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Environmental stress is a major factor that affects courtship behavior and evolutionary fitness. Although mature virgin females of Drosophila melanogaster usually accept a courting male to mate, they may not mate under stressful conditions. Above the temperature optimal for mating (20-25 °C), copulation success of D. melanogaster declines with increasing temperature although we observed vigorous courtship attempts by males, and no copulation takes place at temperatures over 36 °C. We attempted to identify the sensory pathway for detecting heat threat that drives a female to escape rather than to engage in mating that detects hot temperature and suppresses courtship behavior. We found that the artificial activation of warmth-sensitive neurons ('hot cells') in the antennal arista of females completely abrogates female copulation success even at permissive temperatures below 32 °C. Moreover, mutational loss of the GR28b.d thermoreceptor protein caused females to copulate even at 36 °C. These results indicate that antennal hot cells provide the input channel for detecting the high ambient temperature in the control of virgin female mating under stressful conditions.

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.

Desaturase1 (desat1) is one of the few genes known to be involved in the two complementary aspects of sensory communication - signal emission and signal reception - in

Among the sensory modalities involved in controlling mating behavior in

Behavioral changes in evolution have attracted the attention of many evolutionary biologists. Closely related species, or even individuals from different populations within a species, often exhibit remarkably different behaviors. Such behavioral diversification has been implicated as a cause of speciation in some cases, yet the mechanisms that produce and maintain these changes remain largely unknown. Drosophila melanogaster, an outstanding model organism with which to explore the causal link among the gene, neural circuitry, and behavior, provides an excellent entry point for a comparative approach to the origin of behavioral diversification with a single-gene and single-cell resolution. Here we survey studies that attempted to reveal the mechanistic bases for behavioral changes potentially associated with speciation, and we discuss the successfully identified candidate genes or neurons involved in such events. Although evolutionary developmental biology (evo-devo), a field devoted to the developmental and molecular basis of phenotypic diversity, has mostly been focused on morphological traits, the extension of this evo-devo approach to behavioral diversity will provide a comprehensive understanding of the genome-environment interactions underlying adaptive evolution.

In the pupal stage, the fly body undergoes extensive metamorphic remodeling, in which programmed cell death plays a critical role. We studied two of the constituent processes in this remodeling, salivary gland degeneration and breakdown of the eclosion muscle, which are triggered by an increase and a decrease in the circulating steroid hormone ecdysone at the start and end of metamorphosis, respectively. We found that knockdown of zeste (z), a gene encoding a sequence-specific DNA-binding protein implicated in transvection, in salivary gland cells advances the initiation of their degeneration, whereas z knockdown in neurons delays muscle breakdown. We further showed that knockdown of an ecdysone-inducible gene, E74, retards salivary gland degeneration with little effect on eclosion muscle breakdown. We propose that Z tunes the sensitivity of ecdysone targets to this hormone in order to ensure a high safety margin so that the cell death program will be activated when the ecdysone titer is at a sufficiently high level that is reached only at a defined stage during metamorphosis.

fickle is a P-element mutation identified from a screen for defects in courtship behavior and disrupts the fly homolog of Bruton's tyrosine kinase (Btk) gene (Baba et al., 1999). Here, we show that habituation of the olfactory jump reflex also is defective in fickle. Unlike, the prototypical memory mutants, rutabaga and dunce, which habituate more slowly than normal, fickle flies habituate faster than normal. fickle's faster-than-normal response decrement did not appear to be due to sensorimotor fatigue, and dishabituation of the jump response was normal. Based on a long-standing “two opponent process” theory of habituation, these data suggested that behavioral sensitization might be defective in fickle. To test this hypothesis, we designed a olfactory sensitization procedure, using the same stimuli to habituate (odor) and dishabituate (vortexing) flies. Mutant flies failed to show any sensitization with this procedure. Our study reveals a “genetic dissection” of sensitization and dishabituation and, for the first time, provides a biological confirmation of the two opponent process theory of habituation.

We studied, in a genetic model organism, Drosophila melanogaster, the channel mechanisms underlying membrane excitation in the embryonic body wall muscle whose biophysical properties have been poorly characterized. The inward current underlying the action potential was solely mediated by a high-threshold class of voltage-gated Ca(2+) channels, which exhibited slow inactivation, Ca(2+) permeability with saturation at high [Ca(2+)]OUT, and sensitivity to a Ca(2+) channel blocker, Cd(2+). The Ca(2+) current in the embryonic muscle was completely eliminated in Dmca1D mutants, indicating that the Dmca1D-encoded Ca(2+) channel is the major mediator of inward currents in the body wall muscles throughout the embryonic and larval stages.

The muscle of Lawrence (MOL) is a male-specific muscle present in the abdomen of some adult Drosophila species. Formation of the MOL depends on innervation by motoneurons that express fruitless, a neural male determinant. Drosophila melanogaster males carry a pair of MOLs in the 5th abdominal segment, whereas D. subobscura males carry a pair in both the 5th and 4th segments. We hypothesized that the fru gene of D. subobscura but not that of D. melanogaster contains a cis element that directs the formation of the additional pair of MOLs. Successively extended 5' DNA fragments to the P1 promoter of D. subobscura or the corresponding fragments that are chimeric (i.e., containing both melanogaster and subobscura elements) were introduced into D. melanogaster and tested for their ability to induce the MOL to locate the hypothetical cis element. We found that a 1.5-2-kb genomic fragment located 4-6-kb upstream of the P1 promoter in D. subobscura but not that of D. melanogaster permits MOL formation in females, provided this fragment is grafted to the distal ∼4-kb segment from D. melanogaster, demonstrating that this genomic fragment of D. subobscura contains a cis element for the MOL induction.

The lingerer (lig) gene is necessary for initiation and termination of copulatory behavior in Drosophila melanogaster. The lig gene encodes cytoplasmic proteins, and is expressed in the central nervous system (CNS) during the late third-instar larval stage when the lig function is required for normal copulation to occur after adult eclosion. To characterize the lig-expressing cells in the late third-instar larval CNS, we have isolated a genomic fragment containing the promoter/enhancer region of the lig gene, and established transgenic lines in which expression of reporter genes is controlled by the lig promoter/enhancer. In the larval brain, reporter genes were expressed in all of the glial cells and in clusters of neurons that projected contralaterally. In the larval ventral ganglion, reporter genes were expressed in subperineurial glia, peripheral exit glia, and a number of interneurons, but not in motor neurons. In the cloned promoter/enhancer region, we have found the sequence motif for binding of the REPO protein, a transcription factor essential for the differentiation and maintenance of glial cells. The lig gene is thus one of the candidate target genes for the REPO transcription factor.

The lingerer (lig) gene is necessary for initiation and termination of copulatory behavior in Drosophila melanogaster. The lig gene encodes cytoplasmic proteins, and is expressed in the central nervous system (CNS) during the late third-instar larval stage when the lig function is required for normal copulation to occur after adult eclosion. To characterize the lig-expressing cells in the late third-instar larval CNS, we have isolated a genomic fragment containing the promoter/enhancer region of the lig gene, and established transgenic lines in which expression of reporter genes is controlled by the lig promoter/enhancer. In the larval brain, reporter genes were expressed in all of the glial cells and in clusters of neurons that projected contralaterally. In the larval ventral ganglion, reporter genes were expressed in subperineurial glia, peripheral exit glia, and a number of interneurons, but not in motor neurons. In the cloned promoter/enhancer region, we have found the sequence motif for binding of the REPO protein, a transcription factor essential for the differentiation and maintenance of glial cells. The lig gene is thus one of the candidate target genes for the REPO transcription factor.

During courtship, Drosophila melanogaster males usually position themselves behind the target female. To decipher the sensory cues that guide the males to the courting position, we quantitatively analyzed male locomotion traces within a circular observation chamber, at the center of which an immobilized virgin female was placed as a courtship target. Wild-type males preferentially stayed behind the female under not only daylight but also dark conditions, indicating that vision is dispensable for orientation by males. By contrast, olfaction-defective Or83b2 homozygous males often positioned themselves in front of a female to court under dark but not daylight conditions. We conclude that vision and olfaction redundantly guide the male fly to move behind the female to perform courtship actions. The visual and olfactory features that provide a male with cues for differentiating between the front and back end of a female are yet to be determined.

Recently mated Drosophila females were shown to be reluctant to copulate and to exhibit rejecting behavior when courted by a male. Males that experience mate refusal by a mated female subsequently attenuate their courtship effort toward not only mated females but also virgin females. This courtship suppression persists for more than a day, and thus represents long-term memory. The courtship long-term memory has been shown to be impaired in heterozygotes as well as homozygotes of mutants in orb2, a locus encoding a set of CPEB RNA-binding proteins. We show that the impaired courtship long-term memory in orb2-mutant heterozygotes is restored by reducing the activity of lig, another putative RNA-binding protein gene, yet on its own the loss-of-function lig mutation is without effect. We further show that Lig forms a complex with Orb2. We infer that a reduction in the Lig levels compensates the Orb2 deficiency by mitigating the negative feedback for Orb2 expression and thereby alleviating defects in long-term memory.

By screening about 2,000 P-element-insertion lines of Drosophila melanogaster, we isolated a new behavioral mutant line, chaste (chst), the females of which display extraordinarily strong rejection behavior against courting males. The chst mutation is mapped to the muscleblind (mbl) locus at 54B on the right arm of chromosome 2. The reduced sexual receptivity in chst mutant females is reversed to the wild-type level by introducing a transgene, which expresses either the mblB (+) or mblC (+) isoform, demonstrating that chst is an allele of mbl. Among the P-elements inserted upstream of the mbl gene, those inserted in the same orientation as that of mbl express the chst phenotype, whereas a P-element inserted in the opposite orientation does not. This finding implies that the former P-elements induce the mutant phenotype by a mechanism that is sensitive to the direction of transcription (e.g., transcriptional interference). The mbl alleles, with deletions near the transcription start site and/or in part of the exons, complement the chst mutation in the sexual receptivity phenotype, but not in the lethality phenotype of mbl mutations. Such interallelic complementation of the sexual receptivity phenotype in the mbl locus disappears in the presence of a mutant copy of zeste (z), a gene encoding a protein that mediates transvection. We suggest that the mbl gene function is required for the normal development of neural substrates that regulate female sexual receptivity.

The fruitless (fru) gene in Drosophila plays a pivotal role in the formation of neural circuits underlying gender-specific behaviors. Specific labeling of fru-expressing neurons has revealed a core circuit responsible for male courtship behavior. Females with a small number of masculinized neuronal clusters in their brain can initiate male-type courtship behavior. By examining the correlations between the masculinized neurons and behavioral gender type, a male-specific neuronal cluster, named P1, which coexpresses fru and doublesex, was identified as a putative trigger center for male-type courtship behavior. P1 neurons extend dendrite to the lateral horn, where multimodal sensory inputs converge. Molecular studies suggest that fru determines the level of masculinization of neurons by orchestrating the transcription of a set of downstream genes, which remain to be identified.

One major goal of neurobiology is to understand how a neural network produces an appropriate behavior under rapidly changing environments the animal encounters through its active and passive experi...

In Drosophila melanogaster, male-specific forms of the fruitless (fru) gene product, mFru protein, function as a neural sex-determination factors that directs the development of at least two male characteristics, namely courtship and mating behavior and the formation of the muscle of Lawrence (MOL). In D. melanogaster, the male-specific expression of Fru protein in motoneurons is responsible for the male-limited induction of the MOL by such neurons. Although no Drosophila species whose females have the MOL are known, there are many Drosophila species whose males lack the MOL. We performed immunohistochemical staining of the central nervous system (CNS) from 9 Drosophila species to determine whether the mFru expression profile is different between MOL-present and MOL-absent species. In 8 of the 9 species, Fru protein expression in the CNS is strictly male-specific, regardless of the presence or absence of the MOL. The sole exception is D. suzukii, in which females express the Fru protein though less extensively than males do: Fru expression in the CNS of female D. suzukii is restricted to the lamina and ventral ganglia. Expression of Fru protein in the lamina is observed in males of D. virilis and in both sexes of D. suzukii, but not in males and females of the 7 other species. These results indicate that sexually dimorphic expression of the Fru protein has been subjected to species-specific modulation during evolution.

Many declared aims of the genome projects have been achieved. The total genomic sequences of several relatively noncomplex/complex organisms (such as E. coli, yeast, Caenorhabditis, Drosophila) are being determined, and the nucleotide sequencing of the entire human genome will be complete in the near future. However, this achievement is not the end of the road but rather the first step toward the functional understanding of the genome of humans and other organisms. The determined linear nucleotide sequences remain only lists of A, C, G and T, unless they are given functional significance. The coding sequences of genes can be identified in a relatively reliable manner by computational methods, but the exact function of their protein products can rarely be determined without obtaining much additional information, e.g., by biochemical or cell biological methods. Thus, following sequencing, the next step must be to assign functions to the identified genes. The final goal of genome research today may look futuristic, but the knowledge of the function of every single gene and the interactions between them will finally allow us to understand the development and functioning of an organism as a whole. Gene-trapping methodology is a powerful strategy for cloning and identifying functional genes, as it marks a gene with a tag and simultaneously generates a corresponding genetic variation for that particular locus. Therefore, gene trapping is an extremely useful tool for functional genomics, establishing a correlation between the physical and genetic maps of the genome. The relative simplicity of its genome and the availability of huge bodies of genetic and molecular information make Drosophila melanogaster one of the most important model organisms. Its genome will serve as a "reference" for the in-depth analysis of the organization of more complex eukaryotic genomes. Multifaceted approaches to Drosophila functional genomics and the dual-tagging gene trap system newly developed for functional analysis of Drosophila genes are discussed in this review.

Bisexual courtship in male Drosophila melanogaster may be induced in some circumstances. These include ectopic expression of the transformer (tra) gene, ectopic expression of the mini-white (mw) gene, and the homozygous presence of mutant alleles of the fruitless (fru) gene. Experiments were performed to determine if ectopic mw and fru, as well as ectopic tra and fru, acted in the same pathway to control courtship. Male flies homozygous for the frusat allele court females little if at all and males at a low level. When homozygous, the frusat allele suppresses the bisexual courtship induced by both ectopic mw and ectopic tra, indicating that the fru wild-type function is necessary for expression of the ectopic mw and ectopic tra effect. This demonstrates that fru shares a pathway controlling courtship behavior with these ectopically expressed genes.

Natural selection implements an efficient mechanism for the invention of adaptive structure and function, including sexual selection, which plays a role in creating sex-linked traits. These selecti...