The Gene CG6767 Affects Olfactory Behavior in Drosophila melanogaster.

Chemosensory systems mediate some of the most vital animal behaviors. However, our knowledge of the genetic mechanisms that underlie behavioral responses to olfactory cues remains fragmented. Genome-wide association mapping has greatly advanced our ability to identify candidate loci associated with variation in olfactory behavior, but functional validation of these candidates remain a necessary next step in understanding the mechanisms by which these genes influence chemoreception. In previous genome-wide association analyses, a genomic region that spans multiple polymorphic loci on the left arm of the third chromosome was found to be significantly associated with variation in olfactory behavioral responses to the odorant 2,3-butanedione, a volatile compound present in fermenting fruit. In this study, behavioral analysis of flies possessing either the major or minor haplotype for this region confirmed the association between polymorphisms in the region and variation in olfactory behavior. Moreover, functional dissection of the genes within this region using P-element insertional mutagenesis together with targeted RNAi experiments revealed that the gene CG6767, a gene of previously unknown function but predicted to encode an enzyme responsible for the synthesis and metabolism of nucleic acids, affects olfactory behavioral responses to 2,3-butanedione. Specifically, RNAi mediated knockdown of CG6767 expression in different neuroanatomical populations of the olfactory system suggests that this gene functions in local interneurons of the antennal lobe. These results reveal a new role for CG6767 and its importance in olfactory behavior.

Host Preference and Olfaction in Drosophila mojavensis.

Many organisms live in complex environments that vary geographically in resource availability. This environmental heterogeneity can lead to changes within species in their phenotypic traits. For example, in many herbivorous insects, variation in host plant availability has been shown to influence insect host preference behavior. This behavior can be mediated in part through the insect olfactory system and the odor-evoked responses of olfactory sensory neurons (OSNs), which are in turn mediated by their corresponding odorant receptor genes. The desert dwelling fly Drosophila mojavensis is a model species for understanding the mechanisms underlying host preference in a heterogeneous environment. Depending on geographic region, one to multiple host plant species are available. Here, we conducted electrophysiological studies and found variation in responses of ORNs to host plant volatiles both within and between 2 populations-particularly to the odorant 4-methylphenol. Flies from select localities within each population were found to lack a response to 4-methylphenol. Experiments then assessed the extent to which these electrophysiological differences were associated with differences in several odor-mediated behavioral responses. No association between the presence/absence of these odor-evoked responses and short range olfactory behavior or oviposition behavior was observed. However, differences in odor-induced feeding behavior in response to 4-methylphenol were found. Localities that exhibit an odor-evoked response to the odorant had increased feeding behavior in the presence of the odorant. This study sets the stage for future work examining the functional genetics underlying variation in odor perception.

The Drosophila melanogaster Genetic Reference Panel.

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype-phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype-phenotype mapping using the power of Drosophila genetics.

Artificial selection for odor-guided behavior in Drosophila reveals changes in food consumption.

BACKGROUND: The olfactory system enables organisms to detect chemical cues in the environment and can signal the availability of food or the presence of a predator. Appropriate behavioral responses to these chemical cues are therefore important for organismal survival and can influence traits such as organismal life span and food consumption. However, understanding the genetic mechanisms underlying odor-guided behavior, correlated responses in other traits, and how these constrain or promote their evolution, remain an important challenge. Here, we performed artificial selection for attractive and aversive behavioral responses to four chemical compounds, two aromatics (4-ethylguaiacol and 4-methylphenol) and two esters (methyl hexanoate and ethyl acetate), for thirty generations. RESULTS: Artificial selection for odor-guided behavior revealed symmetrical responses to selection for each of the four chemical compounds. We then investigated whether selection for odor-guided behavior resulted in correlated responses in life history traits and/or food consumption. We found changes in food consumption upon selection for behavioral responses to aromatics. In many cases, lines selected for increased attraction to aromatics showed an increase in food consumption. We then performed RNA sequencing of lines selected for responses to 4-ethylguaiacol to identify candidate genes associated with odor-guided behavior and its impact on food consumption. We identified 91 genes that were differentially expressed among lines, many of which were associated with metabolic processes. RNAi-mediated knockdown of select candidate genes further supports their role in odor-guided behavior and/or food consumption. CONCLUSIONS: This study identifies novel genes underlying variation in odor-guided behavior and further elucidates the genetic mechanisms underlying the interrelationship between olfaction and feeding.

Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines.

The Drosophila melanogaster Genetic Reference Panel (DGRP) is a community resource of 205 sequenced inbred lines, derived to improve our understanding of the effects of naturally occurring genetic variation on molecular and organismal phenotypes. We used an integrated genotyping strategy to identify 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants. Our molecular population genomic analyses show higher deletion than insertion mutation rates and stronger purifying selection on deletions. Weaker selection on insertions than deletions is consistent with our observed distribution of genome size determined by flow cytometry, which is skewed toward larger genomes. Insertion/deletion and single nucleotide polymorphisms are positively correlated with each other and with local recombination, suggesting that their nonrandom distributions are due to hitchhiking and background selection. Our cytogenetic analysis identified 16 polymorphic inversions in the DGRP. Common inverted and standard karyotypes are genetically divergent and account for most of the variation in relatedness among the DGRP lines. Intriguingly, variation in genome size and many quantitative traits are significantly associated with inversions. Approximately 50% of the DGRP lines are infected with Wolbachia, and four lines have germline insertions of Wolbachia sequences, but effects of Wolbachia infection on quantitative traits are rarely significant. The DGRP complements ongoing efforts to functionally annotate the Drosophila genome. Indeed, 15% of all D. melanogaster genes segregate for potentially damaged proteins in the DGRP, and genome-wide analyses of quantitative traits identify novel candidate genes. The DGRP lines, sequence data, genotypes, quality scores, phenotypes, and analysis and visualization tools are publicly available.

Population differences in olfaction accompany host shift in Drosophila mojavensis.

Evolutionary shifts in plant-herbivore interactions provide a model for understanding the link among the evolution of behaviour, ecological specialization and incipient speciation. Drosophila mojavensis uses different host cacti across its range, and volatile chemicals emitted by the host are the primary cue for host plant identification. In this study, we show that changes in host plant use between distinct D. mojavensis populations are accompanied by changes in the olfactory system. Specifically, we observe differences in olfactory receptor neuron specificity and sensitivity, as well as changes in sensillar subtype abundance, between populations. Additionally, RNA-seq analyses reveal differential gene expression between populations for members of the odorant receptor gene family. Hence, alterations in host preference are associated with changes in development, regulation and function at the olfactory periphery.

Pleiotropic fitness effects of the Tre1-Gr5a region in Drosophila melanogaster.

The abundance of transposable elements and DNA repeat sequences in mammalian genomes raises the question of whether such insertions represent passive evolutionary baggage or may influence the expression of complex traits. We addressed this question in Drosophila melanogaster, in which the effects of single transposable elements on complex traits can be assessed in genetically identical individuals reared in controlled environments. Here we demonstrate that single P-element insertions in the intergenic region between the gustatory receptor 5a (Gr5a, also known as Tre) and trapped in endoderm 1 (Tre1), which encodes an orphan receptor, exert complex pleiotropic effects on fitness traits, including selective nutrient intake, life span, and resistance to starvation and heat stress. Mutations in this region interact epistatically with downstream components of the insulin signaling pathway. Transposon-induced sex-specific and sex-antagonistic effects further accentuate the complex influences that intergenic transposable elements can contribute to quantitative trait phenotypes.

The genetic architecture of odor-guided behavior in Drosophila: epistasis and the transcriptome.

We combined transcriptional profiling and quantitative genetic analysis to elucidate the genetic architecture of olfactory behavior in Drosophila melanogaster. We applied whole-genome expression analysis to five coisogenic smell-impaired (smi) mutant lines and their control. We used analysis of variance to partition variation in transcript abundance between males and females and between smi genotypes and to determine the genotype-by-sex interaction. A total of 666 genes showed sexual dimorphism in transcript abundance, and 530 genes were coregulated in response to one or more smi mutations, showing considerable epistasis at the level of the transcriptome in response to single mutations. Quantitative complementation tests of mutations at these coregulated genes with the smi mutations showed that in most cases (67%) epistatic interactions for olfactory behavior mirrored epistasis at the level of transcription, thus identifying new candidate genes regulating olfactory behavior.

Novel oxidatively activated agents modify DNA and are enhanced by ercc1 silencing.

Agents that chemically modify DNA form a backbone of many cancer treatments. A key problem for DNA-modifying agents is lack of specificity. To address this issue, we designed novel molecular scaffolds, termed An-Hq and An-Hq(2), which are activated by a hallmark of some cancers: elevated concentrations of reactive oxygen species. Elevated reactive oxygen species are linked to oncogenesis and are found to increase in several aggressive cancers. The agents are quinones that, upon oxidation, form highly electrophilic species. In vitro studies identified the mode of addition to DNA. The aniline portion of An-Hq serves to enhance nucleophilic addition to the ethyl phenyl ether instead of forming common Michael additions. Structural characterization showed that the agents add to 2'-deoxyguanosine at the N2,N3-positions. The product formed is a bulky hydroxy-N2,3-benzetheno-2'-deoxyguanosine adduct. In addition, the oxidatively activated agents added to 2'-deoxyadenosine and 2'-deoxycytidine but not thymidine or 2'-deoxyinosine. These findings are confirmed by primer extension analysis of a 392 base pair DNA. The full-length primer extension product was reduced by 69.0 ± 0.6% upon oxidative activation of An-Hq(2) as compared to controls. Little sequence dependence was observed with 76% of guanine, adenine, and cytosine residues showing an increase in extension stops between 2- and 4-fold above controls. Benzetheno-nucleobase addition to double-stranded DNA was confirmed by LC/MS of a self-complementary oligonucletide. Experiments were carried out to confirm in vivo DNA damage. Because of the lesion identified in vitro, we reasoned that nucleotide excision repair should be involved in reversing the effects of these oxidatively activated agents and enhance toxicity in Drosophila melanogaster. Using an RNAi-based approach, Ercc1 was silenced, and survival was monitored after injection of an agent. As expected, bulky cross-linking DNA-modifying agents, cisplatin and chlorambucil, showed statistically significant enhanced toxicity in Drosophila with silenced Ercc1. In addition, 5-fluorouracil, which does not produce bulky lesions, showed no selective toxicity. An-Hq and An-Hq(2) showed statistically significant toxicity in Drosophila with silenced Ercc1. Examination of cytotoxicity shows renal carcinoma cell lines as a target of these agents with a median IC(50) of 1.8 µM. Taken together, these data show that the designed oxidatively activated agents form distinct, bulky DNA modifications that prove difficult for cancer cells possessing an elevated reactive oxygen species phenotype to overcome. The modification produced is relatively unique among anticancer agents.

Host plant shift differentially alters olfactory sensitivity in female and male Drosophila mojavensis.

Animals may vary in their utilization of plants depending on plant availability, and also on the sex of the animal. Evolutionary adaptations may arise, particularly in specialist animals to the chemistry of the host plants, and these adaptations may differ between the sexes due to differences in their interactions with the plants. Drosophila mojavensis uses different host cacti across its range, and volatile chemicals emitted by the host are the primary cue for host plant identification. In this study, we measured responses of individual olfactory sensory neurons to a large suite of odorants across males and females of the two southern D. mojavensis populations. We show that a switch in host plant is accompanied by changes in the olfactory system, but the effect of this switch is minor compared to that of sex. That is, we observe differences in olfactory receptor neuron specificity and sensitivity to odorants between sexes, and to a lesser extent between populations. The majority of sensory differences are restricted to only three of the 17 sensory neurons measured. Further, we found numerous differences between sexes that only occur within one population, i.e., sex-by-population interactions.

Molecular evidence for color discrimination in the Atlantic sand fiddler crab, Uca pugilator.

Fiddler crabs are intertidal brachyuran crabs that belong to the genus Uca. Approximately 97 different species have been identified, and several of these live sympatrically. Many have species-specific body color patterns that may act as signals for intra- and interspecific communication. To understand the behavioral and ecological role of this coloration we must know whether fiddler crabs have the physiological capacity to perceive color cues. Using a molecular approach, we identified the opsin-encoding genes and determined their expression patterns across the eye of the sand fiddler crab, Uca pugilator. We identified three different opsin-encoding genes (UpRh1, UpRh2 and UpRh3). UpRh1 and UpRh2 are highly related and have similarities in their amino acid sequences to other arthropod long- and medium-wavelength-sensitive opsins, whereas UpRh3 is similar to other arthropod UV-sensitive opsins. All three opsins are expressed in each ommatidium, in an opsin-specific pattern. UpRh3 is present only in the R8 photoreceptor cell, whereas UpRh1 and UpRh2 are present in the R1-7 cells, with UpRh1 expression restricted to five cells and UpRh2 expression present in three cells. Thus, one photoreceptor in every ommatidium expresses both UpRh1 and UpRh2, providing another example of sensory receptor coexpression. These results show that U. pugilator has the basic molecular machinery for color perception, perhaps even trichromatic vision.

Behavioral and Transcriptional Response to Selection for Olfactory Behavior in Drosophila.

The detection, discrimination, and behavioral responses to chemical cues in the environment can have marked effects on organismal survival and reproduction, eliciting attractive or aversive behavior. To gain insight into mechanisms mediating this hedonic valence, we applied thirty generations of divergent artificial selection for Drosophila melanogaster olfactory behavior. We independently selected for positive and negative behavioral responses to two ecologically relevant chemical compounds: 2,3-butanedione and cyclohexanone. We also tested the correlated responses to selection by testing behavioral responses to other odorants and life history traits. Measurements of behavioral responses of the selected lines and unselected controls to additional odorants showed that the mechanisms underlying responses to these odorants are, in some cases, differentially affected by selection regime and generalization of the response to other odorants was only detected in the 2,3-butanedione selection lines. Food consumption and lifespan varied with selection regime and, at times, sex. An analysis of gene expression of both selection regimes identified multiple differentially expressed genes. New genes and genes previously identified in mediating olfactory behavior were identified. In particular, we found functional enrichment of several gene ontology terms, including cell-cell adhesion and sulfur compound metabolic process, the latter including genes belonging to the glutathione S-transferase family. These findings highlight a potential role for glutathione S-transferases in the evolution of hedonic valence to ecologically relevant volatile compounds and set the stage for a detailed investigation into mechanisms by which these genes mediate attraction and aversion.

Mate discrimination among subspecies through a conserved olfactory pathway.

Communication mechanisms underlying the sexual isolation of species are poorly understood. Using four subspecies of Drosophila mojavensis as a model, we identify two behaviorally active, male-specific pheromones. One functions as a conserved male antiaphrodisiac in all subspecies and acts via gustation. The second induces female receptivity via olfaction exclusively in the two subspecies that produce it. Genetic analysis of the cognate receptor for the olfactory pheromone indicates an important role for this sensory pathway in promoting sexual isolation of subspecies, in combination with auditory signals. Unexpectedly, the peripheral sensory pathway detecting this pheromone is conserved molecularly, physiologically, and anatomically across subspecies. These observations imply that subspecies-specific behaviors arise from differential interpretation of the same peripheral cue, reminiscent of sexually conserved detection but dimorphic interpretation of male pheromones in Drosophila melanogaster. Our results reveal that, during incipient speciation, pheromone production, detection, and interpretation do not necessarily evolve in a coordinated manner.

Pleiotropic effects of Drosophila neuralized on complex behaviors and brain structure.

Understanding how genotypic variation influences variation in brain structures and behavioral phenotypes represents a central challenge in behavioral genetics. In Drosophila melanogaster, the neuralized (neur) gene plays a key role in development of the nervous system. Different P-element insertional mutations of neur allow the development of viable and fertile adults with profoundly altered behavioral phenotypes that depend on the exact location of the inserted P element. The neur mutants exhibit reduced responsiveness to noxious olfactory and mechanosensory stimulation and increased aggression when limited food is presented after a period of food deprivation. These behavioral phenotypes are correlated with distinct structural changes in integrative centers in the brain, the mushroom bodies, and the ellipsoid body of the central complex. Transcriptional profiling of neur mutants revealed considerable overlap among ensembles of coregulated genes in the different mutants, but also distinct allele-specific differences. The diverse phenotypic effects arising from nearby P-element insertions in neur provide a new appreciation of the concept of allelic effects on phenotype, in which the wild type and null mutant are at the extreme ends of a continuum of pleiotropic allelic effects.

Quantitative genomics of aggressive behavior in Drosophila melanogaster.

Aggressive behavior is important for animal survival and reproduction, and excessive aggression is an enormous social and economic burden for human society. Although the role of biogenic amines in modulating aggressive behavior is well characterized, other genetic mechanisms affecting this complex behavior remain elusive. Here, we developed an assay to rapidly quantify aggressive behavior in Drosophila melanogaster, and generated replicate selection lines with divergent levels of aggression. The realized heritability of aggressive behavior was approximately 0.10, and the phenotypic response to selection specifically affected aggression. We used whole-genome expression analysis to identify 1,539 probe sets with different expression levels between the selection lines when pooled across replicates, at a false discovery rate of 0.001. We quantified the aggressive behavior of 19 mutations in candidate genes that were generated in a common co-isogenic background, and identified 15 novel genes affecting aggressive behavior. Expression profiling of genetically divergent lines is an effective strategy for identifying genes affecting complex traits.

The early developmental gene Semaphorin 5c contributes to olfactory behavior in adult Drosophila.

Behaviors are complex traits influenced by multiple pleiotropic genes. Understanding the mechanisms that give rise to complex behaviors requires an understanding of how variation in transcriptional regulation shapes nervous system development and how variation in brain structure influences an organism's ability to respond to its environment. To begin to address this problem, we used olfactory behavior in Drosophila melanogaster as a model and showed that a hypomorphic transposon-mediated mutation of the early developmental gene Semaphorin-5c (Sema-5c) results in aberrant behavioral responses to the repellant odorant benzaldehyde. We fine mapped this effect to the Sema-5c locus using deficiency mapping, phenotypic reversion through P-element excision, and transgenic rescue. Morphometric analysis of this Sema-5c allele reveals subtle neuroanatomical changes in the brain with a reduction in the size of the ellipsoid body. High-density oligonucleotide expression microarrays identified 50 probe sets with altered transcriptional regulation in the Sema-5c background and quantitative complementation tests identified epistatic interactions between nine of these coregulated genes and the transposon-disrupted Sema-5c gene. Our results demonstrate how hypomorphic mutation of an early developmental gene results in genomewide transcriptional consequences and alterations in brain structure accompanied by profound impairment of adult behavior.

Evolution of coeloconic sensilla in the peripheral olfactory system of Drosophila mojavensis.

Populations inhabiting habitats with different environmental conditions, such as climate, resource availability, predation, competition, can undergo selection for traits that are adaptive in one habitat and not the other, leading to divergence between populations. Changes in the olfactory systems of insects that rely on different host plants, for example, can occur in response to differences in sensory stimuli between habitats. In this study, we investigate the evolution of host preference by characterizing the coeloconic sensilla in Drosophila mojavensis, a species that breeds on different necrotic cacti across its geographic range. These cactus species differ in the volatile chemicals they emit, a primary sensory cue for host plant discrimination. Analysis of odor-evoked responses identified four coeloconic sensilla that were qualitatively similar to those of Drosophila melanogaster, but varied in the breadth and strength of their olfactory sensory neuron responses to some acids and amines. Variation in responses to certain odorants among D. mojavensis populations was also observed. Compared to D. melanogaster, there was a lack of sensitivity of antennal coeloconic type 3 (ac3) sensilla to primary ligands of OR35a across all populations. Consistent with this result was a lack of detectable Or35a gene expression. Using a comparative approach, we then examined odor specificity of ac3 sensilla for seven additional Drosophila species, and found that OR35a-like sensitivity may be limited to the melanogaster subgroup. The variation in specificity that was observed among species is not clearly attributable to the degree of ecological specialization, nor to the ecological niche.

Population differences in host plant preference and the importance of yeast and plant substrate to volatile composition.

Divergent selection between environments can result in changes to the behavior of an organism. In many insects, volatile compounds are a primary means by which host plants are recognized and shifts in plant availability can result in changes to host preference. Both the plant substrate and microorganisms can influence this behavior, and host plant choice can have an impact on the performance of the organism. In Drosophila mojavensis, four geographically isolated populations each use different cacti as feeding and oviposition substrates and identify those cacti by the composition of the volatile odorants emitted. Behavioral tests revealed D. mojavensis populations vary in their degree of preference for their natural host plant. Females from the Mojave population show a marked preference for their host plant, barrel cactus, relative to other cactus choices. When flies were given a choice between cacti that were not their host plant, the preference for barrel and organ pipe cactus relative to agria and prickly pear cactus was overall lower for all populations. Volatile headspace composition is influenced by the cactus substrate, microbial community, and substrate-by-microorganism interactions. Differences in viability, developmental time, thorax length, and dry body weight exist among populations and depend on cactus substrate and population-by-cactus interactions. However, no clear association between behavioral preference and performance was observed. This study highlights a complex interplay between the insect, host plant, and microbial community and the factors mediating insect host plant preference behavior.

Vanaso is a candidate quantitative trait gene for Drosophila olfactory behavior.

Most animals depend on olfaction for survival and procreation. Odor-guided behavior is a quantitative trait, with phenotypic variation due to multiple segregating quantitative trait loci (QTL). Despite its profound biological importance, the genetic basis of naturally occurring variation in olfactory behavior remains unexplored. Here, we mapped a single Drosophila QTL affecting variation in avoidance response to benzaldehyde, using a population of recombinant inbred lines. Deficiency complementation mapping resolved this region into one female- and one male-specific QTL. Subsequent quantitative complementation tests to all available mutations of positional candidate genes showed that the female-specific QTL failed to complement a P-element insertional mutation, l(3)04276. The P-element insertion was in the intron of a novel gene, Vanaso, which contains a putative guanylate binding protein domain, is highly polymorphic, and is expressed in the third antennal segment, the major olfactory organ of Drosophila. No expression was detected in the fly brain, suggesting that Vanaso plays a role in peripheral chemosensory processes rather than in central integration of olfactory information. QTL mapping followed by quantitative complementation tests to deficiencies and mutations is an effective strategy for gene discovery that allows characterization of effects of recessive lethal genes on adult phenotypes and here enabled identification of a candidate gene that contributes to sex-specific quantitative variation in olfactory behavior.