Deletion of the Drosophila neuronal gene found in neurons disrupts brain anatomy and male courtship.

The fne (found-in-neurons) locus encodes one of the three paralogs of the ELAV gene family of Drosophila melanogaster. Members of this family are found throughout metazoans and encode RNA-binding proteins with primarily neuronal localization, but with remarkably diverse functions given their high level of amino acid sequence conservation. The first identified member of the family, elav of Drosophila is a vital gene. Mutations in the second Drosophila elav paralog, rbp9, are viable but female sterile. No alleles of fne were previously available. FNE protein is normally present in the cytoplasm of all neurons throughout development. Here we describe the generation and characterization of fne(null) mutations by homologous recombination. In contrast to elav and similar to rbp9, fne(null) mutants are viable, but exhibit a specific and fully penetrant fusion of the ß-lobes in their mushroom bodies (MB), a paired neuropil of the central brain involved in a variety of complex behaviors. Mutant males have reduced courtship indices, but normal short- and long-term courtship memory. Our data show that fne has specific functions which are non-overlapping with the other two family members, namely in courtship behavior and in the development of the adult MB. The data further show that courtship memory does not require intact ß-lobes in the MB.

Genetic analysis of cuticular hydrocarbons and their effect on courtship in Drosophila virilis and D. lummei.

Genetical factors controlling the cuticular hydrocarbons (CHC) of Drosophila virilis and D. lummei, and the effect of CHC on female attractivity were studied. A strong sexual dimorphism was found in D. virilis where (Z)-10-heneicosene and (Z)-11-pentacosene were major CHC for males and females, respectively. In D. lummei, (Z)-9-pentacosene was the major CHC for both sexes. These CHC are under a complex genetical control: the level of male (Z)-10-heneicosene was mainly sex-linked; chromosome 2 exerted a marked hypermorphic effect on it as well as on female (Z)-11-pentacosene; other autosomes had additional smaller effects on the CHC. Further, these two species showed differences in the double bond position pattern: Monoenes, with double bonds in (Z)-11 or (Z)-13, were more abundant in D. virilis (monoenes-vi), while monoenes, with double bonds in (Z)-7 or (Z)-9, were more abundant in D. lummei (monoenes-lu). In agreement with previous studies of perfumed dummies, experiments with live females showed that a higher proportion of (Z)-11-pentacosene or of monoenes-vi induced D. virilis males to court sooner. On the other hand, monoenes-lu showed antagonistic effects.

Role of cuticular hydrocarbons in the chemical recognition between ant species in the Pachycondyla villosa species complex.

Cuticular hydrocarbons (HCs) play important roles in insect communication but few studies clearly demonstrate the direct link between HCs and nestmate recognition. Therefore, cuticular lipids were extracted from ants, their HC and non-HC fractions as well as the three principal classes of HCs (n-alkanes, branched alkanes and alkenes) were purified and tested using an immobilizing "joust" device which allowed quantification of early pairwise behavioural responses, mandibular opening and antennal retraction, without occurrence of subsequent damages as in classic dyadic encounters. Chemical recognition of ants was studied at three levels of interactions (intra-colonial, intra-specific and inter-specific). Three closely related species already chemically characterized were used: Pachycondyla villosa (Pv), P. inversa (Pi) and P. subversa (Ps). Each species had its own behavioural responses. Moreover, responses of Pi and Ps towards Pv were significantly longer, than they were between themselves whereas Pv ants were equally aggressive towards Pi and Ps. These differences are in agreement with the results of the cluster analysis of the cuticular HCs profiles that place Pi closer to Ps. In support of the idea that components of cuticular lipids profiles are important for recognition, we found that only the HC fraction and its branched subfraction elicited a behavioural response of Ps workers. It is suggested that internally branched methyl- and dimethylalkanes are involved in recognition behaviour.

Hydrocarbon circulation and colonial signature in Pachycondyla villosa.

In ants, both cuticular and postpharyngeal gland (PPG) hydrocarbons (HCs) have been involved in nestmate recognition. However, no detailed comparison is available. A comparative study including also high density lipophorin (HDLp), an internal HC carrier, was therefore undertaken on Pachycondyla villosa. Purified HDLp is an 820 kDa lipoprotein with a density of 1.114 g/ml and two 245 and 80 kDa apo-proteins. Its hydrocarbon profile is very similar with the cuticular one, in agreement with its hydrocarbon carrier function. Conversely, n-alkanes and externally branched monomethylalkanes are markedly decreased in the PPG. According to their physical properties, this suggests that they are involved in waterproofing on the cuticle. The PPG actually contains only internally branched mono-, dimethylalkanes or monomethylalkenes; their greater fluidity is more adequate for chemical communication. The percentages of some of them are statistically not different between the cuticle and PPG. Their mixtures vary with colonies and they may thus be involved in colonial signature. A scheme for hydrocarbon circulation is discussed, involving lipophorin, cuticle, PPG and self-grooming in one individual, a pathway complementary or alternative to the selective delivery by lipophorin in some other insects. HCs are then distributed between nestmates' cuticles through allo-grooming and physical contacts.

Reproductive isolation in natural populations of Drosophila melanogaster from Brazzaville (Congo).

The aim of this work is to analyze the homogamy previously detected between two natural populations of Drosophila melanogaster from Brazzaville. It is shown that mating isolation was still maintained under laboratory conditions 10 years after the populations samples were trapped. Isolation seemed to be due mainly to premating isolation and we checked for any suggestion of post-mating mortality of hybrids. Pre-mating isolation was not symmetrical, and significant chi2 values were found in 3/4 possible 3-way mating choice experiments. The only exception involved a male from the countryside and two females (one from each population) for which no significant mating preference was detected. Mortality of hybrids was intermediate between those of the parental strains showing a clear maternal effect and the existence of partial dominance. Major differences in the cuticular hydrocarbons were also found and they could account for the isolation. These findings in populations from African breweries indicate that they are closely related to European ones, suggesting that this phenomenon is not a case of sympatric speciation, but probably attributable to the reintroduction of an allopatric population.

Compared ontogenesis of courtship song components of males from the sibling species, D. melanogaster and D. simulans.

The courtship song of Drosophila is known to be an important signal involved in sex and species recognition. It consists of pulse song and sine song, which have been studied in a quantitative way with different parameters. For the first time the setting of both components of the acoustic signaling is described and compared for males belonging to the sibling species D. melanogaster and D. simulans. At early ages, these two species share similar interpulse interval values but maturation establishes the species specificity of this character. For D. melanogaster the variations of several acoustic parameters take place in two successive periods, whereas for D. simulans the majority of the acoustic features does not change much with age. In D. melanogaster, copulation success seems to be linked to the maturation of the acoustic performance, which is not the case for D. simulans.

Control of female pheromones in Drosophila melanogaster by homeotic genes.

We have investigated the role of the Antennapedia and Bithorax complexes (ANT-C and BX-C) on the production of cuticular hydrocarbons in Drosophila melanogaster. In males, there is little, if any, influence of these complexes on the hydrocarbon pattern. In females, there are large and opposite effects of these complexes on diene production: two ANT-C mutations cause an increase in diene production and a reduction of monoenes, whereas most BX-C mutations result in a decrease in dienes and an increase in monoenes, although their sum remains constant. The effect is the highest in Mcp and iab6 females. It is suggested that a factor originating from the prothorax might activate the conversion of monoenes to dienes in females. The abdomen seems to have a crucial role in the production or control of pheromones: abdominal segments four to seven have the main effects, with a most dramatic effect for segments four and five.

A delta 9 desaturase gene with a different substrate specificity is responsible for the cuticular diene hydrocarbon polymorphism in Drosophila melanogaster.

Drosophila melanogaster cuticular pheromones consist of unsaturated hydrocarbons with at least one double bond in position 7: 7 tricosene (T) in males and 7,11 heptacosadiene (HD) in females. However, in many African populations like the Tai strain, females possess low levels of 7,11 HD and high levels of its positional isomer 5,9 HD. We have previously isolated a desaturase gene, desat1, from the Canton-S strain (CS), a 7,11 HD-2-rich morph of D. melanogaster. This desaturase is located in 87C, a locus that has been involved in the difference between 7,11 HD and 5,9 HD morphs. Therefore, we have searched for different desaturase isoforms in both strains. We first cloned desat1 in the Tai strain and report here functional expression of desat1 in CS and Tai. In both strains, the Desat1 enzymes have the same Delta9 specificity and preferentially use palmitate as a substrate, leading to the synthesis of omega7 fatty acids. Also found was a desaturase sequence, named desat2, with a homologous catalytic domain and a markedly different N-terminal domain compared with desat1. In CS genome, it lies 3.8 kb upstream of desat1 and is not transcribed in either sex. In the Tai strain, it is expressed only in females and acts preferentially on myristate, leading to the synthesis of omega5 fatty acids. We suggest, therefore, that desat2 might play a control role in the biosynthesis of 5,9 HD hydrocarbons in Tai females and could explain the dienic hydrocarbon polymorphism in D. melanogaster.

Sexual isolation of genetically differentiated sympatric populations of Drosophila melanogaster in Brazzaville, Congo: the first step towards speciation?

Two sympatric populations of Drosophila melanogaster were collected in the Brazzaville area in Congo, one from the suburban countryside and the other from a brewery located in the city. They were compared for several genetically determined traits including morphology, allozymes, microsatellites, cuticular hydrocarbons, and sexual behaviour. The two populations were similar to other African populations for morphological traits, but differed significantly from each other for all other characters. The countryside population resembled other African populations, whereas the urban population was consistently similar to European populations. Mating choice experiments showed incipient reproductive separation between the populations. In agreement with the hypothesis that D. melanogaster originated in Africa and spread to the rest of the world by invading human-modified habitats, we suggest that man-adapted fruit fly populations have returned 'back to Africa', and remained partially isolated from older native stocks.

Variations of male cuticular hydrocarbons with geoclimatic variables: an adaptative mechanism in Drosophila melanogaster?

7-tricosene (7T) and 7-pentacosene (7P) are the major components of cuticular hydrocarbons in Drosophila simulans and D. melanogaster males. A chemical study of 16 isofemale lines of D. melanogaster sampled at the first and eighth generations in laboratory conditions showed the stability of chromatographical profiles. Then a large scale study of male 7T/7P polymorphism was performed with 85 populations of D. melanogaster and 29 of D. simulans collected all over the world. There were significant correlations of the values of the balanced ratio (7T- 7P)/(7T + 7P) with geo-climatic parameters, such as latitude, longitude, mean temperature, temperature range and vapour pressure. Parallel variations were also reported for the homologous linear alkanes (23 and 25 Carbon atoms) but not for the longer branched alkanes (27 and 29 Carbon atoms). No correlation was significant for the D. simulans populations studied. In this species a similar polymorphism of 7T/7P was found but restricted to a few populations from West Equatorial Africa.

A gene responsible for a cuticular hydrocarbon polymorphism in Drosophila melanogaster.

Drosophila melanogaster is polymorphic for the major cuticular hydrocarbon of females. In most populations this hydrocarbon is 7,11-heptacosadiene, but females from Africa and the Caribbean usually possess low levels of 7,11-heptacosadiene and high quantities of its position isomer 5,9-heptacosadiene. Genetic analysis shows that the difference between these two morphs is due to variation at a single segregating factor located on the right arm of chromosome 3 near map position 51.5 and cytological position 87C-D. This is precisely the position of a desaturase gene previously sequenced using primers derived from yeast and mouse, and localized by in situ hybridization to the polytene chromosomes of D. melanogaster. Alleles of this desaturase gene may therefore be responsible for producing the two hydrocarbon morphs. Mating tests following the transfer of these isomers between females of the two morphs show that, in contrast to previous studies, the hydrocarbon profiles have no detectable effect on mating behaviour or sexual isolation.

Sexual behaviour in Drosophila is irreversibly programmed during a critical period.

Sexual differentiation in Drosophila is controlled by a short cascade of regulatory genes, the expression pattern of which determines all aspects of maleness and femaleness, including complex behaviours displayed by males and females [1-3]. One sex-determining gene is transformer (tra), the activity of which is needed for female development. Flies with a female karyotype (XX) but which are mutant for tra develop and behave as males. In such flies, a female phenotype can be restored by a transgene that carries the female-specific cDNA of tra under the control of a heat-shock promoter. This transgene, called hs[trafem], also transforms XY animals into sterile females [4]. When we raised these XX and XY 'females' at 25 degreesC, however, they displayed vigorous male courtship while at the same time, as a result of their female pheromone pattern, they were attractive to males. Intriguingly, their male courtship behaviour was indiscriminately directed towards both females and males. When we forced expression of tra by heat shock, applied during a limited period around puparium formation, male behaviour was abolished and replaced by female behaviour. We conclude that sexual behaviour is irreversibly programmed during a critical period as a result of the activity or inactivity of a single control gene.

Genetic feminization of pheromones and its behavioral consequences in Drosophila males.

Pheromones are intraspecific chemical signals important for mate attraction and discrimination. In the fruit fly Drosophila melanogaster, hydrocarbons on the cuticular surface of the animal are sexually dimorphic in both their occurrence and their effects: Female-specific molecules stimulate male sexual excitation, whereas the predominant male-specific molecule tends to inhibit male excitation. Complete feminization of the pheromone mixture produced by males was induced by targeted expression of the transformer gene in adult oenocytes (subcuticular abdominal cells) or by ubiquitous expression during early imaginal life. The resulting flies generally exhibited male heterosexual orientation but elicited homosexual courtship from other males.

Genetic dissection of sexual behavior in Drosophila melanogaster.

Mating of Drosophila melanogaster is a sterotypically patterned behavior consisting of a fixed sequence of actions that are primarily under genetic control. Mutations that disrupt specific aspects of mating activities offer a starting point for exploring the molecular machineries underlying sexual behavior. Several genes, identified as causing aberrant sexual behavior when mutated, have been isolated and cloned, providing molecular probes for expression and mosaic analyses that can be used in specifying the cells responsible for the behavior. This review presents current understandings of mating behavior obtained by such molecular and cellular approaches and provides an overview of future directions of research in behavioral genetics.

Incorporation of fatty acids into cuticular hydrocarbons of male and female Drosophila melanogaster.

The biosynthesis of cuticular hydrocarbons was investigated in male and female Drosophila melanogaster (Canton-S strain), especially in those with a pheromonal role i.e. male 7-tricosene and female 7, 11- heptacosadiene. The incorporation of radioactivity was followed after topical application of (14)C-labelled myristic, palmitic and stearic acid and (3)H-labelled cis-vaccenic acid on one to ten day old flies. The incorporation levels into unsaturated hydrocarbons are similar in both sexes, depending markedly on the chain length of the saturated precursor, with a maximum level from myristic acid. Cis-vaccenic acid leads only to unsaturated compounds. With this precursor, there is an enhanced incorporation into female monoenes and dienes, maximum in two to three day old females. The total fatty acid composition shows the highest abundance of fatty acids with 16 carbon atoms and the presence of a major position for double bond, Delta9. Moreover, cis-vaccenic acid and 17-tetracosenoic acid are identified by GC-MS analysis. These data support an elongation-decarboxylation mechanism for the biosynthesis of D. melanogaster cuticular hydrocarbons. Its early steps for male monoenes and female monoenes and dienes might involve a Delta9 desaturase transforming palmitic acid into palmitoleic acid which would then be elongated into vaccenic acid, an important common precursor for all pheromones.

Genetic control of male cuticular hydrocarbons in Drosophila melanogaster.

7-tricosene (7-T) and 7-pentacosene (7-P) are the two main hydrocarbons on the cuticle of male Drosophila melanogaster. These two substances might play a pheromonal role during courtship behaviour. We investigated the genetic basis of the quantitative polymorphism observed in the production of 7-T and 7-P. Strains of different geographic origin, with males producing either predominantly 7-T or predominantly 7-P, were hybridized with strains carrying genetic markers. We found that chromosome II changes the balance between 7-T and 7-P while chromosome III regulates the overall quantity of both 7-monoenes. We have also characterized and roughly mapped sept and smoq, two genetic factors on chromosome II that act additively on the production of both cuticular hydrocarbons. The genetic control of the variation in 7-T and 7-P varies between D. melanogaster strains and between D. melanogaster and its sibling species D. simulans. The possible evolutionary and physiological causes of this variation as well as its functional implication for courtship behaviour are discussed.

World-wide variation in Drosophila melanogaster sex pheromone: behavioural effects, genetic bases and potential evolutionary consequences.

In Drosophila melanogaster, male wing vibration, a key element of courtship behaviour, is most efficiently induced by a female-specific contact pheromone cis, cis 7,11 heptacosadiene (7,11 HD), which is the main mature female cuticular hydrocarbon in the CS laboratory strain. A study of 63 strains from around the world revealed that flies from Sub-Saharan Africa and the Caribbean are unique in showing low levels 7,11 HD and high levels of the position isomer 5,9 HD. This difference maps to chromosome III, perhaps indicating a simple genetic control of the 7,11 HD:5,9 HD ratio. Females from strains with high levels of 7,11 HD showed higher levels of mating and mated more rapidly than females with low levels of 7,11 HD. The results are discussed in light of recent discoveries of genetic differences between D. melanogaster strains from Africa and those from elsewhere around the world.

Purification of adult Drosophila melanogaster lipophorin and its role in hydrocarbon transport.

Lipophorin was isolated from homogenized adult Drosophila melanogaster. It is stained by Sudan Black and has a native molecular mass of 640 kD and a density of 1.12 g/ml. It consists of two glycosylated apoproteins of 240 and 75 kDa. Gas chromatography and mass spectrometry showed that lipophorins isolated separately from virgin 3-day-old male and female flies were associated with specific hydrocarbons, and that these were the same hydrocarbons found in male and female cuticles, respectively. Moreover, a pool of internal hydrocarbons was demonstrated for the first time, with chain lengths similar to those of the cuticular pool. Studies on the fate of the hydrocarbons synthesized de novo after topical applications of radiolabelled fatty acid precursors showed a decrease of the internal pool of hydrocarbons with time, concomitant with an increase of the cuticular pool. These results suggest that hydrocarbons synthesized at an internal site, possibly in oenocytes, may be transported to the cuticle of the flies by lipophorin.

Molecular basis ofMorinda citrifolia (L.): Toxicity on drosophila.

The ripe fruit ofMorinda citrifolia, host plant forDrosophila sechellia is highly toxic for three closely related species (D. melanogaster, D. simulans, andD. mauritiana). Green and rotten fruits are not toxic for all species tested. Short fatty acids were found to be present in large quantities in the extract of the ripe fruit. The most abundant (octanoic acid) was tested pure for its toxicity in a dose-dependent manner;D. sechellia is five to six times more resistant thanD. melanogaster to octanoic acid. Octanoic acid alone seems to be sufficient to explain the toxic effect of the pulp. It is less abundant in the rotten fruit and absent in the green fruit.