Micro‐geographical scale variation in Drosophila melanogaster larval olfactory behaviour is associated with host fruit heterogeneity

Organisms utilize environmental cues to deal with heterogeneous environments. In this sense, behaviours that mediate interactions between organisms and their environment are complex traits, especially sensitive to environmental conditions. In animals, olfaction is a critical sensory system that allows them to acquire chemical information from the environment. The genetic basis and physiological mechanisms of the olfactory system of Drosophila melanogaster Meigen (Diptera: Drosophilidae) are well known, but the effects of ecological factors on the olfactory system have received less attention. In this study, we analysed the effect of environmental heterogeneity (different host fruits) on variation in larval olfactory behaviour in a natural population of D. melanogaster. We generated half‐sib lines of D. melanogaster derived from two nearby fruit plantations, Vitis vinifera L. (Vitaceae) (‘grape’) and Prunus persica L. (Rosaceae) (‘peach’), and measured, using a simple behavioural assay, larval olfactory response to natural olfactory stimuli. Results indicate that patterns of variation for this trait depend on host fruit plantation where lines were collected. In fact, only lines derived from ‘grape’ showed phenotypic plasticity for larval olfaction, whereas a genotype*environment interaction was detected solely in lines derived from ‘peach’. Therefore, our results demonstrate the existence of genetic differences in D. melanogaster larval olfactory behaviour at a micro‐geographical scale and also reveal that the trait studied presents a dynamic genetic architecture which is strongly influenced by the environment.     

Behavioral analyses of mutants for two odorant-binding protein genes, Obp57d and Obp57e, in Drosophila melanogaster

The odorant-binding protein (OBP) functions in chemosensation in insects. Two OBP genes, Obp57d and Obp57e, are involved in the evolution of the unique host-plant preference in Drosophila sechellia. Comparative analysis of the Obp57d/e genomic sequences in the Drosophila melanogaster species group has revealed that the rapid evolution of Obp57d and Obp57e has resulted in functional divergence between the two genes. Here, using D. melanogaster knockout strains generated by gene targeting, we examined the roles of Obp57d and Obp57e in behavioral response to a series of fatty acids. In the taste-based oviposition-site preference assay, the knockout flies showed stronger preference for acids than wild-type flies, indicating that the normal functions of Obp57d and Obp57e are associated with the suppression of positive preference for C6-C9 acids. Heterozygotes for each knockout haplotype also exhibited a significantly different behavioral response compared with wild-type flies, suggesting that Obp57d and Obp57e have a gene dosage effect on behavior. In contrast, the wild-type and knockout flies exhibited similar responses in the feeding assay and the odor-based free-walking assay, suggesting that the two OBPs' contribution to feeding and olfactory behaviors is small. Taken together, our results demonstrated that each of Obp57d and Obp57e in D. melanogaster contributes to the determination of reproductive sites, suggesting that the two OBP genes play an important role in ecological adaptation of Drosophila.     

Natural variation in decision-making behavior in Drosophila melanogaster

There has been considerable recent interest in using Drosophila melanogaster to investigate the molecular basis of decision-making behavior. Deciding where to place eggs is likely one of the most important decisions for a female fly, as eggs are vulnerable and larvae have limited motility. Here, we show that many natural genotypes of D. melanogaster prefer to lay eggs near nutritious substrate, rather than in nutritious substrate. These preferences are highly polymorphic in both degree and direction, with considerable heritability (0.488) and evolvability.Relative preferences are modulated by the distance between options and the overall concentration of ethanol, suggesting Drosophila integrate many environmental factors when making oviposition decisions. As oviposition-related decisions can be efficiently assessed by simply counting eggs, oviposition behavior is an excellent model for understanding information processing in insects. Associating natural genetic polymorphisms with decision-making variation will shed light on the molecular basis of host choice behavior, the evolutionary maintenance of genetic variation, and the mechanistic nature of preference variation in general.     

Phenotype and transmission efficiency of artificial and natural male-killing Spiroplasma infections in Drosophila melanogaster

Many insect species carry inherited Spiroplasma bacteria which act as important partners and antagonists. The nature of symbioses between Spiroplasma and insects has been most extensively studied in the interaction between male-killing Spiroplasma infection and Drosophila melanogaster. For historical reasons, these studies have largely focussed on the Spiroplasma strain known as NSRO, derived from Drosophila nebulosa and transinfected into D. melanogaster. More recently, D. melanogaster naturally infected with Spiroplasma were discovered. Whilst the well studied strain NSRO is closely related to that found natively in D. melanogaster, it is unclear whether strains from D. nebulosa reflect a natural interaction when placed in D. melanogaster. In this paper, we determine if NSRO has similar or different properties from strains of Spiroplasma naturally infecting D. melanogaster in terms of transmission efficiency and the strength and timing of male-killing. Native infections were observed to have higher transmission efficiency than introduced NSRO infections during the early phases of host reproduction, but not during late reproduction. The timing and intensity of male-killing did not differ between infection classes. As a precautionary measure, it is proposed that future work seeking to reveal the nature of coevolved Spiroplasma-Drosophila interactions use the native strain.     

How different are recently diverged species? More than 150 phenotypic differences have been reported for the D. melanogaster species subgroup.

The nine species of the Drosophila melanogaster subgroup are currently one of the best systems for studying the genetic basis of species differences and speciation. However, despite the availability of complete genome sequences from most of these species, no comprehensive accounting of their phenotypic differences has been made. We gathered data from about 200 papers published between 1919 and 2008 that report phenotypic differences between species of the D. melanogaster species subgroup and we compiled 163 morphological, physiological and behavioral traits that differ between at least two species of the D. melanogaster subgroup. Our compilation is available online at www.normalesup.org/~vorgogoz/FlyPhenomics. The FlyPhenomics database provides a useful starting point for Drosophila researchers who aim to identify the mutations underlying phenotypic variation.     

A cluster of candidate odorant receptors from the malaria vector mosquito,Anopheles gambiae

Olfaction is critical to the host preference selection behavior of many disease-transmitting insects, including the mosquito Anopheles gambiae sensu stricto (hereafter A. gambiae), one of the major vectors for human malaria. In order to more fully understand the molecular biology of olfaction in this insect, we have previously identified several members member of a family of candidate odorant receptor proteins from A. gambiae (AgORs). Here we report the cloning and characterization of an additional AgOR gene, denoted as AgOr5, which shows significant similarity to putative odorant receptors in A. gambiae and Drosophila melanogaster and which is selectively expressed in olfactory organs. AgOr5 is tightly clustered within the A. gambiae genome to two other highly homologous candidate odorant receptors, suggesting that these genes are derived from a common ancestor. Analysis of the developmental expression within members of this AgOR gene cluster reveals considerable variation between these AgORs as compared to candidate odorant receptors from D. melanogaster.     

Do parasitoid preferences for different host species match virulence?

Abstract.  Leptopilina boulardi is a parasitoid wasp specialist of Drosophila larvae of the melanogaster subgroup. In Mediterranean areas, natural populations are highly virulent against their main host Drosophila melanogaster . In Congo, populations are less virulent against D. melanogaster but are able to develop successfully inside the tropical African species Drosophila yakuba . Host preferences are compared between two laboratory isofemale lines of L. boulardi , obtained from populations of Congo and Tunisia, respectively, and differing in virulence levels against D. melanogaster and D. yakuba . Host selection is studied by offering female parasitoids a choice between larvae of the two host species. In agreement with optimal foraging models, the line highly virulent against D. melanogaster shows a clear preference for this host species. The other line, less virulent against D. melanogaster but more virulent against D. yakuba , prefers to oviposit on D. yakuba . Such preferences can be observed after a period of host-patch exploitation only, suggesting that experience plays an important role in the host-selection process. These results evidence the existence of intraspecific variability in preference between two host species in L. boulardi , a major requisite in theoretical models of parasite specialization by the host. They also sustain the hypothesis that intraspecific variation in parasitoid preferences between host species might mirror intraspecific variation in virulence.     

Wolbachia-mediated antibacterial protection and immune gene regulation in Drosophila

The outcome of microbial infection of insects is dependent not only on interactions between the host and pathogen, but also on the interactions between microbes that co-infect the host. Recently the maternally inherited endosymbiotic bacteria Wolbachia has been shown to protect insects from a range of microbial and eukaryotic pathogens. Mosquitoes experimentally infected with Wolbachia have upregulated immune responses and are protected from a number of pathogens including viruses, bacteria, Plasmodium and filarial nematodes. It has been hypothesised that immune upregulation underpins Wolbachia-mediated protection. Drosophila is a strong model for understanding host-Wolbachia-pathogen interactions. Wolbachia-mediated antiviral protection in Drosophila has been demonstrated for a number of different Wolbachia strains. In this study we investigate whether Wolbachia-infected flies are also protected against pathogenic bacteria. Drosophila simulans lines infected with five different Wolbachia strains were challenged with the pathogenic bacteria Pseudomonas aeruginosa PA01, Serratia marcescens and Erwinia carotovora and mortality compared to paired lines without Wolbachia. No difference in mortality was observed in the flies with or without Wolbachia. Similarly no antibacterial protection was observed for D. melanogaster infected with Wolbachia. Interestingly, D. melanogaster Oregon RC flies which are naturally infected with Wolbachia showed no upregulation of the antibacterial immune genes TepIV, Defensin, Diptericin B, PGRP-SD, Cecropin A1 and Attacin D compared to paired flies without Wolbachia. Taken together these results indicate that Wolbachia-mediated antibacterial protection is not ubiquitous in insects and furthermore that the mechanisms of antibacterial and antiviral protection are independent. We suggest that the immune priming and antibacterial protection observed in Wolbachia-infected mosquitoes may be a consequence of the recent artificial introduction of the symbiont into insects that normally do not carry Wolbachia and that antibacterial protection is unlikely to be found in insects carrying long-term Wolbachia infections.     

基于四臂嗅觉仪内轨迹跟踪的 昆虫嗅觉行为分析体系

【目的】结合四臂嗅觉仪与轨迹跟踪分析系统,通过分析不同试验条件下黑腹果蝇Drosophila melanogaster的嗅觉行为,建立适合于果蝇等小型昆虫的嗅觉行为实验技术体系。【方法】采用Ethovision XT软件追踪黑腹果蝇在四臂嗅觉仪内的活动轨迹,分析各区域内的累计停留时长、运动速度;同时,考察气流速度和味源设置对苹果醋引诱及蓝桉Eucalyptus globulus叶油驱避黑腹果蝇效果的影响。【结果】适合于黑腹果蝇嗅觉行为分析的气流速度为300~1 200 mL/min;300 mL/min时黑腹果蝇个体间运动速度最为均一,因此推荐最适气流速度大小为300 mL/min。味源设置不会改变黑腹果蝇对味源物的嗅觉行为反应,但对味源物的引诱指数有一定的影响,单一臂放置味源物试验效果更佳。单头测试的实验效果优于多头试验。【结论】本研究建立的基于轨迹跟踪的嗅觉行为分析体系可用于昆虫信息化合物有效成分的筛选鉴定。在合适的气流条件下,单一臂设置味源化合物逐头进行测试试验效果更佳;多头试验可用于化合物的初步筛选。     

Cytogenetic analysis and breakpoint distribution of radiation-induced interchanges in Hylemya antiqua Meigen

Three sibling species of Drosophila, D. melanogaster, D. simulans and D. mauritiana, still without complete reproductive isolation, show specific fluorescent patterns. The difference is so striking that a tubular lamp of 365 nm U.V. light and a binocular are sufficient for routine observations. Besides those differences, it has been concluded from the comparison with totally different species like Drosophila busckii and D. littoralis that the basic pattern formations of the three species are broadly the same. Observations of behaviour under U.V. light suggest that the visual stimulation is integrated in communication in addition to olfactory, aural and tactile stimuli. Our preliminary results suggest that fluorescence is widely present in insects, perhaps even in arthropods in general. Such fluorescent patterns can perhaps shed a new light upon the status of sibling species. The results suggest that fluorescence is dependent on redox-sensitive pteridines.     

Wolbachia infection alters olfactory-cued locomotion in Drosophila spp

Wolbachia pipientis is an endosymbiotic bacterium present in diverse insect species. Although it is well studied for its dramatic effects on host reproductive biology, little is known about its effects on other aspects of host biology, despite its presence in a wide array of host tissues. This study examined the effects of three Wolbachia strains on two different Drosophila species, using a laboratory performance assay for insect locomotion in response to olfactory cues. The results demonstrate that Wolbachia infection can have significant effects on host responsiveness that vary with respect to the Wolbachia strain-host species combination. The wRi strain, native to Drosophila simulans, increases the basal activity level of the host insect as well as its responsiveness to food cues. In contrast, the wMel strain and the virulent wMelPop strain, native to Drosophila melanogaster, cause slight decreases in responsiveness to food cues but do not alter basal activity levels in the host. Surprisingly, the virulent wMelPop strain has very little impact on host responsiveness in D. simulans. This novel strain-host relationship was artificially created previously by transinfection. These findings have implications for understanding the evolution and spread of Wolbachia infections in wild populations and for Wolbachia-based vector-borne disease control strategies currently being developed.     

Odorant-specific requirements for arrestin function in Drosophila olfaction

The ability to modulate olfactory sensitivity is necessary to detect chemical gradients and discriminate among a multitude of odor stimuli. Desensitization of odorant receptors has been postulated to occur when arrestins prevent the activation of downstream second messengers. A paucity of in vivo data on olfactory desensitization prompts use of Drosophila melanogaster genetics to investigate arrestins' role in regulating olfactory signaling pathways. Physiological analysis of peripheral olfactory sensitivity reveals decreased responsiveness to a host of chemically distinct odorants in flies deficient for arrestin1 (arr1), arrestin2 (arr2), or both. These phenotypes are manifest in odorant- and dose- dependent fashions. Additionally, mutants display altered adaptive properties under a prolonged exposure paradigm. Behaviorally, arr1 mutants are impaired in olfactory-based orientation towards attractive odor sources. As the olfactory deficits vary according to chemical identity and concentration, they indicate that a spectrum of arrestin activity is essential for odor processing depending upon the particular olfactory pathway involved. Arrestin mutant phenotypes are hypothesized to be a consequence of down-regulation of olfactory signaling to avoid cellular excitotoxicity. Importantly, phenotypic rescue of olfactory defects in arr1(1) mutants is achieved through transgenic expression of wild-type arr1. Taken together, these data clearly indicate that arrestins are required in a stimulus-specific manner for wild type olfactory function and add another level of complexity to peripheral odor coding mechanisms that ultimately impact olfactory behavior.     

Comparative pathology of bacteria in the genus Providencia to a natural host, Drosophila melanogaster

Bacteria in the genus Providencia are pathogens of many organisms, including humans and insects. We and colleagues have isolated five different strains belonging to four distinct Providencia species as natural infections of Drosophila melanogaster captured in the wild. We found that these isolates vary considerably in pathology to infected D. melanogaster, differing in the level of mortality they cause, their ability to replicate within the host and the level that the fly's immune response is elicited. One interesting bacterium was Providencia sneebia, which causes nearly complete mortality and reaches large numbers in the fly but does not elicit a comparably strong immune response. Through coinfection experiments, we determined that P. sneebia avoids recognition by the immune system. We tested for biofilm formation and replication within D. melanogaster cells as possible mechanisms for P. sneebia escape from host immunity, but did not find evidence for either. D. melanogaster and Providencia provide a powerful system for studying general host-pathogen interactions, and for understanding how the well-studied immune model host D. melanogaster interacts with its natural bacterial pathogens.     

Effects of extreme temperatures on phenotypic variation and developmental stability in Drosophila melanogaster and Drosophila buzzatii

The effect of stressful and nonstressful rearing temperatures on phenotypic variation of four quantitative characters (thorax length, wing length, number of sternopleural chaetae, number of arista branches) and on developmental stability (fluctuating asymmetry) of the three latter characters was estimated in two Drosophila species: Drosophila melanogaster and Drosophila buzzatii . In both species, a general trend for increasing of phenotypic variation and fluctuating asymmetry at stress temperatures was observed; in fluctuating asymmetry, this effect was more pronounced. An increase of phenotypic variation under stress was shown for all characters examined except sternopleural chaeta number in D. buzzatii . Comparison of species responses suggests that the increase of variation in D. melanogaster was somewhat higher than in D. buzzatii .     

Variable fitness effects of Wolbachia infection in Drosophila melanogaster

Maternally inherited Wolbachia bacteria are extremely widespread among insects and their presence is usually associated with parasitic modifications of host fitness. Wolbachia pipientis infects Drosophila melanogaster populations from all continents, but their persistence in this species occurs despite any strong parasitic effects. Here, we have investigated the symbiosis between Wolbachia and D. melanogaster and found that Wolbachia infection can have significant survival and fecundity effects. Relative to uninfected flies, infected females from three fly strains showed enhanced survival or fecundity associated with Wolbachia infection, one strain showed both and one strain responded positively to Wolbachia removal. We found no difference in egg hatch rates (cytoplasmic incompatibility) for crosses between infected males and uninfected females, although there were fecundity differences. Females from this cross consistently produced fewer eggs than infected females and these fecundity differences could promote the spread of infection just like cytoplasmic incompatibility. More surprising, we found that infected females often had the greatest fecundity when mated to uninfected males. This could also promote the spread of Wolbachia infection, though here the fitness benefits would also help to spread infection when Wolbachia are rare. We suggest that variable fitness effects, in both sexes, and which interact strongly with the genetic background of the host, could increase cytoplasmic drive rates in some genotypes and help explain the widespread persistence of Wolbachia bacteria in D. melanogaster populations. These interactions may further explain why many D. melanogaster populations are polymorphic for Wolbachia infection. We discuss our results in the context of host-symbiont co-evolution.     

PUPARIATION SITE PREFERENCE WITHIN AND BETWEEN DROSOPHILA SIBLING SPECIES

Holometabolous insects pass through a sedentary pupal stage and often choose a location for pupation that is different from the site of larval feeding. We have characterized a difference in pupariation site choice within and between sibling species of Drosophila. We found that, in nature, Drosophila sechellia pupariate within their host fruit, Morinda citrifolia, and that they perform this behavior in laboratory assays. In contrast, in the laboratory, geographically diverse strains of Drosophila simulans vary in their pupariation site preference; D. simulans lines from the ancestral range in southeast Africa pupariate on fruit, or a fruit substitute, whereas populations from Europe or the New World select sites off of fruit. We explored the genetic basis for the evolved preference in puariation site preference by performing quantitative trait locus mapping within and between species. We found that the interspecific difference is controlled largely by loci on chromosomes X and II. In contrast, variation between two strains of D. simulans appears to be highly polygenic, with the majority of phenotypic effects due to loci on chromosome III. These data address the genetic basis of how new traits arise as species diverge and populations disperse.     

A simple vision-based algorithm for decision making in flying Drosophila

Animals must quickly recognize objects in their environment and act accordingly. Previous studies indicate that looming visual objects trigger avoidance reflexes in many species [1-5]; however, such reflexes operate over a close range and might not detect a threatening stimulus at a safe distance. We analyzed how fruit flies (Drosophila melanogaster) respond to simple visual stimuli both in free flight and in a tethered-flight simulator. Whereas Drosophila, like many other insects, are attracted toward long vertical objects [6-10], we found that smaller visual stimuli elicit not weak attraction but rather strong repulsion. Because aversion to small spots depends on the vertical size of a moving object, and not on looming, it can function at a much greater distance than expansion-dependent reflexes. The opposing responses to long stripes and small spots reflect a simple but effective object classification system. Attraction toward long stripes would lead flies toward vegetative perches or feeding sites, whereas repulsion from small spots would help them avoid aerial predators or collisions with other insects. The motion of flying Drosophila depends on a balance of these two systems, providing a foundation for studying the neural basis of behavioral choice in a genetic model organism.     

Patterns of epoxide metabolism by epoxide hydrolase and glutathione S-transferase associated with age and genotype in Drosophila melanogaster

Epoxide hydrolase and glutathione S-transferase activities toward trans- and cis-stilbene oxides were measured in 3 strains of Drosophila melanogaster. Differences in age dependence, substrate selectivity and subcellular location were detected suggesting the presence of multiple forms of these enzymes. In addition, interstrain differences indicate the presence of genetic variation for epoxide hydrolase and glutathione transferase activities. These results illustrate a potential use of these assays in D. melanogaster to complement existing tests (e.g. recessive lethal tests or Ames assays) for evaluating the relationship between epoxide hydrolase and glutathione S-transferase levels and the genotoxicity of epoxides.     

Competition may determine the diversity of transposable elements

Transposable elements are genomic parasites that replicate independently from their hosts. They harm their hosts by causing mutations or genomic rearrangements, and most organisms have evolved various mechanisms to suppress their activity. The evolutionary dynamics of transposons in insects, fish, birds and mammals are dramatically different. Mammalian genomes contain few, very abundant but relatively inactive transposon strains, while Drosophila and fish species harbour diverse strains, which typically have low abundance but are much more virulent. We hypothesise that the variation in the diversity and activity of transposable elements between various animal genomes is caused by the differences in the host defence mechanisms against transposon activity. In recent years RNAi, a mechanism capable of gene, virus and transposon silencing has been discovered. We model RNAi as a density dependant mechanism of defence, which can cause competition among transposons depending on its specificity, and test its predictions using the complete Caenorhabditis elegans, Drosophila melanogaster, Fugu rubripes, chicken, mouse, rat and human genome sequences.