Impact of transgenerational host switch on gut bacterial assemblage in generalist pest, Spodoptera littoralis (Lepidoptera: Noctuidae).

Diet composition is vital in shaping gut microbial assemblage in many insects. Minimal knowledge is available about the influence of transgenerational diet transition on gut microbial community structure and function in polyphagous pests. This study investigated transgenerational diet-induced changes in Spodoptera littoralis larval gut bacteriome using 16S ribosomal sequencing. Our data revealed that 88% of bacterial populations in the S. littoralis larval gut comprise Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. The first diet transition experiment from an artificial diet (F0) to a plant diet (F1), cabbage and cotton, caused an alteration of bacterial communities in the S. littoralis larval gut. The second transgenerational diet switch, where F1 larvae feed on the same plant in the F2 generation, displayed a significant variation suggesting further restructuring of the microbial communities in the Spodoptera larval gut. F1 larvae were also challenged with the plant diet transition at the F2 generation (cabbage to cotton or cotton to cabbage). After feeding on different plant diets, the microbial assemblage of F2 larvae pointed to considerable differences from other F2 larvae that continued on the same diet. Our results showed that S. littoralis larval gut bacteriome responds rapidly and inexplicably to different diet changes. Further experiments must be conducted to determine the developmental and ecological consequences of such changes. Nevertheless, this study improves our perception of the impact of transgenerational diet switches on the resident gut bacteriome in S. littoralis larvae and could facilitate future research to understand the importance of symbiosis in lepidopteran generalists better.

Drosophila Free-Flight Odor Tracking is Altered in a Sex-Specific Manner By Preimaginal Sensory Exposure.

In insects such as Drosophila melanogaster, flight guidance is based on converging sensory information provided by several modalities, including chemoperception. Drosophila flies are particularly attracted by complex odors constituting volatile molecules from yeast, pheromones and microbe-metabolized food. Based on a recent study revealing that adult male courtship behavior can be affected by early preimaginal exposure to maternally transmitted egg factors, we wondered whether a similar exposure could affect free-flight odor tracking in flies of both sexes. Our main experiment consisted of testing flies differently conditioned during preimaginal development in a wind tunnel. Each fly was presented with a dual choice of food labeled by groups of each sex of D. melanogaster or D. simulans flies. The combined effect of food with the cis-vaccenyl acetate pheromone (cVA), which is involved in aggregation behavior, was also measured. Moreover, we used the headspace method to determine the "odorant" identity of the different labeled foods tested. We also measured the antennal electrophysiological response to cVA in females and males resulting from the different preimaginal conditioning procedures. Our data indicate that flies differentially modulated their flight response (take off, flight duration, food landing and preference) according to sex, conditioning and food choice. Our headspace analysis revealed that many food-derived volatile molecules diverged between sexes and species. Antennal responses to cVA showed clear sex-specific variation for conditioned flies but not for control flies. In summary, our study indicates that preimaginal conditioning can affect Drosophila free flight behavior in a sex-specific manner.

G. sinense and P. notoginseng Extracts Improve Healthspan of Aging Flies and Provide Protection in A Huntington Disease Model.

In the last decades, the strong increase in the proportion of older people worldwide, and the increased prevalence of age associated degenerative diseases, have put a stronger focus on aging biology. In spite of important progresses in our understanding of the aging process, an integrative view is still lacking and there is still need for efficient anti-aging interventions that could improve healthspan, reduce incidence of age-related disease and, eventually, increase the lifespan. Interestingly, some compounds from traditional medicine have been found to possess anti-oxidative and anti-inflammatory properties, suggesting that they could play a role as anti-aging compounds, although in depth in vivo investigations are still scarce. In this study we used one the major aging model organisms, Drosophila melanogaster, to investigate the ability of four herb extracts (HEs: Dendrobium candidum, Ophiopogon japonicum, Ganoderma sinense and Panax notoginseng) widely used in traditional Chinese medicine (TCM) to slow down aging and improve healthspan of aged animals. Combining multiple approaches (stress resistance assays, lifespan and metabolic measurements, functional heart characterizations and behavioral assays), we show that these four HEs provide in vivo protection from various insults, albeit with significant compound-specific differences. Importantly, extracts of P. notoginseng and G. sinense increase the healthspan of aging animals, as shown by increased activity during aging and improved heart function. In addition, these two compounds also provide protection in a Drosophila model of Huntington's disease (HD), suggesting that, besides their anti-aging properties in normal individuals, they could be also efficient in the protection against age-related diseases.

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.

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.

Flight motor networks modulate primary olfactory processing in the moth Manduca sexta.

Nervous systems must distinguish sensory signals derived from an animal's own movements (reafference) from environmentally derived sources (exafference). To accomplish this, motor networks producing reafference transmit motor information, via a corollary discharge circuit (CDC), to affected sensory networks, modulating sensory function during behavior. While CDCs have been described in most sensory modalities, none have been observed projecting to an olfactory pathway. In moths, two mesothoracic to deutocerebral histaminergic neurons (MDHns) project from flight sensorimotor centers in the mesothoracic neuromere to the antennal lobe (AL), where they provide the sole source of histamine (HA), but whether they represent a CDC is unknown. We demonstrate that MDHn spiking activity is positively correlated with wing-motor output and increased before bouts of motor activity, suggesting that MDHns communicate global locomotor state, rather than providing a precisely timed motor copy. Within the AL, HA application sharpened entrainment of projection neuron responses to odor stimuli embedded within simulated wing-beat-induced flows, whereas MDHn axotomy or AL HA receptor (HA-r) blockade reduced entrainment. This finding is consistent with higher-order CDCs, as the MDHns enhanced rather than filtered entrainment of AL projection neurons. Finally, HA-r blockade increased odor detection and discrimination thresholds in behavior assays. These results establish MDHns as a CDC that modulates AL temporal resolution, enhancing odor-guided behavior. MDHns thus appear to represent a higher-order CDC to an insect olfactory pathway; this CDC's unique nature highlights the importance of motor-to-sensory signaling as a context-specific mechanism that fine-tunes sensory function.

Gene Regulation and Species-Specific Evolution of Free Flight Odor Tracking in Drosophila.

The flying ability of insects has coevolved with the development of organs necessary to take-off from the ground, generate, and modulate lift during flight in complex environments. Flight orientation to the appropriate food source and mating partner depends on the perception and integration of multiple chemical signals. We used a wind tunnel-based assay to investigate the natural and molecular evolution of free flight odor tracking in Drosophila. First, the comparison of female and male flies of several populations and species revealed substantial sex-, inter-, and intra-specific variations for distinct flight features. In these flies, we compared the molecular structure of desat1, a fast-evolving gene involved in multiple aspects of Drosophila pheromonal communication. We manipulated desat1 regulation and found that both neural and nonneural tissues affect distinct flight features. Together, our data suggest that desat1 is one of the genes involved in the evolution of free-flight odor tracking behaviors in Drosophila.

Free flight odor tracking in Drosophila: Effect of wing chemosensors, sex and pheromonal gene regulation.

The evolution of powered flight in insects had major consequences for global biodiversity and involved the acquisition of adaptive processes allowing individuals to disperse to new ecological niches. Flies use both vision and olfactory input from their antennae to guide their flight; chemosensors on fly wings have been described, but their function remains mysterious. We studied Drosophila flight in a wind tunnel. By genetically manipulating wing chemosensors, we show that these structures play an essential role in flight performance with a sex-specific effect. Pheromonal systems are also involved in Drosophila flight guidance: transgenic expression of the pheromone production and detection gene, desat1, produced low, rapid flight that was absent in control flies. Our study suggests that the sex-specific modulation of free-flight odor tracking depends on gene expression in various fly tissues including wings and pheromonal-related tissues.

Antennal lobe representations are optimized when olfactory stimuli are periodically structured to simulate natural wing beat effects.

Animals use behaviors to actively sample the environment across a broad spectrum of sensory domains. These behaviors discretize the sensory experience into unique spatiotemporal moments, minimize sensory adaptation, and enhance perception. In olfaction, behaviors such as sniffing, antennal flicking, and wing beating all act to periodically expose olfactory epithelium. In mammals, it is thought that sniffing enhances neural representations; however, the effects of insect wing beating on representations remain unknown. To determine how well the antennal lobe (AL) produces odor dependent representations when wing beating effects are simulated, we used extracellular methods to record neural units and local field potentials (LFPs) from moth AL. We recorded responses to odors presented as prolonged continuous stimuli or periodically as 20 and 25 Hz pulse trains designed to simulate the oscillating effects of wing beating around the antennae during odor guided flight. Using spectral analyses, we show that ~25% of all recorded units were able to entrain to "pulsed stimuli"; this includes pulsed blanks, which elicited the strongest overall entrainment. The strength of entrainment to pulse train stimuli was dependent on molecular features of the odorants, odor concentration, and pulse train duration. Moreover, units showing pulse tracking responses were highly phase locked to LFPs during odor stimulation, indicating that unit-LFP phase relationships are stimulus-driven. Finally, a Euclidean distance-based population vector analysis established that AL odor representations are more robust, peak more quickly, and do not show adaptation when odors were presented at the natural wing beat frequency as opposed to prolonged continuous stimulation. These results suggest a general strategy for optimizing olfactory representations, which exploits the natural rhythmicity of wing beating by integrating mechanosensory and olfactory cues at the level of the AL.

Genes involved in sex pheromone discrimination in Drosophila melanogaster and their background-dependent effect.

Mate choice is based on the comparison of the sensory quality of potential mating partners, and sex pheromones play an important role in this process. In Drosophila melanogaster, contact pheromones differ between male and female in their content and in their effects on male courtship, both inhibitory and stimulatory. To investigate the genetic basis of sex pheromone discrimination, we experimentally selected males showing either a higher or lower ability to discriminate sex pheromones over 20 generations. This experimental selection was carried out in parallel on two different genetic backgrounds: wild-type and desat1 mutant, in which parental males showed high and low sex pheromone discrimination ability respectively. Male perception of male and female pheromones was separately affected during the process of selection. A comparison of transcriptomic activity between high and low discrimination lines revealed genes not only that varied according to the starting genetic background, but varied reciprocally. Mutants in two of these genes, Shaker and quick-to-court, were capable of producing similar effects on discrimination on their own, in some instances mimicking the selected lines, in others not. This suggests that discrimination of sex pheromones depends on genes whose activity is sensitive to genetic context and provides a rare, genetically defined example of the phenomenon known as "allele flips," in which interactions have reciprocal effects on different genetic backgrounds.

Incipient speciation in Drosophila melanogaster involves chemical signals.

The sensory and genetic bases of incipient speciation between strains of Drosophila melanogaster from Zimbabwe and those from elsewhere are unknown. We studied mating behaviour between eight strains - six from Zimbabwe, together with two cosmopolitan strains. The Zimbabwe strains showed significant sexual isolation when paired with cosmopolitan males, due to Zimbabwe females discriminating against these males. Our results show that flies' cuticular hydrocarbons (CHs) were involved in this sexual isolation, but that visual and acoustic signals were not. The mating frequency of Zimbabwe females was highly significantly negatively correlated with the male's relative amount of 7-tricosene (%7-T), while the mating of cosmopolitan females was positively correlated with %7-T. Variation in transcription levels of two hydrocarbon-determining genes, desat1 and desat2, did not correlate with the observed mating patterns. Our study represents a step forward in our understanding of the sensory processes involved in this classic case of incipient speciation.

Expression of a desaturase gene, desat1, in neural and nonneural tissues separately affects perception and emission of sex pheromones in Drosophila.

Animals often use sex pheromones for mate choice and reproduction. As for other signals, the genetic control of the emission and perception of sex pheromones must be tightly coadapted, and yet we still have no worked-out example of how these two aspects interact. Most models suggest that emission and perception rely on separate genetic control. We have identified a Drosophila melanogaster gene, desat1, that is involved in both the emission and the perception of sex pheromones. To explore the mechanism whereby these two aspects of communication interact, we investigated the relationship between the molecular structure, tissue-specific expression, and pheromonal phenotypes of desat1. We characterized the five desat1 transcripts-all of which yielded the same desaturase protein-and constructed transgenes with the different desat1 putative regulatory regions. Each region was used to target reporter transgenes with either (i) the fluorescent GFP marker to reveal desat1 tissue expression, or (ii) the desat1 RNAi sequence to determine the effects of genetic down-regulation on pheromonal phenotypes. We found that desat1 is expressed in a variety of neural and nonneural tissues, most of which are involved in reproductive functions. Our results suggest that distinct desat1 putative regulatory regions independently drive the expression in nonneural and in neural cells, such that the emission and perception of sex pheromones are precisely coordinated in this species.

Effect of laboratory acclimation on the variation of reproduction-related characters in Drosophila melanogaster.

The natural variation of sex-specific characters between populations can favor their behavioral isolation, eventually leading to the formation of new species. Marked variations for male courtship, mating and the production of sex pheromones - three complex characters potentially inducing sexual isolation - were found between Drosophila melanogaster populations of various origins acclimated for many generations in research laboratories. However, the natural variation of these three characters between natural populations and their evolution after long-term acclimation in the laboratory remains unknown. We measured many traits involved in these characters in six stocks initiated with distinct populations sampled in a restricted geographic area. Several sex-specific traits varied between stocks freshly brought back to the laboratory. After 100 generations spent in the laboratory without any experimental selection, traits varied in a strain-dependent manner. This variation was not related to a reduction of their variance except for copulation duration. This indicates that reproduction-related characters can diverge between neighboring D. melanogaster populations, and differently adapt to stable laboratory conditions.

The consequences of regulation of desat1 expression for pheromone emission and detection in Drosophila melanogaster.

Sensory communication depends on the precise matching between the emission and the perception of sex- and species-specific signals; understanding both the coevolutionary process and the genes involved in both production and detection is a major challenge. desat1 determines both aspects of communication-a mutation in desat1 simultaneously alters both sex pheromone emission and perception in Drosophila melanogaster flies. We investigated whether the alteration of pheromonal perception is a consequence of the altered production of pheromones or if the two phenotypes are independently controlled by the same locus. Using several genetic tools, we were able to separately manipulate the two pheromonal phenotypes, implying that desat1 is the sole gene responsible, exerting a pleiotropic effect on both transmission and detection. The levels of the five desat1 trancripts, measured in the head and body of manipulated flies, were related to variation in pheromone production. This suggests that the pleiotropic action of desat1 on pheromonal communication depends on the fine regulation of its transcriptional activity.

desat1 and the evolution of pheromonal communication in Drosophila.

The evolution of communication is a fundamental biological problem. The genetic control of the signal and its reception must be tightly coadapted, especially in interindividual sexual communication. However, there is very little experimental evidence for tight genetic linkage connecting the emission of a signal and its reception. In Drosophila melanogaster, desat1 is the first known gene that simultaneously affects the emission and the perception of sex pheromones. Our experiments show that both aspects of pheromonal communication (the emission and the perception of sex pheromones) depend on distinct genetic control and may result from tissue-specific expression of different transcripts, all coding for the same desaturase. Therefore, and given the high conservation of its coding region, the pleiotropic activity of the desat1 gene may have arisen from an evolutionary process that shaped its regulatory regions.

Effect of genes, social experience, and their interaction on the courtship behaviour of transgenic Drosophila males.

Behaviour depends (a) on genes that specify the neural and non-neural elements involved in the perception of and responses to sensory stimuli and (b) on experience that can modulate the fine development of these elements. We exposed transgenic and control Drosophila melanogaster males, and their hybrids, to male siblings during adult development and measured the contribution of genes and of experience to their courtship behaviour. The transgene CheB42a specifically targets male gustatory sensillae and alters the perception of male inhibitory pheromones which leads to frequent male-male interactions. The age at which social experience occurred and the genotype of tester males induced a variable effect on the intensity of male homo- and heterosexual courtship. The strong interaction between the effects of genes and of social experience reveals the plasticity of the apparently stereotyped elements involved in male courtship behaviour. Finally, a high intensity of homosexual courtship was found only in males that simultaneously carried a mutation in their white gene and the CheB42a transgene.