Esterase-6 and the pheromonal effects of cis-vaccenyl acetate in Drosophila melanogaster

In Drosophila melanogaster the polymorphic enzyme Est-6 and a male specific lipid, cis-vaccenyl acetate (cVA), are components of the seminal fluid. It has been suggested that cVA could be a physiological substrate for Est-6, constituting, together with the hypothetical hydrolytic product, cis-vaccenyl alcohol (cVOH), a pheromonal system active in the regulation of the sexual attractiveness of mated females. However, some doubt exists concerning the physiological conversion of cVA to cVOH in females. Est-6 is also known to affect sperm motility, sperm use and female fecundity. The results of a study on qualitative and quantitative effects of topical treatment of females with cis-vaccenyl acetate and cis-vaccenyl alcohol on courtship behaviour are presented. The inhibition of courtship produced by cVOH was more persistent; this was possibly related to its lower volatility. The hypothesis was tested that different Est-6 allozymes could produce different amounts of cis-vaccenyl alcohol in vivo by differential hydrolysis of cis-vaccenyl acetate, and thus affect the timing of remating. For this purpose highly homogeneous Est-6^S and Est-6^F homozygous lines, with almost certainly identical levels of Est-6 and cVA, were used. They were obtained after 100 generations of inbreeding and selection of the heterozygotes at each generation. The Est-6^S and Est-6^F products of the Est-6 structural gene did not differentially affect the timing of remating. This could imply that the two allozymes have similar catalytic properties. However, such results could be obtained even if cVA is not converted to cVOH in vivo by Est-6. The effects of Est-6 allozymes on sperm use after remating were also investigated. The general effect of second males was a very high elimination of first male fertilizations. No evidence for a different contribution of the two allozymes to the fitness through sperm predominance was obtained. Remating and sperm predominance experiments were performed at three temperatures: 18, 25 and 30° C. A more general—not species specific—role of cis-vaccenyl esters is suggested by preliminary results on their presence in the ejaculate of other Drosophila species.     

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.     

Automated analysis of courtship suppression learning and memory in Drosophila melanogaster

Study of the fruit fly, Drosophila melanogaster, has yielded important insights into the underlying molecular mechanisms of learning and memory. Courtship conditioning is a well-established behavioral assay used to study Drosophila learning and memory. Here, we describe the development of software to analyze courtship suppression assay data that correctly identifies normal or abnormal learning and memory traits of individual flies. Development of this automated analysis software will significantly enhance our ability to use this assay in large-scale genetic screens and disease modeling. The software increases the consistency, objectivity, and types of data generated.     

Social facilitation of long-lasting memory is mediated by CO2 in Drosophila

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The courtship tracking of Drosophila melanogaster

The courtship of male Drosophila melanogaster, which occurs so far as is known exclusively on the ground, may be regarded as a tracking process upon which are superimposed specific elements of courtship behaviour. Two methods were utilised to study this process, both of which involved the use of a fixed female courtship stimulus. The male courted this female whilst 1. running on the moving floor of a revolving chamber (female stationary) and 2. tracking her as she was rotated on a radius in a stationary chamber. Behaviour in the first apparatus was recorded via an electronic eye watching the flies and a keyboard operated by the experimenter. Behaviour in the second apparatus was filmed and subsequently analysed frame by frame. The patterning of courtship around the female was shown to be limited by the velocity at which the male had to run in order to court the female. At lower velocities more excursions (circling) to the side of the female were made. The mean distance of the male behind the female increased with velocity. The slope of this effect provides an estimate of the reciprocal feedback gain of the control system for tracking. Strain and velocity dependent differences in courtship variables were revealed. The proportion of wing extension emitted in tracking varied widely, the lowest value being shown by the strain which tracked the fastest (WT Kapelle). It is proposed that the relation between these two variables is of fundamental importance in determining the outcome of courtship. Using females moving sideways it was demonstrated that males orientate to the abdomen in close range of the female (<4 mm centre to centre), and more centrally at greater distance, where visual stimuli probably dominate as cues to orientation. Sinusoidal modulation of the female's velocity enabled a preliminary stimulus-response analysis of the linear aspects of courtship tracking. For males tracking females whose speed was modulated at a mean of 1.2 Hz a phase lag of 148±30ms was determined for the translatory component and 21±55ms for the rotatory component of the male's response. A strain difference between wild type Kapelle and Canton-S strains in tracking efficiency permits the conclusion that courtship tracking ability may provide a basis for selective mating in Drosophila.     

Oxygen regulation of airway branching in Drosophila is mediated by branchless FGF.

The Drosophila tracheal (respiratory) system is a tubular epithelial network that delivers oxygen to internal tissues. Sprouting of the major tracheal branches is stereotyped and controlled by hard-wired developmental cues. Here we show that ramification of the fine terminal branches is variable and regulated by oxygen, and that this process is controlled by a local signal or signals produced by oxygen-starved cells. We provide evidence that the critical signal is Branchless (Bnl) FGF, the same growth factor that patterns the major branches during embryogenesis. During larval life, oxygen deprivation stimulates expression of Bnl, and the secreted growth factor functions as a chemoattractant that guides new terminal branches to the expressing cells. Thus, a single growth factor is reiteratively used to pattern each level of airway branching, and the change in branch patterning results from a switch from developmental to physiological control of its expression.     

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.     

Phosphatidylcholine hydrolysis stimulated by phorbol myristate acetate is mediated principally by phospholipase D in endothelial cells

The mechanism of phosphatidylcholine (PC) degradation stimulated by phorbol myristate acetate (PMA) was investigated in bovine pulmonary artery endothelial cells prelabeled with [methyl-3H]choline ([3H]choline) or [9,10-3H]myristic acid ([3H]myristic acid). Both labels were selectively incorporated into PC, and addition of PMA stimulated comparable losses of 3H from PC in cells prelabeled with [3H]choline or [3H]myristate. In cells prelabeled with [3H]choline, the loss of 3H from PC correlated with a rapid increase in intracellular free [3H]choline. The increase in intracellular [3H]choline stimulated by PMA was not preceded by an increase in any other 3H-labeled PC degradation product. PMA did not stimulate the formation of PC deacylation products in cells prelabeled with [3H]choline. In permeabilized cells prelabeled with [3H]choline, PMA stimulated the formation of [3H]choline but not [3H]phosphocholine. In intact cells prelabeled with [3H]myristate, the loss of 3H from PC induced by PMA correlated with the formation of [3H]phosphatidic acid ([3H]PA) and [3H]diacylglycerol. In the presence of ethanol, PMA stimulated the formation of [3H]phosphatidylethanol ([3H]PEt) at the expense of [3H]PA. The time-course of [3H]PEt formation was similar to the time-course of intracellular [3H]choline formation in cells stimulated with PMA. These data taken together support the notion that PC degradation in endothelial cells stimulated with PMA is mediated principally by phospholipase D. PC breakdown via phospholipase D was not observed in cells treated with phorbol esters incapable of interacting with protein kinase C. Activation of phospholipase D by phorbol esters was inhibited by long-term pretreatment of cells with PMA to down-regulate protein kinase C and by pretreatment of the cells with staurosporine. These data support the notion that activation of phospholipase D by phorbol esters is dependent upon protein kinase C.     

The transmission of yeasts by Drosophila buzzatii during courtship and mating

The transmission of yeasts from generation to generation for Drosophila buzzatii was shown to occur vertically through the pupal stage and horizontally during mating. Males and females transmitted yeasts to the opposite sex most often during mating but also during courtship. Yeasts transferred from the male to the female were more often associated with the abdomens of the females, while yeasts transferred from the female to the male were associated with both the head and the abdomen of the male. Mate choice was affected by the previous yeast diet of both males and females. Yeast transmission during courtship and mating represents the potential for parental care because mate choice is affected by previous yeast diet, and progeny development is a function of the yeasts inoculated onto the larval substrate by the adults.     

Sexual dimorphism and courtship behavior in Drosophila prolongata

Sexual dimorphism is often derived from sexual selection. In sexually dimorphic Drosophila species, exaggerated male structures are used for specific behaviors in male-to-male competition or courtship toward females. In Drosophila prolongata, a member of the melanogaster species group, males have enlarged forelegs whereas females do not. However, the adaptive role of the enlarged forelegs is unclear because little is known about the behavior of D. prolongata. In this study, the courtship behavior of D. prolongata was investigated in comparison with closely related species. Males of D. prolongata use their forelegs in a specific behavior, “leg vibration”, in which the male vigorously vibrates the female’s abdomen by extending his forelegs from in front of her. Leg vibration was observed immediately before “attempting copulation”, indicating that it has an adaptive role in the mating process. In contrast, leg vibration was not observed in closely related species. Because the large forelegs are necessary to accomplish leg vibration, it was suggested that the sexual dimorphism of D. prolongata forelegs is currently under the influence of sexual selection in courtship behavior.     

fruitless splicing specifies male courtship behavior in Drosophila

All animals exhibit innate behaviors that are specified during their development. Drosophila melanogaster males (but not females) perform an elaborate and innate courtship ritual directed toward females (but not males). Male courtship requires products of the fruitless (fru) gene, which is spliced differently in males and females. We have generated alleles of fru that are constitutively spliced in either the male or the female mode. We show that male splicing is essential for male courtship behavior and sexual orientation. More importantly, male splicing is also sufficient to generate male behavior in otherwise normal females. These females direct their courtship toward other females (or males engineered to produce female pheromones). The splicing of a single neuronal gene thus specifies essentially all aspects of a complex innate behavior.     

Kin discrimination in sticklebacks is mediated by social learning rather than innate recognition

Theory predicts several advantages for animals to recognize kin. These include inbreeding avoidance and an increase in inclusive fitness. In shoaling species, kin recognition may lead to an increased amount of altruism among shoal members. Adult, non‐reproductive three‐spined sticklebacks, Gasterosteus aculeatus, prefer to shoal with kin. This preference was shown for familiar as well as for unfamiliar individuals. However, whether it is based on learned cues of familiar individuals or on innate mechanisms like self‐referent phenotype matching or ‘true’ kin recognition through recognition alleles remains unknown. In our experiments, juvenile fish were given the choice between shoals that differed in relatedness and familiarity. The number of testfish who joined each group indicated that sticklebacks prefer to shoal with familiar kin when the alternative shoal was composed of unfamiliar non‐kin. When one shoal consisted of familiar kin while the second consisted of familiar non‐kin testfish did not show any preference. Kin recognition in sticklebacks is thus most likely mediated by social learning.     

Regulation of the Drosophila epidermal growth factor-ligand vein is mediated by multiple domains

Vein (Vn), a ligand for the Drosophila epidermal growth factor receptor (Egfr), has a complex structure including a PEST, Ig, and EGF domain. We analyzed the structure-function relationships of Vn by assaying deletion mutants. The results show that each conserved domain influences Vn activity. A PEST deletion increases Vn potency and genetic evidence suggests that Vn is regulated by proteasomal degradation. The Ig deletion causes toxic effects not seen following expression of native Vn, but the Ig domain is not required for Vn localization or for the activation of Egfr signaling in wing vein patterning. Remarkably, when the EGF domain is deleted, Vn functions as a dominant negative ligand, implying that Vn normally physically interacts with another factor to promote its activity. We identified additional highly conserved sequences and found several regions that affect Vn potency and one that may mediate the effect of dominant negative Vn molecules. Together the results show that the activity of Vn is controlled both positively and negatively, demonstrating the existence of additional levels at which Egfr signaling can be regulated.     

A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship

Propagation in higher animals requires the efficient and accurate display of innate mating behaviors. In Drosophila melanogaster, male courtship consists of a stereotypic sequence of behaviors involving multiple sensory modalities, such as vision, audition, and chemosensation. For example, taste bristles located in the male forelegs and the labial palps are thought to recognize nonvolatile pheromones secreted by the female. Here, we report the identification of the putative pheromone receptor GR68a, which is expressed in chemosensory neurons of about 20 male-specific gustatory bristles in the forelegs. Gr68a expression is dependent on the sex determination gene doublesex, which controls many aspects of sexual differentiation and is necessary for normal courtship behavior. Tetanus toxin-mediated inactivation of Gr68a-expressing neurons or transgene-mediated RNA interference of Gr68a RNA leads to a significant reduction in male courtship performance, suggesting that GR68a protein is an essential component of pheromone-driven courtship behavior in Drosophila.     

Social context-dependent modification of courtship behaviour in Drosophila prolongata

Induction of alternative mating tactics by surrounding conditions, such as the presence of conspecific males, is observed in many animal species. Satellite behaviour is a remarkable example in which parasitic males exploit the reproductive investment by other males. Despite the abundance of parasitic mating tactics, however, few examples are known in which males alter courtship behaviour as a counter tactic against parasitic rivals. The fruit fly Drosophila prolongata shows prominent sexual dimorphism in the forelegs. When courting females, males of D. prolongata perform ‘leg vibration’, in which a male vibrates the female''s body with his enlarged forelegs. In this study, we found that leg vibration increased female receptivity, but it also raised a risk of interception of the female by rival males. Consequently, in the presence of rivals, males of D. prolongata shifted their courtship behaviour from leg vibration to ‘rubbing’, which was less vulnerable to interference by rival males. These results demonstrated that the males of D. prolongata adjust their courtship behaviour to circumvent the social context-dependent risk of leg vibration.