake.