Which Sugar to Take and How Much to Take? Two Distinct Decisions Mediated by Separate Sensory Channels.

In Drosophila melanogaster, gustatory receptor neurons (GRNs) for sugar taste coexpress various combinations of gustatory receptor (Gr) genes and are found in multiple sites in the body. To determine whether diverse sugar GRNs expressing different combinations of Grs have distinct behavioral roles, we examined the effects on feeding behavior of genetic manipulations which promote or suppress functions of GRNs that express either or both of the sugar receptor genesGr5a (Gr5a+ GRNs) and Gr61a (Gr61a+ GRNs). Cell-population-specific overexpression of the wild-type form of Gr5a (Gr5a+ ) in the Gr5a mutant background revealed that Gr61a+ GRNs localized on the legs and internal mouthpart critically contribute to food choice but not to meal size decisions, while Gr5a+ GRNs, which are broadly expressed in many sugar-responsive cells across the body with an enrichment in the labella, are involved in both food choice and meal size decisions. The legs harbor two classes of Gr61a expressing GRNs, one with Gr5a expression (Gr5a+/Gr61a+ GRNs) and the other without Gr5aexpression (Gr5a-/Gr61a+ GRNs). We found that blocking the Gr5a+ class in the entire body reduced the preference for trehalose and blocking the Gr5a- class reduced the preference for fructose. These two subsets of GRNsare also different in their central projections: axons of tarsal Gr5a+/Gr61a+ GRNs terminate exclusively in the ventral nerve cord, while some axons of tarsal Gr5a-/Gr61a+ GRNs ascend through the cervical connectives to terminate in the subesophageal ganglion. We propose that tarsal Gr5a+/Gr61a+ GRNs and Gr5a-/Gr61a+ GRNs represent functionally distinct sensory pathways that function differently in food preference and meal-size decisions.

Optogenetic Activation of the fruitless-Labeled Circuitry in Drosophila subobscura Males Induces Mating Motor Acts.

It remains an enigma how the nervous system of different animal species produces different behaviors. We studied the neural circuitry for mating behavior in Drosophila subobscura, a species that displays unique courtship actions not shared by other members of the genera including the genetic model D. melanogaster, in which the core courtship circuitry has been identified. We disrupted the D. subobscura fruitless (fru) gene, a master regulator for the courtship circuitry formation in D. melanogaster, resulting in complete loss of mating behavior. We also generated frusoChrimV , which expresses the optogenetic activator Chrimson fused with a fluorescent marker under the native fru promoter. The fru-labeled circuitry in D. subobscura visualized by frusoChrimV revealed differences between females and males, optogenetic activation of which in males induced mating behavior including attempted copulation. These findings provide a substrate for neurogenetic dissection and manipulation of behavior in non-model animals, and will help to elucidate the neural basis for behavioral diversification.SIGNIFICANCE STATEMENT How did behavioral specificity arise during evolution? Here we attempted to address this question by comparing the parallel genetically definable neural circuits controlling the courtship behavior of Drosophila melanogaster, a genetic model, and its relative, D. subobscura, which exhibits a courtship behavioral pattern unique to it, including nuptial gift transfer. We found that the subobscura fruitless circuit, which is required for male courtship behavior, was slightly but clearly different from its melanogaster counterpart, and that optogenetic activation of this circuit induced subobscura-specific behavior, i.e., regurgitating crop contents, a key element of transfer of nuptial gift. Our study will pave the way for determining how and which distinctive cellular elements within the fruitless circuit determine the species-specific differences in courtship behavior.

Quantitative analysis of visually induced courtship elements in Drosophila subobscura.

We developed a new paradigm for quantitative analysis of courtship behavior in flies, Fly Motion-detector with an Actuator-Coupled Stimulator (FlyMacs), in which the stimulation of a fly with a moving visual target and recording of induced behaviors are automated under computer control. We employ FlyMacs for the identification of motion features that trigger specific courtship elements in Drosophila subobscura, whose mating is suggested to be strongly vision dependent. A female abdomen attached to the actuator, when moved in an appropriate pattern, evokes in the test male tapping-like foreleg motions, midleg swing and proboscis extension, which are considered to be elementary actions in male courtship behavior. Tapping is primarily induced when the target is moving, whereas midleg swing and proboscis extension are most frequently observed after the target stops moving. In contrast to midleg swing, which tends to occur immediately after target cessation (∼3000 ms), the incidence of proboscis extension gradually increases with time after target cessation, reaching a plateau at 3000 ms. The results suggest that tapping, midleg swing and proboscis extension are each induced by different movement features of the visual target. These findings do not support the view that a single key stimulus induces the entire courtship ritual. Rather, courtship behaviors in D. subobscura are correlated with movement and position of the target, which suggests that D. subobscura uses sensory information to pattern its courtship.

What does the fruitless gene tell us about nature vs. nurture in the sex life of Drosophila?

The fruitless (fru) gene in Drosophila has been proposed to play a master regulator role in the formation of neural circuitries for male courtship behavior, which is typically considered to be an innate behavior composed of a fixed action pattern as generated by the central pattern generator. However, recent studies have shed light on experience-dependent changes and sensory-input-guided plasticity in courtship behavior. For example, enhanced male-male courtship, a fru mutant "hallmark," disappears when fru-mutant males are raised in isolation. The fact that neural fru expression is induced by neural activities in the adult invites the supposition that Fru as a chromatin regulator mediates experience-dependent epigenetic modification, which underlies the neural and behavioral plasticity.

Visually induced initiation of Drosophila innate courtship-like following pursuit is mediated by central excitatory state.

The courtship ritual of male Drosophila represents an innate behaviour that is initiated by female-derived sensory stimuli. Here we report that moving light spots can induce courtship-like following pursuit in tethered wild-type male flies provided the fly is primed by optogenetic stimulation of specific dsx-expressing neuronal clusters in the lateral protocerebrum (LPR). Namely, stimulation of the pC1 neuronal cluster initiates unilateral wing extension and vibration of both sides, whereas stimulation of the pC2l cluster initiates only contralateral wing displays. In addition, stimulation of pC2l but not pC1 neurons induced abdominal bending and proboscis extension. Ca(2+) imaging of the pC1 cluster revealed periodic Ca(2+) rises, each corresponding to a turn of the male fly during courtship. In contrast, group-reared fru mutant males exhibit light spot-induced courtship pursuit without optogenetic priming. Ca(2+) imaging revealed enhanced responses of LPR neurons to visual stimuli in the mutants, suggesting a neural correlate of the light spot-induced courtship behaviour.

Insect pheromone behavior: fruit fly.

The amenability to genetics of Drosophila melanogaster has made this organism one of the best-suited models for studying the neurobiology of pheromone-guided behavior. Single-male assays use the minigene encoding the thermosensitive channel dTrpA1 to activate neurons expressing fruitless (fru), a major courtship regulator gene, and thereby induce most of the elementary courtship acts in a solitary male exposed to temperature increase. Tethered male assays allow Ca(2+)-imaging of neuronal activities of a male fly displaying courtship behavior on a treadmill when stimulated with a female or pheromones. Here we describe technical details of these assays.

Female contact activates male-specific interneurons that trigger stereotypic courtship behavior in Drosophila.

We determined the cellular substrate for male courtship behavior by quasinatural and artificial stimulation of brain neurons. Activation of fruitless (fru)-expressing neurons via stimulation of thermosensitive dTrpA1 channels induced an entire series of courtship acts in male Drosophila placed alone without any courting target. By reducing the number of neurons expressing dTrpA1 by MARCM, we demonstrated that the initiation of courtship behavior is significantly correlated with the activation of the transmidline P1 interneurons, the descending P2b interneurons, or both, indicating that these interneurons trigger courtship. Using an experimental paradigm in which a tethered male can be stimulated to initiate courtship by touching his foreleg tarsus to a female's abdomen, we found that P1 neurites of tethered males showed a transient Ca(2+) rise after tarsal stimulation with the female-associated sensory cues. These observations strongly suggest that P1 neurons are the prime components of the neural circuitry that initiates male courtship.

Gsalpha is involved in sugar perception in Drosophila melanogaster.

In Drosophila melanogaster, gustatory receptor genes (Grs) encode G-protein-coupled receptors (GPCRs) in gustatory receptor neurons (GRNs) and some olfactory receptor neurons. One of the Gr genes, Gr5a, encodes a sugar receptor that is expressed in a subset of GRNs and has been most extensively studied both molecularly and physiologically, but the G-protein alpha subunit (Galpha) that is coupled to this sugar receptor remains unknown. Here, we propose that Gs is the Galpha that is responsible for Gr5a-mediated sugar-taste transduction, based on the following findings: First, immunoreactivities against Gs were detected in a subset of GRNs including all Gr5a-expressing neurons. Second, trehalose-intake is reduced in flies heterozygous for null mutations in DGsalpha, a homolog of mammalian Gs, and trehalose-induced electrical activities in sugar-sensitive GRNs were depressed in those flies. Furthermore, expression of wild-type DGsalpha in sugar-sensitive GRNs in heterozygotic DGsalpha mutant flies rescued those impairments. Third, expression of double-stranded RNA for DGsalpha in sugar-sensitive GRNs depressed both behavioral and electrophysiological responses to trehalose. Together, these findings indicate that DGsalpha is involved in trehalose perception. We suggest that sugar-taste signals are processed through the Gsalpha-mediating signal transduction pathway in sugar-sensitive GRNs in Drosophila.