Love Is Blind: Indiscriminate Female Mating Responses to Male Courtship Pheromones in Newts (Salamandridae)

Internal fertilization without copulation or prolonged physical contact is a rare reproductive mode among vertebrates. In many newts (Salamandridae), the male deposits a spermatophore on the substrate in the water, which the female subsequently takes up with her cloaca. Because such an insemination requires intense coordination of both sexes, male newts have evolved a courtship display, essentially consisting of sending pheromones under water by tail-fanning towards their potential partner. Behavioral experiments until now mostly focused on an attractant function, i.e. showing that olfactory cues are able to bring both sexes together. However, since males start their display only after an initial contact phase, courtship pheromones are expected to have an alternative function. Here we developed a series of intraspecific and interspecific two-female experiments with alpine newt (Ichthyosaura alpestris) and palmate newt (Lissotriton helveticus) females, comparing behavior in male courtship water and control water. We show that male olfactory cues emitted during tail-fanning are pheromones that can induce all typical features of natural female mating behavior. Interestingly, females exposed to male pheromones of their own species show indiscriminate mating responses to conspecific and heterospecific females, indicating that visual cues are subordinate to olfactory cues during courtship.     

Pheromonal and behavioral cues trigger male-to-female aggression in Drosophila

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.     

The survival advantage of olfaction in a competitive environment

Olfaction is generally assumed to be critical for survival because this sense allows animals to detect food and pheromonal cues. Although the ability to sense sex pheromones [1, 2, 3] is likely to be important for insects, the contribution of general odor detection to survival is unknown. We investigated the extent to which the olfactory system confers a survival advantage on Drosophila larvae foraging for food under conditions of limited resources and competition from other larvae.     

Olfaction and olfactory learning in Drosophila: recent progress

The olfactory system of Drosophila resembles that of vertebrates in its overall anatomical organization, but is considerably reduced in terms of cell number, making it an ideal model system to investigate odor processing in a brain [Vosshall LB, Stocker RF: Molecular architecture of smell and taste in Drosophila. Annu Rev Neurosci 2007, 30:505-533]. Recent studies have greatly increased our knowledge about odor representation at different levels of integration, from olfactory receptors to 'higher brain centers'. In addition, Drosophila represents a favourite model system to study the neuronal basis of olfactory learning and memory, and considerable progress during the last years has been made in localizing the structures mediating olfactory learning and memory [Davis RL: Olfactory memory formation in Drosophila: from molecular to systems neuroscience. Annu Rev Neurosci 2005, 28:275-302; Gerber B, Tanimoto H, Heisenberg M: An engram found? Evaluating the evidence from fruit flies. Curr Opin Neurobiol 2004, 14:737-744; Keene AC, Waddell S: Drosophila olfactory memory: single genes to complex neural circuits. Nat Rev Neurosci 2007, 8:341-354]. This review summarizes recent progress in analyzing olfactory processing and olfactory learning in Drosophila.     

Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate

Reproductive behavior in Drosophila has both stereotyped and plastic components that are driven by age- and sex-specific chemical cues. Males who unsuccessfully court virgin females subsequently avoid females that are of the same age as the trainer. In contrast, males trained with mature mated females associate volatile appetitive and aversive pheromonal cues and learn to suppress courtship of all females. Here we show that the volatile aversive pheromone that leads to generalized learning with mated females is (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA). cVA is a major component of the male cuticular hydrocarbon profile, but it is not found on virgin females. During copulation, cVA is transferred to the female in ejaculate along with sperm and peptides that decrease her sexual receptivity. When males sense cVA (either synthetic or from mated female or male extracts) in the context of female pheromone, they develop a generalized suppression of courtship. The effects of cVA on initial courtship of virgin females can be blocked by expression of tetanus toxin in Or65a, but not Or67d neurons, demonstrating that the aversive effects of this pheromone are mediated by a specific class of olfactory neuron. These findings suggest that transfer of cVA to females during mating may be part of the male's strategy to suppress reproduction by competing males.     

Release of male courtship display in Periplaneta americana: evidence for female contact sex pheromone

Male American cockroaches (Periplaneta americana) are attracted to virgin females by volatile sex pheromones. After antennal contact with the female they turn through 180° and spread their wings in courtship display. A chemical contact stimulus releasing male courtship is demonstrated in the female cuticle. Experiments with standardized olfactory stimulation by volatile sex pheromones revealed that the contact stimulus is sex-specific and species-specific. It can be washed off the cuticle with non-polar solvents and was successfully transferred to glass dummies. However, it is not effective in the absence of volatile sex pheromones. Thus volatile sex pheromones are responsible for male attraction and sexual motivation, while mate recognition is accomplished through the contact pheromone.     

Courtship initiation is stimulated by acoustic signals in Drosophila melanogaster

Finding a mating partner is a critical task for many organisms. It is in the interest of males to employ multiple sensory modalities to search for females. In Drosophila melanogaster, vision is thought to be the most important courtship stimulating cue at long distance, while chemosensory cues are used at relatively short distance. In this report, we show that when visual cues are not available, sounds produced by the female allow the male to detect her presence in a large arena. When the target female was artificially immobilized, the male spent a prolonged time searching before starting courtship. This delay in courtship initiation was completely rescued by playing either white noise or recorded fly movement sounds to the male, indicating that the acoustic and/or seismic stimulus produced by movement stimulates courtship initiation, most likely by increasing the general arousal state of the male. Mutant males expressing tetanus toxin (TNT) under the control of Gr68a-GAL4 had a defect in finding active females and a delay in courtship initiation in a large arena, but not in a small arena. Gr68a-GAL4 was found to be expressed pleiotropically not only in putative gustatory pheromone receptor neurons but also in mechanosensory neurons, suggesting that Gr68a-positive mechanosensory neurons, not gustatory neurons, provide motion detection necessary for courtship initiation. TNT/Gr68a males were capable of discriminating the copulation status and age of target females in courtship conditioning, indicating that female discrimination and formation of olfactory courtship memory are independent of the Gr68a-expressing neurons that subserve gustation and mechanosensation. This study suggests for the first time that mechanical signals generated by a female fly have a prominent effect on males' courtship in the dark and leads the way to studying how multimodal sensory information and arousal are integrated in behavioral decision making.     

Smelling on the fly: sensory cues and strategies for olfactory navigation in Drosophila

Navigating toward (or away from) a remote odor source is a challenging problem that requires integrating olfactory information with visual and mechanosensory cues. Drosophila melanogaster is a useful organism for studying the neural mechanisms of these navigation behaviors. There are a wealth of genetic tools in this organism, as well as a history of inventive behavioral experiments. There is also a large and growing literature in Drosophila on the neural coding of olfactory, visual, and mechanosensory stimuli. Here we review recent progress in understanding how these stimulus modalities are encoded in the Drosophila nervous system. We also discuss what strategies a fly might use to navigate in a natural olfactory landscape while making use of all these sources of sensory information. We emphasize that Drosophila are likely to switch between multiple strategies for olfactory navigation, depending on the availability of various sensory cues. Finally, we highlight future research directions that will be important in understanding the neural circuits that underlie these behaviors.     

Testing for Odor Discrimination and Habituation in Mice

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal’s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.     

Crossmodal visual input for odor tracking during fly flight

Flies generate robust and high-performance olfactory and visual behaviors. Adult fruit flies can distinguish small differences in odor concentration across antennae separated by less than 1 mm [1], and a single olfactory sensory neuron is sufficient for near-normal gradient tracking in larvae [2]. During flight a male housefly chasing a female executes a corrective turn within 40 ms after a course deviation by its target [3]. The challenges imposed by flying apparently benefit from the tight integration of unimodal sensory cues. Crossmodal interactions reduce the discrimination threshold for unimodal memory retrieval by enhancing stimulus salience [4], and dynamic crossmodal processing is required for odor search during free flight because animals fail to locate an odor source in the absence of rich visual feedback [5]. The visual requirements for odor localization are unknown. We tethered a hungry fly in a magnetic field, allowing it to yaw freely, presented odor plumes, and examined how visual cues influence odor tracking. We show that flies are unable to use a small-field object or landmark to assist plume tracking, whereas odor activates wide-field optomotor course control to enable accurate orientation toward an attractive food odor.     

Reversing Stimulus Timing in Visual Conditioning Leads to Memories with Opposite Valence in Drosophila

Animals need to associate different environmental stimuli with each other regardless of whether they temporally overlap or not. Drosophila melanogaster displays olfactory trace conditioning, where an odor is followed by electric shock reinforcement after a temporal gap, leading to conditioned odor avoidance. Reversing the stimulus timing in olfactory conditioning results in the reversal of memory valence such that an odor that follows shock is later on approached (i.e. relief conditioning). Here, we explored the effects of stimulus timing on memory in another sensory modality, using a visual conditioning paradigm. We found that flies form visual memories of opposite valence depending on stimulus timing and can associate a visual stimulus with reinforcement despite being presented with a temporal gap. These results suggest that associative memories with non-overlapping stimuli and the effect of stimulus timing on memory valence are shared across sensory modalities.     

Associative learning and memory in Drosophila: beyond olfactory conditioning

The associative learning abilities of the fruit fly, Drosophila melanogaster, have been demonstrated in both classical and operant conditioning paradigms. Efforts to identify the neural pathways and cellular mechanisms of learning have focused largely on olfactory classical conditioning. Results derived from various genetic and molecular manipulations provide considerable evidence that this form of associative learning depends critically on neural activity and cAMP signaling in brain neuropil structures called mushroom bodies. Three other behavioral learning paradigms in Drosophila serve as the main subject of this review. These are (1) visual and motor learning of flies tethered in a flight simulator, (2) a form of spatial learning that is independent of visual and olfactory cues, and (3) experience-dependent changes in male courtship behavior. The present evidence suggests that at least some of these modes of learning are independent of mushroom bodies. Applying targeted genetic manipulations to these behavioral paradigms should allow for a more comprehensive understanding of neural mechanisms responsible for diverse forms of associative learning and memory.     

Conditioning Mutations in DROSOPHILA MELANOGASTER Affect an Experience-Dependent Behavioral Modification in Courting Males

One aspect of courtship in male Drosophila melanogaster has been reported to be experience dependent. Males that have courted fertilized females are virtually unresponsive to virgin females for 2–3 hr. Here, this response was utilized as an assay for the effects of conditioning mutations on experience-dependent courtship. Seven strains expressing conditioning mutations (previously isolated and characterized for learning or memory defects in an electrical shock-odor association paradigm, independent of courtship) were all found to be mutant in expression of this experience-dependent change in courtship behavior. By comparison, three control strains that were unselected for conditioning defects all expressed normal experience-dependent courtship. Other results indicate that males of the conditioning-defective strains are able to elicit necessary cues from fertilized females, yet do not then modify their courtship with virgin females. Thus, it is suggested that experience-dependent modification of courtship and the previously reported associative olfactory conditioning with electric shock share common elements of processing. The possibility that experience-dependent courtship represents adaptive behavior is discussed.     

A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship

Propagation in higher animals requires the efficient and accurate display of innate mating behaviors. In Drosophila melanogaster, male courtship consists of a stereotypic sequence of behaviors involving multiple sensory modalities, such as vision, audition, and chemosensation. For example, taste bristles located in the male forelegs and the labial palps are thought to recognize nonvolatile pheromones secreted by the female. Here, we report the identification of the putative pheromone receptor GR68a, which is expressed in chemosensory neurons of about 20 male-specific gustatory bristles in the forelegs. Gr68a expression is dependent on the sex determination gene doublesex, which controls many aspects of sexual differentiation and is necessary for normal courtship behavior. Tetanus toxin-mediated inactivation of Gr68a-expressing neurons or transgene-mediated RNA interference of Gr68a RNA leads to a significant reduction in male courtship performance, suggesting that GR68a protein is an essential component of pheromone-driven courtship behavior in Drosophila.     

The Influence of Sequential Male Courtship Behaviors on Courtship Success and Duration in a Terrestrial Salamander,Plethodon shermani

Most organisms use multiple cues across many modalities to assess potential mates. Salamanders in the genus Plethodon have an elaborate courtship display, yet previous studies primarily have concentrated on the influence of olfactory cues on courtship success. In this study, we tested whether two different cues (one chemical and one visual) affected courtship success and duration in the red‐legged salamander, Plethodon shermani. By staging and observing over 180 courtship trials, we found that ‘foot‐dancing’, a male visual cue performed in the earliest stages of courtship, significantly increased the likelihood that a male and female would progress to subsequent stages of courtship and, thus, the likelihood of insemination. In contrast, delivery of biologically relevant levels of a non‐volatile pheromone (produced in a large gland on the male's chin) during courtship trials did not increase overall insemination rate success, but did significantly reduce the duration of one of the later stages of courtship, tail‐straddling walk. Thus, we propose that early use of visual cues in Plethodon courtship, may (1) increase the detectability of the male message by increasing the range at which the male can be perceived by the female and (2) indicate the context of the male's approach and subsequent behaviors. In short, this study draws attention to the need to examine behavioral traits affecting courtship success at multiple stages and across several sensory modalities.     

A Drosophila protein specific to pheromone-sensing gustatory hairs delays males' copulation attempts

In insects, increasing evidence suggests that small secreted pheromone binding proteins (PBPs) and odorant binding proteins (OBPs) are important for normal olfactory detection of airborne pheromones and odorants far from their source. In contrast, it is unknown whether extracellular ligand binding proteins participate in perception of less volatile chemicals, including many pheromones, that are detected by direct contact with chemosensory organs. CheB42a, a small Drosophila melanogaster protein unrelated to known PBPs or OBPs, is expressed and likely secreted in only a small subset of gustatory sensilla on males' front legs, the site of gustatory perception of contact pheromones. Here we show that CheB42a is expressed specifically in the sheath cells surrounding the taste neurons expressing Gr68a, a putative gustatory pheromone receptor for female cuticular hydrocarbons that stimulate male courtship. Surprisingly, however, CheB42a mutant males attempt to copulate with females earlier and more frequently than control males. Furthermore, CheB42a mutant males also attempt to copulate more frequently with other males that secrete female-specific cuticular hydrocarbon pheromones, but not with females lacking cuticular hydrocarbons. Together, these data indicate that CheB42a is required for a normal gustatory response to female cuticular hydrocarbon pheromones that modulate male courtship.     

Innate attractiveness and associative learnability of odors can be dissociated in larval Drosophila

We investigate olfactory associative learning in larval Drosophila. A reciprocal training design is used, such that one group of animals receives a reward in the presence of odor X but not in the presence of odor Y (Train: X+ // Y), whereas another group is trained reciprocally (Train: X // Y+). After training, differences in odor preference between these reciprocally trained groups in a choice test (Test: X - Y) reflect associative learning. The current study, after showing which odor pairs can be used for such learning experiments, 1) introduces a one-odor version of such reciprocal paradigm that allows estimating the learnability of single odors. Regarding this reciprocal one-odor paradigm, we show that 2) paired presentations of an odor with a reward increase odor preference above baseline, whereas unpaired presentations of odor and reward decrease odor preference below baseline; this suggests that odors can become predictive either of reward or of reward absence. Furthermore, we show that 3) innate attractiveness and associative learnability can be dissociated. These data deepen our understanding of odor-reward learning in larval Drosophila on the behavioral level, and thus foster its neurogenetic analysis.     

Courtship, aggression and avoidance: pheromones, receptors and neurons for social behaviors in Drosophila

Chemical communication between individual Drosophila is extremely important for social behaviors required for survival and reproduction, such as con-specific recognition, courtship, aggression and avoidance of odor from "stressed" flies. Characterization of the receptors and neural circuits that detect pheromone cues and an understanding of how these circuits are modulated by the social interactions are fundamental questions about the neurobiology of social behaviors. Recent years have seen important advances in the identification of chemoreceptors and sensory neurons that are involved in sensing pheromones. Here we present a brief review of the current understanding of the peripheral chemosensory systems that are involved in social behaviors.     

果蝇嗅觉分子机理研究进展

黑腹果蝇Drosophila melanogaster是生物学研究的重要模式生物,也是探索研究生物体嗅觉奥秘的理想材料。近年来,由于分子生物学技术在神经科学领域的广泛应用,黑腹果蝇嗅觉机理研究取得了许多重大突破, 对气味分子受体及其识别机理、 嗅觉神经电信号的产生和传递、嗅觉信息的加工、编码以及记忆等方面都有了深入的了解。研究表明, 果蝇约1 300个嗅神经元(olfactory receptor neurons, ORNs)共表达62种不同的气味受体蛋白（olfactory receptor proteins, ORs）, 用以检测和识别其所感受的所有化学气味分子。许多OR所识别的气味分子配体已鉴定出来,普通的气味（如水果的气味）由数种不同的OR组合来识别,而信息素（pheromone）分子则由单种特定的OR来检测。气味信息在嗅神经元内转换成神经电信号,嗅觉电信号沿嗅神经元的轴突传递到触角叶, 再经投射神经元（projection neurons, PNs）将信息送至高级中枢如蘑菇体（mushroom body, MB）和侧角（lateral horn, LH）,最终引发行为反应。在黑腹果蝇嗅觉信息传递通路中,某些蛋白如Dock,N-cadherin,Fruitless等起着重要作用,缺失这些蛋白会导致嗅觉异常。本文对这些研究进展作一综述。     

Divergence of premating behaviors in the closely related species Drosophila subquinaria and D. recens

Most animal species use distinctive courship patterns to choose among potential mates. Over time, the sensory signaling and preferences used during courtship can diverge among groups that are reproductively isolated. This divergence of signal traits and preferences is thought to be an important cause of behavioral isolation during the speciation process. Here, we examine the sensory modalities used in courtship by two closely related species, Drosophila subquinaria and Drosophila recens, which overlap in geographic range and are incompletely reproductively isolated. We use observational studies of courtship patterns and manipulation of male and female sensory modalities to determine the relative roles of visual, olfactory, gustatory, and auditory signals during conspecific mate choice. We find that sex‐specific, species‐specific, and population‐specific cues are used during mate acquisition within populations of D. subquinaria and D. recens. We identify shifts in both male and female sensory modalities between species, and also between populations of D. subquinaria. Our results indicate that divergence in mating signals and preferences have occurred on a relatively short timescale within and between these species. Finally, we suggest that because olfactory cues are essential for D. subquinaria females to mate within species, they may also underlie variation in behavioral discrimination across populations and species.