Base Recording: A Technique for Analyzing Responses of Taste Neurons in Drosophila.

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential food source, compounds from the food source enter through the pore and activate neurons within. For over 50 years, these responses have been recorded using a technique called tip recording. However, this method has major limitations, including the inability to measure neural activity before or after stimulus contact and the requirement for tastants to be soluble in aqueous solutions. We describe here a technique that we call base recording, which overcomes these limitations. Base recording allows the measurement of taste neuron activity before, during, and after the stimulus. Thus, it allows extensive analysis of OFF responses that occur after a taste stimulus. It can be used to study hydrophobic compounds such as long-chain pheromones that have very low solubility in water. In summary, base recording offers the advantages of single-sensillum electrophysiology as a means of measuring neuronal activity - high spatial and temporal resolution, without the need for genetic tools - and overcomes key limitations of the traditional tip recording technique.

The subapical labial sensory organ of spotted lanternfly Lycorma delicatula.

Deciphering how spotted lanternfly (SLF), an invasive polyphagous planthopper in North America, engages with its environment is a pressing issue with fundamental biological significance and economic importance. This interaction primarily depends on olfaction. However, the cellular basis of olfaction in SLF remains elusive. Here we investigate the neuronal and functional organization of the subapical labial sensory organ using scanning electron microscopy and electrophysiological recordings. This organ is believed to supply planthoppers with crucial sensory information that influences their subsequent feeding behaviour. We find in SLF that this organ comprises two identical placoid sensilla, each housing two distinct neurons. The A neuron displays a remarkable sensitivity to changes in airflow speed. Importantly, the same neuron also exhibits robust excitatory responses exclusively to three aldehydes out of a diverse pool of 85 tested odorants and inhibitory responses to 62 other odorants. By contrast, the B neuron solely serves as an olfactory detector, showing strong excitatory responses to 17 odorants and inhibitory responses to only three. The results provide a potential cellular basis for the behavioural responses of SLF to its ecologically relevant stimuli. Our study also identifies new odorants that may be useful for managing this serious pest.

Diverse mechanisms of taste coding in Drosophila.

Taste systems encode chemical cues that drive vital behaviors. We have elucidated noncanonical features of taste coding using an unconventional kind of electrophysiological analysis. We find that taste neurons of Drosophila are much more sensitive than previously thought. They have a low spontaneous firing frequency that depends on taste receptors. Taste neurons have a dual function as olfactory neurons: They are activated by most tested odorants, including N,N-diethyl-meta-toluamide (DEET), at a distance. DEET can also inhibit certain taste neurons, revealing that there are two modes of taste response: activation and inhibition. We characterize electrophysiological OFF responses and find that the tastants that elicit them are related in structure. OFF responses link tastant identity to behavior: the magnitude of the OFF response elicited by a tastant correlated with the egg laying behavior it elicited. In summary, the sensitivity and coding capacity of the taste system are much greater than previously known.

Hany K.M. Dweck.

Interview with Hany Dweck, who works on Drosophila chemoreception at Connecticut Agricultural Experiment Station.

Cross-generation pheromonal communication drives Drosophila oviposition site choice.

In a heterogeneous and changing environment, oviposition site selection strongly affects the survival and fitness of the offspring.1,2 Similarly, competition between larvae affects their prospects.3 However, little is known about the involvement of pheromones in regulating these processes.4,5,6,7,8 Here, we show that mated females of Drosophila melanogaster prefer to lay eggs on substrates containing extracts of conspecific larvae. After analyzing these extracts chemically, we test each compound in an oviposition assay and find that mated females display a dose-dependent preference to lay eggs on substrates spiked with (Z)-9-octadecenoic acid ethyl ester (OE). This egg-laying preference relies on gustatory receptor Gr32a and tarsal sensory neurons expressing this receptor. The concentration of OE also regulates larval place choice in a dose-dependent manner. Physiologically, OE activates female tarsal Gr32a+ neurons. In conclusion, our results reveal a cross-generation communication strategy essential for oviposition site selection and regulation of larval density.

A volatile sex attractant of tsetse flies.

Tsetse flies transmit trypanosomes-parasites that cause devastating diseases in humans and livestock-across much of sub-Saharan Africa. Chemical communication through volatile pheromones is common among insects; however, it remains unknown if and how such chemical communication occurs in tsetse flies. We identified methyl palmitoleate (MPO), methyl oleate, and methyl palmitate as compounds that are produced by the tsetse fly Glossina morsitans and elicit strong behavioral responses. MPO evoked a behavioral response in male-but not virgin female-G. morsitans. G. morsitans males mounted females of another species, Glossina fuscipes, when they were treated with MPO. We further identified a subpopulation of olfactory neurons in G. morsitans that increase their firing rate in response to MPO and showed that infecting flies with African trypanosomes alters the flies' chemical profile and mating behavior. The identification of volatile attractants in tsetse flies may be useful for reducing disease spread.

Sugar sensation and mechanosensation in the egg-laying preference shift of Drosophila suzukii.

The agricultural pest Drosophila suzukii differs from most other Drosophila species in that it lays eggs in ripe, rather than overripe, fruit. Previously, we showed that changes in bitter taste sensation accompanied this adaptation (Dweck et al., 2021). Here, we show that D. suzukii has also undergone a variety of changes in sweet taste sensation. D. suzukii has a weaker preference than Drosophila melanogaster for laying eggs on substrates containing all three primary fruit sugars: sucrose, fructose, and glucose. Major subsets of D. suzukii taste sensilla have lost electrophysiological responses to sugars. Expression of several key sugar receptor genes is reduced in the taste organs of D. suzukii. By contrast, certain mechanosensory channel genes, including no mechanoreceptor potential C, are expressed at higher levels in the taste organs of D. suzukii, which has a higher preference for stiff substrates. Finally, we find that D. suzukii responds differently from D. melanogaster to combinations of sweet and mechanosensory cues. Thus, the two species differ in sweet sensation, mechanosensation, and their integration, which are all likely to contribute to the differences in their egg-laying preferences in nature.

Ir56b is an atypical ionotropic receptor that underlies appetitive salt response in Drosophila.

Salt taste is one of the most ancient of all sensory modalities. However, the molecular basis of salt taste remains unclear in invertebrates. Here, we show that the response to low, appetitive salt concentrations in Drosophila depends on Ir56b, an atypical member of the ionotropic receptor (Ir) family. Ir56b acts in concert with two coreceptors, Ir25a and Ir76b. Mutation of Ir56b virtually eliminates an appetitive behavioral response to salt. Ir56b is expressed in neurons that also sense sugars via members of the Gr (gustatory receptor) family. Misexpression of Ir56b in bitter-sensing neurons confers physiological responses to appetitive doses of salt. Ir56b is unique among tuning Irs in containing virtually no N-terminal region, a feature that is evolutionarily conserved. Moreover, Ir56b is a "pseudo-pseudogene": its coding sequence contains a premature stop codon that can be replaced with a sense codon without loss of function. This stop codon is conserved among many Drosophila species but is absent in a number of species associated with cactus in arid regions. Thus, Ir56b serves the evolutionarily ancient function of salt detection in neurons that underlie both salt and sweet taste modalities.

Larval sites of the mosquito Aedes aegypti formosus in forest and domestic habitats in Africa and the potential association with oviposition evolution.

Adaptations to anthropogenic domestic habitats contribute to the success of the mosquito Aedes aegypti as a major global vector of several arboviral diseases. The species inhabited African forests before expanding into domestic habitats and spreading to other continents. Despite a well-studied evolutionary history, how this species initially moved into human settlements in Africa remains unclear. During this initial habitat transition, African Ae. aegypti switched their larval sites from natural water containers like tree holes to artificial containers like clay pots. Little is known about how these natural versus artificial containers differ in their characteristics. Filling this knowledge gap could provide valuable information for studying the evolution of Ae. aegypti associated with larval habitat changes. As an initial effort, in this study, we characterized the microenvironments of Ae. aegypti larval sites in forest and domestic habitats in two African localities: La Lopé, Gabon, and Rabai, Kenya. Specifically, we measured the physical characteristics, microbial density, bacterial composition, and volatile chemical profiles of multiple larval sites. In both localities, comparisons between natural containers in the forests and artificial containers in the villages revealed significantly different microenvironments. We next examined whether the between-habitat differences in larval site microenvironments lead to differences in oviposition, a key behavior affecting larval distribution. Forest Ae. aegypti readily accepted the artificial containers we placed in the forests. Laboratory choice experiments also did not find distinct oviposition preferences between forest and village Ae. aegypti colonies. These results suggested that African Ae. aegypti are likely generalists in their larval site choices. This flexibility to accept various containers with a wide range of physical, microbial, and chemical conditions might allow Ae. aegypti to use human-stored water as fallback larval sites during dry seasons, which is hypothesized to have initiated the domestic evolution of Ae. aegypti.

Large-scale characterization of sex pheromone communication systems in Drosophila.

Insects use sex pheromones as a reproductive isolating mechanism to attract conspecifics and repel heterospecifics. Despite the profound knowledge of sex pheromones, little is known about the coevolutionary mechanisms and constraints on their production and detection. Using whole-genome sequences to infer the kinship among 99 drosophilids, we investigate how phylogenetic and chemical traits have interacted at a wide evolutionary timescale. Through a series of chemical syntheses and electrophysiological recordings, we identify 52 sex-specific compounds, many of which are detected via olfaction. Behavioral analyses reveal that many of the 43 male-specific compounds are transferred to the female during copulation and mediate female receptivity and/or male courtship inhibition. Measurement of phylogenetic signals demonstrates that sex pheromones and their cognate olfactory channels evolve rapidly and independently over evolutionary time to guarantee efficient intra- and inter-specific communication systems. Our results show how sexual isolation barriers between species can be reinforced by species-specific olfactory signals.

Mate discrimination among subspecies through a conserved olfactory pathway.

Communication mechanisms underlying the sexual isolation of species are poorly understood. Using four subspecies of Drosophila mojavensis as a model, we identify two behaviorally active, male-specific pheromones. One functions as a conserved male antiaphrodisiac in all subspecies and acts via gustation. The second induces female receptivity via olfaction exclusively in the two subspecies that produce it. Genetic analysis of the cognate receptor for the olfactory pheromone indicates an important role for this sensory pathway in promoting sexual isolation of subspecies, in combination with auditory signals. Unexpectedly, the peripheral sensory pathway detecting this pheromone is conserved molecularly, physiologically, and anatomically across subspecies. These observations imply that subspecies-specific behaviors arise from differential interpretation of the same peripheral cue, reminiscent of sexually conserved detection but dimorphic interpretation of male pheromones in Drosophila melanogaster. Our results reveal that, during incipient speciation, pheromone production, detection, and interpretation do not necessarily evolve in a coordinated manner.

Molecular Logic and Evolution of Bitter Taste in Drosophila.

Taste systems detect a vast diversity of toxins, which are perceived as bitter. When a species adapts to a new environment, its taste system must adapt to detect new death threats. We deleted each of six commonly expressed bitter gustatory receptors (Grs) from Drosophila melanogaster. Systematic analysis revealed that requirements for these Grs differed for the same tastant in different neurons and for different tastants in the same neuron. Responses to some tastants in some neurons required four Grs, including Gr39a. Deletions also produced increased or novel responses, supporting a model of Gr-Gr inhibitory interactions. Coexpression of four Grs conferred several bitter responses to a sugar neuron. We then examined bitter coding in three other Drosophila species. We found major evolutionary shifts. One shift depended on the concerted activity of seven Grs. This work shows how the complex logic of bitter coding provides the capacity to detect innumerable hazards and the flexibility to adapt to new ones.

The Olfactory Logic behind Fruit Odor Preferences in Larval and Adult Drosophila.

Despite the comprehensive knowledge on odor coding, our understanding of the relationship between sensory input and behavioral output in Drosophila remains weak. Here, we measure the behavioral responses generated by larval and adult flies in response to 34 fruit odors and find that larval preference for fruit odors differs from that of adult flies. Next, we provide a functional analysis of the full repertoire of the peripheral olfactory system using the same comprehensive stimulus spectrum. We find that 90% and 53% of larval and adult olfactory receptors tested here, respectively, are involved in evaluating these fruit odors. Finally, we find that the total amount of olfactory neuronal activity correlates strongly positively with behavioral output in larvae and correlates weakly negatively in adult flies. Our results suggest that larval and adult flies have evolved different mechanisms for detection and computation of fruit odors, mechanisms likely mirroring the different lifestyles of both developmental stages.

A Drosophila female pheromone elicits species-specific long-range attraction via an olfactory channel with dual specificity for sex and food.

BACKGROUND: Mate finding and recognition in animals evolves during niche adaptation and involves social signals and habitat cues. Drosophila melanogaster and related species are known to be attracted to fermenting fruit for feeding and egg-laying, which poses the question of whether species-specific fly odours contribute to long-range premating communication. RESULTS: We have discovered an olfactory channel in D. melanogaster with a dual affinity to sex and food odorants. Female flies release a pheromone, (Z)-4-undecenal (Z4-11Al), that elicits flight attraction in both sexes. Its biosynthetic precursor is the cuticular hydrocarbon (Z,Z)-7,11-heptacosadiene (7,11-HD), which is known to afford reproductive isolation between the sibling species D. melanogaster and D. simulans during courtship. Twin olfactory receptors, Or69aB and Or69aA, are tuned to Z4-11Al and food odorants, respectively. They are co-expressed in the same olfactory sensory neurons, and feed into a neural circuit mediating species-specific, long-range communication; however, the close relative D. simulans, which shares food resources with D. melanogaster, does not respond to Z4-11Al. CONCLUSION: The Or69aA and Or69aB isoforms have adopted dual olfactory traits. The underlying gene yields a collaboration between natural and sexual selection, which has the potential to drive speciation.

Olfactory coding from the periphery to higher brain centers in the Drosophila brain.

BACKGROUND: Odor information is processed through multiple receptor-glomerular channels in the first order olfactory center, the antennal lobe (AL), then reformatted into higher brain centers and eventually perceived by the fly. To reveal the logic of olfaction, it is fundamental to map odor representations from the glomerular channels into higher brain centers. RESULTS: We characterize odor response profiles of AL projection neurons (PNs) originating from 31 glomeruli using whole cell patch-clamp recordings in Drosophila melanogaster. We reveal that odor representation from olfactory sensory neurons to PNs is generally conserved, while transformation of odor tuning curves is glomerulus-dependent. Reconstructions of PNs reveal that attractive and aversive odors are represented in different clusters of glomeruli in the AL. These separate representations are preserved into higher brain centers, where attractive and aversive odors are segregated into two regions in the lateral horn and partly separated in the mushroom body calyx. CONCLUSIONS: Our study reveals spatial representation of odor valence coding from the AL to higher brain centers. These results provide a global picture of the olfactory circuit design underlying innate odor-guided behavior.

Elucidating the Neuronal Architecture of Olfactory Glomeruli in the Drosophila Antennal Lobe.

Olfactory glomeruli are morphologically conserved spherical compartments of the olfactory system, distinguishable solely by their chemosensory repertoire, anatomical position, and volume. Little is known, however, about their numerical neuronal composition. We therefore characterized their neuronal architecture and correlated these anatomical features with their functional properties in Drosophila melanogaster. We quantitatively mapped all olfactory sensory neurons (OSNs) innervating each glomerulus, including sexually dimorphic distributions. Our data reveal the impact of OSN number on glomerular dimensions and demonstrate yet unknown sex-specific differences in several glomeruli. Moreover, we quantified uniglomerular projection neurons for each glomerulus, which unraveled a glomerulus-specific numerical innervation. Correlation between morphological features and functional specificity showed that glomeruli innervated by narrowly tuned OSNs seem to possess a larger number of projection neurons and are involved in less lateral processing than glomeruli targeted by broadly tuned OSNs. Our study demonstrates that the neuronal architecture of each glomerulus encoding crucial odors is unique.

Activation of Latent Courtship Circuitry in the Brain of Drosophila Females Induces Male-like Behaviors.

Courtship in Drosophila melanogaster offers a powerful experimental paradigm for the study of innate sexually dimorphic behaviors [1, 2]. Fruit fly males exhibit an elaborate courtship display toward a potential mate [1, 2]. Females never actively court males, but their response to the male's display determines whether mating will actually occur. Sex-specific behaviors are hardwired into the nervous system via the actions of the sex determination genes doublesex (dsx) and fruitless (fru) [1]. Activation of male-specific dsx/fru(+) P1 neurons in the brain initiates the male's courtship display [3, 4], suggesting that neurons unique to males trigger this sex-specific behavior. In females, dsx(+) neurons play a pivotal role in sexual receptivity and post-mating behaviors [1, 2, 5-9]. Yet it is still unclear how dsx(+) neurons and dimorphisms in these circuits give rise to the different behaviors displayed by males and females. Here, we manipulated the function of dsx(+) neurons in the female brain to investigate higher-order neurons that drive female behaviors. Surprisingly, we found that activation of female dsx(+) neurons in the brain induces females to behave like males by promoting male-typical courtship behaviors. Activated females display courtship toward conspecific males or females, as well other Drosophila species. We uncovered specific dsx(+) neurons critical for driving male courtship and identified pheromones that trigger such behaviors in activated females. While male courtship behavior was thought to arise from male-specific central neurons, our study shows that the female brain is equipped with latent courtship circuitry capable of inducing this male-specific behavioral program.

Drosophila Avoids Parasitoids by Sensing Their Semiochemicals via a Dedicated Olfactory Circuit.

Detecting danger is one of the foremost tasks for a neural system. Larval parasitoids constitute clear danger to Drosophila, as up to 80% of fly larvae become parasitized in nature. We show that Drosophila melanogaster larvae and adults avoid sites smelling of the main parasitoid enemies, Leptopilina wasps. This avoidance is mediated via a highly specific olfactory sensory neuron (OSN) type. While the larval OSN expresses the olfactory receptor Or49a and is tuned to the Leptopilina odor iridomyrmecin, the adult expresses both Or49a and Or85f and in addition detects the wasp odors actinidine and nepetalactol. The information is transferred via projection neurons to a specific part of the lateral horn known to be involved in mediating avoidance. Drosophila has thus developed a dedicated circuit to detect a life-threatening enemy based on the smell of its semiochemicals. Such an enemy-detecting olfactory circuit has earlier only been characterized in mice and nematodes.

Pheromones mediating copulation and attraction in Drosophila.

Intraspecific olfactory signals known as pheromones play important roles in insect mating systems. In the model Drosophila melanogaster, a key part of the pheromone-detecting system has remained enigmatic through many years of research in terms of both its behavioral significance and its activating ligands. Here we show that Or47b-and Or88a-expressing olfactory sensory neurons (OSNs) detect the fly-produced odorants methyl laurate (ML), methyl myristate, and methyl palmitate. Fruitless (fru(M))-positive Or47b-expressing OSNs detect ML exclusively, and Or47b- and Or47b-expressing OSNs are required for optimal male copulation behavior. In addition, activation of Or47b-expressing OSNs in the male is sufficient to provide a competitive mating advantage. We further find that the vigorous male courtship displayed toward oenocyte-less flies is attributed to an oenocyte-independent sustained production of the Or47b ligand, ML. In addition, we reveal that Or88a-expressing OSNs respond to all three compounds, and that these neurons are necessary and sufficient for attraction behavior in both males and females. Beyond the OSN level, information regarding the three fly odorants is transferred from the antennal lobe to higher brain centers in two dedicated neural lines. Finally, we find that both Or47b- and Or88a-based systems and their ligands are remarkably conserved over a number of drosophilid species. Taken together, our results close a significant gap in the understanding of the olfactory background to Drosophila mating and attraction behavior; while reproductive isolation barriers between species are created mainly by species-specific signals, the mating enhancing signal in several Drosophila species is conserved.