Virulent bacterial infection improves aversive learning performance in Drosophila melanogaster.

Virulent infections are expected to impair learning ability, either as a direct consequence of stressed physiological state or as an adaptive response that minimizes diversion of energy from immune defense. This prediction has been well supported for mammals and bees. Here, we report an opposite result in Drosophila melanogaster. Using an odor-mechanical shock conditioning paradigm, we found that intestinal infection with bacterial pathogens Pseudomonas entomophila or Erwinia c. carotovora improved flies' learning performance after a 1h retention interval. Infection with P. entomophila (but not E. c. carotovora) also improved learning performance after 5 min retention. No effect on learning performance was detected for intestinal infections with an avirulent GacA mutant of P. entomophila or for virulent systemic (hemocoel) infection with E. c. carotovora. Assays of unconditioned responses to odorants and shock do not support a major role for changes in general responsiveness to stimuli in explaining the changes in learning performance, although differences in their specific salience for learning cannot be excluded. Our results demonstrate that the effects of pathogens on learning performance in insects are less predictable than suggested by previous studies, and support the notion that immune stress can sometimes boost cognitive abilities.

Evolution and functional characterisation of the IR chemosensory receptors in the Drosophila larval gustatory system

Chemosensory receptor gene families encode divergent proteins capable of detecting a huge diversity of environmental stimuli that are constantly changing over evolutionary time as organisms adapt to distinct ecological niches. While olfaction is dedicated to the detection of volatile compounds, taste is key to assess food quality for nutritional value and presence of toxic substances. The sense of taste also provides initial signals to mediate endocrine regulation of appetite and food metabolism and plays a role in kin recognition. The fruit fly Drosophila melanogaster is a very good model for studying smell and taste because these senses are very important in insects and because a broad variety of genetic tools are available in Drosophila. Recently, a family of 66 chemosensory receptors, the Ionotropic Receptors (IRs) was described in fruit flies. IRs are distantly related to ionotropic glutamate receptors (iGluRs), but their evolutionary origin from these synaptic receptors is unclear. While 16 IRs are expressed in the olfactory system, nothing is known about the other members of this repertoire. In this thesis, I describe bioinformatic, expression and functional analyses of the IRs aimed at understanding how these receptors have evolved, and at characterising the role of the non-olfactory IRs. I show that these have emerged at the basis of the protostome lineage and probably have acquired their sensory function very early. Moreover, although several IRs are conserved across insects, there are rapid and dramatic changes in the size and divergence of IR repertoires across species. I then performed a comprehensive analysis of IR expression in the larva of Drosophila melanogaster, which is a good model to study taste and feeding mechanisms as it spends most of its time eating or foraging. I found that most of the divergent members of the IR repertoire are expressed in both peripheral and internal gustatory neurons, suggesting that these are involved in taste perception. Finally, through the establishment of a new neurophysiological assay in larvae, I identified for the first time subsets of IR neurons that preferentially detect sugars and amino acids, indicating that IRs might be involved in sensing these compounds. Together, my results indicate that IRs are an evolutionarily dynamic and functionally versatile family of receptors. In contrast to the olfactory IRs that are well-conserved, gustatory IRs are rapidly evolving species-specific receptors that are likely to be involved in detecting a wide variety of tastants. - La plupart des animaux possèdent de grandes familles de récepteurs chimiosensoriels dont la fonction est de détecter l'immense diversité de composés chimiques présents dans l'environnement. Ces récepteurs évoluent en même temps que les organismes s'adaptent à leur écosystème. Il existe deux manières de percevoir ces signaux chimiques : l'olfaction et le goût. Alors que le système olfactif perçoit les composés volatiles, le sens du goût permet d'évaluer, par contact, la qualité de la nourriture, de détecter des substances toxiques et de réguler l'appétit et le métabolisme. L'un des organismes modèles les plus pertinents pour étudier le sens du goût est le stade larvaire de la mouche du vinaigre Drosophila melanogaster. En effet, la principale fonction du stade larvaire est de trouver de la nourriture et de manger. De plus, il est possible d'utiliser tous les outils génétiques développés chez la drosophile. Récemment, une nouvelle famille de 66 récepteurs chimiosensoriels appelés Récepteurs Ionotropiques (IRs) a été découverte chez la drosophile. Bien que leur orogine soit peu claire, ces récepteurs sont similaires aux récepteurs ionotropiques glutamatergiques impliqués dans la transmission synaptique. 16 IRs sont exprimés dans le système olfactif de la mouche adulte, mais pour l'instant on ne connaît rien des autres membres de cette famille. Durant ma thèse, j'ai effectué des recherches sur l'évolution de ces récepteurs ainsi que sur l'expression et la fonction des IRs non olfactifs. Je démontre que les IRs sont apparus chez l'ancêtre commun des protostomiens et ont probablement acquis leur fonction sensorielle très rapidement. De plus, bien qu'un certain nombre d'IRs olfactifs soient conservés chez les insectes, d'importantes variations dans la taille et la divergence des répertoires d'IRs entre les espèces ont été constatées. J'ai également découvert qu'un grand nombre d'IRs non olfactifs sont exprimés dans différents organes gustatifs, ce qui leur confère probablement une fonction dans la perception des goûts. Finalement, pour la première fois, des neurones exprimant des IRs ont été identifiés pour leur fonction dans la perception de sucres et d'acides aminés chez la larve. Mes résultats présentent les IRs comme une famille très dynamique, aux fonctions très variées, qui joue un rôle tant dans l'odorat que dans le goût, et dont la fonction est restée importante tout au long de l'évolution. De plus, l'identification de neurones spécialisés dans la perception de certains composés permettra l'étude des circuits neuronaux impliqués dans le traitement de ces informations.

Prepupal Building Behavior in Drosophila melanogaster and Its Evolution under Resource and Time Constraints.

Structures built by animals are a widespread and ecologically important 'extended phenotype'. While its taxonomic diversity has been well described, factors affecting short-term evolution of building behavior within a species have received little experimental attention. Here we describe how, given the opportunity, wandering Drosophila melanogaster larvae often build long tunnels in agar substrates and embed their pupae within them. These embedded larvae are characterized by a longer egg-to-pupariation developmental time than larvae that pupate on the surface. Assuming that such building behaviors are likely to be energetically costly and/or time consuming, we hypothesized that they should evolve to be less pronounced under resource or time limitation. In accord with this prediction, larvae from populations evolved for 160 generations under a regime that combines larval malnutrition with limited developmental time dug shorter tunnels than larvae from control unselected populations. However, the proportion of larvae that embedded before pupation did not differ between the malnutrition-adapted and control populations, suggesting that tunnel length and likelihood of embedding before pupation are controlled by different genetic loci. The behaviors exhibited by wandering larvae of Drosophila melanogaster prior to pupation offer a model system to study evolution of animal building behaviors because the tunneling and embedding phenotypes are simple, facultative and highly variable.

Drosophila rely on learning while foraging under semi-natural conditions

Learning is predicted to affect manifold ecological and evolutionary processes, but the extent to which animals rely on learning in nature remains poorly known, especially for short-lived non-social invertebrates. This is in particular the case for Drosophila, a favourite laboratory system to study molecular mechanisms of learning. Here we tested whether Drosophila melanogaster use learned information to choose food while free-flying in a large greenhouse emulating the natural environment. In a series of experiments flies were first given an opportunity to learn which of two food odours was associated with good versus unpalatable taste; subsequently, their preference for the two odours was assessed with olfactory traps set up in the greenhouse. Flies that had experienced palatable apple-flavoured food and unpalatable orange-flavoured food were more likely to be attracted to the odour of apple than flies with the opposite experience. This was true both when the flies first learned in the laboratory and were then released and recaptured in the greenhouse, and when the learning occurred under free-flying conditions in the greenhouse. Furthermore, flies retained the memory of their experience while exploring the greenhouse overnight in the absence of focal odours, pointing to the involvement of consolidated memory. These results support the notion that even small, short lived insects which are not central-place foragers make use of learned cues in their natural environments.

Robustness of Drosophila early embryo and wing imaginal disc development

Within a developing organism, cells require information on where they are in order to differentiate into the correct cell-type. Pattern formation is the process by which cells acquire and process positional cues and thus determine their fate. This can be achieved by the production and release of a diffusible signaling molecule, called a morphogen, which forms a concentration gradient: exposure to different morphogen levels leads to the activation of specific signaling pathways. Thus, in response to the morphogen gradient, cells start to express different sets of genes, forming domains characterized by a unique combination of differentially expressed genes. As a result, a pattern of cell fates and specification emerges.Though morphogens have been known for decades, it is not yet clear how these gradients form and are interpreted in order to yield highly robust patterns of gene expression. During my PhD thesis, I investigated the properties of Bicoid (Bcd) and Decapentaplegic (Dpp), two morphogens involved in the patterning of the anterior-posterior axis of Drosophila embryo and wing primordium, respectively. In particular, I have been interested in understanding how the pattern proportions are maintained across embryos of different sizes or within a growing tissue. This property is commonly referred to as scaling and is essential for yielding functional organs or organisms. In order to tackle these questions, I analysed fluorescence images showing the pattern of gene expression domains in the early embryo and wing imaginal disc. After characterizing the extent of these domains in a quantitative and systematic manner, I introduced and applied a new scaling measure in order to assess how well proportions are maintained. I found that scaling emerged as a universal property both in early embryos (at least far away from the Bcd source) and in wing imaginal discs (across different developmental stages). Since we were also interested in understanding the mechanisms underlying scaling and how it is transmitted from the morphogen to the target genes down in the signaling cascade, I also quantified scaling in mutant flies where this property could be disrupted. While scaling is largely conserved in embryos with altered bcd dosage, my modeling suggests that Bcd trapping by the nuclei as well as pre-steady state decoding of the morphogen gradient are essential to ensure precise and scaled patterning of the Bcd signaling cascade. In the wing imaginal disc, it appears that as the disc grows, the Dpp response expands and scales with the tissue size. Interestingly, scaling is not perfect at all positions in the field. The scaling of the target gene domains is best where they have a function; Spalt, for example, scales best at the position in the anterior compartment where it helps to form one of the anterior veins of the wing. Analysis of mutants for pentagone, a transcriptional target of Dpp that encodes a secreted feedback regulator of the pathway, indicates that Pentagone plays a key role in scaling the Dpp gradient activity.

Ionotropic receptors (IRs): chemosensory ionotropic glutamate receptors in Drosophila and beyond.

Ionotropic Receptors (IRs) are a recently characterized family of olfactory receptors in the fruit fly, Drosophila melanogaster. IRs are not related to insect Odorant Receptors (ORs), but rather have evolved from ionotropic glutamate receptors (iGluRs), a conserved family of synaptic ligand-gated ion channels. Here, we review the expression and function of IRs in Drosophila, highlighting similarities and differences with iGluRs. We also briefly describe the organization of the neuronal circuits in which IRs function, comparing and contrasting them with the sensory pathways expressing ORs. Finally, we summarize the bioinformatic identification and initial characterization of IRs in other species, which imply an evolutionarily conserved role for these receptors in chemosensation in insects and other protostomes.

Global Diversity Lines-A Five-Continent Reference Panel of Sequenced Drosophila melanogaster Strains.

Reference collections of multiple Drosophila lines with accumulating collections of "omics" data have proven especially valuable for the study of population genetics and complex trait genetics. Here we present a description of a resource collection of 84 strains of Drosophila melanogaster whose genome sequences were obtained after 12 generations of full-sib inbreeding. The initial rationale for this resource was to foster development of a systems biology platform for modeling metabolic regulation by the use of natural polymorphisms as perturbations. As reference lines, they are amenable to repeated phenotypic measurements, and already a large collection of metabolic traits have been assayed. Another key feature of these strains is their widespread geographic origin, coming from Beijing, Ithaca, Netherlands, Tasmania, and Zimbabwe. After obtaining 12.5× coverage of paired-end Illumina sequence reads, SNP and indel calls were made with the GATK platform. Thorough quality control was enabled by deep sequencing one line to >100×, and single-nucleotide polymorphisms and indels were validated using ddRAD-sequencing as an orthogonal platform. In addition, a series of preliminary population genetic tests were performed with these single-nucleotide polymorphism data for assessment of data quality. We found 83 segregating inversions among the lines, and as expected these were especially abundant in the African sample. We anticipate that this will make a useful addition to the set of reference D. melanogaster strains, thanks to its geographic structuring and unusually high level of genetic diversity.

Spatially varying selection shapes life history clines among populations of Drosophila melanogaster from sub-Saharan Africa.

Clines in life history traits, presumably driven by spatially varying selection, are widespread. Major latitudinal clines have been observed, for example, in Drosophila melanogaster, an ancestrally tropical insect from Africa that has colonized temperate habitats on multiple continents. Yet, how geographic factors other than latitude, such as altitude or longitude, affect life history in this species remains poorly understood. Moreover, most previous work has been performed on derived European, American and Australian populations, but whether life history also varies predictably with geography in the ancestral Afro-tropical range has not been investigated systematically. Here, we have examined life history variation among populations of D. melanogaster from sub-Saharan Africa. Viability and reproductive diapause did not vary with geography, but body size increased with altitude, latitude and longitude. Early fecundity covaried positively with altitude and latitude, whereas lifespan showed the opposite trend. Examination of genetic variance-covariance matrices revealed geographic differentiation also in trade-off structure, and QST -FST analysis showed that life history differentiation among populations is likely shaped by selection. Together, our results suggest that geographic and/or climatic factors drive adaptive phenotypic differentiation among ancestral African populations and confirm the widely held notion that latitude and altitude represent parallel gradients.

Genomic evidence for role of inversion 3RP of Drosophila melanogaster in facilitating climate change adaptation.

Chromosomal inversion polymorphisms are common in animals and plants, and recent models suggest that alternative arrangements spread by capturing different combinations of alleles acting additively or epistatically to favour local adaptation. It is also thought that inversions typically maintain favoured combinations for a long time by suppressing recombination between alternative chromosomal arrangements. Here, we consider patterns of linkage disequilibrium and genetic divergence in an old inversion polymorphism in Drosophila melanogaster (In(3R)Payne) known to be associated with climate change adaptation and a recent invasion event into Australia. We extracted, karyotyped and sequenced whole chromosomes from two Australian populations, so that changes in the arrangement of the alleles between geographically separated tropical and temperate areas could be compared. Chromosome-wide linkage disequilibrium (LD) analysis revealed strong LD within the region spanned by In(3R)Payne. This genomic region also showed strong differentiation between the tropical and the temperate populations, but no differentiation between different karyotypes from the same population, after controlling for chromosomal arrangement. Patterns of differentiation across the chromosome arm and in gene ontologies were enhanced by the presence of the inversion. These data support the notion that inversions are strongly selected by bringing together combinations of genes, but it is still not clear if such combinations act additively or epistatically. Our data suggest that climatic adaptation through inversions can be dynamic, reflecting changes in the relative abundance of different forms of an inversion and ongoing evolution of allelic content within an inversion.

Idiosyncratic evolution of maternal effects in response to juvenile malnutrition in Drosophila.

Maternal effects often affect fitness traits, but there is little experimental evidence pertaining to their contribution to response to selection imposed by novel environments. We studied the evolution of maternal effects in Drosophila populations selected for tolerance to chronic larval malnutrition. To this end, we performed pairwise reciprocal F1 crosses between six selected (malnutrition tolerant) populations and six unselected control populations and assessed the effect of cross direction on larval growth and developmental rate, adult weight and egg-to-adult viability expressed under the malnutrition regime. Each pair of reciprocal crosses revealed large maternal effects (possibly including cytoplasmic genetic effects) on at least one trait, but the magnitude, sign and which traits were affected varied among populations. Thus, maternal effects contributed significantly to the response to selection imposed by the malnutrition regime, but these changes were idiosyncratic, suggesting a rugged adaptive landscape. Furthermore, although the selected populations evolved both faster growth and higher viability, the maternal effects on growth rate and viability were negatively correlated across populations. Thus, genes mediating maternal effects can evolve to partially counteract the response to selection mediated by the effects of alleles on their own carriers' phenotype, and maternal effects may contribute to evolutionary trade-offs between components of offspring fitness.

Genome sequence of the Drosophila melanogaster male-killing Spiroplasma strain MSRO endosymbiont.

Spiroplasmas are helical and motile members of a cell wall-less eubacterial group called Mollicutes. Although all spiroplasmas are associated with arthropods, they exhibit great diversity with respect to both their modes of transmission and their effects on their hosts; ranging from horizontally transmitted pathogens and commensals to endosymbionts that are transmitted transovarially (i.e., from mother to offspring). Here we provide the first genome sequence, along with proteomic validation, of an endosymbiotic inherited Spiroplasma bacterium, the Spiroplasma poulsonii MSRO strain harbored by Drosophila melanogaster. Comparison of the genome content of S. poulsonii with that of horizontally transmitted spiroplasmas indicates that S. poulsonii has lost many metabolic pathways and transporters, demonstrating a high level of interdependence with its insect host. Consistent with genome analysis, experimental studies showed that S. poulsonii metabolizes glucose but not trehalose. Notably, trehalose is more abundant than glucose in Drosophila hemolymph, and the inability to metabolize trehalose may prevent S. poulsonii from overproliferating. Our study identifies putative virulence genes, notably, those for a chitinase, the H2O2-producing glycerol-3-phosphate oxidase, and enzymes involved in the synthesis of the eukaryote-toxic lipid cardiolipin. S. poulsonii also expresses on the cell membrane one functional adhesion-related protein and two divergent spiralin proteins that have been implicated in insect cell invasion in other spiroplasmas. These lipoproteins may be involved in the colonization of the Drosophila germ line, ensuring S. poulsonii vertical transmission. The S. poulsonii genome is a valuable resource to explore the mechanisms of male killing and symbiont-mediated protection, two cardinal features of many facultative endosymbionts. IMPORTANCE: Most insect species, including important disease vectors and crop pests, harbor vertically transmitted endosymbiotic bacteria. These endosymbionts play key roles in their hosts' fitness, including protecting them against natural enemies and manipulating their reproduction in ways that increase the frequency of symbiont infection. Little is known about the molecular mechanisms that underlie these processes. Here, we provide the first genome draft of a vertically transmitted male-killing Spiroplasma bacterium, the S. poulsonii MSRO strain harbored by D. melanogaster. Analysis of the S. poulsonii genome was complemented by proteomics and ex vivo metabolic experiments. Our results indicate that S. poulsonii has reduced metabolic capabilities and expresses divergent membrane lipoproteins and potential virulence factors that likely participate in Spiroplasma-host interactions. This work fills a gap in our knowledge of insect endosymbionts and provides tools with which to decipher the interaction between Spiroplasma bacteria and their well-characterized host D. melanogaster, which is emerging as a model of endosymbiosis.

Drosophila Ionotropic Receptor 25a mediates circadian clock resetting by temperature.

Circadian clocks are endogenous timers adjusting behaviour and physiology with the solar day. Synchronized circadian clocks improve fitness and are crucial for our physical and mental well-being. Visual and non-visual photoreceptors are responsible for synchronizing circadian clocks to light, but clock-resetting is also achieved by alternating day and night temperatures with only 2-4 °C difference. This temperature sensitivity is remarkable considering that the circadian clock period (~24 h) is largely independent of surrounding ambient temperatures. Here we show that Drosophila Ionotropic Receptor 25a (IR25a) is required for behavioural synchronization to low-amplitude temperature cycles. This channel is expressed in sensory neurons of internal stretch receptors previously implicated in temperature synchronization of the circadian clock. IR25a is required for temperature-synchronized clock protein oscillations in subsets of central clock neurons. Extracellular leg nerve recordings reveal temperature- and IR25a-dependent sensory responses, and IR25a misexpression confers temperature-dependent firing of heterologous neurons. We propose that IR25a is part of an input pathway to the circadian clock that detects small temperature differences. This pathway operates in the absence of known 'hot' and 'cold' sensors in the Drosophila antenna, revealing the existence of novel periphery-to-brain temperature signalling channels.

Fluctuation-Driven Neural Dynamics Reproduce Drosophila Locomotor Patterns.

The neural mechanisms determining the timing of even simple actions, such as when to walk or rest, are largely mysterious. One intriguing, but untested, hypothesis posits a role for ongoing activity fluctuations in neurons of central action selection circuits that drive animal behavior from moment to moment. To examine how fluctuating activity can contribute to action timing, we paired high-resolution measurements of freely walking Drosophila melanogaster with data-driven neural network modeling and dynamical systems analysis. We generated fluctuation-driven network models whose outputs-locomotor bouts-matched those measured from sensory-deprived Drosophila. From these models, we identified those that could also reproduce a second, unrelated dataset: the complex time-course of odor-evoked walking for genetically diverse Drosophila strains. Dynamical models that best reproduced both Drosophila basal and odor-evoked locomotor patterns exhibited specific characteristics. First, ongoing fluctuations were required. In a stochastic resonance-like manner, these fluctuations allowed neural activity to escape stable equilibria and to exceed a threshold for locomotion. Second, odor-induced shifts of equilibria in these models caused a depression in locomotor frequency following olfactory stimulation. Our models predict that activity fluctuations in action selection circuits cause behavioral output to more closely match sensory drive and may therefore enhance navigation in complex sensory environments. Together these data reveal how simple neural dynamics, when coupled with activity fluctuations, can give rise to complex patterns of animal behavior.

Evolution of reduced post-copulatory molecular interactions in Drosophila populations lacking sperm competition.

In many species with internal fertilization, molecules transferred in the male ejaculate trigger and interact with physiological changes in females. It is controversial to what extent these interactions between the sexes act synergistically to mediate the female switch to a reproductive state or instead reflect sexual antagonism evolved as a by product of sexual selection on males. To address this question, we eliminated sexual selection by enforcing monogamy in populations of Drosophila melanogaster for 65 generations and then measured the expression of male seminal fluid protein genes and genes involved in the female response to mating. In the absence of sperm competition, male and female reproductive interests are perfectly aligned and any antagonism should be reduced by natural selection. Consistent with this idea, males from monogamous populations showed reduced expression of seminal fluid protein genes, 16% less on average than in polygamous males. Further, we identified 428 genes that responded to mating in females. After mating, females with an evolutionary history of monogamy exhibited lower relative expression of genes that were up regulated in response to mating and higher expression of genes that were down-regulated - in other words, their post-mating transcriptome appeared more virgin-like. Surprisingly, these genes showed a similar pattern even before mating, suggesting that monogamous females evolved to be less poised for mating and the accompanying receipt of male seminal fluid proteins. This reduced investment by both monogamous males and females in molecules involved in post-copulatory interactions points to a pervasive role of sexual conflict in shaping these interactions.