The effect of neurospecific knockdown of candidate genes for locomotor behavior and sound production in Drosophila melanogaster

Molecular mechanisms underlying the functioning of central pattern generators (CPGs) are poorly understood. Investigations using genetic approaches in the model organism Drosophila may help to identify unknown molecular players participating in the formation or control of motor patterns. Here we report Drosophila genes as candidates for involvement in the neural mechanisms responsible for motor functions, such as locomotion and courtship song. Twenty-two Drosophila lines, used for gene identification, were isolated from a previously created collection of 1064 lines, each carrying a P element insertion in one of the autosomes. The lines displayed extreme deviations in locomotor and/or courtship song parameters compared with the whole collection. The behavioral consequences of CNS-specific RNAi-mediated knockdowns for 10 identified genes were estimated. The most prominent changes in the courtship song interpulse interval (IPI) were seen in flies with Sps2 or CG15630 knockdown. Glia-specific knockdown of these genes produced no effect on the IPI. Estrogen-induced knockdown of CG15630 in adults reduced the IPI. The product of the CNS-specific gene, CG15630 (a predicted cell surface receptor), is likely to be directly involved in the functioning of the CPG generating the pulse song pattern. Future studies should ascertain its functional role in the neurons that constitute the song CPG. Other genes (Sps2, CG34460), whose CNS-specific knockdown resulted in IPI reduction, are also worthy of detailed examination.     

The function of courtship song rhythms in Drosophila

The courtship songs of Drosophila are produced by the male's wing vibration and consist of a series of pulses, with an inter-pulse interval (IPI) of 34 ms for D. melanogaster and 48 ms for D. simulans. The IPI's are not constant in length during courtship, but oscillate sinusoidally with 55-s cycles in the former species and 35-s cycles in the latter. We have stimulated D. melanogaster females with artificially generated courtship songs, and have observed that they mate fastest when the song incorporates a 55-s oscillation superimposed on a 34-ms IPI. D. simulans females, on the other hand, mate fastest with a 48-ms IPI and a 35-s oscillation period. Consequently these newly-discovered song cycles produce significant mating enhancement in these species, with the females showing a preference for songs which carry both the species-specific IPI and the species-specific IPI rhythm.     

The courtship song of African Drosophila melanogaster

For many years it was thought that Drosophila melanogaster was relatively panmictic, without differentiation in the Mate Recognition System. Recent studies have demonstrated that flies from Africa vary in pheromones and assortative mating. Strains from Zimbabwe show strong sexual isolation from others. We show that the interpulse interval (IPI) of courtship song, an important mating signal, is unusually short among African flies. Zimbabwean flies have the shortest IPI, but there is no correlation with assortative mating, suggesting little direct role in sexual isolation. Chromosome replacements show that the IPI difference is largely due to genes on chromosome III, with significant interactions involving other chromosomes. Several traits potentially influencing sexual isolation among the melanogaster group of Drosophila seem to be localized to this chromosome. A concentration of important genetic differences might mean that the interaction effects reflect secondary coadaptation of the genetic background to changes associated with chromosome III.     

Social effects on fruit fly courtship song

Courtship behavior in Drosophila has often been described as a classic innate behavioral repertoire, but more recently extensive plasticity has been described. In particular, prior exposure to acoustic signals of con‐ or heterspecific males can change courtship traits in both sexes that are liable to be important in reproductive isolation. However, it is unknown whether male courtship song itself is socially plastic. We examined courtship song plasticity of two species in the Drosophila melanogaster subgroup. Sexual isolation between the species is influenced by two male song traits, the interpulse interval (IPI) and sinesong frequency (SSF). Neither of these showed plasticity when males had prior experience of con‐ and heterospecific social partners. However, males of both species produced longer bursts of song during courtship when they were exposed to social partners (either con‐ or heterospecific) than when they were reared in isolation. D. melanogaster carrying mutations affecting short‐ or medium‐term memory showed a similar response to the social environment, not supporting a role for learning. Our results demonstrate that the amount of song a male produces during courtship is plastic depending on the social environment, which might reflect the advantage of being able to respond to variation in intrasexual competition, but that song structure itself is relatively inflexible, perhaps due to strong selection against hybridization.     

Courtship Behavior of Zaprionus indianus (Gupta) (Diptera: Drosophilidae) from Populations Colonizing South America

We describe for the first time the sexual behavior and the courtship song of males of the African fly Zaprionus indianus (Gupta), a recent invader of South America. The male courtship song is formed by monocyclic pulses and the courtship behavior is simple when compared to that of species of Drosophila. Two interpulse interval (IPI) distributions were observed: pre-mounting and mounting. No significant difference was observed between the pre-mounting IPIs of males that descended from three geographical populations from South America. We also observed the songs produced by females and the homosexual behavior exhibited by males. A sequence of bursts is produced by females as a refusal signal against males, while males emit a characteristic song that identifies sex genus, which differs from the courtship song. The short courtship and mating latencies recorded reveal vigorous males and receptive females, respectively.     

Failure to confirm rhythms in Drosophila courtship song

The importance of courtship song rhythms in Drosophila to molecular and behaviour genetics led to an attempt to verify them. Two songs considered rhythmic by one of the two workers who first reported this behaviour were found to be arrhythmic because the analysis used earlier was inappropriate for identifying song cycles. Using time series analyses by spectral analysis and autocorrelation, no fly showed the sinusoidal pattern in its song claimed by the earlier workers. These findings require recent research concerned with Drosophila song rhythms to be re-evaluated.     

Sexual selection,sexual isolation and the evolution of song in the Drosophila repleta group of species

The evolution of male courtship song pattern in fruit flies of the repleta group within the genus Drosophila is described. We suggest that the archetypal song was composed of two distinct components, an ‘A’ song with short, regular pulse trains which occurred at the beginning of courtship, and a ‘B’ song consisting of longer pulse trains of more widely spaced pulses occurring later in courtship. During evolution of the repleta group some species have lost the A song, others the B song and, in many species, the latter has become less regular and more complex in form. A songs appear to be involved primarily in species recognition and have been subject to stabilizing selection while the B songs sexually stimulate females and have therefore evolved rapidly through the action of sexual selection. Other factors influencing the evolution of song patterns have been certain physiological and energetic constraints.     

Genome-wide RNA interference screening for the clock-related gene of ATP-binding cassette transporters in <Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">melanogaster</Emphasis> (Diptera: Drosophilidae)

ATP-binding cassette (ABC) transporters are widespread among organisms, and 56 genes encode ABC transporters in Drosophila melanogaster (Meigen). Their functions are thought to be divergent. In this study, we examined whether there is a clock-related ABC transporter by performing genome-wide screening using tissue-specific RNA interference. We obtained five candidates when we used tim(UAS)-gal4, which expresses in virtually all clock-related cells. Because their phenotypes were principally reproducible even when we used pdf-gal4, which expresses in a subset of pacemaker neurons only, those transporters were presumed to function in pacemaker neurons. Those five candidates can be categorized into two groups according to the phenotype of the knockdown flies. In one group, CG9281 and CG15410 (E23), the circadian period of knockdown flies was altered. In the other group, CG5944, CG6052, and CG18633, some of the knockdown flies became arrhythmic whereas for others rhythmicity remained intact. Our results suggest that some ABC transporters that have a significant function in the Drosophila circadian system.     

Drosophila male courtship behavior is modulated by ecdysteroids

Temperature-dependent induction of ecdysteroid deficiency in the ecdysoneless mutant ecd¹ adult Drosophilamelanogaster results in altered courtship behavior in males. Ecdysteroid deficiency brings about significantly elevated male-male courtship behavior including song production resembling that directed toward females. Supplementation with dietary 20-hydroxyecdysone reduces male-male attraction, but does not change motor activity, courtship patterns or attraction to females. These observations support the hypothesis that reduced levels of ecdysteroids increase the probability that male fruit flies will display courtship behaviors to male stimuli.     

Temporal analysis of tone pulses within the courtship songs of two siblingDrosophila species,their interspecific hybrid,and behavioral mutants ofD. melanogaster (Diptera: Drosophilidae)

Temporal analyses were applied to the tone pulses within the courtship songs of Drosophila melanogaster, D. simulans,their interspecific hybrid, and behavioral mutants of D. melanogaster.Linear regression was performed on various parameters of the song pulses (cycles per pulse, absolute peak amplitude, intrapulse frequency, number of peaks in fast Fourier transform, width of the primary frequency peak, and interpulse interval), as a function of their positions within pulse trains. Significant differences in the slope values of these two species and of the mutant genotypes allowed for discriminative quantification of temporal changes within trains. These results are discussed in relation to previous kinds of temporal analyses of Drosophilacourtship songs and also with regard to the mechanisms of song production.     

Copulation Song in Drosophila: Do Females Sing to Change Male Ejaculate Allocation and Incite Postcopulatory Mate Choice?

Drosophila males sing a courtship song to achieve copulations with females. Females were recently found to sing a distinct song during copulation, which depends on male seminal fluid transfer and delays female remating. Here, it is hypothesized that female copulation song is a signal directed at the copulating male and changes ejaculate allocation. This may alter female remating and sperm usage, and thereby affect postcopulatory mate choice. Mechanisms of how female copulation song is elicited, how males respond to copulation song, and how remating is modulated, are considered. The potential adaptive value of female signaling during copulation is discussed with reference to vertebrate copulation calls and their proposed function in eliciting mate guarding. Female copulation song may be widespread within the Drosophila genus. This newly discovered behavior opens many interesting avenues for future research, including investigation of how sexually dimorphic neuronal circuits mediate communication between nervous system and reproductive organs.     

The Effect of Male Competition on the Courtship Song of Drosophila melanogaster

The courtship song of Drosophila has been extensively used as a model system for studies of sexual selection and species recognition. Traditionally, the courtship song has been recorded from males placed individually with a female. However, under natural conditions females are exposed to multiple courting males, and the effect of their joint signal on mate recognition by the female is not yet understood. Here, we recorded the courtship behavior of D. melanogaster males singing either individually to a female lpar;1:1) or in the presence of an additional male lpar;2:1). We compared the structure of the male song in the two experimental designs. Our results show that when two males courted a female their songs could overlap each other. Males produced a significantly different signal in the presence of competition; the duration of each song component was significantly shorter and the rate of singing was markedly lower. The present study demonstrates that male competition can dramatically alter the acoustic signal detected by the female.     

Nucleotide and repeat length variation at the nonA gene of the Drosophila virilis group species and its effects on male courtship song

Genes found to affect male courtship song characters in Drosophila melanogaster are good candidates when tracing genes responsible for species-specific songs in other Drosophila species. It has previously been shown that Thr–Gly repeat length variation at the period gene affects song traits in D. melanogaster, which gives the repetitive regions a special interest. In this work, we have characterised the patterns of nucleotide variation for gene regions containing two Gly and one Gln–Ala repeat in another D. melanogaster song gene, no-on-transient A, in D. virilis group species. The levels of nucleotide variability in D. virilis nonA were similar to those found for other genes of the species, and the gene sequences showed no signs of deviation from neutrality. The Gly 2 repeat preceding the central domain of the gene exhibited length variation, which did not, however, correlate with song variation either within D. virilis or between the species of D. virilis group. The Gly 3 repeat located on the other side of the central domain showed amino acid divergence parallel to the consensus phylogeny of the D. virilis group species. The species of the virilis subgroup having Asn after the first three glycines in this repeat have simple songs with no species-specificity, while the species of the montana subgroup having two Gly or Asn–Ser in this site have unique courtship songs. Amino acid differences between the species in this repeat may, however, reflect species phylogeny rather than have an effect on song divergence per se.     

Role of cell death in the formation of sexual dimorphism in the Drosophila central nervous system

Currently, sex differences in behavior are believed to result from sexually dimorphic neural circuits in the central nervous system (CNS). Drosophila melanogaster is a common model organism for studying the relationship between brain structure, behavior, and genes. Recent studies of sex‐specific reproductive behaviors in D. melanogaster have addressed the contribution of sexual differences in the CNS to the control of sex‐specific behaviors and the development of sexual dimorphism. For example, sexually dimorphic regions of the CNS are involved in the initiation of male courtship behavior, the generation of the courtship song, and the induction of male‐specific muscles in D. melanogaster. In this review, I discuss recent findings about the contribution of cell death to the formation of sexually dimorphic neural circuitry and the regulation of sex‐specific cell death by two sex determination factors, Fruitless and Doublesex, in Drosophila.     

果蝇nasuta亚群求爱歌的种间识别与进化遗传学研究

果蝇ｎａｓｕｔａ亚群由１４个种、亚种和分类群组成,广泛分布于印度－太平洋区域。本文首次记录了ｎａｓｕｔａ亚群种的求爱歌,测量了脉冲歌时域模式的参数：脉冲串间隔（ＩＢＩ）、脉冲间隔（ＩＰＩ）、脉冲串时间长度（ＰＴＬ）、每个脉冲串的脉冲数（ＰＮ）、脉冲时间长度（ＰＬ）、波动周期时间长度（ＣＬ）。采用计算机声谱分析技术,作出求爱歌信号的三维数字功率谱图,进行频率分析。发现Ｄ．ｐｕｌａｕｎａ和Ｔａｘｏｎ－Ｆ不发出求爱歌声信号,视觉在交配中可能起重要作用。对其余种、亚种和分类群的求爱歌分析表明,ｎａｓｕｔａ亚群种的求爱歌分为脉冲歌和正弦歌。对部分种的正反交Ｆ１求爱歌分析表明,脉冲歌时域参数,如ＩＰＩ平均值为Ｘ染色体连锁或常染色体多基因控制,正弦歌频率偏向母方。根据不同种、亚种和分类群脉冲歌的时域模式构建ｎａｓｕｔａ亚群的系统树,对亚群中不同种、亚种和分类群的亲缘关系进行讨论。     

Different sexual traits show covariation among genotypes: implications for sexual selection

An unresolved question in sexual selection research is whetherdifferent secondary sexual traits are developmentally independentor instead whether their degree of expression is a manifestationof a general resource pool (i.e., condition) within the organism.If degree of expression of different sexual traits reflectsability to accumulate condition, then covariation should existacross genotypes in the expression of these traits, even ifthey are very different in kind. Here we present evidence forpredicted covariation between morphological (sex comb size)and behavioral (courtship song) sexual traits among geneticlines of Drosophila bipectinata Duda extracted from a naturalpopulation. There is evidence that both these traits in Drosophilaare condition dependent and subject to sexual selection. Wedetected significant body size–independent differencesin comb size among 32 lines. Replicate lines exhibiting relativelyhigh and low values of comb size were then subjected to analysesof courtship song. High sex comb lines exhibited shorter meanburst period and shorter mean burst duration than low sex comblines. These song differences occurred only during the distantpursuit phase of male courtship and existed despite factoringout individual variations in sex comb size, the trait on thebasis of which test lines were originally chosen. The resultsverify the prediction of an association between condition-dependentsecondary sexual traits across genotypes and, therefore, supportthe existence of an overall genetic quality related to conditionacquisition.     

Divergence of a speciation trait through artificial selection: Insights into constraints,by-product effects and sexual isolation

Speciation and sexual isolation often occur when divergent female mating preferences target male secondary sexual traits. Despite the importance of such male signals, little is known about their evolvability and genetic linkage to other traits during speciation. To answer these questions, we imposed divergent artificial selection for 10 non-overlapping generations on the Inter-Pulse-Interval (IPI) of male courtship songs; which has been previously shown to be a major species recognition trait for females in the Drosophila athabasca species complex. Focusing on one of the species, Drosophila mahican (previously known as EA race), we examined IPI's: (1) rate of divergence, (2) response to selection in different directions, (3) genetic architecture of divergence and (4) by-product effects on other traits that have diverged in the species complex. We found rapid and consistent response for higher IPI but less response to lower IPI; implying asymmetrical constraints. Genetic divergence in IPI differed from natural species in X versus autosome contribution and in dominance, suggesting that evolution may take different paths. Finally, selection on IPI did not alter other components of male songs, or other ecological traits, and did not cause divergence in female preferences, as evidenced by lack of sexual isolation. This suggests that divergence of male courtship song IPI is unconstrained by genetic linkage with other traits in this system. This lack of linkage between male signals and other traits implies that female preferences or ecological selection can co-opt and mould specific male signals for species recognition free of genetic constraints from other traits.     

Species and sex-specific songs and courtship behaviour in the genus Zaprionus (Diptera-Drosophilidae)

Courtship behaviour and songs of six Zaprionus species show differences not only between species but also between sexes. Courtship behaviour differs from that in the related genus Drosophila. In most species, males produce two song types which may alternate or be repeated. Singing occurs during courtship displays, mounting, after copulation and briefly during male-to-male interactions. Females produce a loud whine and body rocking movements in refusal but have a species-specific pulsed song produced during normal courtship which differs from the conspecific male song. One species produces an irregular male song but a regular female song. Sex-specific songs may have selective advantages but pose problems for the sensory template hypothesis.     

The Drosophila dopamine 2-like receptor D2R (Dop2R) is required in the blood brain barrier for male courtship

The blood brain barrier (BBB) has the essential function to protect the brain from potentially hazardous molecules while also enabling controlled selective uptake. How these processes and signaling inside BBB cells control neuronal function is an intense area of interest. Signaling in the adult Drosophila BBB is required for normal male courtship behavior and relies on male-specific molecules in the BBB. Here we show that the dopamine receptor D2R is expressed in the BBB and is required in mature males for normal mating behavior. Conditional adult male knockdown of D2R in BBB cells causes courtship defects. The courtship defects observed in genetic D2R mutants can be rescued by expression of normal D2R specifically in the BBB of adult males. Drosophila BBB cells are glial cells. Our findings thus identify a specific glial function for the DR2 receptor and dopamine signaling in the regulation of a complex behavior.