Song production in auditory mutants of Drosophila: the role of sensory feedback

To test the role of sensory feedback in song production. we analyzed the courtship songs of Drosophila males expressing auditory mutations. We compared the courtship songs of atonal (ato), beethoven (btv) and touch-insensitive-larva-B (tilB) to wild-type songs. These mutations have in common the fact that the chordotonal organs are disrupted. Since chordotonal organs subserve both hearing (in the antenna) and proprioception (from the wing), these two potential routes for sensory feedback are defective in the mutant flies. We measured six song characters: pulse number within a train, inter-pulse interval, pulse duration, sine burst duration, the carrier frequency of the sine song and the relative amplitude of the sine song. Using multivariate analysis, we found significant differences between mutant and normal songs. In addition many mutant flies exhibit an unusual wing position during singing. The results indicate that sensory feedback plays an important role in shaping the courtship song of Drosophila.     

Towards a molecular understanding of Drosophila hearing

The Drosophila auditory system is presented as a powerful new genetic model system for understanding the molecular aspects of development and physiology of hearing organs. The fly's ear resides in the antenna, with Johnston's organ serving as the mechanoreceptor. New approaches using electrophysiology and laser vibrometry have provided useful tools to apply to the study of mutations that disrupt hearing. The fundamental developmental processes that generate the peripheral nervous system are fairly well understood, although specific variations of these processes for chordotonal organs (CHO) and especially for Johnston's organ require more scrutiny. In contrast, even the fundamental physiologic workings of mechanosensitive systems are still poorly understood, but rapid recent progress is beginning to shed light. The identification and analysis of mutations that affect auditory function are summarized here, and prospects for the role of the Drosophila auditory system in understanding both insect and vertebrate hearing are discussed.     

Divergence in Multiple Courtship Song Traits between Drosophila santomea and D. yakuba

The courtship song of Drosophila is useful for species recognition and sexual selection. A new species of the melanogaster group of Drosophila , D. santomea , has recently been described from the island of São Tomé in the Gulf of Guinea. We describe the courtship song of D. santomea and compare it with that of its sibling species D. yakuba . Both species have a relatively unusual song pattern for melanogaster-group species, in that they have two types of pulse song but no sine song. There are large differences in the inter-pulse interval of both types of song, but no major differences in pulse shape or intrapulse frequency between the species. The song of D. yakuba is similar in lines from the African mainland (allopatric to D. santomea ) and from São Tomé (sympatric). We test if song pattern might influence sexual isolation by examining the mating success of wingless males with homo- and hetero-specific females. We show that song pattern contributes to sexual stimulation, but the differences in song patterns alone are unlikely to explain patterns of sexual isolation such as the asymmetrical isolation seen between species.     

Inheritance of species differences in female receptivity and song requirement between Drosophila virilis and D. montana

Females of two Drosophila virilis group species, D. virilis and D. montana, have different requirements for the courting males. In the present study we have examined species differences in female receptivity and male courtship song requirement using females' acceptance signal instead of copulation for measuring female readiness to mate. Behavior of D. virilis and D. montana females and F1 and backcross hybrid females was observed in a single-pair courtships with D. virilis and D. montana males and normal and wingless (mute) F1 hybrid males. D. virilis females were very receptive and they commonly accepted the courtship of males unable to produce courtship song. D. montana females, on the contrary, had a low receptivity and these females accepted the courting male only after hearing his song. Interspecific F1 and backcross (BCm) females resembled D. virilis more than D. montana in their receptivity. These females, however, resembled D. montana in their song requirement. These findings suggest that female song requirement and female receptivity are determined by different genetic factors.     

Two distinct genomic regions, harbouring the period and fruitless genes, affect male courtship song in Drosophila montana

Acoustic signals often have a significant role in pair formation and in species recognition. Determining the genetic basis of signal divergence will help to understand signal evolution by sexual selection and its role in the speciation process. An earlier study investigated quantitative trait locus for male courtship song carrier frequency (FRE) in Drosophila montana using microsatellite markers. We refined this study by adding to the linkage map markers for 10 candidate genes known to affect song production in Drosophila melanogaster. We also extended the analyses to additional song characters (pulse train length (PTL), pulse number (PN), interpulse interval, pulse length (PL) and cycle number (CN)). Our results indicate that loci in two different regions of the genome control distinct features of the courtship song. Pulse train traits (PTL and PN) mapped to the X chromosome, showing significant linkage with the period gene. In contrast, characters related to song pulse properties (PL, CN and carrier FRE) mapped to the region of chromosome 2 near the candidate gene fruitless, identifying these genes as suitable loci for further investigations. In previous studies, the pulse train traits have been found to vary substantially between Drosophila species, and so are potential species recognition signals, while the pulse traits may be more important in intra-specific mate choice.     

Adult bengalese finches (Lonchura striata var. domestica) require real-time auditory feedback to produce normal song syntax

Songbirds develop their songs by imitating songs of adults. For song learning to proceed normally, the bird's hearing must remain intact throughout the song development process. In many species, song learning takes place during one period early in life, and no more new song elements are learned thereafter. In these so-called close-ended learners, it has long been assumed that once song development is complete, audition is no longer necessary to maintain the motor patterns of full song. However, many of these close-ended learners maintain plasticity in overall song organization; the number and the sequence of song elements included in a song of an individual vary from one utterance to another, although no new song elements are added or lost in adulthood. It is conceivable that these species rely on continued auditory feedback to produce normal song syntax. The Bengalese finch is a close-ended learner that produces considerably variable songs as an adult. In the present study, we found that Bengalese finches require real-time auditory feedback for motor control even after song learning is complete; deafening adult finches resulted in development of abnormal song syntax in as little as 5 days. We also found that there was considerable individual variation in the degree of song deterioration after deafening. The neural mechanisms underlying adult song production in different species of songbirds may be more diverse than has been traditionally considered. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 343–356, 1997     

The function of pulse song and sine song in the courtship of Drosophila melanogaster

Drosophila melanogaster females were subjected to pulse song before being allowed to mix with males. Sine song increases female receptivity, pulse song does not. Pulse song does however increase receptivity if the females are subjected to it while being courted by males which are deaf and which cannot produce any auditory stimulation themselves. It is suggested that sine song is summated and has a priming effect on female receptivity whereas pulse song functions as a species recognition signal in a trigger-like fashion.     

Emerging technologies for gene manipulation in Drosophila melanogaster

The popularity of Drosophila melanogaster as a model for understanding eukaryotic biology over the past 100 years has been accompanied by the development of numerous tools for manipulating the fruitfly genome. Here we review some recent technologies that will allow Drosophila melanogaster to be manipulated more easily than any other multicellular organism. These developments include the ability to create molecularly designed deletions, improved genetic mapping technologies, strategies for creating targeted mutations, new transgenic approaches and the means to clone and modify large fragments of DNA.     

Courtship song analysis of Drosophila muscle mutants

As part of the mating ritual, males of Drosophila species produce species-specific courtship songs through wing vibrations generated by the thoracic musculature. While previous studies have shown that indirect flight muscles (IFM) are neurally activated during courtship song production, the precise role of these muscles in song production has not been investigated. Fortunately, IFM mutants abound in Drosophila melanogaster and studies spanning several decades have shed light on the role of muscle proteins in IFM-powered flight. Analysis of courtship songs in these mutants offers the opportunity to uncover the role of the IFM in a behavior distinct than flight and subject to different evolutionary selection regimes. Here, we describe protocols for the recording and analysis of courtship behavior and mating song of D. melanogaster muscle transgenic and mutant strains. To record faint acoustic signal of courtship songs, an insulated mating compartment was used inside a recording device (INSECTAVOX) equipped with a modified electret microphone, a low-noise power supply, and noise filters. Songs recorded in the INSECTAVOX are digitized using Goldwave, whose several features enable extraction of critical song parameters, including carrier frequencies for pulse song and sine song. We demonstrate the utility of this approach by showing that deletion of the N-terminal region of the myosin regulatory light chain, a mutation known to decrease wing beat frequency and flight power, affects courtship song parameters.     

Analysis of temperature-sensitive mutants reveals new genes involved in the courtship song of Drosophila.

cacophony (cac), a mutation affecting the courtship song in Drosophila melanogaster, is revealed to cause temperature-sensitive (TS) abnormalities. When exposed to high temperatures (37 degrees), cac flies show frequent convulsions and pronounced locomotor defects. This TS phenotype seems consistent with the idea that cac is a mutation in a calcium-channel gene; it maps to the same X-chromosomal locus that encodes the polypeptide comprising the alpha-1 subunit of this membrane protein. Analysis of the courtship song of some TS physiological mutants showed that slowpoke mutations, which affect a calcium-activated potassium channel, cause severe song abnormalities. Certain additional TS mutants, in particular para(ts1) and nap(ts1), exhibit subtler song defects. The results therefore suggest that genes involved in ion-channel function are a potential source of intraspecific genetic variation for song parameters, such as the number of cycles present in "pulses" of tone or the rate at which pulses are produced by the male''s courtship wing vibrations. The implications of these findings from the perspective of interspecific lovesong variations in Drosophila are discussed.     

Buzzwords in Females’ Ears? The Use of Buzz Songs in the Communication of Nightingales (Luscinia megarhynchos)

Differences in individual male birds’ singing may serve as honest indicators of male quality in male-male competition and female mate choice. This has been shown e.g. for overall song output and repertoire size in many bird species. More recently, differences in structural song characteristics such as the performance of physically challenging song components were analysed in this regard. Here we show that buzz elements in the song of nightingales (Luscinia megarhynchos) hold the potential to serve as indicators of male quality and may therefore serve a communicative function. Buzzes were produced with considerable differences between males. The body weight of the males was correlated with one measure of these buzzes, namely the repetition rate of the buzz subunits, and individuals with larger repertoires sang buzzes at higher subunit-rates. A model of buzz performance constraints suggested that buzzes were sung with different proficiencies. In playback experiments, female nightingales showed more active behaviour when hearing buzz songs. The results support the idea that performance differences in the acoustic fine structure of song components are used in the communication of a large repertoire species such as the nightingale.     

Influence of group rearing on sexual behavior of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> males

Social interactions are able to strongly influence animal physiology and behavior. As is known, social experience can lead to changes in sexual and aggressive behavior, circadian rhythms and composition of cuticular hydrocarbons in Drosophila. Previously, we have shown that housing Drosophila males in monosexual groups of 20 individuals for 3 days after eclosion leads to a strong and long-term suppression of locomotor activity as revealed at individual testing, in contrast to males kept separately. The present research addressed courtship behavior, and specifically song production, in Drosophila males reared under similar conditions. It was found that rearing males in monosexual groups leads to a suppression of courtship and song production as well as to a simultaneous increase in locomotor activity when tested with a moving female. The latter effect was due to the strong urge of males to avoid interindividual contacts that prevented triggering the courtship ritual. It was suggested that intermale aggression caused by group rearing generates a state similar to conditioned fear.     

Rapid evolution of courtship song pattern in Drosophila willistoni sibling species

Courtship behaviours may provide a more reliable means of identifying reproductively isolated taxa than traits such as morphology or many genetic markers. Here we describe the courtship songs of the Drosophila willistoni sibling species group, which consists of several species and subspecies. We find that song pattern is species-specific, despite significant differences among strains within species. D. paulistorum has the most variable song pattern, which reflects this species' traditional subdivision into semispecies. All the other species could be unambiguously identified by song. The major differences among these species was in the interpulse interval, as has been found in other studies of fly song. However, the interpulse intervals of the species studied here were often multimodal. This was partly due to the presence of multiple song types within the courtship repertoire, but it also reflected changes in interpulse interval within a song type by some males. Unusually, some species had distinctively patterned variation in interpulse interval. Song must have evolved rapidly within the species complex, probably due to sexual selection.     

Drosophila auditory organ genes and genetic hearing defects

The Drosophila auditory organ shares equivalent transduction mechanisms with vertebrate hair cells, and both are specified by atonal family genes. Using a whole-organ knockout strategy based on atonal, we have identified 274 Drosophila auditory organ genes. Only four of these genes had previously been associated with fly hearing, yet one in five of the genes that we identified has a human cognate that is implicated in hearing disorders. Mutant analysis of 42 genes shows that more than half of them contribute to auditory organ function, with phenotypes including hearing loss, auditory hypersusceptibility, and ringing ears. We not only discover ion channels and motors important for hearing, but also show that auditory stimulus processing involves chemoreceptor proteins as well as phototransducer components. Our findings demonstrate mechanosensory roles for ionotropic receptors and visual rhodopsins and indicate that different sensory modalities utilize common signaling cascades.     

Drosophila virilis and its cosmopolitan relatives in urban islands

Populations of brewery Drosophila have been studied for 2 years. Species distributions diner between breweries, and are very different from those found in woodland and fruitmarkets. It is suggested that breweries form 'urban islands'. One of the commonest species found in breweries appears to be Drosophila virilis , which has not previously been found in Britain. Tests on various characteristics, including courtship song, have shown conclusively that this species is a strain of D. virilis.     

Behavioral Sequence Leading to Sexual Isolation Between <Emphasis Type="Italic">Drosophila ananassae</Emphasis> and <Emphasis Type="Italic">D. pallidosa</Emphasis>

Drosophila ananassae and D. pallidosa are closely related, sympatric species that lack postmating isolation. Sexual isolation has been considered important in maintaining them as independent species. To clarify the behavioral processes leading to sexual isolation, we analyzed behavioral sequences and examined the effect of courtship song on mating success and on behaviors of both sexes by surgically removing male wings (song generators), female aristae (song receivers), or female wings (means of fluttering). We found that heterospecific courtship songs evoked female wing fluttering, whereas conspecific courtship song did not. Furthermore, female wing fluttering made courting males discontinue courtship. These findings suggest that strong sexual isolation is achieved through the following behavioral sequence: heterospecific song→female wing fluttering→courtship discontinuation.     

Ultrasonic singing by the blue-throated hummingbird: a comparison between production and perception

Blue-throated hummingbirds produce elaborate songs extending into the ultrasonic frequency range, up to 30 kHz. Ultrasonic song elements include harmonics and extensions of audible notes, non-harmonic components of audible syllables, and sounds produced at frequencies above 20 kHz without corresponding hearing range sound. To determine whether ultrasonic song elements function in intraspecific communication, we tested the hearing range of male and female blue-throated hummingbirds. We measured auditory thresholds for tone pips ranging from 1 kHz to 50 kHz using auditory brainstem responses. Neither male nor female blue-throated hummingbirds appear to be able to hear above 7 kHz. No auditory brainstem responses could be detected between 8 and 50 kHz at 90 dB. This high-frequency cutoff is well within the range reported for other species of birds. These results suggest that high-frequency song elements are not used in intraspecific communication. We propose that the restricted hummingbird hearing range may exemplify a phylogenetic constraint.     

Factors limiting song acquisition in adult zebra finches

Song learning takes place in two separate or partially overlapping periods, a sensory phase in which a tutor song is memorized and a sensorimotor phase in which a copy of the model is produced. The stage of song development where song becomes stable and stereotyped is called crystallization. Adult birds usually do not learn new song in many species including the zebra finch. However, it is not known whether song crystallization as such or aging impedes adult learning. Exposure to loud noises prevents birds from developing and crystallizing their song, because they cannot control their voice by auditory feedback. Zebra finches even without previous experience of hearing or singing a song failed to learn a song model provided in adulthood. Thus, neither the absence of a tutor song nor the lack of song crystallization enables new song learning in adulthood, but age per se limits the ability or motivation to learn song. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Memory in the making: localized brain activation related to song learning in young songbirds

Songbird males learn to sing their songs from an adult ‘tutor’ early in life, much like human infants learn to speak. Similar to humans, in the songbird brain there are separate neural substrates for vocal production and for auditory memory. In adult songbirds, the caudal pallium, the avian equivalent of the auditory association cortex, has been proposed to contain the neural substrate of tutor song memory, while the song system is involved in song production as well as sensorimotor learning. If this hypothesis is correct, there should be neuronal activation in the caudal pallium, and not in the song system, while the young bird is hearing the tutor song. We found increased song-induced molecular neuronal activation, measured as the expression of an immediate early gene, in the caudal pallium of juvenile zebra finch males that were in the process of learning to sing their songs. No such activation was found in the song system. Molecular neuronal activation was significantly greater in response to tutor song than to novel song or silence in the medial part of the caudomedial nidopallium (NCM). In the caudomedial mesopallium, there was significantly greater molecular neuronal activation in response to tutor song than to silence. In addition, in the NCM there was a significant positive correlation between spontaneous molecular neuronal activation and the strength of song learning during sleep. These results suggest that the caudal pallium contains the neural substrate for tutor song memory, which is activated during sleep when the young bird is in the process of learning its song. The findings provide insight into the formation of auditory memories that guide vocal production learning, a process fundamental for human speech acquisition.