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1.  Rapid Evolution of Sex Pheromone-Producing Enzyme Expression in Drosophila 
PLoS Biology  2009;7(8):e1000168.
Rapid evolution of gene expression patterns responsible for pheromone production in 24 species of Drosophila was mapped to simple mutations within the regulatory domain of the desatF gene.
A wide range of organisms use sex pheromones to communicate with each other and to identify appropriate mating partners. While the evolution of chemical communication has been suggested to cause sexual isolation and speciation, the mechanisms that govern evolutionary transitions in sex pheromone production are poorly understood. Here, we decipher the molecular mechanisms underlying the rapid evolution in the expression of a gene involved in sex pheromone production in Drosophilid flies. Long-chain cuticular hydrocarbons (e.g., dienes) are produced female-specifically, notably via the activity of the desaturase DESAT-F, and are potent pheromones for male courtship behavior in Drosophila melanogaster. We show that across the genus Drosophila, the expression of this enzyme is correlated with long-chain diene production and has undergone an extraordinary number of evolutionary transitions, including six independent gene inactivations, three losses of expression without gene loss, and two transitions in sex-specificity. Furthermore, we show that evolutionary transitions from monomorphism to dimorphism (and its reversion) in desatF expression involved the gain (and the inactivation) of a binding-site for the sex-determination transcription factor, DOUBLESEX. In addition, we documented a surprising example of the gain of particular cis-regulatory motifs of the desatF locus via a set of small deletions. Together, our results suggest that frequent changes in the expression of pheromone-producing enzymes underlie evolutionary transitions in chemical communication, and reflect changing regimes of sexual selection, which may have contributed to speciation among Drosophila.
Author Summary
Mate selection is a complex process involving communication between potential partners on many levels, such as visual, aural, and olfactory cues. Many animals use chemical signals in the form of pheromones to communicate and correctly recognize individuals of the appropriate species and sex during reproduction. Evolutionary changes in the production of these chemicals have been suggested to contribute to speciation. Yet, the molecular mechanisms governing these transitions have seldom been addressed. Here, we show that expression of the gene desatF, which encodes an enzyme involved in the production of the Drosophila pheromones known as dienes, is highly variable and rapidly evolving across Drosophila species. Changes in desatF gene expression correlate with changes in sex- and species-specific production of dienes. Further, these changes in diene production can be explained by simple modifications in the regulatory regions of the desatF gene, providing a molecular level understanding of the evolution of pheromone production in Drosophila.
doi:10.1371/journal.pbio.1000168
PMCID: PMC2711336  PMID: 19652700
2.  A Drosophila male pheromone affects female sexual receptivity 
Sex pheromones are chemical signals frequently required for mate choice, but their reciprocal role on mate preference has rarely been shown in both sexes. In Drosophila melanogaster flies, the predominant cuticular hydrocarbons (CHs) are sexually dimorphic: only females produce 7,11-dienes, whereas 7-tricosene (7-T) is the principal male CH. Males generally prefer females with 7,11-dienes, but the role of 7-T on female behaviour remains unclear. With perfumed males, control females mated faster and more often with males carrying increased levels of 7-T showing that this CH acts as a chemical stimulant for D. melanogaster females. Control females—but not antenna-less females—could detect small variation of 7-T. Finally, our finding that desat1 mutant female showed altered response towards 7-T provides an additional role for this gene which affects the production and the perception of pheromones involved in mate choice, in both sexes.
doi:10.1098/rspb.2005.3332
PMCID: PMC1560049  PMID: 16543174
male pheromone; 7-tricosene; female receptivity; antenna; desat1; Drosophila
3.  Gametic incompatibilities between races of Drosophila melanogaster. 
Reproductive-isolating mechanisms between nascent species may involve sperm-egg recognition and have been best described in externally fertilizing organisms where such recognition is essential in preventing undesirable fertilizations. However, reproductive barriers in internally fertilizing species differ in significant ways, and a direct role for sperm-egg interactions has yet to be demonstrated. Females of many strains of Drosophila melanogaster from Zimbabwe, Africa, do not mate readily with cosmopolitan males. This polymorphism in mate choice is postulated to represent incipient speciation. We now report that, in one direction, crosses between the above populations produce far fewer offspring than reciprocal crosses due to a lower rate of egg hatch. We established that egg inviability in these crosses was due to defects in fertilization. Thus, even in taxa with internal fertilization, gametic incompatibility may be a mechanism relevant to reproductive isolation during incipient speciation.
doi:10.1098/rspb.2000.1420
PMCID: PMC1088670  PMID: 11345322
4.  Cuticular Hydrocarbon Content that Affects Male Mate Preference of Drosophila melanogaster from West Africa 
Intraspecific variation in mating signals and preferences can be a potential source of incipient speciation. Variable crossability between Drosophila melanogaster and D. simulans among different strains suggested the abundance of such variations. A particular focus on one combination of D. melanogaster strains, TW1(G23) and Mel6(G59), that showed different crossabilities to D. simulans, revealed that the mating between females from the former and males from the latter occurs at low frequency. The cuticular hydrocarbon transfer experiment indicated that cuticular hydrocarbons of TW1 females have an inhibitory effect on courtship by Mel6 males. A candidate component, a C25 diene, was inferred from the gas chromatography analyses. The intensity of male refusal of TW1 females was variable among different strains of D. melanogaster, which suggested the presence of variation in sensitivity to different chemicals on the cuticle. Such variation could be a potential factor for the establishment of premating isolation under some conditions.
doi:10.1155/2012/278903
PMCID: PMC3321289  PMID: 22536539
5.  Peripheral, Central and Behavioral Responses to the Cuticular Pheromone Bouquet in Drosophila melanogaster Males 
PLoS ONE  2011;6(5):e19770.
Pheromonal communication is crucial with regard to mate choice in many animals including insects. Drosophila melanogaster flies produce a pheromonal bouquet with many cuticular hydrocarbons some of which diverge between the sexes and differently affect male courtship behavior. Cuticular pheromones have a relatively high weight and are thought to be — mostly but not only — detected by gustatory contact. However, the response of the peripheral and central gustatory systems to these substances remains poorly explored. We measured the effect induced by pheromonal cuticular mixtures on (i) the electrophysiological response of peripheral gustatory receptor neurons, (ii) the calcium variation in brain centers receiving these gustatory inputs and (iii) the behavioral reaction induced in control males and in mutant desat1 males, which show abnormal pheromone production and perception. While male and female pheromones induced inhibitory-like effects on taste receptor neurons, the contact of male pheromones on male fore-tarsi elicits a long-lasting response of higher intensity in the dedicated gustatory brain center. We found that the behavior of control males was more strongly inhibited by male pheromones than by female pheromones, but this difference disappeared in anosmic males. Mutant desat1 males showed an increased sensitivity of their peripheral gustatory neurons to contact pheromones and a behavioral incapacity to discriminate sex pheromones. Together our data indicate that cuticular hydrocarbons induce long-lasting inhibitory effects on the relevant taste pathway which may interact with the olfactory pathway to modulate pheromonal perception.
doi:10.1371/journal.pone.0019770
PMCID: PMC3098836  PMID: 21625481
6.  Female Drosophila melanogaster Gene Expression and Mate Choice: The X Chromosome Harbours Candidate Genes Underlying Sexual Isolation 
PLoS ONE  2011;6(2):e17358.
Background
The evolution of female choice mechanisms favouring males of their own kind is considered a crucial step during the early stages of speciation. However, although the genomics of mate choice may influence both the likelihood and speed of speciation, the identity and location of genes underlying assortative mating remain largely unknown.
Methods and Findings
We used mate choice experiments and gene expression analysis of female Drosophila melanogaster to examine three key components influencing speciation. We show that the 1,498 genes in Zimbabwean female D. melanogaster whose expression levels differ when mating with more (Zimbabwean) versus less (Cosmopolitan strain) preferred males include many with high expression in the central nervous system and ovaries, are disproportionately X-linked and form a number of clusters with low recombination distance. Significant involvement of the brain and ovaries is consistent with the action of a combination of pre- and postcopulatory female choice mechanisms, while sex linkage and clustering of genes lead to high potential evolutionary rate and sheltering against the homogenizing effects of gene exchange between populations.
Conclusion
Taken together our results imply favourable genomic conditions for the evolution of reproductive isolation through mate choice in Zimbabwean D. melanogaster and suggest that mate choice may, in general, act as an even more important engine of speciation than previously realized.
doi:10.1371/journal.pone.0017358
PMCID: PMC3046225  PMID: 21386982
7.  Contribution of oenocytes and pheromones to courtship behaviour in Drosophila 
BMC Biochemistry  2009;10:21.
Background
In Drosophila, cuticular sex pheromones are long-chain unsaturated hydrocarbons synthesized from fatty acid precursors in epidermal cells called oenocytes. The species D. melanogaster shows sex pheromone dimorphism, with high levels of monoenes in males, and of dienes in females. Some biosynthesis enzymes are expressed both in fat body and oenocytes, rendering it difficult to estimate the exact role of oenocytes and of the transport of fatty acids from fat body to oenocytes in pheromone elaboration. To address this question, we RNAi silenced two main genes of the biosynthesis pathway, desat1 and desatF, in the oenocytes of D. melanogaster, without modifying their fat body expression.
Results
Inactivation of desat1 in oenocytes resulted in a 96% and 78% decrease in unsaturated hydrocarbons in males and females, respectively. Female pheromones (dienes) showed a decrease of 90%. Inactivation of desatF, which is female-specific and responsible for diene formation, resulted in a dramatic loss of pheromones (-98%) paralleled with a two-fold increase in monoenes. Courtship parameters (especially courtship latency) from wild-type males were more affected by desat1 knocked-down females (courtship latency increased by four fold) than by desatF knocked-down ones (+65% of courtship latency).
The number of transcripts in oenocytes was estimated at 0.32 and 0.49 attomole/μg for desat1 in males and females, respectively, about half of the total transcripts in a fly. There were only 0.06 attomole/μg desatF transcripts in females, all located in the oenocytes.
Conclusion
Knock-down results for desat1 suggest that there must be very little transport of unsaturated precursors from fat body to the oenocytes, so pheromone synthesis occurs almost entirely through the action of biosynthesis enzymes within the oenocytes. Courtship experiments allow us to discuss the behavioral role of diene pheromones, which, under special conditions, could be replaced by monoenes in D. melanogaster. A possible explanation is given of how pheromones could have evolved in species such as D. simulans, which only synthesize monoenes.
doi:10.1186/1471-2091-10-21
PMCID: PMC2734525  PMID: 19671131
8.  A single mutation alters production and discrimination of Drosophila sex pheromones 
The evolution of communication is a fundamental biological problem. The genetic control of the signal and its reception must be tightly coadapted, especially in inter-individual sexual communication. However, there is very little experimental evidence for tight genetic linkage connecting the emission of a signal and its reception. We found that a single genomic transposon inserted in the desat1 gene of Drosophila melanogaster simultaneously affected the emission and the perception of sex-specific signals. This mutation greatly decreased the production of unsaturated hydrocarbons on the cuticle of mature flies of both sexes. These substances represent the sex pheromones necessary for mate discrimination: control males could not discriminate the sex of mutant desat1 flies. Moreover, mutant males were unable to discriminate the sex pheromones of control flies. Expression of desat1 was found in the peripheral tissues that produce and detect sex pheromones. Excision of the transposon rescued both the production and discrimination phenotypes, but the two effects did not always coincide. This indicates that the emission and perception of pheromones are coded by different products of the same gene, reflecting the pleiotropic activity of desat1.
doi:10.1098/rspb.2004.2971
PMCID: PMC1634977  PMID: 15705556
pheromonal communication; mate discrimination; desaturase; Drosophila; PGal4
9.  Molecular Evolution and Functional Diversification of Fatty Acid Desaturases after Recurrent Gene Duplication in Drosophila 
Molecular Biology and Evolution  2009;26(7):1447-1456.
Frequent gene duplications in the genome incessantly supply new genetic materials for functional innovation presumably driven by positive Darwinian selection. This mechanism in the desaturase gene family has been proposed to be important in triggering the pheromonal diversification in insects. With the recent completion of a dozen Drosophila genomes, a genome-wide perspective is possible. In this study, we first identified homologs of desaturase genes in 12 Drosophila species and noted that while gene duplication events are relatively frequent, gene losses are not scarce, especially in the desat1–desat2–desatF clade. By reconciling the gene tree with species phylogeny and the chromosomal synteny of the sequenced Drosophila genomes, at least one gene loss in desat2 and a minimum of six gene gains (resulting in seven desatF homologs, α-η), three gene losses and one relocation in desatF were inferred. Upon branching off the ancestral desat1 lineage, both desat2 and desatF gained novel functions through accelerating protein evolution. The amino acid residues under positive selection located near the catalytic sites and the C-terminal region might be responsible for altered substrate selectivity between closely related species. The association between the expression pattern of desatF-α and the chemical composition of cuticular hydrocarbons implies that the ancestral function of desatF-α is the second desaturation at the four carbons after the first double bond in diene synthesis, and the shift from bisexual to female-specific expression in desatF-α occurred in the ancestral lineage of Drosophila melanogaster subgroup. A relationship between the number of expressed desatF homologs and the diene diversification has also been observed. These results suggest that the molecular diversification of fatty acid desaturases after recurrent gene duplication plays an important role in pheromonal diversity in Drosophila.
doi:10.1093/molbev/msp057
PMCID: PMC2693736  PMID: 19307313
cuticular hydrocarbon; fatty acid desaturase; gene duplication; pheromonal diversity; positive selection
10.  Variation in the Male Pheromones and Mating Success of Wild Caught Drosophila melanogaster 
PLoS ONE  2011;6(8):e23645.
Drosophila melanogaster males express two primary cuticular hydrocarbons (male-predominant hydrocarbons). These act as sex pheromones by influencing female receptivity to mating. The relative quantities of these hydrocarbons vary widely among natural populations and can contribute to variation in mating success. We tested four isofemale lines collected from a wild population to assess the effect of intrapopulation variation in male-predominant hydrocarbons on mating success. The receptivity of laboratory females to males of the four wild-caught lines varied significantly, but not consistently in the direction predicted by variation in male-predominant hydrocarbons. Receptivity of the wild-caught females to laboratory males also varied significantly, but females from lines with male-predominant hydrocarbon profiles closer to a more cosmopolitan one did not show a correspondingly strong mating bias toward a cosmopolitan male. Among wild-caught lines, the male-specific ejaculatory bulb lipid, cis-vaccenyl acetate, varied more than two-fold, but was not associated with variation in male mating success. We observed a strong inverse relationship between the receptivity of wild-caught females and the mating success of males from their own lines, when tested with laboratory flies of the opposite sex.
doi:10.1371/journal.pone.0023645
PMCID: PMC3157407  PMID: 21858189
11.  Hydrocarbon divergence and reproductive isolation in Timema stick insects 
Background
Individuals commonly prefer certain trait values over others when choosing their mates. If such preferences diverge between populations, they can generate behavioral reproductive isolation and thereby contribute to speciation. Reproductive isolation in insects often involves chemical communication, and cuticular hydrocarbons, in particular, serve as mate recognition signals in many species. We combined data on female cuticular hydrocarbons, interspecific mating propensity, and phylogenetics to evaluate the role of cuticular hydrocarbons in diversification of Timema walking-sticks.
Results
Hydrocarbon profiles differed substantially among the nine analyzed species, as well as between partially reproductively-isolated T. cristinae populations adapted to different host plants. In no-choice trials, mating was more likely between species with similar than divergent hydrocarbon profiles, even after correcting for genetic divergences. The macroevolution of hydrocarbon profiles, along a Timema species phylogeny, fits best with a punctuated model of phenotypic change concentrated around speciation events, consistent with change driven by selection during the evolution of reproductive isolation.
Conclusion
Altogether, our data indicate that cuticular hydrocarbon profiles vary among Timema species and populations, and that most evolutionary change in hydrocarbon profiles occurs in association with speciation events. Similarities in hydrocarbon profiles between species are correlated with interspecific mating propensities, suggesting a role for cuticular hydrocarbon profiles in mate choice and speciation in the genus Timema.
doi:10.1186/1471-2148-13-151
PMCID: PMC3728149  PMID: 23855797
12.  Sexual and reproductive behaviour of Drosophila melanogaster from a microclimatically interslope differentiated population of "Evolution Canyon" (Mount Carmel, Israel). 
The strong microscale interslope environmental differences in "Evolution Canyon" provide an excellent natural model for sympatric speciation. Our previous studies revealed significant slope-specific differences for a fitness complex of Drosophila. This complex involved either adaptation traits (tolerance to high temperature, different viability and longevity pattern) or behavioural differentiation, manifested in habitat choice and non-random mating. This remarkable differentiation has evolved despite a very small interslope distance (a few hundred metres only). Our hypothesis is that strong interslope microclimatic contrast caused differential selection for fitness-related traits accompanied by behavioural differentiation and reinforced by some sexual isolation, which started incipient speciation. Here we describe the results of a systematic analysis of sexual behaviour in a non-choice situation and several reproductive parameters of D. melanogaster populations from the opposite slopes of "Evolution Canyon". The evidence indicates that: (i) mate choice derives from differences in mating propensity and discrimination; (ii) females from the milder north-facing slope discriminate strongly against males of the opposite slope; (iii) both sexes of the south-facing slope display distinct reproductive and behavioural patterns with females showing increased fecundity, shorter time before remating and relatively higher receptivity, and males showing higher mating propensity. These patterns represent adaptive life strategies contributing to higher fitness.
doi:10.1098/rspb.2001.1822
PMCID: PMC1088888  PMID: 11703877
13.  Interaction between temperature and male pheromone in sexual isolation in Drosophila melanogaster 
Journal of Evolutionary Biology  2013;26(9):2008-2020.
In Drosophila, female hydrocarbons are known to be involved in premating isolation between different species and pheromonal races. The role of male-specific hydrocarbon polymorphism is not as well documented. The dominant cuticular hydrocarbon (CHC) in male D. melanogaster is usually 7-tricosene (7-T), with the exception of African populations, in which 7-pentacosene (7-P) is dominant. Here, we took advantage of a population from the Comoro Islands (Com), in which males fell on a continuum of low to high levels of 7-T, to perform temperature selection and selection on CHCs’ profiles. We conducted several experiments on the selected Com males to study the plasticity of their CHCs in response to temperature shift, their role in resistance to desiccation and in sexual selection. We then compared the results obtained for selected lines to those from three common laboratory strains with different and homogenous hydrocarbon profiles: CS, Cot and Tai. Temperature selection modified the CHC profiles of the Com males in few generations of selection. We showed that the 7-P/7-T ratio depends on temperature with generally more 7-P at higher temperatures and observed a relationship between chain length and resistance to desiccation in both temperature- and phenotypically selected Com lines. There was partial sexual isolation between the flies with clear-cut phenotypes within the phenotypically selected lines and the laboratory strains. These results indicate that the dominant male pheromones are under environmental selection and may have played a role in reproductive isolation.
doi:10.1111/jeb.12206
PMCID: PMC4217391  PMID: 23944628
adaptation; desiccation resistance; mating; selection; temperature
14.  An Inhibitory Sex Pheromone Tastes Bitter for Drosophila Males 
PLoS ONE  2007;2(8):e661.
Sexual behavior requires animals to distinguish between the sexes and to respond appropriately to each of them. In Drosophila melanogaster, as in many insects, cuticular hydrocarbons are thought to be involved in sex recognition and in mating behavior, but there is no direct neuronal evidence of their pheromonal effect. Using behavioral and electrophysiological measures of responses to natural and synthetic compounds, we show that Z-7-tricosene, a Drosophila male cuticular hydrocarbon, acts as a sex pheromone and inhibits male-male courtship. These data provide the first direct demonstration that an insect cuticular hydrocarbon is detected as a sex pheromone. Intriguingly, we show that a particular type of gustatory neurons of the labial palps respond both to Z-7-tricosene and to bitter stimuli. Cross-adaptation between Z-7-tricosene and bitter stimuli further indicates that these two very different substances are processed by the same neural pathways. Furthermore, the two substances induced similar behavioral responses both in courtship and feeding tests. We conclude that the inhibitory pheromone tastes bitter to the fly.
doi:10.1371/journal.pone.0000661
PMCID: PMC1937024  PMID: 17710124
15.  Proteomics Reveals Novel Drosophila Seminal Fluid Proteins Transferred at Mating 
PLoS Biology  2008;6(7):e178.
Across diverse taxa, seminal fluid proteins (Sfps) transferred at mating affect the reproductive success of both sexes. Such reproductive proteins often evolve under positive selection between species; because of this rapid divergence, Sfps are hypothesized to play a role in speciation by contributing to reproductive isolation between populations. In Drosophila, individual Sfps have been characterized and are known to alter male sperm competitive ability and female post-mating behavior, but a proteomic-scale view of the transferred Sfps has been missing. Here we describe a novel proteomic method that uses whole-organism isotopic labeling to detect transferred Sfps in mated female D. melanogaster. We identified 63 proteins, which were previously unknown to function in reproduction, and confirmed the transfer of dozens of predicted Sfps. Relative quantification of protein abundance revealed that several of these novel Sfps are abundant in seminal fluid. Positive selection and tandem gene duplication are the prevailing forces of Sfp evolution, and comparative proteomics with additional species revealed lineage-specific changes in seminal fluid content. We also report a proteomic-based gene discovery method that uncovered 19 previously unannotated genes in D. melanogaster. Our results demonstrate an experimental method to identify transferred proteins in any system that is amenable to isotopic labeling, and they underscore the power of combining proteomic and evolutionary analyses to shed light on the complex process of Drosophila reproduction.
Author Summary
Across many species, males transfer both sperm and seminal proteins to their mates. These proteins increase male reproductive success by improving sperm competitive ability and modifying female behavior. In Drosophila, seminal proteins increase female rates of egg-laying and sperm storage and reduce a female's willingness to mate with subsequent suitors. Several male seminal proteins have been extensively characterized, and others have been predicted based on gene expression patterns, yet the full set of proteins that is transferred to females has not been defined. Here we introduce a new proteomic method that identifies transferred seminal proteins in recently mated females and quantifies their relative abundance. We confirm many of the predicted seminal proteins and discover a number of novel seminal fluid components. Some of these proteins show elevated rates of evolution, consistent with their involvement in sexual selection or sexual conflict, and many have arisen by tandem gene duplication. By using this method in three species of Drosophila, we identified lineage-specific components of seminal fluid. Additionally, we developed and validated a method to identify completely new genes in the D. melanogaster genome. These transferred proteins are now targets for follow-up genetic, biochemical, and evolutionary analysis.
Seminal fluid proteins are critical for male reproductive success. This paper describes a new method to identify these proteins in recently mated females and details their dynamic evolutionary histories.
doi:10.1371/journal.pbio.0060178
PMCID: PMC2486302  PMID: 18666829
16.  Divergent Selection and the Evolution of Signal Traits and Mating Preferences 
PLoS Biology  2005;3(11):e368.
Mating preferences are common in natural populations, and their divergence among populations is considered an important source of reproductive isolation during speciation. Although mechanisms for the divergence of mating preferences have received substantial theoretical treatment, complementary experimental tests are lacking. We conducted a laboratory evolution experiment, using the fruit fly Drosophila serrata, to explore the role of divergent selection between environments in the evolution of female mating preferences. Replicate populations of D. serrata were derived from a common ancestor and propagated in one of three resource environments: two novel environments and the ancestral laboratory environment. Adaptation to both novel environments involved changes in cuticular hydrocarbons, traits that predict mating success in these populations. Furthermore, female mating preferences for these cuticular hydrocarbons also diverged among populations. A component of this divergence occurred among treatment environments, accounting for at least 17.4% of the among-population divergence in linear mating preferences and 17.2% of the among-population divergence in nonlinear mating preferences. The divergence of mating preferences in correlation with environment is consistent with the classic by-product model of speciation in which premating isolation evolves as a side effect of divergent selection adapting populations to their different environments.
Experimentally manipulating the resource environment of Drosophila serrata reveals that mating preferences can evolve, at least in part, as a result of environmentally-based divergent natural selection.
doi:10.1371/journal.pbio.0030368
PMCID: PMC1262626  PMID: 16231971
17.  Feminization of pheromone-sensing neurons affects mating decisions in Drosophila males 
Biology Open  2014;3(2):152-160.
Summary
The response of individual animals to mating signals depends on the sexual identity of the individual and the genetics of the mating targets, which represent the mating social context (social environment). However, how social signals are sensed and integrated during mating decisions remains a mystery. One of the models for understanding mating behaviors in molecular and cellular terms is the male courtship ritual in the fruit fly (Drosophila melanogaster). We have recently shown that a subset of gustatory receptor neurons (GRNs) that are enriched in the male appendages and express the ion channel ppk23 play a major role in the initiation and maintenance of male courtship via the perception of cuticular contact pheromones, and are likely to represent the main chemosensory pathway that influences mating decisions by males. Here we show that genetic feminization of ppk23-expressing GRNs in male flies resulted in a significant increase in male–male sexual attraction without an apparent impact on sexual attraction to females. Furthermore, we show that this increase in male–male sexual attraction is sensory specific, which can be modulated by variable social contexts. Finally, we show that feminization of ppk23-expressing sensory neurons lead to major transcriptional shifts, which may explain the altered interpretation of the social environment by feminized males. Together, these data indicate that the sexual cellular identity of pheromone sensing GRNs plays a major role in how individual flies interpret their social environment in the context of mating decisions.
doi:10.1242/bio.20147369
PMCID: PMC3925318  PMID: 24463366
Fruit fly; Courtship; ppk23; Poxn; transformer; DEG/ENaC
18.  Sexual selection on song and cuticular hydrocarbons in two distinct populations of Drosophila montana 
Ecology and Evolution  2012;2(1):80-94.
Sexual selection has the potential to contribute to population divergence and speciation. Most studies of sexual selection in Drosophila have concentrated on a single signaling modality, usually either courtship song or cuticular hydrocarbons (CHCs), which can act as contact pheromones. We have examined the relationship between both signal types and reproductive success using F1–3 offspring of wild-collected flies, raised in the lab. We used two populations of the Holarctic species Drosophila montana that represent different phylogeographic clades that have been separate for ca. 0.5 million years (MY), and differ to some extent in both traits. Here, we characterize the nature and identify the targets of sexual selection on song, CHCs, and both traits combined within the populations. Three measures of courtship outcome were used as fitness proxies. They were the probability of mating, mating latency, and the production of rejection song by females, and showed patterns of association with different traits that included both linear and quadratic selection. Courtship song predicted courtship outcome better than CHCs and the signal modalities acted in an additive rather than synergistic manner. Selection was generally consistent in direction and strength between the two populations and favored males that sang more vigorously. Sexual selection differed in the extent, strength, and nature on some of the traits between populations. However, the differences in the directionality of selection detected were not a good predictor of population differences. In addition, a character previously shown to be important for species recognition, interpulse interval, was found to be under sexual selection. Our results highlight the complexity of understanding the relationship between within-population sexual selection and population differences. Sexual selection alone cannot predict differences between populations.
doi:10.1002/ece3.75
PMCID: PMC3297180  PMID: 22408728
Courtship song; cuticular hydrocarbons; Drosophila montana; selection analysis; sexual selection
19.  Functional coupling of acoustic and chemical signals in the courtship behaviour of the male Drosophila melanogaster. 
During courtship, the male Drosophila melanogaster sends signals to the female through two major sensory channels: chemical and acoustic. These signals are involved in the stimulation of the female to accept copulation. In order to determine the respective importance in the courtship of these signals, their production was controlled using genetical and surgical techniques. Males deprived of the ability to emit both signals are unable to mate, demonstrating that other (e.g. visual or tactile) signals are not sufficient to stimulate the female. If either acoustic or chemical signals are lacking, the courtship success is strongly reduced, the lack of the former having significantly more drastic effects. However, the accelerated matings of males observed with males bearing wild-type hydrocarbons compared with defective ones, whichever the modality of acoustic performance (wing vibration or playback), strongly support the role of cuticular compounds to stimulate females. We can conclude that among the possible factors involved in communication during courtship, acoustic and chemical signals may act in a synergistic way and not separately in D. melanogaster.
doi:10.1098/rspb.2001.1919
PMCID: PMC1690944  PMID: 11934360
20.  Using RNA sequencing to characterize female reproductive genes between Z and E Strains of European Corn Borer moth (Ostrinia nubilalis) 
BMC Genomics  2014;15:189.
Background
Reproductive proteins often evolve rapidly and are thought to be subject to strong sexual selection, and thus may play a key role in reproductive isolation and species divergence. However, our knowledge of reproductive proteins has been largely limited to males and model organisms with sequenced genomes. With advances in sequencing technology, Lepidoptera are emerging models for studies of sexual selection and speciation. By profiling the transcriptomes of the bursa copulatrix and bursal gland from females of two incipient species of moth, we characterize reproductive genes expressed in the primary reproductive tissues of female Lepidoptera and identify candidate genes contributing to a one-way gametic incompatibility between Z and E strains of the European corn borer (Ostrinia nubilalis).
Results
Using RNA sequencing we identified transcripts from ~37,000 and ~36,000 loci that were expressed in the bursa copulatrix or the bursal gland respectively. Of bursa copulatrix genes, 8% were significantly differentially expressed compared to the female thorax, and those that were up-regulated or specific to the bursa copulatrix showed functional biases toward muscle activity and/or organization. In the bursal gland, 9% of genes were differentially expressed compared to the thorax, with many showing reproduction or gamete production functions. Of up-regulated bursal gland genes, 46% contained a transmembrane region and 16% possessed secretion signal peptides. Divergently expressed genes in the bursa copulatrix were exclusively biased toward protease-like functions and 51 proteases or protease inhibitors were divergently expressed overall.
Conclusions
This is the first comprehensive characterization of female reproductive genes in any lepidopteran system. The transcriptome of the bursa copulatrix supports its role as a muscular sac that is the primary site for disruption of the male ejaculate. We find that the bursal gland acts as a reproductive secretory body that might also interact with male ejaculate. In addition, differential expression of proteases between strains supports a potential role for these tissues in contributing to reproductive isolation. Our study provides new insight into how male ejaculate is processed by female Lepidoptera, and paves the way for future work on interactions between post-mating sexual selection and speciation.
doi:10.1186/1471-2164-15-189
PMCID: PMC4007636  PMID: 24621199
Female reproductive genes; Speciation; Next-generation sequencing; Lepidoptera
21.  Infectious Speciation Revisited: Impact of Symbiont-Depletion on Female Fitness and Mating Behavior of Drosophila paulistorum 
PLoS Pathogens  2010;6(12):e1001214.
The neotropical Drosophila paulistorum superspecies, consisting of at least six geographically overlapping but reproductively isolated semispecies, has been the object of extensive research since at least 1955, when it was initially trapped mid-evolution in flagrant statu nascendi. In this classic system females express strong premating isolation patterns against mates belonging to any other semispecies, and yet uncharacterized microbial reproductive tract symbionts were described triggering hybrid inviability and male sterility. Based on theoretical models and limited experimental data, prime candidates fostering symbiont-driven speciation in arthropods are intracellular bacteria belonging to the genus Wolbachia. They are maternally inherited symbionts of many arthropods capable of manipulating host reproductive biology for their own benefits. However, it is an ongoing debate as to whether or not reproductive symbionts are capable of driving host speciation in nature and if so, to what extent. Here we have reevaluated this classic case of infectious speciation by means of present day molecular approaches and artificial symbiont depletion experiments. We have isolated the α-proteobacteria Wolbachia as the maternally transmitted core endosymbionts of all D. paulistorum semispecies that have coevolved towards obligate mutualism with their respective native hosts. In hybrids, however, these mutualists transform into pathogens by overreplication causing embryonic inviability and male sterility. We show that experimental reduction in native Wolbachia titer causes alterations in sex ratio, fecundity, and mate discrimination. Our results indicate that formerly designated Mycoplasma-like organisms are most likely Wolbachia that have evolved by becoming essential mutualistic symbionts in their respective natural hosts; they have the potential to trigger pre- and postmating isolation. Furthermore, in light of our new findings, we revisit the concept of infectious speciation and discuss potential mechanisms that can restrict or promote symbiont-induced speciation at post- and prezygotic levels in nature and under artificial laboratory conditions.
Author Summary
The Drosophila paulistorum species complex serves as a well-studied model system for evaluating the impact of symbiosis on host speciation since they evolve rapidly and comprise an ancestral, but highly dynamic, reservoir of microbial symbionts. Theory and some experimental evidence suggest that in evolutionary longterm host-symbiont interactions, reproductive parasites might evolve a more benign lifestyle towards mutualism, manipulate sexual mating behavior, and foster host speciation. However, it is an ongoing debate as to whether or not microbial symbionts are capable of driving host speciation in nature and if so, to what extent. Prime candidates are Wolbachia, inherited, endosymbiotic bacteria of many arthropods, presently attracting attention as potential biocontrol agents since they affect host reproductive biology. Here we document that all D. paulistorum semispecies harbor Wolbachia that provide significant fitness benefits to their natural hosts. In semispecies hybrids, however, mutualistic Wolbachia turn into pathogens, triggering embryonic lethality and male sterility via overreplication. Besides their impacts on post-mating isolation, we show that in their native D. paulistorum hosts Wolbachia manipulate sexual behavior by triggering pre-mating isolation via selective mate avoidance, i.e. avoiding mates harboring another, incompatible symbiont variant. Our study reveals that endosymbionts can coevolve rapidly with their native hosts and play a significant role in driving natural host speciation.
doi:10.1371/journal.ppat.1001214
PMCID: PMC2996333  PMID: 21151959
22.  Divergence in Sex Steroid Hormone Signaling between Sympatric Species of Japanese Threespine Stickleback 
PLoS ONE  2011;6(12):e29253.
Sex steroids mediate the expression of sexually dimorphic or sex-specific traits that are important both for mate choice within species and for behavioral isolation between species. We investigated divergence in sex steroid signaling between two sympatric species of threespine stickleback (Gasterosteus aculeatus): the Japan Sea form and the Pacific Ocean form. These sympatric forms diverge in both male display traits and female mate choice behaviors, which together contribute to asymmetric behavioral isolation in sympatry. Here, we found that plasma levels of testosterone and 17β-estradiol differed between spawning females of the two sympatric forms. Transcript levels of follicle-stimulating hormone-β (FSHβ) gene were also higher in the pituitary gland of spawning Japan Sea females than in the pituitary gland of spawning Pacific Ocean females. By contrast, none of the sex steroids examined were significantly different between nesting males of the two forms. However, combining the plasma sex steroid data with testis transcriptome data suggested that the efficiency of the conversion of testosterone into 11-ketotestosterone has likely diverged between forms. Within forms, plasma testosterone levels in males were significantly correlated with male body size, a trait important for female mate choice in the two sympatric species. These results demonstrate that substantial divergence in sex steroid signaling can occur between incipient sympatric species. We suggest that investigation of the genetic and ecological mechanisms underlying divergence in hormonal signaling between incipient sympatric species will provide a better understanding of the mechanisms of speciation in animals.
doi:10.1371/journal.pone.0029253
PMCID: PMC3247238  PMID: 22216225
23.  Evidence for no sexual isolation between Drosophila albomicans and D. nasuta 
Ecology and Evolution  2013;3(7):2061-2074.
Sexual isolation, the reduced tendency to mate, is one of the reproductive barriers that prevent gene flow between different species. Various species-specific signals during courtship contribute to sexual isolation between species. Drosophila albomicans and D. nasuta are closely related species of the nasuta subgroup within the Drosophila immigrans group and are distributed in allopatry. We analyzed mating behavior and courtship as well as cuticular hydrocarbon profiles within and between species. Here, we report that these two species randomly mated with each other. We did not observe any sexual isolation between species or between strains within species by multiple-choice tests. Significant difference in the courtship index was detected between these two species, but males and females of both species showed no discrimination against heterospecific partners. Significant quantitative variations in cuticular hydrocarbons between these two species were also found, but the cuticular hydrocarbons appear to play a negligible role in both courtship and sexual isolation between these two species. In contrast to the evident postzygotic isolation, the lack of sexual isolation between these two species suggests that the evolution of premating isolation may lag behind that of the intergenomic incompatibility, which might be driven by intragenomic conflicts.
doi:10.1002/ece3.619
PMCID: PMC3728947  PMID: 23919152
Courtship; cuticular hydrocarbons; D. albomicans; D. nasuta; mating behavior; speciation
24.  Pedigrees, assortative mating and speciation in Darwin's finches 
Pedigree analysis is a useful tool in the study of speciation. It can reveal trans-generational influences on the choice of mates. We examined mating patterns in a population of Darwin's medium ground finches (Geospiza fortis) on Daphne Major Island to improve our understanding of how a barrier to the exchange of genes between populations arises in evolution. Body sizes of mates were weakly correlated. In one year, the smallest females were paired non-randomly with the males of similar size, and in another year the largest males were paired with the largest females. An influence of parental morphology on the choice of mates, as expected from sexual imprinting theory, was found; the body size of mates was predicted by the body sizes of both parents, and especially strongly by the father's. These associations imply that the seeds of reproductive isolation between species are present within a single variable population. The implication was subject to a natural test: two exceptionally large birds of the study species, apparently immigrants, bred with each other, as did their offspring, and not with the members of the resident population. The intense inbreeding represents incipient speciation. It parallels a similar phenomenon when another species, the large ground finch, immigrated to Daphne and established a new population without interbreeding with the resident medium ground finches.
doi:10.1098/rspb.2007.0898
PMCID: PMC2596835  PMID: 18211884
beak and body size; Geospiza fortis; inbreeding; mating patterns; reproductive isolation; sexual imprinting
25.  Pheromonal and Behavioral Cues Trigger Male-to-Female Aggression in Drosophila 
PLoS Biology  2010;8(11):e1000541.
By genetically manipulating both pheromonal profiles and behavioral patterns, we find that Drosophila males showed a complete reversal in their patterns of aggression towards other males and females
Appropriate displays of aggression rely on the ability to recognize potential competitors. As in most species, Drosophila males fight with other males and do not attack females. In insects, sex recognition is strongly dependent on chemosensory communication, mediated by cuticular hydrocarbons acting as pheromones. While the roles of chemical and other sensory cues in stimulating male to female courtship have been well characterized in Drosophila, the signals that elicit aggression remain unclear. Here we show that when female pheromones or behavior are masculinized, males recognize females as competitors and switch from courtship to aggression. To masculinize female pheromones, a transgene carrying dsRNA for the sex determination factor transformer (traIR) was targeted to the pheromone producing cells, the oenocytes. Shortly after copulation males attacked these females, indicating that pheromonal cues can override other sensory cues. Surprisingly, masculinization of female behavior by targeting traIR to the nervous system in an otherwise normal female also was sufficient to trigger male aggression. Simultaneous masculinization of both pheromones and behavior induced a complete switch in the normal male response to a female. Control males now fought rather than copulated with these females. In a reciprocal experiment, feminization of the oenocytes and nervous system in males by expression of transformer (traF) elicited high levels of courtship and little or no aggression from control males. Finally, when confronted with flies devoid of pheromones, control males attacked male but not female opponents, suggesting that aggression is not a default behavior in the absence of pheromonal cues. Thus, our results show that masculinization of either pheromones or behavior in females is sufficient to trigger male-to-female aggression. Moreover, by manipulating both the pheromonal profile and the fighting patterns displayed by the opponent, male behavioral responses towards males and females can be completely reversed. Therefore, both pheromonal and behavioral cues are used by Drosophila males in recognizing a conspecific as a competitor.
Author Summary
As in other species, the fruit fly Drosophila melanogaster uses chemical signals in the form of pheromones to recognize the species and sex of another individual. Males typically fight with other males and do not attack females. While the roles of pheromonal and other sensory cues in stimulating courtship towards females have been extensively studied, the signals that elicit aggression towards other males remain unclear. In this work, we use genetic tools to show that masculinization of female pheromones is sufficient to trigger aggression from wild type males towards females. Surprisingly, males also attacked females that displayed male patterns of aggression, even if they show normal female pheromonal profiles, indicating that pheromones are not the only cues important for identifying another animal as an opponent. By simultaneously manipulating pheromones and behavioral patterns of opponents, we can completely switch the behavioral response of males towards females and males. These results demonstrate that not only pheromonal but also behavioral cues can serve as triggers of aggression, underlining the importance of behavioral feedback in the manifestation of social behaviors.
doi:10.1371/journal.pbio.1000541
PMCID: PMC2990703  PMID: 21124886

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