Molecular identification of the first SIFamide receptor

SIFamide is the short name and also the C terminus of the Drosophila neuropeptide AYRKPPFNGSIFamide. SIFamide has been isolated or predicted from various insects and crustaceans, and appears to be extremely well conserved among these arthropods. However, the function of this neuropeptide is still enigmatic. Here, we have identified the Drosophila gene (CG10823) coding for the SIFamide receptor. When expressed in Chinese hamster ovary cells, the receptor is only activated by Drosophila SIFamide (EC(50), 2x10(-8)M) and not by a library of 32 other insect neuropeptides and eight biogenic amines. Database searches revealed SIFamide receptor orthologues in the genomes from the malaria mosquito Anopheles gambiae, the silkworm Bombyx mori, the red flour beetle Tribolium castaneum, and the honey bee Apis mellifera. An alignment of the five insect SIFamide or SIFamide-like receptors showed, again, an impressive sequence conservation (67-77% amino acid sequence identities between the seven-transmembrane areas; 82-87% sequence similarities). The identification of well-conserved SIFamide receptor orthologues in all other insects with a sequenced genome, suggests that the SIFamide/receptor couple must have an essential function in arthropods. This paper is the first report on the identification of a SIFamide receptor.     

The hector G-protein coupled receptor is required in a subset of fruitless neurons for male courtship behavior

Male courtship behavior in Drosophila melanogaster is controlled by two main regulators, fruitless (fru) and doublesex (dsx). Their sex-specific expression in brain neurons has been characterized in detail, but little is known about the downstream targets of the sex-specific FRU and DSX proteins and how they specify the function of these neurons. While sexual dimorphism in the number and connections of fru and dsx expressing neurons has been observed, a majority of the neurons that express the two regulators are present in both sexes. This poses the question which molecules define the sex-specific function of these neurons. Signaling molecules are likely to play a significant role. We have identified a predicted G-protein coupled receptor (GPCR), CG4395, that is required for male courtship behavior. The courtship defect in the mutants can be rescued by expression of the wildtype protein in fru neurons of adult males. The GPCR is expressed in a subset of fru-positive antennal glomeruli that have previously been shown to be essential for male courtship. Expression of 4395-RNAi in GH146 projection neurons lowers courtship. This suggests that signaling through the CG4395 GPCR in this subset of fru neurons is critical for male courtship behavior.     

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.     

Drosophila TRPA channel painless inhibits male-male courtship behavior through modulating olfactory sensation

The Drosophila melanogaster TRPA family member painless, expressed in a subset of multidendritic neurons embeding in the larval epidermis, is necessary for larval nociception of noxious heat or mechanical stimuli. However, the function of painless in adult flies remains largely unknown. Here we report that mutation of painless leads to a defect in male-male courtship behavior and alteration in olfaction sensitivity in adult flies. Specific downregulation of the expression of the Painless protein in the olfactory projection neurons (PNs) of the antennal lobes (ALs) resulted in a phenotype resembling that found in painless mutant flies, whereas overexpression of Painless in PNs of painless mutant males suppressed male-male courtship behavior. The downregulation of Painless exclusively during adulthood also resulted in male-male courtship behavior. In addition, mutation of the painless gene in flies caused changes in olfaction, suggesting a role for this gene in olfactory processing. These results indicate that functions of painless in the adult central nervous system of Drosophila include modulation of olfactory processing and inhibition of male-male courtship behavior.     

Drosophila female precopulatory behavior is modulated by ecdysteroids

The effect of ecdysteroid signaling on Drosophila female precopulatory behavior was investigated using two types of mutants with either globally reduced ecdysteroid availability or reduced expression of ecdysone receptors in fruitless neurons, known to control sexual behavior. While being courted by males, mutant females performed significantly less full ovipositor extrusion behavior to reject male copulation attempts. Ecdysteroid depleted females (ecdysoneless(1)) performed male-like courtship behaviors, including unilateral wing extension and song production with patterns very similar to male courtship song. These results support the hypothesis that ecdysteroids modulate female sexual behavior, perhaps acting as a regulator of sexual motivation, and as a component affecting the performance of sex specific behavior patterns.     

Turning males on: activation of male courtship behavior in Drosophila melanogaster

The innate sexual behaviors of Drosophila melanogaster males are an attractive system for elucidating how complex behavior patterns are generated. The potential for male sexual behavior in D. melanogaster is specified by the fruitless (fru) and doublesex (dsx) sex regulatory genes. We used the temperature-sensitive activator dTRPA1 to probe the roles of fru(M)- and dsx-expressing neurons in male courtship behaviors. Almost all steps of courtship, from courtship song to ejaculation, can be induced at very high levels through activation of either all fru(M) or all dsx neurons in solitary males. Detailed characterizations reveal different roles for fru(M) and dsx in male courtship. Surprisingly, the system for mate discrimination still works well when all dsx neurons are activated, but is impaired when all fru(M) neurons are activated. Most strikingly, we provide evidence for a fru(M)-independent courtship pathway that is primarily vision dependent.     

Octopamine neuromodulatory effects on a social behavior decision-making network in Drosophila males

Situations requiring rapid decision-making in response to dynamic environmental demands occur repeatedly in natural environments. Neuromodulation can offer important flexibility to the output of neural networks in coping with changing conditions, but the contribution of individual neuromodulatory neurons in social behavior networks remains relatively unknown. Here we manipulate the Drosophila octopaminergic system and assay changes in adult male decision-making in courtship and aggression paradigms. When the functional state of OA neural circuits is enhanced, males exhibit elevated courtship behavior towards other males in both behavioral contexts. Eliminating the expression of the male form of the neural sex determination factor, Fruitless (Fru(M)), in three OA suboesophageal ganglia (SOG) neurons also leads to increased male-male courtship behavior in these same contexts. We analyzed the fine anatomical structure through confocal examination of labeled single neurons to determine the arborization patterns of each of the three Fru(M)-positive OA SOG neurons. These neurons send processes that display mirror symmetric, widely distributed arbors of endings within brain regions including the ventrolateral protocerebra, the SOG and the peri-esophageal complex. The results suggest that a small subset of OA neurons have the potential to provide male selective modulation of behavior at a single neuron level.     

Drosophila retained/dead ringer is necessary for neuronal pathfinding, female receptivity and repression of fruitless independent male courtship behaviors

Mutations in the Drosophila retained/dead ringer (retn) gene lead to female behavioral defects and alter a limited set of neurons in the CNS. retn is implicated as a major repressor of male courtship behavior in the absence of the fruitless (fru) male protein. retn females show fru-independent male-like courtship of males and females, and are highly resistant to courtship by males. Males mutant for retn court with normal parameters, although feminization of retn cells in males induces bisexuality. Alternatively spliced RNAs appear in the larval and pupal CNS, but none shows sex specificity. Post-embryonically, retn RNAs are expressed in a limited set of neurons in the CNS and eyes. Neural defects of retn mutant cells include mushroom body beta-lobe fusion and pathfinding errors by photoreceptor and subesophageal neurons. We posit that some of these retn-expressing cells function to repress a male behavioral pathway activated by fruM.     

Functional significance of the copper transporter ATP7 in peptidergic neurons and endocrine cells in Drosophila melanogaster

The Drosophila ATP7 copper transporter has sequence homology to the human copper transporters ATP7A and ATP7B, which are defective in Menkes and Wilson disease, respectively. We show here that in Drosophila ATP7 is expressed by many peptidergic neurons. As C-terminal amidation of neuropeptides depends on the copper-containing enzyme PHM, it seemed likely that in the absence of ATP7 the activity of PHM might be compromised. Indeed, inhibition of ATP7 expression by RNAi led to a decrease in mature amidated neuropeptides and the appearance of C-terminally Gly-extended neuropeptides. The strength of this effect differed from one cell type to another; it was very pronounced for AKH and corazonin, but much less so for SIFamide and myosuppressin. Nevertheless, down-regulation of ATP7 specifically in the SIFamide-expressing neurons resulted in male-male courtship behavior.     

Neural circuitry that governs Drosophila male courtship behavior

Male-specific fruitless (fru) products (Fru(M)) are both necessary and sufficient to "hardwire" the potential for male courtship behavior into the Drosophila nervous system. Fru(M) is expressed in approximately 2% of neurons in the male nervous system, but not in the female. We have targeted the insertion of GAL4 into the fru locus, allowing us to visualize and manipulate the Fru(M)-expressing neurons in the male as well as their counterparts in the female. We present evidence that these neurons are directly and specifically involved in male courtship behavior and that at least some of them are interconnected in a circuit. This circuit includes olfactory neurons required for the behavioral response to sex pheromones. Anatomical differences in this circuit that might account for the dramatic differences in male and female sexual behavior are not apparent.     

SIFamide and SIFamide Receptor Define a Novel Neuropeptide Signaling to Promote Sleep in Drosophila

SIFamide receptor (SIFR) is a Drosophila G protein-coupled receptor for the neuropeptide SIFamide (SIFa). Although the sequence and spatial expression of SIFa are evolutionarily conserved among insect species, the physiological function of SIFa/SIFR signaling remains elusive. Here, we provide genetic evidence that SIFa and SIFR promote sleep in Drosophila. Either genetic ablation of SIFa-expressing neurons in the pars intercerebralis (PI) or pan-neuronal depletion of SIFa expression shortened baseline sleep and reduced sleep-bout length, suggesting that it caused sleep fragmentation. Consistently, RNA interference-mediated knockdown of SIFR expression caused short sleep phenotypes as observed in SIFa-ablated or depleted flies. Using a panel of neuron-specific Gal4 drivers, we further mapped SIFR effects to subsets of PI neurons. Taken together, these results reveal a novel physiological role of the neuropeptide SIFa/SIFR pathway to regulate sleep through sleep-promoting neural circuits in the PI of adult fly brains.     

Diversification of takeout, a male-biased gene family in Drosophila

The display of courtship behavior has evolved in response to sexual selection driven by competition to obtain mates. Sexually dimorphic mate selection rituals are likely controlled at least in part by genes with sex-biased patterns of expression. In Drosophila melanogaster, male courtship behavior has been well described and consists of a series of stereotyped behaviors. The takeout gene is predominantly expressed in males and affects male courtship behavior. In this study, we examine the patterns of expression and evolution in takeout and the family of related proteins. We show that a number of genes in the takeout gene family show male-biased expression in D. melanogaster, largely in non-reproductive tissues. Phylogenetic analysis reveals that this gene family is conserved across insects. As expected for genes with male-biased expression, we also find evidence of positive selection in some lineages. Our results suggest that the genes in this family may have evolutionarily conserved sex specific roles in male mating behavior across insects.     

Differential roles of two major brain structures, mushroom bodies and central complex, for Drosophila male courtship behavior

Drosophila male courtship is a complex and robust behavior, the potential for which is genetically built into specific neural circuits in the central nervous system. Previous studies using male-female mosaics and the flies with defects in particular brain structures implicated the critical central regions involved in male courtship behavior. However, their acute physiological roles in courtship regulation still largely remain unknown. Using the temperature-sensitive Dynamin mutation, shibire(ts1), here we demonstrate the significance of two major brain structures, the mushroom bodies and the central complex, in experience-independent aspects of male courtship. We show that blocking of synaptic transmission in the mushroom body intrinsic neurons significantly delays courtship initiation and reduces the courtship activity by shortening the courtship bout length when virgin females are used as a sexual target. Interestingly, however, the same treatment affects neither initiation nor maintenance of courtship toward young males that release courtship-stimulating pheromones different from those of virgin females. In contrast, blocking of synaptic transmission in a central complex substructure, the fan-shaped body, slightly but significantly reduces courtship activity toward both virgin females and young males with little effect on courtship initiation. Taken together, our results indicate that the neuronal activity in the mushroom bodies plays an important role in responding to female-specific sex pheromones that stimulate initiation and maintenance of male courtship behavior, whereas the fan-shaped body neurons are involved in maintenance of male courtship regardless of the nature of courtship-stimulating cues.     

Mushroom bodies are not required for courtship behavior by normal and sexually mosaic Drosophila

To elucidate the effect of feminization of male Drosophila brain cells on courtship control, we performed a large scale screening of expression drivers that can suppress male-specific behavior with transformer gene expression. Two drivers caused essentially total courtship suppression. The expression pattern of these drivers did not show any correlation with the mushroom bodies or the antennal lobes, the regions that have been suggested to play important roles in courtship. Ablation of mushroom bodies using hydroxyurea treatment did not affect this courtship suppression. The ablation did not change either wild-type heterosexual behavior or bisexual behavior caused by transformer expression driven by the same drivers used in the previous studies to suggest the involvement of the mushroom bodies in courtship. Our results show that feminization of different nonoverlapping cells in other parts of the protocerebrum was sufficient to cause the same bisexual or suppressed-courtship phenotype. Thus, contrary to previous assumptions, the mushroom bodies are not required for the control of courtship. Present evidence supports its mediation by other distributed protocerebral regions.     

Pheromonal and behavioral cues trigger male-to-female aggression in Drosophila

Appropriate displays of aggression rely on the ability to recognize potential competitors. As in most species, Drosophila males fight with other males and do not attack females. In insects, sex recognition is strongly dependent on chemosensory communication, mediated by cuticular hydrocarbons acting as pheromones. While the roles of chemical and other sensory cues in stimulating male to female courtship have been well characterized in Drosophila, the signals that elicit aggression remain unclear. Here we show that when female pheromones or behavior are masculinized, males recognize females as competitors and switch from courtship to aggression. To masculinize female pheromones, a transgene carrying dsRNA for the sex determination factor transformer (traIR) was targeted to the pheromone producing cells, the oenocytes. Shortly after copulation males attacked these females, indicating that pheromonal cues can override other sensory cues. Surprisingly, masculinization of female behavior by targeting traIR to the nervous system in an otherwise normal female also was sufficient to trigger male aggression. Simultaneous masculinization of both pheromones and behavior induced a complete switch in the normal male response to a female. Control males now fought rather than copulated with these females. In a reciprocal experiment, feminization of the oenocytes and nervous system in males by expression of transformer (traF) elicited high levels of courtship and little or no aggression from control males. Finally, when confronted with flies devoid of pheromones, control males attacked male but not female opponents, suggesting that aggression is not a default behavior in the absence of pheromonal cues. Thus, our results show that masculinization of either pheromones or behavior in females is sufficient to trigger male-to-female aggression. Moreover, by manipulating both the pheromonal profile and the fighting patterns displayed by the opponent, male behavioral responses towards males and females can be completely reversed. Therefore, both pheromonal and behavioral cues are used by Drosophila males in recognizing a conspecific as a competitor.     

Copulatory courtship behavior and sine song as a mate recognition cue in Drosophila lini and its sibling species

Most Drosophila species sing species-specific pulse songs during their "precopulatory courtship." Three sibling species of the Drosophila montium species subgroup performed "copulatory courtship": males generated courtship songs by vibrating either wing only after mounting and during copulation. In these three species, strong sexual isolation was detected between D. ohnishii and D. lini and between D. ohnishii and D. ogumai, but not between D. lini and D. ogumai. Female showed strong repelling behavior when they were mounted by a heterospecific male in the species combinations including D. ohnishii, resulting in failure of the copulation attempt of the male. Acoustic analyses of courtship songs revealed that the pulse song was irregular, without any species-specific parameters, but that the frequency of the sine song was different among the three species in accordance with the modes of sexual isolation between them; it was significantly lower in D. ohnishii (mean ± SE = 193.0 ± 1.7 Hz) but higher in D. lini (253.4 ± 2.7 Hz) and D. ogumai (246.7 ± 5.3 Hz). We suggest that this difference in the sine song frequency is a sexual signal in the Specific Mate Recognition System (SMRS) among these three Drosophila species.