Population Density Affects Drosophila Male Pheromones in Laboratory-Acclimated and Natural Lines.

In large groups of vertebrates and invertebrates, aggregation can affect biological characters such as gene expression, physiological, immunological and behavioral responses. The insect cuticle is covered with hydrocarbons (cuticular hydrocarbons; CHCs) which reduce dehydration and increase protection against xenobiotics. Drosophila melanogaster and D. simulans flies also use some of their CHCs as contact pheromones. In these two sibling species, males also produce the volatile pheromone 11-cis-Vaccenyl acetate (cVa). To investigate the effect of insect density on the production of CHCs and cVa we compared the level of these male pheromones in groups of different sizes. These compounds were measured in six lines acclimated for many generations in our laboratory - four wild-type and one CHC mutant D. melanogaster lines plus one D. simulans line. Increasing the group size substantially changed pheromone amounts only in the four D. melanogaster wild-type lines. To evaluate the role of laboratory acclimation in this effect, we measured density-dependent pheromonal production in 21 lines caught in nature after 1, 12 and 25 generations in the laboratory. These lines showed varied effects which rarely persisted across generations. Although increasing group size often affected pheromone production in laboratory-established and freshly-caught D. melanogaster lines, this effect was not linear, suggesting complex determinants.

Natural Diversity of Cuticular Pheromones in a Local Population of Drosophila after Laboratory Acclimation.

Experimental studies of insects are often based on strains raised for many generations in constant laboratory conditions. However, laboratory acclimation could reduce species diversity reflecting adaptation to varied natural niches. Hydrocarbons covering the insect cuticle (cuticular hydrocarbons; CHCs) are reliable adaptation markers. They are involved in dehydration reduction and protection against harmful factors. CHCs can also be involved in chemical communication principally related to reproduction. However, the diversity of CHC profiles in nature and their evolution in the laboratory have rarely been investigated. Here, we sampled CHC natural diversity in Drosophila melanogaster flies from a particular location in a temperate region. We also measured cis-Vaccenyl acetate, a male-specific volatile pheromone. After trapping flies using varied fruit baits, we set up 21 D. melanogaster lines and analysed their pheromones at capture and after 1 to 40 generations in the laboratory. Under laboratory conditions, the broad initial pheromonal diversity found in male and female flies rapidly changed and became more limited. In some females, we detected CHCs only reported in tropical populations: the presence of flies with a novel CHC profile may reflect the rapid adaptation of this cosmopolitan species to global warming in a temperate area.

Colorimetric surface lipid quantification in Drosophila.

Insects are covered with free neutral cuticular hydrocarbons (CHC) that may be linear, branched, and unsaturated and vary in their chain length. The CHC composition is species-specific and contributes to the adaptation of the animal to its ecological niche. Commonly, CHCs contribute substantially to the inward and outward barrier function of the cuticle and serve pheromonal communication. They are generally determined by gas-chromatography, a time-consuming method requiring detailed expertize, but it is not available in many laboratories. Here, we report on the establishment of a colorimetric method allowing semi-quantitative determination of unsaturated CHCs in Drosophila flies. This method is based on the in vitro reaction of vanillin with double bounds in lipid molecules in an acidic solution to generate a reddish color. We found a robust correlation between gas chromatographic and vanillin-colorimetric data on unsaturated CHCs amounts in single flies. As the role of unsaturated CHCs in the performance of insects in their environment is only partly understood, we think that this novel method would allow fast and broad analyses of this type of CHCs in insects both in the field and in laboratories and thereby contribute to a substantial improvement in the investigation of this matter.

Replenishment of Drosophila Male Pheromone After Mating.

Insect exocrine gland products can be involved in sexual communication, defense, territory labelling, aggregation and alarm. In the vinegar fly Drosophila melanogaster the ejaculatory bulb synthesizes and releases 11-cis-Vaccenyl acetate (cVa). This pheromone, transferred to the female during copulation, affects aggregation, courtship and male-male aggressive behaviors. To determine the ability of male flies to replenish their cVa levels, males of a control laboratory strain and from the desat1 pheromone-defective mutant strain were allowed to mate successively with several females. We measured mating frequency, duration and latency, the amount of cVa transferred to mated females and the residual cVa in tested males. Mating duration remained constant with multiple matings, but we found that the amount of cVa transferred to females declined with multiple matings, indicating that, over short, biologically-relevant periods, replenishment of the pheromone does not keep up with mating frequency, resulting in the transfer of varying quantities of cVa. Adult responses to cVa are affected by early developmental exposure to this pheromone; our revelation of quantitative variation in the amount of cVa transferred to females in the event of multiple matings by a male suggests variable responses to cVa shown by adults produced by such matings. This implies that the natural role of this compound may be richer than suggested by laboratory experiments that study only one mating event and its immediate behavioral or neurobiological consequences.

Factors affecting the biosynthesis and emission of a Drosophila pheromone.

The most studied pheromone in Drosophila melanogaster, cis-vaccenyl acetate (cVA), is synthesized in the male ejaculatory bulb and transferred to the female during copulation. Combined with other chemicals, cVA can modulate fly aggregation, courtship, mating and fighting. We explored the mechanisms underlying both cVA biosynthesis and emission in males of two wild types and a pheromonal mutant line. The effects of ageing, adult social interaction, and maternally transmitted cVA and microbes - both associated with the egg chorion - on cVA biosynthesis and emission were measured. While ageing and genotype changed both biosynthesis and emission in similar ways, early developmental exposure to maternally transmitted cVA and microbes strongly decreased cVA emission but not the biosynthesis of this molecule. This indicates that the release - but not the biosynthesis - of this sex pheromone strongly depends on early developmental context. The mechanism by which the preimaginal effects occur is unknown, but reinforces the significance of development in determining adult physiology and behaviour.

Aging-Related Variation of Cuticular Hydrocarbons in Wild Type and Variant Drosophila melanogaster.

The cuticle of all insects is covered with hydrocarbons which have multiple functions. Cuticular hydrocarbons (CHCs) basically serve to protect insects against environmental harm and reduce dehydration. In many species, some CHCs also act as pheromones. CHCs have been intensively studied in Drosophila species and more especially in D. melanogaster. In this species, flies produce about 40 CHCs forming a complex sex- and species-specific bouquet. The quantitative and qualitative pattern of the CHC bouquet was characterized during the first days of adult life but remains unexplored in aging flies. Here, we characterized CHCs during the whole-or a large period of-adult life in males and females of several wild type and transgenic lines. Both types of lines included standard and variant CHC profiles. Some of the genotypes tested here showed very dramatic and unexpected aging-related variation based on their early days' profile. This study provides a concrete dataset to better understand the mechanisms underlying the establishment and maintenance of CHCs on the fly cuticle. It could be useful to determine physiological parameters, including age and response to climate variation, in insects collected in the wild.

Experimental Introgression To Evaluate the Impact of Sex Specific Traits on Drosophila melanogaster Incipient Speciation.

Sex specific traits are involved in speciation but it is difficult to determine whether their variation initiates or reinforces sexual isolation. In some insects, speciation depends of the rapid change of expression in desaturase genes coding for sex pheromones. Two closely related desaturase genes are involved in Drosophila melanogaster pheromonal communication: desat1 affects both the production and the reception of sex pheromones while desat2 is involved in their production in flies of Zimbabwe populations. There is a strong asymmetric sexual isolation between Zimbabwe populations and all other "Cosmopolitan" populations: Zimbabwe females rarely copulate with Cosmopolitan males whereas Zimbabwe males readily copulate with all females. All populations express desat1 but only Zimbabwe strains show high desat2 expression. To evaluate the impact of sex pheromones, female receptivity and desat expression on the incipient speciation process between Zimbabwe and Cosmopolitan populations, we introgressed the Zimbabwe genome into a Cosmopolitan genome labeled with the white mutation, using a multi-generation procedure. The association between these sex-specific traits was determined during the procedure. The production of pheromones was largely dissociated between the sexes. The copulation frequency (but not latency) was highly correlated with the female-but not with the male-principal pheromones. We finally obtained two stable white lines showing Zimbabwe-like sex pheromones, copulation discrimination and desat expression. Our study indicates that the variation of sex pheromones and mating discrimination depend of distinct-yet overlapping-sets of genes in each sex suggesting that their cumulated effects participate to reinforce the speciation process.

Desiccation resistance: effect of cuticular hydrocarbons and water content in Drosophila melanogaster adults.

BACKGROUND: The insect cuticle covers the whole body and all appendages and has bi-directionnal selective permeability: it protects against environmental stress and pathogen infection and also helps to reduce water loss. The adult cuticle is often associated with a superficial layer of fatty acid-derived molecules such as waxes and long chain hydrocarbons that prevent rapid dehydration. The waterproofing properties of cuticular hydrocarbons (CHs) depend on their chain length and desaturation number. Drosophila CH biosynthesis involves an enzymatic pathway including several elongase and desaturase enzymes. METHODS: The link between desiccation resistance and CH profile remains unclear, so we tested (1) experimentally selected desiccation-resistant lines, (2) transgenic flies with altered desaturase expression and (3) natural and laboratory-induced CH variants. We also explored the possible relationship between desiccation resistance, relative water content and fecundity in females. RESULTS: We found that increased desiccation resistance is linked with the increased proportion of desaturated CHs, but not with their total amount. Experimentally-induced desiccation resistance and CH variation both remained stable after many generations without selection. Conversely, flies with a higher water content and a lower proportion of desaturated CHs showed reduced desiccation resistance. This was also the case in flies with defective desaturase expression in the fat body. DISCUSSION: We conclude that rapidly acquired desiccation resistance, depending on both CH profile and water content, can remain stable without selection in a humid environment. These three phenotypes, which might be expected to show a simple relationship, turn out to have complex physiological and genetic links.

Maternally-transmitted microbiota affects odor emission and preference in Drosophila larva.

Experimental studies show that early sensory experience often affects subsequent sensory preference, suggesting that the heterogeneity of sensory cues in nature could induce significant inter-individual behavioral variation, potentially contributing to maintain intraspecific diversity. To test this hypothesis, we explored the behavioral effect induced by variation in the levels of a self-produced chemical, acetoin, and its link with intraspecific diversity. Acetoin is a pheromone-like substance produced by gut-associated microorganisms in Drosophila. Using wild-type Drosophila melanogaster populations producing variable acetoin levels, we (i) characterized factors involved in this variation and (ii) manipulated some of these factors to affect acetoin responses in larvae. We found that increased and decreased variations in acetoin levels were caused by microorganisms associated with the outside and inside of the egg, respectively. Wild-type larvae preferred acetoin-rich food only when they both produced and were exposed to substantial amounts of acetoin. The removal of the outside of the egg or the genetic alteration of olfaction abolished this preference. In contrast, larvae exposed to high doses of synthetic acetoin were repulsed by acetoin. The similar effects obtained with freshly caught wild-type lines suggest that this acetoin "production-preference" link underlies the diversity of acetoin-producing microorganisms among natural D. melanogaster populations.

Drosophila Food-Associated Pheromones: Effect of Experience, Genotype and Antibiotics on Larval Behavior.

Animals ubiquitously use chemical signals to communicate many aspects of their social life. These chemical signals often consist of environmental cues mixed with species-specific signals-pheromones-emitted by conspecifics. During their life, insects can use pheromones to aggregate, disperse, choose a mate, or find the most suitable food source on which to lay eggs. Before pupariation, larvae of several Drosophila species migrate to food sources depending on their composition and the presence of pheromones. Some pheromones derive from microbiota gut activity and these food-associated cues can enhance larval attraction or repulsion. To explore the mechanisms underlying the preference (attraction/repulsion) to these cues and clarify their effect, we manipulated factors potentially involved in larval response. In particular, we found that the (i) early exposure to conspecifics, (ii) genotype, and (iii) antibiotic treatment changed D. melanogaster larval behavior. Generally, larvae-tested either individually or in groups-strongly avoided food processed by other larvae. Compared to previous reports on larval attractive pheromones, our data suggest that such attractive effects are largely masked by food-associated compounds eliciting larval aversion. The antagonistic effect of attractive vs. aversive compounds could modulate larval choice of a pupariation site and impact the dispersion of individuals in nature.

When Choice Makes Sense: Menthol Influence on Mating, Oviposition and Fecundity in Drosophila melanogaster.

The environment to which insects have been exposed as larvae and adults can affect subsequent behaviors, such as mating, oviposition, food preference or fitness. Experience can change female preference for oviposition, particularly in phytophagous insects. In Drosophila melanogaster, females avoid laying eggs on menthol rich-food when given the choice. Exposure to menthol during larval development reduces this aversion. However, this observation was not reproduced in the following generation. Recently, we have shown that oviposition-site preference (OSP) differs between wild-type D. melanogaster lines freely or forcibly exposed to menthol. After 12 generations, menthol "forced" lines still exhibit a persistent aversion to menthol whereas 'free-choice' lines show a decreased aversion for menthol rich-food. Here, we compare courtship behavior, mating and female fecundity in "forced" and "free-choice" lines, raised either on menthol rich-food (Menthol-lines) or on menthol-free food (Plain-lines). "Forced" males did not discriminate between decapitated virgin females of the two lines. They courted and mated with intact females of both "forced" lines in a comparable rate. However "forced" M-line males did mate significantly more rapidly with "forced" M-line females. In the "free-choice" procedure, P-line males show a similar pattern as "forced" males for discrimination ability and courtship. M-line males courted significantly more M-line females. Both 'free-choice' lines males mated significantly more with females of their own line. Female fecundity was assessed during 10 days in 'free-choice' lines. Menthol-line females laid more eggs during the first 4 days than female Plain-lines and parental control-line. The total number of eggs laid during the first 10 days of female adult life is comparable in M-line and parental control line. However, Menthol-line females laid eggs earlier than both parental control and Plain-lines. Our findings show that in D. melanogaster, as for OSP, mating and fecundity are more rapidly influenced when flies have a choice between alternative resources compared to flies permanently exposed to menthol.

Drosophila adult and larval pheromones modulate larval food choice.

Insects use chemosensory cues to feed and mate. In Drosophila, the effect of pheromones has been extensively investigated in adults, but rarely in larvae. The colonization of natural food sources by Drosophila buzzatii and Drosophila simulans species may depend on species-specific chemical cues left in the food by larvae and adults. We identified such chemicals in both species and measured their influence on larval food preference and puparation behaviour. We also tested compounds that varied between these species: (i) two larval volatile compounds: hydroxy-3-butanone-2 and phenol (predominant in D. simulans and D. buzzatii, respectively), and (ii) adult cuticular hydrocarbons (CHs). Drosophila buzzatii larvae were rapidly attracted to non-CH adult conspecific cues, whereas D. simulans larvae were strongly repulsed by CHs of the two species and also by phenol. Larval cues from both species generally reduced larval attraction and pupariation on food, which was generally--but not always--low, and rarely reflected larval response. As these larval and adult pheromones specifically influence larval food search and the choice of a pupariation site, they may greatly affect the dispersion and survival of Drosophila species in nature.

Choice alters Drosophila oviposition site preference on menthol.

Food choice and preference relies on multiple sensory systems that are under the control of genes and sensory experience. Exposure to specific nutrients and nutrient-related molecules can change food preference in vertebrates and invertebrates. For example, larval exposure of several holometabolous insects to menthol can change their adult response to this molecule. However, studies involving Drosophila melanogaster exposure to menthol produced controversial results due maybe to methodological differences. Here, we compared the oviposition-site preference of wild-type D. melanogaster lines freely or forcibly exposed to menthol-rich food. After 12 generations, oviposition-site preference diverged between the two lines. Counterintuitively, menthol 'forced' lines showed a persistent aversion to menthol whereas 'free choice' lines exhibited a decreased aversion to menthol-rich food. This effect was specific to menthol since the 'free choice' lines showed unaltered responses to caffeine and sucrose. This suggests that the genetic factors underlying Drosophila oviposition site preference are more rapidly influenced when flies have a choice between alternative sources compared to flies permanently exposed to the same aversive substance.

An olfactory receptor for food-derived odours promotes male courtship in Drosophila.

Many animals attract mating partners through the release of volatile sex pheromones, which can convey information on the species, gender and receptivity of the sender to induce innate courtship and mating behaviours by the receiver. Male Drosophila melanogaster fruitflies display stereotyped reproductive behaviours towards females, and these behaviours are controlled by the neural circuitry expressing male-specific isoforms of the transcription factor Fruitless (FRU(M)). However, the volatile pheromone ligands, receptors and olfactory sensory neurons (OSNs) that promote male courtship have not been identified in this important model organism. Here we describe a novel courtship function of Ionotropic receptor 84a (IR84a), which is a member of the chemosensory ionotropic glutamate receptor family, in a previously uncharacterized population of FRU(M)-positive OSNs. IR84a-expressing neurons are activated not by fly-derived chemicals but by the aromatic odours phenylacetic acid and phenylacetaldehyde, which are widely found in fruit and other plant tissues that serve as food sources and oviposition sites for drosophilid flies. Mutation of Ir84a abolishes both odour-evoked and spontaneous electrophysiological activity in these neurons and markedly reduces male courtship behaviour. Conversely, male courtship is increased--in an IR84a-dependent manner--in the presence of phenylacetic acid but not in the presence of another fruit odour that does not activate IR84a. Interneurons downstream of IR84a-expressing OSNs innervate a pheromone-processing centre in the brain. Whereas IR84a orthologues and phenylacetic-acid-responsive neurons are present in diverse drosophilid species, IR84a is absent from insects that rely on long-range sex pheromones. Our results suggest a model in which IR84a couples food presence to the activation of the fru(M) courtship circuitry in fruitflies. These findings reveal an unusual but effective evolutionary solution to coordinate feeding and oviposition site selection with reproductive behaviours through a specific sensory pathway.