Fruit flies can learn non-elemental olfactory discriminations.

Associative learning allows animals to establish links between stimuli based on their concomitance. In the case of Pavlovian conditioning, a single stimulus A (the conditional stimulus, CS) is reinforced unambiguously with an unconditional stimulus (US) eliciting an innate response. This conditioning constitutes an 'elemental' association to elicit a learnt response from A+ without US presentation after learning. However, associative learning may involve a 'complex' CS composed of several components. In that case, the compound may predict a different outcome than the components taken separately, leading to ambiguity and requiring the animal to perform so-called non-elemental discrimination. Here, we focus on such a non-elemental task, the negative patterning (NP) problem, and provide the first evidence of NP solving in Drosophila. We show that Drosophila learn to discriminate a simple component (A or B) associated with electric shocks (+) from an odour mixture composed either partly (called 'feature-negative discrimination' A+ versus AB-) or entirely (called 'NP' A+B+ versus AB-) of the shock-associated components. Furthermore, we show that conditioning repetition results in a transition from an elemental to a configural representation of the mixture required to solve the NP task, highlighting the cognitive flexibility of Drosophila.

Drosophila females trade off good nutrition with high-quality oviposition sites when choosing foods.

Animals, from insects to humans, select foods to regulate their acquisition of key nutrients in amounts and balances that maximise fitness. In species in which the nutrition of juveniles depends on parents, adults must make challenging foraging decisions that simultaneously address their own nutrient needs as well as those of their progeny. Here, we examined how the fruit fly Drosophila melanogaster, a species in which individuals eat and lay eggs in decaying fruits, integrate feeding decisions (individual nutrition) and oviposition decisions (offspring nutrition) when foraging. Using cafeteria assays with artificial diets varying in concentrations and ratios of protein to carbohydrates, we show that D. melanogaster females exhibit complex foraging patterns, alternating between laying eggs on high carbohydrate foods and feeding on foods with different nutrient contents depending on their own nutritional state. Although larvae showed faster development on high protein foods, both survival and learning performance were higher on balanced foods. We suggest that the apparent mismatch between the oviposition preference of females for high carbohydrate foods and the high performances of larvae on balanced foods reflects a natural situation where high carbohydrate ripened fruits gradually enrich in proteinaceous yeast as they start rotting, thereby yielding optimal nutrition for the developing larvae. Our findings that animals with rudimentary parental care uncouple feeding and egg-laying decisions in order to balance their own diet and provide a nutritionally optimal environment to their progeny reveal unsuspected levels of complexity in the nutritional ecology of parent-offspring interactions.

Behavioral consequences of dopamine deficiency in the Drosophila central nervous system.

The neuromodulatory function of dopamine (DA) is an inherent feature of nervous systems of all animals. To learn more about the function of neural DA in Drosophila, we generated mutant flies that lack tyrosine hydroxylase, and thus DA biosynthesis, selectively in the nervous system. We found that DA is absent or below detection limits in the adult brain of these flies. Despite this, they have a lifespan similar to WT flies. These mutants show reduced activity, extended sleep time, locomotor deficits that increase with age, and they are hypophagic. Whereas odor and electrical shock avoidance are not affected, aversive olfactory learning is abolished. Instead, DA-deficient flies have an apparently "masochistic" tendency to prefer the shock-associated odor 2 h after conditioning. Similarly, sugar preference is absent, whereas sugar stimulation of foreleg taste neurons induces normal proboscis extension. Feeding the DA precursor L-DOPA to adults substantially rescues the learning deficit as well as other impaired behaviors that were tested. DA-deficient flies are also defective in positive phototaxis, without alteration in visual perception and optomotor response. Surprisingly, visual tracking is largely maintained, and these mutants still possess an efficient spatial orientation memory. Our findings show that flies can perform complex brain functions in the absence of neural DA, whereas specific behaviors involving, in particular, arousal and choice require normal levels of this neuromodulator.

Mate copying requires the coincidence detector Rutabaga in the mushroom bodies of Drosophila melanogaster.

Mate choice constitutes a major fitness-affecting decision often involving social learning leading to copying the preference of other individuals (i.e., mate copying). While mate copying exists in many taxa, its underlying neurobiological mechanisms remain virtually unknown. Here, we show in Drosophila melanogaster that the rutabaga gene is necessary to support mate copying. Rutabaga encodes an adenylyl cyclase (AC-Rut+) acting as a coincidence detector in associative learning. Since the brain localization requirements for AC-Rut+ expression differ in classical and operant learning, we determine the functional localization of AC-Rut+ for mate copying by artificially rescuing the expression of AC-Rut+ in neural subsets of a rutabaga mutant. We found that AC-Rut+ has to be expressed in the mushroom bodies' Kenyon cells (KCs), specifically in the γ-KCs subset. Thus, this form of discriminative social learning requires the same KCs as non-social Pavlovian learning, suggesting that pathways of social and asocial learning overlap significantly.

Conformity in mate choice, the overlooked social component of animal and human culture.

Although conformity as a major driver for human cultural evolution is a well-accepted and intensely studied phenomenon, its importance for non-human animal culture has been largely overlooked until recently. This limited for decades the possibility of studying the roots of human culture. Here, we provide a historical review of the study of conformity in both humans and non-human animals. We identify gaps in knowledge and propose an evolutionary route towards the sophisticated cultural processes that characterize humanity. A landmark in the study of conformity is Solomon Asch's famous experiment on humans in 1955. By contrast, interest in conformity among evolutionary biologists has only become salient since the turn of the new millennium. A striking result of our review is that, although studies of conformity have examined many biological contexts, only one looked at mate choice. This is surprising because mate choice is probably the only context in which conformity has self-reinforcing advantages across generations. Within a metapopulation, i.e. a group of subpopulations connected by dispersing individuals, dispersers able to conform to the local preference for a given type of mate have a strong and multigenerational fitness advantage. This is because once females within one subpopulation locally show a bias for one type of males, immigrant females who do not conform to the local trend have sons, grandsons, etc. of the non-preferred phenotype, which negatively and cumulatively affects fitness over generations in a process reminiscent of the Fisher runaway process. This led us to suggest a sex-driven origin of conformity, indicating a possible evolutionary route towards animal and human culture that is rooted in the basic, and thus ancient, social constraints acting on mating preferences within a metapopulation. In a generic model, we show that dispersal among subpopulations within a metapopulation can effectively maintain independent Fisher runaway processes within subpopulations, while favouring the evolution of social learning and conformity at the metapopulation scale; both being essential for the evolution of long-lasting local traditions. The proposed evolutionary route to social learning and conformity casts surprising light on one of the major processes that much later participated in making us human. We further highlight several research avenues to define the spectrum of conformity better, and to account for its complexity. Future studies of conformity should incorporate experimental manipulation of group majority. We also encourage the study of potential links between conformity and mate copying, animal aggregations, and collective actions. Moreover, validation of the sex-driven origin of conformity will rest on the capacity of human and evolutionary sciences to investigate jointly the origin of social learning and conformity. This constitutes a stimulating common agenda and militates for a rapprochement between these two currently largely independent research areas.

2-D sex images elicit mate copying in fruit flies.

Although the environment is three-dimensional (3-D), humans are able to extract subtle information from two-dimensional (2-D) images, particularly in the domain of sex. However, whether animals with simpler nervous systems are capable of such information extraction remains to be demonstrated, as this ability would suggest a functional generalisation capacity. Here, we performed mate-copying experiments in Drosophila melanogaster using 2-D artificial stimuli. Mate copying occurs when naïve females observe the mating success of potential mates and use that social information to build their own mating preference. By replacing live demonstrations with (i) photos or (ii) simplified images of copulating pairs, we found that even crudely simplified images of sexual intercourse still elicit mate copying, suggesting that Drosophila is able to extract sex-related information even from a degraded image. This new method constitutes a powerful tool to further investigate mate copying in that species and sexual preferences in general.

Social facilitation of long-lasting memory is mediated by CO2 in Drosophila.

How social interactions influence cognition is a fundamental question, yet rarely addressed at the neurobiological level. It is well established that the presence of conspecifics affects learning and memory performance, but the neural basis of this process has only recently begun to be investigated. In the fruit fly Drosophila melanogaster, the presence of other flies improves retrieval of a long-lasting olfactory memory. Here, we demonstrate that this is a composite memory composed of two distinct elements. One is an individual memory that depends on outputs from the α'ß' Kenyon cells (KCs) of the mushroom bodies (MBs), the memory center in the insect brain. The other is a group memory requiring output from the αß KCs, a distinct sub-part of the MBs. We show that social facilitation of memory increases with group size and is triggered by CO2 released by group members. Among the different known neurons carrying CO2 information in the brain, we establish that the bilateral ventral projection neuron (biVPN), which projects onto the MBs, is necessary for social facilitation. Moreover, we demonstrate that CO2-evoked memory engages a serotoninergic pathway involving the dorsal-paired medial (DPM) neurons, revealing a new role for this pair of serotonergic neurons. Overall, we identified both the sensorial cue and the neural circuit (biVPN>αß>DPM>αß) governing social facilitation of memory in flies. This study provides demonstration that being in a group recruits the expression of a cryptic memory and that variations in CO2 concentration can affect cognitive processes in insects.

Beyond social learning.

Cultural evolution requires the social transmission of information. For this reason, scholars have emphasized social learning when explaining how and why culture evolves. Yet cultural evolution results from many mechanisms operating in concert. Here, we argue that the emphasis on social learning has distracted scholars from appreciating both the full range of mechanisms contributing to cultural evolution and how interactions among those mechanisms and other factors affect the output of cultural evolution. We examine understudied mechanisms and other factors and call for a more inclusive programme of investigation that probes multiple levels of the organization, spanning the neural, cognitive-behavioural and populational levels. To guide our discussion, we focus on factors involved in three core topics of cultural evolution: the emergence of culture, the emergence of cumulative cultural evolution and the design of cultural traits. Studying mechanisms across levels can add explanatory power while revealing gaps and misconceptions in our knowledge. This article is part of the theme issue 'Foundations of cultural evolution'.

Response to Comment on "Cultural flies: Conformist social learning in fruitflies predicts long-lasting mate-choice traditions".

Thornquist and Crickmore claim that systematic experimental error may explain the results of Danchin and colleagues. Their claim rests on mistakes in their analyses, for which we provide corrections. We reassert that conformity in fruitflies predicts long-lasting mate-preference traditions.

Effects of a sex ratio gradient on female mate-copying and choosiness in Drosophila melanogaster.

In many sexually reproducing species, individuals can gather information about potential mates by observing their mating success. This behavioral pattern, that we call mate-copying, was reported in the fruit fly Drosophila melanogaster where females choosing between 2 males of contrasting phenotypes can build a preference for males of the phenotype they previously saw being chosen by a demonstrator female. As sex ratio is known to affect mate choice, our goal was to test whether mate-copying is also affected by encountered sex ratios. Thus, we created a gradient of sex ratio during demonstrations of mate-copying experiments by changing the number of females observing from a central arena 6 simultaneous demonstrations unfolding in 6 peripheral compartments of a hexagonal device. We also tested whether the sex ratio experienced by females during demonstrations affected their choosiness (male courtship duration and double courtship rate) in subsequent mate-choice tests. Experimental male:female sex ratio during demonstrations did not affect mate-copying indices, but positively affected the proportion of both males courting the female during mate-choice tests, as well as male courtship duration, the latter potentially explaining the former relationship. As expected, the sex ratio affected female choosiness positively, and Drosophila females seem to have evolved a mate-copying ability independently of sex ratio, and a capacity to adapt their choosiness to male availability. This suggests that, as in many animal species, individuals, especially females, can adapt their mate choice depending on the current sex ratio.

Dopamine and Serotonin Are Both Required for Mate-Copying in Drosophila melanogaster.

Mate-copying is a form of social learning in which the mate-choice decision of an individual (often a female) is influenced by the mate-choice of conspecifics. Drosophila melanogaster females are known to perform such social learning, and in particular, to mate-copy after a single observation of one conspecific female mating with a male of one phenotype, while the other male phenotype is rejected. Here, we show that this form of social learning is dependent on serotonin and dopamine. Using a pharmacological approach, we reduced dopamine or serotonin synthesis in adult virgin females with 3-iodotyrosine (3-IY) and DL-para-chlorophenylalanine (PCPA), respectively, and then tested their mate-copying performance. We found that, while control females without drug treatment copied the choice of the demonstrator, drug-treated females with reduced dopamine or serotonin chose randomly. To ensure the specificity of the drugs, the direct precursors of the neurotransmitters, either the dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) or the serotonin precursor 5-L-hydroxytryptophan (5-HTP) were given together with the drug, (respectively 3-IY and PCPA) resulting in a full rescue of the mate-copying defects. This indicates that dopamine and serotonin are both required for mate-copying. These results give a first insight into the mechanistic pathway underlying this form of social learning in D. melanogaster.

Cultural flies: Conformist social learning in fruitflies predicts long-lasting mate-choice traditions.

Despite theoretical justification for the evolution of animal culture, empirical evidence for it beyond mammals and birds remains scant, and we still know little about the process of cultural inheritance. In this study, we propose a mechanism-driven definition of animal culture and test it in the fruitfly. We found that fruitflies have five cognitive capacities that enable them to transmit mating preferences culturally across generations, potentially fostering persistent traditions (the main marker of culture) in mating preference. A transmission chain experiment validates a model of the emergence of local traditions, indicating that such social transmission may lead initially neutral traits to become adaptive, hence strongly selecting for copying and conformity. Although this situation was suggested decades ago, it previously had little empirical support.

Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory.

Efficient energy use has constrained the evolution of nervous systems. However, it is unresolved whether energy metabolism may resultantly regulate major brain functions. Our observation that Drosophila flies double their sucrose intake at an early stage of long-term memory formation initiated the investigation of how energy metabolism intervenes in this process. Cellular-resolution imaging of energy metabolism reveals a concurrent elevation of energy consumption in neurons of the mushroom body, the fly's major memory centre. Strikingly, upregulation of mushroom body energy flux is both necessary and sufficient to drive long-term memory formation. This effect is triggered by a specific pair of dopaminergic neurons afferent to the mushroom bodies, via the D5-like DAMB dopamine receptor. Hence, dopamine signalling mediates an energy switch in the mushroom body that controls long-term memory encoding. Our data thus point to an instructional role for energy flux in the execution of demanding higher brain functions.

The role of cAMP response element-binding protein in Drosophila long-term memory.

In Drosophila, the transcription factor cAMP response element-binding protein 2 (dCREB2) has been reported to modulate the formation of long-term olfactory memory (LTM). Overexpression of a repressor isoform of CREB (dCREB2-b) under the control of a heat-shock promoter was reported to block LTM, whereas overexpression of an activator isoform (dCREB2-a) was reported to enhance LTM. A ratiometric model based on these results predicts that the balance of functional dCREB2-a and dCREB2-b provides a switch for memories to remain labile or to become enduring. We show here that the dCREB2-a transgene originally reported to enhance LTM carries a mutation that produces a translational reading-frame shift with the consequent formation of a stop codon at predicted amino acid position 79. Overexpression of this mutant dCREB2-a transgene or a corrected dCREB2-a transgene failed to show any enhancement of LTM. Overexpression of the dCREB2-b repressor transgene, in contrast, produced the anticipated block in LTM formation. We discuss the implications of these findings and propose an alternative model for the role of dCREB in Drosophila LTM.

GABAergic feedback signaling into the calyces of the mushroom bodies enables olfactory reversal learning in honey bees.

In reversal learning, subjects first learn to respond to a reinforced stimulus A and not to a non-reinforced stimulus B (A(+) vs. B(-)) and then have to learn the opposite when stimulus contingencies are reversed (A(-) vs. B(+)). This change in stimulus valence generates a transitory ambiguity at the level of stimulus outcome that needs to be overcome to solve the second discrimination. Honey bees (Apis mellifera) efficiently master reversal learning in the olfactory domain. The mushroom bodies (MBs), higher-order structures of the insect brain, are required to solve this task. Here we aimed at uncovering the neural circuits facilitating reversal learning in honey bees. We trained bees using the olfactory conditioning of the proboscis extension reflex (PER) coupled with localized pharmacological inhibition of Gamma-AminoButyric Acid (GABA)ergic signaling in the MBs. We show that inhibition of ionotropic but not metabotropic GABAergic signaling into the MB calyces impairs reversal learning, but leaves intact the capacity to perform two consecutive elemental olfactory discriminations with ambiguity of stimulus valence. On the contrary, inhibition of ionotropic GABAergic signaling into the MB lobes had no effect on reversal learning. Our results are thus consistent with a specific requirement of the feedback neurons (FNs) providing ionotropic GABAergic signaling from the MB lobes to the calyces for counteracting ambiguity of stimulus valence in reversal learning.

Slow oscillations in two pairs of dopaminergic neurons gate long-term memory formation in Drosophila.

A fundamental duty of any efficient memory system is to prevent long-lasting storage of poorly relevant information. However, little is known about dedicated mechanisms that appropriately trigger production of long-term memory (LTM). We examined the role of Drosophila dopaminergic neurons in the control of LTM formation and found that they act as a switch between two exclusive consolidation pathways leading to LTM or anesthesia-resistant memory (ARM). Blockade, after aversive olfactory conditioning, of three pairs of dopaminergic neurons projecting on mushroom bodies, the olfactory memory center, enhanced ARM, whereas their overactivation conversely impaired ARM. Notably, blockade of these neurons during the intertrial intervals of a spaced training precluded LTM formation. Two pairs of these dopaminergic neurons displayed sustained calcium oscillations in naive flies. Oscillations were weakened by ARM-inducing massed training and were enhanced during LTM formation. Our results indicate that oscillations of two pairs of dopaminergic neurons control ARM levels and gate LTM.

Mushroom body efferent neurons responsible for aversive olfactory memory retrieval in Drosophila.

Aversive olfactory memory is formed in the mushroom bodies in Drosophila melanogaster. Memory retrieval requires mushroom body output, but the manner in which a memory trace in the mushroom body drives conditioned avoidance of a learned odor remains unknown. To identify neurons that are involved in olfactory memory retrieval, we performed an anatomical and functional screen of defined sets of mushroom body output neurons. We found that MB-V2 neurons were essential for retrieval of both short- and long-lasting memory, but not for memory formation or memory consolidation. MB-V2 neurons are cholinergic efferent neurons that project from the mushroom body vertical lobes to the middle superiormedial protocerebrum and the lateral horn. Notably, the odor response of MB-V2 neurons was modified after conditioning. As the lateral horn has been implicated in innate responses to repellent odorants, we propose that MB-V2 neurons recruit the olfactory pathway involved in innate odor avoidance during memory retrieval.

Expression of the mu opioid receptor in Drosophila and its effects on trehalose and glycogen when expressed by the AKH neuroendocrine cells.

We made Drosophila which express the mu opioid receptor under control of UAS in order to inactivate neurons or neuroendocrine cells expressing this receptor with opioid agonists. However, while exposing flies expressing the mu opioid receptor in the SIFamide neurons to opioid agonists was expected to induce male-male courtship behavior, this did not occur. Furthermore, flies which expressed the mu opioid receptor in the AKH or corazonin endocrine cells increased rather than decreased trehalose levels and this was independent of opioid agonists. When the mu opioid receptor is expressed in AKH endocrine cells whole body glycogen also increases, which is no longer the case if the expression of the AKH gene is suppressed by RNAi. It appears that mu opioid receptors expressed in AKH or corazonin endocrine cells are constitutively active and facilitate release of neurohormones. The simultaneous increase in both glycogen and trehalose in these flies suggested that they consumed more food. Indeed, when normally fed males are offered sucrose, those that express this receptor in AKH cells consumed more sucrose, suggesting that AKH increases the motivation to feed. These pharmacological effects of the mu opioid receptor are not limited to neuroendocrine cells; expressing it in the fat body also leads to an increase in trehalose. Thus in Drosophila the mu opioid receptors appear to change the base line activity in the cells in which it is expressed, not unlike to what has been found in transgenic mice expressing receptors activated solely by synthetic ligands with significant constitutive activity.

Social facilitation of long-lasting memory retrieval in Drosophila.

Recent studies demonstrate that social interactions can have a profound influence on Drosophila melanogaster behavior and cuticular pheromone patterns. Olfactory memory performance has mostly been investigated in groups, and previous studies have reported that grouped flies do not interact with each other and behave in the same way as individual flies during short-term memory retrieval. However, the influence of social effects on the two known forms of Drosophila long-lasting associative memory, anesthesia-resistant memory (ARM) and long-term memory (LTM), has never been reported. We show here that ARM is displayed by individual flies but is socially facilitated; flies trained for ARM interact within a group to improve their conditioned performance. In contrast, testing shows LTM improvement in individual flies rather than in a group. We show that the social facilitation of ARM during group testing is independent of the social context of training and does not involve nonspecific aggregation. Furthermore, we demonstrate that social interactions facilitate ARM retrieval. We also show that social interactions necessary for this facilitation are specifically generated by trained flies: when single flies trained for ARM are mixed with groups of naive flies, they display poor retrieval, whereas mixing with groups trained either for ARM or LTM enhances performance.

AKH-producing neuroendocrine cell ablation decreases trehalose and induces behavioral changes in Drosophila.

Adipokinetic hormone (AKH) is a metabolic neuropeptide principally known for its mobilization of energy substrates, notably lipid and trehalose during energy-requiring activities, such as flight and locomotion. Drosophila melanogaster AKH cell localization in corpora cardiaca, as in other insect species, was confirmed by immunoreactivity and by a genetic approach using the UAS/GAL4 system. To assess AKH general physiological rules, we ablated AKH endocrine cells by specifically driving the expression of apoptosis transgenes in AKH cells. Trehalose levels were decreased in larvae and starved adults, when the stimulation by AKH of the production of trehalose from fat body glycogen is no longer possible. Moreover, we show that these adults without AKH cells become progressively hypoactive. Finally, under starvation conditions, those hypoactive AKH-knockout cell flies survived approximately 50% longer than control wild-type flies, suggesting that the slower rate at which AKH-ablated flies mobilize their energy resources extends their survival.