The transplantation of the gut microbiome of fat-1 mice protects against colonic mucus layer disruption and endoplasmic reticulum stress induced by high fat diet.

High-fat diets alter gut barrier integrity, leading to endotoxemia by impacting epithelial functions and inducing endoplasmic reticulum (ER) stress in intestinal secretory goblet cells. Indeed, ER stress, which is an important contributor to many chronic diseases such as obesity and obesity-related disorders, leads to altered synthesis and secretion of mucins that form the protective mucus barrier. In the present study, we investigated the relative contribution of omega-3 polyunsaturated fatty acid (PUFAs)-modified microbiota to alleviating alterations in intestinal mucus layer thickness and preserving gut barrier integrity. Male fat-1 transgenic mice (exhibiting endogenous omega-3 PUFAs tissue enrichment) and wild-type (WT) littermates were fed either an obesogenic high-fat diet (HFD) or a control diet. Unlike WT mice, HFD-fed fat-1 mice were protected against mucus layer alterations as well as an ER stress-mediated decrease in mucin expression. Moreover, cecal microbiota transferred from fat-1 to WT mice prevented changes in the colonic mucus layer mainly through colonic ER stress downregulation. These findings highlight a novel feature of the preventive effects of omega-3 fatty acids against intestinal permeability in obesity-related conditions.

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.

Transcriptional Control of Quality Differences in the Lipid-Based Cuticle Barrier in Drosophila suzukii and Drosophila melanogaster.

Cuticle barrier efficiency in insects depends largely on cuticular lipids. To learn about the evolution of cuticle barrier function, we compared the basic properties of the cuticle inward and outward barrier function in adults of the fruit flies Drosophila suzukii and Drosophila melanogaster that live on fruits sharing a similar habitat. At low air humidity, D. suzukii flies desiccate faster than D. melanogaster flies. We observed a general trend indicating that in this respect males are less robust than females in both species. Xenobiotics penetration occurs at lower temperatures in D. suzukii than in D. melanogaster. Likewise, D. suzukii flies are more susceptible to contact insecticides than D. melanogaster flies. Thus, both the inward and outward barriers of D. suzukii are less efficient. Consistently, D. suzukii flies have less cuticular hydrocarbons (CHC) that participate as key components of the cuticle barrier. Especially, the relative amounts of branched and desaturated CHCs, known to enhance desiccation resistance, show reduced levels in D. suzukii. Moreover, the expression of snustorr (snu) that encodes an ABC transporter involved in barrier construction and CHC externalization, is strongly suppressed in D. suzukii. Hence, species-specific genetic programs regulate the quality of the lipid-based cuticle barrier in these two Drosophilae. Together, we conclude that the weaker inward and outward barriers of D. suzukii may be partly explained by differences in CHC composition and by a reduced Snu-dependent transport rate of CHCs to the surface. In turn, this suggests that snu is an ecologically adjustable and therefore relevant gene in cuticle barrier efficiency.

Dysfunction of Oskyddad causes Harlequin-type ichthyosis-like defects in Drosophila melanogaster.

Prevention of desiccation is a constant challenge for terrestrial organisms. Land insects have an extracellular coat, the cuticle, that plays a major role in protection against exaggerated water loss. Here, we report that the ABC transporter Oskyddad (Osy)-a human ABCA12 paralog-contributes to the waterproof barrier function of the cuticle in the fruit fly Drosophila melanogaster. We show that the reduction or elimination of Osy function provokes rapid desiccation. Osy is also involved in defining the inward barrier against xenobiotics penetration. Consistently, the amounts of cuticular hydrocarbons that are involved in cuticle impermeability decrease markedly when Osy activity is reduced. GFP-tagged Osy localises to membrane nano-protrusions within the cuticle, likely pore canals. This suggests that Osy is mediating the transport of cuticular hydrocarbons (CHC) through the pore canals to the cuticle surface. The envelope, which is the outermost cuticle layer constituting the main barrier, is unaffected in osy mutant larvae. This contrasts with the function of Snu, another ABC transporter needed for the construction of the cuticular inward and outward barriers, that nevertheless is implicated in CHC deposition. Hence, Osy and Snu have overlapping and independent roles to establish cuticular resistance against transpiration and xenobiotic penetration. The osy deficient phenotype parallels the phenotype of Harlequin ichthyosis caused by mutations in the human abca12 gene. Thus, it seems that the cellular and molecular mechanisms of lipid barrier assembly in the skin are conserved during evolution.

Evolution of sexually dimorphic pheromone profiles coincides with increased number of male-specific chemosensory organs in Drosophila prolongata.

Binary communication systems that involve sex-specific signaling and sex-specific signal perception play a key role in sexual selection and in the evolution of sexually dimorphic traits. The driving forces and genetic changes underlying such traits can be investigated in systems where sex-specific signaling and perception have emerged recently and show evidence of potential coevolution. A promising model is found in Drosophila prolongata, which exhibits a species-specific increase in the number of male chemosensory bristles. We show that this transition coincides with recent evolutionary changes in cuticular hydrocarbon (CHC) profiles. Long-chain CHCs that are sexually monomorphic in the closest relatives of D. prolongata (D. rhopaloa, D. carrolli, D. kurseongensis, and D. fuyamai) are strongly male-biased in this species. We also identify an intraspecific female-limited polymorphism, where some females have male-like CHC profiles. Both the origin of sexually dimorphic CHC profiles and the female-limited polymorphism in D. prolongata involve changes in the relative amounts of three mono-alkene homologs, 9-tricosene, 9-pentacosene, and 9-heptacosene, all of which share a common biosynthetic origin and point to a potentially simple genetic change underlying these traits. Our results suggest that pheromone synthesis may have coevolved with chemosensory perception and open the way for reconstructing the origin of sexual dimorphism in this communication system.

Flying Drosophila show sex-specific attraction to fly-labelled food.

Animals searching for food and sexual partners often use odourant mixtures combining food-derived molecules and pheromones. For orientation, the vinegar fly Drosophila melanogaster uses three types of chemical cues: (i) the male volatile pheromone 11-cis-vaccenyl acetate (cVA), (ii) sex-specific cuticular hydrocarbons (CHs; and CH-derived compounds), and (iii) food-derived molecules resulting from microbiota activity. To evaluate the effects of these chemicals on odour-tracking behaviour, we tested Drosophila individuals in a wind tunnel. Upwind flight and food preference were measured in individual control males and females presented with a choice of two food sources labelled by fly lines producing varying amounts of CHs and/or cVA. The flies originated from different species or strains, or their microbiota was manipulated. We found that (i) fly-labelled food could attract-but never repel-flies; (ii) the landing frequency on fly-labelled food was positively correlated with an increased flight duration; (iii) male-but not female or non-sex-specific-CHs tended to increase the landing frequency on fly-labelled food; (iv) cVA increased female-but not male-preference for cVA-rich food; and (v) microbiota-derived compounds only affected male upwind flight latency. Therefore, sex pheromones interact with food volatile chemicals to induce sex-specific flight responses in Drosophila.

Live yeast in juvenile diet induces species-specific effects on Drosophila adult behaviour and fitness.

The presence and the amount of specific yeasts in the diet of saprophagous insects such as Drosophila can affect their development and fitness. However, the impact of different yeast species in the juvenile diet has rarely been investigated. Here, we measured the behavioural and fitness effects of three live yeasts (Saccharomyces cerevisiae = SC; Hanseniaspora uvarum = HU; Metschnikowia pulcherrima = MP) added to the diet of Drosophila melanogaster larvae. Beside these live yeast species naturally found in natural Drosophila populations or in their food sources, we tested the inactivated "drySC" yeast widely used in Drosophila research laboratories. All flies were transferred to drySC medium immediately after adult emergence, and several life traits and behaviours were measured. These four yeast diets had different effects on pre-imaginal development: HU-rich diet tended to shorten the "egg-to-pupa" period of development while MP-rich diet induced higher larval lethality compared to other diets. Pre- and postzygotic reproduction-related characters (copulatory ability, fecundity, cuticular pheromones) varied according to juvenile diet and sex. Juvenile diet also changed adult food choice preference and longevity. These results indicate that specific yeast species present in natural food sources and ingested by larvae can affect their adult characters crucial for fitness.

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.

The desaturase1 gene affects reproduction before, during and after copulation in Drosophila melanogaster.

Desaturase1 (desat1) is one of the few genes known to be involved in the two complementary aspects of sensory communication - signal emission and signal reception - in Drosophila melanogaster. In particular, desat1 is necessary for the biosynthesis of major cuticular pheromones in both males and females. It is also involved in the male ability to discriminate sex pheromones. Each of these two sensory communication aspects depends on distinct desat1 putative regulatory regions. Here, we used (i) mutant alleles resulting from the insertion/excision of a transposable genomic element inserted in a desat1 regulatory region, and (ii) transgenics made with desat1 regulatory regions used to target desat1 RNAi. These genetic variants were used to study several reproduction-related phenotypes. In particular, we compared the fecundity of various mutant and transgenic desat1 females with regard to the developmental fate of their progeny. We also compared the mating performance in pairs of flies with altered desat1 expression in various desat1-expressing tissues together with their inability to disengage at the end of copulation. Moreover, we investigated the developmental origin of altered sex pheromone discrimination in male flies. We attempted to map some of the tissues involved in these reproduction-related phenotypes. Given that desat1 is expressed in many brain neurons and in non-neuronal tissues required for varied aspects of reproduction, our data suggest that the regulation of this gene has evolved to allow the optimal reproduction and a successful adaptation to varied environments in this cosmopolitan species.

Pre-imaginal conditioning alters adult sex pheromone response in Drosophila.

Pheromones are chemical signals that induce innate responses in individuals of the same species that may vary with physiological and developmental state. In Drosophila melanogaster, the most intensively studied pheromone is 11-cis-vaccenyl acetate (cVA), which is synthezised in the male ejaculatory bulb and is transferred to the female during copulation. Among other effects, cVA inhibits male courtship of mated females. We found that male courtship inhibition depends on the amount of cVA and this effect is reduced in male flies derived from eggs covered with low to zero levels of cVA. This effect is not observed if the eggs are washed, or if the eggs are laid several days after copulation. This suggests that courtship suppression involves a form of pre-imaginal conditioning, which we show occurs during the early larval stage. The conditioning effect could not be rescued by synthetic cVA, indicating that it largely depends on conditioning by cVA and other maternally-transmitted factor(s). These experiments suggest that one of the primary behavioral effects of cVA is more plastic and less stereotypical than had hitherto been realised.

Yeast quality in juvenile diet affects Drosophila melanogaster adult life traits.

Diet quality is critical for animal development and survival. Fungi can provide nutrients that are essential to organisms that are unable to synthetize them, such as ergosterol in Drosophila melanogaster. Drosophila studies examining the influence of yeast quality in the diet have generally either provided the diet over the whole life span (larva to adult) or during the adult stage and have rarely focussed on the juvenile diet. Here, we tested the effect of yeast quality in the larval diet on pre-adult development and adult weight, survival, reproduction and food preference. The yeast Saccharomyces cerevisiae was added in three forms in three treatments-live, heated or dried-to food used as the juvenile diet or was not added (empty treatment). Adults resulting from the larvae raised on these four juvenile diets were all maintained on a similar standard laboratory food diet. Our data indicate that yeast quality in the juvenile diet affects larva-to-pupa-but not pupa-to-adult-development. Importantly, adult survival, food preference, mating behaviour and cuticular pheromones strongly varied with the juvenile diet. Therefore, the variation of yeast quality in the pre-adult Drosophila diet affects key adult life traits involved in food search, reproduction and survival.

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.

Regulation of cuticular hydrocarbon profile maturation by Drosophila tanning hormone, bursicon, and its interaction with desaturase activity.

Shortly after emergence the exoskeleton (cuticle) of adult insects is rapidly expanded, hardened (sclerotized), and pigmented (melanized). In parallel with this process, the oenocytes, which are large polyploid cells located below the abdominal epidermis, secrete onto the cuticle a cocktail of cuticular hydrocarbons (CHs) and waxes. These improve the waterproofing of the cuticle, and also provide important chemosensory and pheromonal cues linked with gender, age, and species differentiation. The hardening and pigmentation of the new cuticle are controlled by the neurohormone, bursicon, and its receptor, encoded by the DLGR2 receptor, rickets (rk); by contrast, little is known about the timecourse of changes in CH profile and about the role of bursicon in this process. Here we show in Drosophila that rk function is also required for the normal maturation of the fly's CH profile, with flies mutant for rk function showing dramatically elevated levels of CHs. Interestingly, this effect is mostly abrogated by mutations in the Δ9 desaturase encoded by the desaturase1 gene, which introduces a first double bond into elongated fatty-acid chains, suggesting that desaturase1 acts downstream of rk. In addition, flies mutant for rk showed changes in the absolute and relative levels of specific 7-monoenes (in males) and 7,11-dienes (in females). The fact that these differences in CH amounts were obtained using extractions of very different durations suggests that the particular CH profile of flies mutant for rk is not simply due to their unsclerotized cuticle but that bursicon may be involved in the process of CH biosynthesis itself.

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.

Dietary rescue of altered metabolism gene reveals unexpected Drosophila mating cues.

To develop and reproduce, animals need long-chain MUFAs and PUFAs. Although some unsaturated FAs (UFAs) can be synthesized by the organism, others must be provided by the diet. The gene, desat1, involved in Drosophila melanogaster UFA metabolism, is necessary for both larval development and for adult sex pheromone communication. We first characterized desat1 expression in larval tissues. Then, we found that larvae in which desat1 expression was knocked down throughout development died during the larval stages when raised on standard food. By contrast pure MUFAs or PUFAs, but not saturated FAs, added to the larval diet rescued animals to adulthood with the best effect being obtained with oleic acid (C18:1). Male and female mating behavior and fertility were affected very differently by preimaginal UFA-rich diet. Adult diet also strongly influenced several aspects of reproduction: flies raised on a C18:1-rich diet showed increased mating performance compared with flies raised on standard adult diet. Therefore, both larval and adult desat1 expression control sex-specific mating signals. A similar nutrigenetics approach may be useful in other metabolic mutants to uncover cryptic effects otherwise masked by severe developmental defects.

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.

Transient and permanent experience with fatty acids changes Drosophila melanogaster preference and fitness.

Food and host-preference relies on genetic adaptation and sensory experience. In vertebrates, experience with food-related cues during early development can change adult preference. This is also true in holometabolous insects, which undergo a drastic nervous system remodelling during their complete metamorphosis, but remains uncertain in Drosophila melanogaster. We have conditioned D. melanogaster with oleic (C18:1) and stearic (C18:0) acids, two common dietary fatty acids, respectively preferred by larvae and adult. Wild-type individuals exposed either during a transient period of development-from embryo to adult-or more permanently-during one to ten generation cycles-were affected by such conditioning. In particular, the oviposition preference of females exposed to each fatty acid during larval development was affected without cross-effect indicating the specificity of each substance. Permanent exposure to each fatty acid also drastically changed oviposition preference as well as major fitness traits (development duration, sex-ratio, fecundity, adult lethality). This suggests that D. melanogaster ability to adapt to new food sources is determined by its genetic and sensory plasticity both of which may explain the success of this generalist-diet species.

Volatile Drosophila cuticular pheromones are affected by social but not sexual experience.

Recognition of conspecifics and mates is based on a variety of sensory cues that are specific to the species, sex and social status of each individual. The courtship and mating activity of Drosophila melanogaster flies is thought to depend on the olfactory perception of a male-specific volatile pheromone, cis-vaccenyl acetate (cVA), and the gustatory perception of cuticular hydrocarbons (CHs), some of which are sexually dimorphic. Using two complementary sampling methods (headspace Solid Phase Micro-Extraction [SPME] and solvent extraction) coupled with GC-MS analysis, we measured the dispersion of pheromonal CHs in the air and on the substrate around the fly. We also followed the variations in CHs that were induced by social and sexual interactions. We found that all CHs present on the fly body were deposited as a thin layer on the substrate, whereas only a few of these molecules were also detected in the air. Moreover, social experience during early adult development and in mature flies strongly affected male volatile CHs but not cVA, whereas sexual interaction only had a moderate influence on dispersed CHs. Our study suggests that, in addition to their role as contact cues, CHs can influence fly behavior at a distance and that volatile, deposited and body pheromonal CHs participate in a three-step recognition of the chemical identity and social status of insects.

Fatty-acid preference changes during development in Drosophila melanogaster.

Fatty-acids (FAs) are required in the diet of many animals throughout their life. However, the mechanisms involved in the perception of and preferences for dietary saturated and unsaturated FAs (SFAs and UFAs, respectively) remain poorly explored, especially in insects. Using the model species Drosophila melanogaster, we measured the responses of wild-type larvae and adults to pure SFAs (14, 16, and 18 carbons) and UFAs (C18 with 1, 2, or 3 double-bonds). Individual and group behavioral tests revealed different preferences in larvae and adults. Larvae preferred UFAs whereas SFAs tended to induce both a strong aversion and a persistent aggregation behavior. Adults generally preferred SFAs, and laid more eggs and had a longer life span when ingesting these substances as compared to UFAs. Our data suggest that insects can discriminate long-chain dietary FAs. The developmental change in preference shown by this species might reflect functional variation in use of FAs or stage-specific nutritional requirements, and may be fundamental for insect use of these major dietary components.