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.     

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.     

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.     

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.     

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.     

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.     

Genetic basis of natural variation in body pigmentation in Drosophila melanogaster

Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at bab1, t and e affect variation in abdominal pigmentation in several populations of Drosophila melanogaster. Recently, we performed a genome wide association (GWA) analysis of variation in abdominal pigmentation using the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP). We confirmed the large effects of regulatory variants in bab1, t and e; identified 81 additional candidate genes; and validated 17 candidate genes (out of 28 tested) using RNAi knockdown of gene expression and mutant alleles. However, these analyses are imperfect proxies for the effects of segregating variants. Here, we describe the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. We replicated the effects on pigmentation of 28 genes implicated by the DGRP GWA study, including bab1, t and e and 7 genes previously validated by RNAi and/or mutant analyses. We also identified many additional loci. The genetic architecture of Drosophila pigmentation is complex, with a few major genes and many other loci with smaller effects.     

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.     

Dissecting the Biological Role of Mucin-type O-Glycosylation Using RNA Interference in Drosophila Cell Culture

Mucin type O-glycosylation is a highly conserved form of post-translational modification initiated by the family of enzymes known as the polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs in mammals and PGANTs in Drosophila). To address the cellular functions of the many PGANT family members, RNA interference (RNAi) to each pgant gene was performed in two independent Drosophila cell culture lines. We demonstrate that RNAi to individual pgant genes results in specific reduction in gene expression without affecting the expression of other family members. Cells with reduced expression of individual pgant genes were then examined for changes in viability, morphology, adhesion, and secretion to assess the contribution of each family member to these cellular functions. Here we find that RNAi to pgant3, pgant6, or pgant7 resulted in reduced secretion, further supporting a role for O-glycosylation in proper secretion. Additionally, RNAi to pgant3 or pgant6 resulted in altered Golgi organization, suggesting a role for each in establishing or maintaining proper secretory apparatus structure. Other subcellular effects observed included multinucleated cells seen after RNAi to either pgant2 or pgant35A, suggesting a role for these genes in the completion of cytokinesis. These studies demonstrate the efficient and specific knockdown of pgant gene expression in two Drosophila cell culture systems, resulting in specific morphological and functional effects. Our work provides new information regarding the biological roles of O-glycosylation and illustrates a new platform for interrogating the cellular and subcellular effects of this form of post-translational modification.     

Dopamine Dynamics and Signaling in Drosophila: An Overview of Genes,Drugs and Behavioral Paradigms

Changes in dopamine (DA) signaling have been implicated in a number of human neurologic and psychiatric disorders. Similarly, defects in DA signaling in the fruit fly, Drosophila melanogaster, have also been associated with several behavioral defects. As most genes involved in DA synthesis, transport, secretion, and signaling are conserved between species, Drosophila is a powerful genetic model organism to study the regulation of DA signaling in vivo. In this review, we will provide an overview of the genes and drugs that regulate DA biology in Drosophila. Furthermore, we will discuss the behavioral paradigms that are regulated by DA signaling in flies. By analyzing the genes and neuronal circuits that govern such behaviors using sophisticated genetic, pharmacologic, electrophysiologic, and imaging approaches in Drosophila, we will likely gain a better understanding about how this neuromodulator regulates motor tasks and cognition in humans.     

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.     

Immune anticipation of mating in Drosophila: Turandot M promotes immunity against sexually transmitted fungal infections

Although it is well known that mating increases the risk of infection, we do not know how females mitigate the fitness costs of sexually transmitted infections (STIs). It has recently been shown that female fruitflies, Drosophila melanogaster, specifically upregulate two members of the Turandot family of immune and stress response genes, Turandot M and Turandot C (TotM and TotC), when they hear male courtship song. Here, we use the Gal4/UAS RNAi gene knockdown system to test whether the expression of these genes provides fitness benefits for females infected with the entomopathogenic fungus, Metarhizium robertsii under sexual transmission. As a control, we also examined the immunity conferred by Dorsal-related immunity factor (Dif), a central component of the Toll signalling pathway thought to provide immunity against fungal infections. We show that TotM, but not TotC or Dif, provides survival benefits to females following STIs, but not after direct topical infections. We also show that though the expression of TotM provides fecundity benefits for healthy females, it comes at a cost to their survival, which helps to explain why TotM is not constitutively expressed. Together, these results show that the anticipatory expression of TotM promotes specific immunity against fungal STIs and suggest that immune anticipation is more common than currently appreciated.     

Syntrophin-2 is required for eye development in Drosophila

Syntrophins are components of the dystrophin glycoprotein complex (DGC), which is encoded by causative genes of muscular dystrophies. The DGC is thought to play roles not only in linking the actin cytoskeleton to the extracellular matrix, providing stability to the cell membrane, but also in signal transduction. Because of their binding to a variety of different molecules, it has been suggested that syntrophins are adaptor proteins recruiting signaling proteins to membranes and the DGC. However, critical roles in vivo remain elusive. Drosophila Syntrophin-2 (Syn2) is an orthologue of human γ1/γ2-syntrophins. Western immunoblot analysis here showed Syn2 to be expressed throughout development, with especially high levels in the adult head. Morphological aberrations were observed in Syn2 knockdown adult flies, with lack of retinal elongation and malformation of rhabdomeres. Furthermore, Syn2 knockdown flies exhibited excessive apoptosis in third instar larvae and alterations in the actin localization in the pupal retinae. Genetic crosses with a collection of Drosophila deficiency stocks allowed us to identify seven genomic regions, deletions of which caused enhancement of the rough eye phenotype induced by Syn2 knockdown. This information should facilitate identification of Syn2 regulators in Drosophila and clarification of roles of Syn2 in eye development.     

The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis

Deubiquitinating enzymes (DUBs) counteract ubiquitin ligases to modulate the ubiquitination and stability of target signaling molecules. In Drosophila, the ubiquitin–proteasome system has a key role in the regulation of apoptosis, most notably, by controlling the abundance of the central apoptotic regulator DIAP1. Although the mechanism underlying DIAP1 ubiquitination has been extensively studied, the precise role of DUB(s) in controlling DIAP1 activity has not been fully investigated. Here we report the identification of a DIAP1-directed DUB using two complementary approaches. First, a panel of putative Drosophila DUBs was expressed in S2 cells to determine whether DIAP1 could be stabilized, despite treatment with death-inducing stimuli that would induce DIAP1 degradation. In addition, RNAi fly lines were used to detect modifiers of DIAP1 antagonist-induced cell death in the developing eye. Together, these approaches identified a previously uncharacterized protein encoded by CG8830, which we named DeUBiquitinating-Apoptotic-Inhibitor (DUBAI), as a novel DUB capable of preserving DIAP1 to dampen Drosophila apoptosis. DUBAI interacts with DIAP1 in S2 cells, and the putative active site of its DUB domain (C367) is required to rescue DIAP1 levels following apoptotic stimuli. DUBAI, therefore, represents a novel locus of apoptotic regulation in Drosophila, antagonizing cell death signals that would otherwise result in DIAP1 degradation.