In many insects, mate finding relies on female-released sex pheromones, which have to be deciphered by the male olfactory system within an odorous background of plant volatiles present in the environment of a calling female. With respect to pheromone-mediated mate localization, plant odorants may be neutral, favorable, or disturbing. Here we examined the impact of plant odorants on detection and coding of the major sex pheromone component, (Z)-11-hexadecenal (Z11-16:Ald) in the noctuid moth Heliothis virescens. By in vivo imaging the activity in the male antennal lobe (AL), we monitored the interference at the level of olfactory sensory neurons (OSN) to illuminate mixture interactions. The results show that stimulating the male antenna with Z11-16:Ald and distinct plant-related odorants simultaneously suppressed pheromone-evoked activity in the region of the macroglomerular complex (MGC), where Z11-16:Ald-specific OSNs terminate. Based on our previous findings that antennal detection of Z11-16:Ald involves an interplay of the pheromone binding protein (PBP) HvirPBP2 and the pheromone receptor (PR) HR13, we asked if the plant odorants may interfere with any of the elements involved in pheromone detection. Using a competitive fluorescence binding assay, we found that the plant odorants neither bind to HvirPBP2 nor affect the binding of Z11-16:Ald to the protein. However, imaging experiments analyzing a cell line that expressed the receptor HR13 revealed that plant odorants significantly inhibited the Z11-16:Ald-evoked calcium responses. Together the results indicate that plant odorants can interfere with the signaling process of the major sex pheromone component at the receptor level. Consequently, it can be assumed that plant odorants in the environment may reduce the firing activity of pheromone-specific OSNs in H. virescens and thus affect mate localization.
pheromone detection; antennal lobe; pheromone receptor; pheromone binding protein; olfaction
The antennal imaginal disc was transplanted between pre-metamorphic male larvae of two different Lepidopteran moth species. Following adult eclosion, electrophysiological recordings were made from 33 central olfactory neurons in the antennal lobes of both Helicoverpa zea donor to Heliothis virescens recipient (Z-V) and reciprocal (V-Z) transplants. Under the influence of sensory neuron input derived from the transplanted antennal imaginal disc, most antennal lobe projection neurons (29/33) were classified as belonging to physiological categories encountered previously in donor species males. Furthermore, when stained, many of these neurons had dendritic arbors restricted to donor-induced glomerular locations predicted by their physiology. However, some neurons with unexpected physiological profiles were also identified (4/33), but only in V-Z transplants. These profiles help to explain why some V-Z bilateral transplants were able to respond to both pheromone blends in flight tunnel bioassays, an unforeseen result counter to the assumption that a donor antenna develops a normal donor antennal olfactory receptor neuron compliment. Stainings of several neurons in V-Z transplant males also revealed unusual morphological features including multiglomerular dendritic arbors and “incorrect” glomerular locations. These results indicate a developmental plasticity in the final dendritic arborization pattern of central olfactory neurons including an ability to colonize and integrate inputs across topographically novel donor glomeruli, different from those found in the normal recipient antennal lobe.
olfaction; antennal lobe; glomerulus; Heliothis virescens; Helicoverpa zea; behavior; pheromone; imaginal disc
The insect's olfactory system is so selective that male moths, for example, can discriminate female-produced sex pheromones from compounds with minimal structural modifications. Yet, there is an exception for this “lock-and-key” tight selectivity. Formate analogs can be used as replacement for less chemically stable, long-chain aldehyde pheromones, because male moths respond physiologically and behaviorally to these parapheromones. However, it remained hitherto unknown how formate analogs interact with aldehyde-sensitive odorant receptors (ORs). Neuronal responses to semiochemicals were investigated with single sensillum recordings. Odorant receptors (ORs) were cloned using degenerate primers, and tested with the Xenopus oocyte expression system. Quality, relative quantity, and purity of samples were evaluated by gas chromatography and gas chromatography-mass spectrometry. We identified olfactory receptor neurons (ORNs) housed in trichoid sensilla on the antennae of male navel orangeworm that responded equally to the main constituent of the sex pheromone, (11Z,13Z)-hexadecadienal (Z11Z13-16Ald), and its formate analog, (9Z,11Z)-tetradecen-1-yl formate (Z9Z11-14OFor). We cloned an odorant receptor co-receptor (Orco) and aldehyde-sensitive ORs from the navel orangeworm, one of which (AtraOR1) was expressed specifically in male antennae. AtraOR1•AtraOrco-expressing oocytes responded mainly to Z11Z13-16Ald, with moderate sensitivity to another component of the sex pheromone, (11Z,13Z)-hexadecadien-1-ol. Surprisingly, this receptor was more sensitive to the related formate than to the natural sex pheromone. A pheromone receptor from Heliothis virescens, HR13 ( = HvirOR13) showed a similar profile, with stronger responses elicited by a formate analog than to the natural sex pheromone, (11Z)-hexadecenal thus suggesting this might be a common feature of moth pheromone receptors.
Both males and females of heliothine moths utilize sex-pheromones during the mating process. Females produce and release a sex pheromone for the long–range attraction of males for mating. Production of sex pheromone in females is controlled by the peptide hormone (pheromone biosynthesis activating neuropeptide, PBAN). This review will highlight what is known about the role PBAN plays in controlling pheromone production in female moths. Male moths produce compounds associated with a hairpencil structure associated with the aedaegus that are used as short-range aphrodisiacs during the mating process. We will discuss the role that PBAN plays in regulating male production of hairpencil pheromones.
pheromone; PBAN; Heliothis; Helicoverpa; moth
In the olfactory pathway of Drosophila, a GABAB receptor mediated presynaptic gain control mechanism at the first synapse between olfactory sensory neurons (OSNs) and projection neurons has been suggested to play a critical role in setting the sensitivity and detection range of the sensory system. To approach the question if such a mechanism may be realized in the pheromone recognition system of male moths in this study attempts were made to explore if moth's pheromone-responsive cells express a GABAB- receptor. Employing a combination of genome analysis, RT-PCR experiments and screening of an antennal cDNA library we have identified a cDNA which encodes the GABAB-R1 receptor of Heliothis virescens. Moreover, based on the HvirGABAB-R1 sequence we could predict a GABAB-R1 protein from genome sequences of the silkmoth Bombyx mori. To assess whether HvirGABAB-R1 is expressed in OSNs of male antenna we performed whole-mount in situ hybridization (WM-ISH) experiments. Several HvirGABAB-R1 positive cells were visualized under long sensilla trichodea, known to contain pheromone-responsive OSNs. In parallel it was shown that cells under long trichoid hairs were labelled with pheromone receptor specific probes. In addition, the HvirGABAB-R1 specific probe also labelled several cells under shorter olfactory sensilla, but never stained cells under mechanosensory/gustatory sensilla chaetica. Together, the results indicate that a GABAB receptor is expressed in pheromone-responsive OSNs of H. virescens and suggest a presynaptic gain control mechanism in the axon terminals of these cells.
moth; olfaction; GABA; pheromone; in situ hybridization.
The relative proportions of components in a pheromone blend play a major role in sexual recognition in moths. Two sympatric species, Helicoverpa armigera and Helicoverpa assulta, use (Z)-11-hexadecenal (Z11–16: Ald) and (Z)-9-hexadecenal (Z9–16: Ald) as essential sex pheromone components but in very different ratios, 97∶3 and 7∶93 respectively. Using wind tunnel tests, single sensillum recording and in vivo calcium imaging, we comparatively studied behavioral responses and physiological activities at the level of antennal sensilla and antennal lobe (AL) in males of the two species to blends of the two pheromone components in different ratios (100∶0, 97∶3, 50∶50, 7∶93, 0∶100). Z11–16: Ald and Z9–16: Ald were recognized by two populations of olfactory sensory neurons (OSNs) in different trichoid sensilla on antennae of both species. The ratios of OSNs responding to Z11–16:Ald and Z9–16:Ald OSNs were 100∶28.9 and 21.9∶100 in H. armigera and H. assulta, respectively. The Z11–16:Ald OSNs in H. armigera exhibited higher sensitivity and efficacy than those in H. assulta, while the Z9–16:Ald OSNs in H. armigera had the same sensitivity but lower efficacy than those in H. assulta. At the dosage of 10 µg, Z11–16: Ald and Z9–16: Ald evoked calcium activity in 8.5% and 3.0% of the AL surface in H. armigera, while 5.4% and 8.6% of AL in H. assulta, respectively. The calcium activities in the AL reflected the peripheral input signals of the binary pheromone mixtures and correlated with the behavioral output. These results demonstrate that the binary pheromone blends were precisely coded by the firing frequency of individual OSNs tuned to Z11–16: Ald or Z9–16: Ald, as well as their population sizes. Such information was then accurately reported to ALs of H. armigera and H. assulta, eventually producing different behaviors.
The olfactory pathway in the insect brain is anatomically well described from the antennal lobe (AL) to the mushroom bodies and the lateral protocerebrum (LP) in several species. Less is known about the further connections of the olfactory network in protocerebrum and how information about relevant plant odorants and mixtures are represented in this network, resulting in output information mediated by descending neurons. In the present study we have recorded intracellularly followed by dye injections from neurons in the LP and superior protocerebrum (SP) of the moth, Heliothis virescens. As relevant stimuli, we have used selected primary plant odorants and mixtures of them. The results provide the morphology and physiological responses of neurons involved in a putative circuit connecting the mushroom body lobes, the SP, and the LP, as well as input to SP and LP by one multiglomerular AL neuron and output from the LP by one descending neuron. All neurons responded to a particular mixture of ten primary plant odorants, some of them also to single odorants of the mixture. Altogether, the physiological data indicate integration in protocerebral neurons of information from several of the receptor neuron types functionally described in this species.
insect olfaction; protocerebral neurons; antenno-protocerebral tracts; lateral protocerebrum; superior protocerebrum; descending neuron and odor mixture
The survival of an animal often depends on an innate response to a particular sensory stimulus. For an adult male moth, two categories of odors are innately attractive: pheromone released by conspecific females, and the floral scents of certain, often co-evolved, plants. These odors consist of multiple volatiles in characteristic mixtures. Here, we review evidence that both categories of odors are processed as sensory objects, and we suggest a mechanism in the primary olfactory center, the antennal lobe (AL), that encodes the configuration of these mixtures and may underlie recognition of innately attractive odors. In the pheromone system, mixtures of two or three volatiles elicit upwind flight. Peripheral changes are associated with behavioral changes in speciation, and suggest the existence of a pattern recognition mechanism for pheromone mixtures in the AL. Moths are similarly innately attracted to certain floral scents. Though floral scents consist of multiple volatiles that activate a broad array of receptor neurons, only a smaller subset, numerically comparable to pheromone mixtures, is necessary and sufficient to elicit behavior. Both pheromone and floral scent mixtures that produce attraction to the odor source elicit synchronous action potentials in particular populations of output (projection) neurons (PNs) in the AL. We propose a model in which the synchronous output of a population of PNs encodes the configuration of an innately attractive mixture, and thus comprises an innate mechanism for releasing odor-tracking behavior. The particular example of olfaction in moths may inform the general question of how sensory objects trigger innate responses.
floral scent; moths; neuroethology; olfaction; pheromone; sensory coding; sensory object; synchrony
Antennal olfaction is extremely important for insect survival, mediating key behaviors such as host preference, mate choice, and oviposition site selection. Multiple antennal proteins are involved in olfactory signal transduction pathways. Of these, odorant receptors (ORs) and ionotropic receptors (IRs) confer specificity on olfactory sensory neuron responses. In this study, we identified the olfactory gene repertoire of the economically important agricultural pest moth, Helicoverpa armigera, by assembling the adult male and female antennal transcriptomes. Within the male and female antennal transcriptomes we identified a total of 47 OR candidate genes containing 6 pheromone receptor candidates. Additionally, 12 IR genes as well as 26 odorant-binding proteins and 12 chemosensory proteins were annotated. Our results allow a systematic functional analysis across much of conventional ORs repertoire and newly reported IRs mediating the key olfaction-mediated behaviors of H. armigera.
Tuning of the olfactory system of male moths to conspecific female sex pheromones is crucial for correct species recognition; however, little is known about the genetic changes that drive speciation in this system. Moths of the genus Ostrinia are good models to elucidate this question, since significant differences in pheromone blends are observed within and among species. Odorant receptors (ORs) play a critical role in recognition of female sex pheromones; eight types of OR genes expressed in male antennae were previously reported in Ostrinia moths.
We screened an O. nubilalis bacterial artificial chromosome (BAC) library by PCR, and constructed three contigs from isolated clones containing the reported OR genes. Fluorescence in situ hybridization (FISH) analysis using these clones as probes demonstrated that the largest contig, which contained eight OR genes, was located on the Z chromosome; two others harboring two and one OR genes were found on two autosomes. Sequence determination of BAC clones revealed the Z-linked OR genes were closely related and tandemly arrayed; moreover, four of them shared 181-bp direct repeats spanning exon 7 and intron 7.
This is the first report of tandemly arrayed sex pheromone receptor genes in Lepidoptera. The localization of an OR gene cluster on the Z chromosome agrees with previous findings for a Z-linked locus responsible for O. nubilalis male behavioral response to sex pheromone. The 181-bp direct repeats might enhance gene duplications by unequal crossovers. An autosomal locus responsible for male response to sex pheromone in Heliothis virescens and H. subflexa was recently reported to contain at least four OR genes. Taken together, these findings support the hypothesis that generation of additional copies of OR genes can increase the potential for male moths to acquire altered specificity for pheromone components, and accordingly, facilitate differentiation of sex pheromones.
For some moth species, especially those closely interrelated and sympatric, recognizing a specific pheromone component concentration ratio is essential for males to successfully locate conspecific females. We propose and determine the properties of a minimalist competition-based feed-forward neuronal model capable of detecting a certain ratio of pheromone components independently of overall concentration. This model represents an elementary recognition unit for the ratio of binary mixtures which we propose is entirely contained in the macroglomerular complex (MGC) of the male moth. A set of such units, along with projection neurons (PNs), can provide the input to higher brain centres. We found that (1) accuracy is mainly achieved by maintaining a certain ratio of connection strengths between olfactory receptor neurons (ORN) and local neurons (LN), much less by properties of the interconnections between the competing LNs proper. An exception to this rule is that it is beneficial if connections between generalist LNs (i.e. excited by either pheromone component) and specialist LNs (i.e. excited by one component only) have the same strength as the reciprocal specialist to generalist connections. (2) successful ratio recognition is achieved using latency-to-first-spike in the LN populations which, in contrast to expectations with a population rate code, leads to a broadening of responses for higher overall concentrations consistent with experimental observations. (3) when longer durations of the competition between LNs were observed it did not lead to higher recognition accuracy.
Calling female moths attract their mates late at night with intermittent release of a species-specific sex-pheromone blend. Mean frequency of pheromone filaments encodes distance to the calling female. In their zig-zagging upwind search male moths encounter turbulent pheromone blend filaments at highly variable concentrations and frequencies. The male moth antennae are delicately designed to detect and distinguish even traces of these sex pheromones amongst the abundance of other odors. Its olfactory receptor neurons sense even single pheromone molecules and track intermittent pheromone filaments of highly variable frequencies up to about 30 Hz over a wide concentration range. In the hawkmoth Manduca sexta brief, weak pheromone stimuli as encountered during flight are detected via a metabotropic PLCβ-dependent signal transduction cascade which leads to transient changes in intracellular Ca2+ concentrations. Strong or long pheromone stimuli, which are possibly perceived in direct contact with the female, activate receptor-guanylyl cyclases causing long-term adaptation. In addition, depending on endogenous rhythms of the moth's physiological state, hormones such as the stress hormone octopamine modulate second messenger levels in sensory neurons. High octopamine levels during the activity phase maximize temporal resolution cAMP-dependently as a prerequisite to mate location. Thus, I suggest that sliding adjustment of odor response threshold and kinetics is based upon relative concentration ratios of intracellular Ca2+ and cyclic nucleotide levels which gate different ion channels synergistically. In addition, I propose a new hypothesis for the cyclic nucleotide-dependent ion channel formed by insect olfactory receptor/coreceptor complexes. Instead of being employed for an ionotropic mechanism of odor detection it is proposed to control subthreshold membrane potential oscillation of sensory neurons, as a basis for temporal encoding of odors.
insect olfaction; second messengers; octopamine; circadian rhythms; signal transduction cascades; field potentials; subthreshold membrane potential oscillations; temporal encoding
Sex pheromones are essential in moth mate communication. Information on pheromone biosynthetic genes and enzymes is needed to comprehend the mechanisms that contribute to specificity of pheromone signals. Most heliothine moths use sex pheromones with (Z)–11–hexadecenal as the major component in combination with minor fatty aldehydes and alcohols. In this study we focus on four closely related species, Heliothis virescens, Heliothis subflexa, Helicoverpa armigera and Helicoverpa assulta, which use (Z)–11–hexadecenal, (Z)–9–tetradecanal, and (Z)–9–hexadecenal in different ratios in their pheromone blend. The components are produced from saturated fatty acid precursors by desaturation, β–oxidation, reduction and oxidation.
We analyzed the composition of fatty acyl pheromone precursors and correlated it to the pheromone composition. Next, we investigated whether the downstream fatty–acyl reduction step modulates the ratio of alcohol intermediates before the final oxidation step. By isolating and functionally characterizing the Fatty Acyl Reductase (pgFAR) from each species we found that the pgFARs were active on a broad set of C8 to C16 fatty acyl substrates including the key pheromone precursors, Z9–14, Z9–16 and Z11–16:acyls. When presenting the three precursors in equal ratios to yeast cultures expressing any of the four pgFARs, all reduced (Z)–9–tetradecenoate preferentially over (Z)–11–hexadecenoate, and the latter over (Z)–9–hexadecenoate. Finally, when manipulating the precursor ratios in vitro, we found that the pgFARs display small differences in the biochemical activity on various substrates.
We conclude that a pgFAR with broad specificity is involved in heliothine moth pheromone biosynthesis, functioning as a semi–selective funnel that produces species–specific alcohol product ratios depending on the fatty–acyl precursor ratio in the pheromone gland. This study further supports the key role of these in pheromone biosynthesis and emphasizes the interplay between the pheromone fatty acyl precursors and the Lepidoptera specific pgFARs in shaping the pheromone composition.
Male moths can accurately perceive the sex pheromone emitted from conspecific females by their highly accurate and specific olfactory sensory system. Pheromone receptors are of special importance in moth pheromone reception because of their central role in chemosensory signal transduction processes that occur in olfactory receptor neurons in the male antennae. There are a number of pheromone receptor genes have been cloned, however, only a few have been functionally characterized. Here we cloned six full-length pheromone receptor genes from Helicoverpa armigera male antennae. Real-time PCR showing all genes exhibited male-biased expression in adult antennae. Functional analyses of the six pheromone receptor genes were then conducted in the heterologous expression system of Xenopus oocytes. HarmOR13 was found to be a specific receptor for the major sex pheromone component Z11-16:Ald. HarmOR6 was equally tuned to both of Z9-16: Ald and Z9-14: Ald. HarmOR16 was sensitively tuned to Z11-16: OH. HarmOR11, HarmOR14 and HarmOR15 failed to respond to the tested candidate pheromone compounds. Our experiments elucidated the functions of some pheromone receptor genes of H. armigera. These advances may provide remarkable evidence for intraspecific mating choice and speciation extension in moths at molecular level.
A biophysical model of receptor potential generation in the male moth olfactory receptor neuron is presented. It takes into account all pre-effector processes—the translocation of pheromone molecules from air to sensillum lymph, their deactivation and interaction with the receptors, and the G-protein and effector enzyme activation—and focuses on the main post-effector processes. These processes involve the production and degradation of second messengers (IP3 and DAG), the opening and closing of a series of ionic channels (IP3-gated Ca2+ channel, DAG-gated cationic channel, Ca2+-gated Cl− channel, and Ca2+- and voltage-gated K+ channel), and Ca2+ extrusion mechanisms. The whole network is regulated by modulators (protein kinase C and Ca2+-calmodulin) that exert feedback inhibition on the effector and channels. The evolution in time of these linked chemical species and currents and the resulting membrane potentials in response to single pulse stimulation of various intensities were simulated. The unknown parameter values were fitted by comparison to the amplitude and temporal characteristics (rising and falling times) of the experimentally measured receptor potential at various pheromone doses. The model obtained captures the main features of the dose–response curves: the wide dynamic range of six decades with the same amplitudes as the experimental data, the short rising time, and the long falling time. It also reproduces the second messenger kinetics. It suggests that the two main types of depolarizing ionic channels play different roles at low and high pheromone concentrations; the DAG-gated cationic channel plays the major role for depolarization at low concentrations, and the Ca2+-gated Cl− channel plays the major role for depolarization at middle and high concentrations. Several testable predictions are proposed, and future developments are discussed.
All sensory neurons transduce their natural stimulus, whether a molecule, a photon, or a mechanical force, in an electrical current flowing through their sensory membrane via similar molecular and ionic mechanisms. Olfactory receptor neurons (ORNs), whose stimuli are volatile molecules, are no exception, including one of the best known: the exquisitely sensitive ORNs of male moths that detect the sexual pheromone released by conspecific females. We provide a detailed computational model of the intracellular molecular mechanisms at work in this ORN type. We describe qualitatively and quantitatively how the initial event, the interaction of pheromone molecules with specialized receptors at the ORN surface, is amplified through a sequence of linked biochemical and electrical events into a whole cell response, the receptor potential. We detail the respective roles of the upward activating reactions involving a cascade of ionic channels permeable to cations, chloride and potassium, their control by feedback inactivating mechanisms, and the central regulatory role of calcium. This computational model contributes to an integrated understanding of this signalling pathway, provides testable hypotheses, and suggests new experimental approaches.
The primary olfactory centres of most vertebrates and most neopteran insects are characterized by the presence of spherical neuropils, glomeruli, where synaptic interactions between olfactory receptor neurons and second-order neurons take place. In the neopteran insect taxa investigated so far, receptor neurons of a specific physiological identity target one glomerulus and thus bestow a functional identity on the glomerulus. In moths, input from pheromone-specific receptor neurons is received in a male-specific structure of the antennal lobe, called the macroglomerular complex (MGC), which consists of a number of specialized glomeruli. Each glomerulus of the complex receives a set of peripheral sensory afferents that encode one of several compounds involved in sexual communication. The complex is also innervated by dendritic branches of antennal lobe output neurons called projection neurons, which transfer information from the antennal lobe to higher centres of the brain. A hypothesis stemming from earlier work on moths claims that the receptor neuron innervation pattern of the MGC should be reflected in the pattern of dendrites of projection neurons invading the different MGC glomeruli. In this study we show that in the noctuid moth Trichoplusia ni, as in several other noctuid moth species, this hypothesis does not hold. The degree of matching between axon terminals of receptor neurons and the dendritic branches of identified projection neurons that express similar physiological specificity is very low.
Most animals rely on olfaction to find sexual partners, food or a habitat. The olfactory system faces the challenge of extracting meaningful information from a noisy odorous environment. In most moth species, males respond to sex pheromone emitted by females in an environment with abundant plant volatiles. Plant odours could either facilitate the localization of females (females calling on host plants), mask the female pheromone or they could be neutral without any effect on the pheromone. Here we studied how mixtures of a behaviourally-attractive floral odour, heptanal, and the sex pheromone are encoded at different levels of the olfactory pathway in males of the noctuid moth Agrotis ipsilon. In addition, we asked how interactions between the two odorants change as a function of the males' mating status. We investigated mixture detection in both the pheromone-specific and in the general odorant pathway. We used a) recordings from individual sensilla to study responses of olfactory receptor neurons, b) in vivo calcium imaging with a bath-applied dye to characterize the global input response in the primary olfactory centre, the antennal lobe and c) intracellular recordings of antennal lobe output neurons, projection neurons, in virgin and newly-mated males. Our results show that heptanal reduces pheromone sensitivity at the peripheral and central olfactory level independently of the mating status. Contrarily, heptanal-responding olfactory receptor neurons are not influenced by pheromone in a mixture, although some post-mating modulation occurs at the input of the sexually isomorphic ordinary glomeruli, where general odours are processed within the antennal lobe. The results are discussed in the context of mate localization.
Digital three dimensional standard brain atlases (SBAs) are valuable tools for integrating neuroimaging data of different preparations. In insects, SBAs of five species are available, including the atlas of the female Heliothis virescens moth brain. Like for the other species, the antennal lobes (ALs) of the moth brain atlas were integrated as one material identity without internal structures. Different from the others, the H. virescens SBA exclusively included the glomerular layer of the AL. This was an advantage in the present study for performing a direct registration of the glomerular layer of individual preparations into the standard brain. We here present the H. virescens female SBA with a new model of the AL glomeruli integrated into the atlas, i.e. with each of the 66 glomeruli identified and labelled with a specific number. The new model differs from the previous H. virescens AL model both in respect to the number of glomeruli and the numbering system; the latter according to the system used for the AL atlases of two other heliothine species. For identifying female specific glomeruli comparison with the male AL was necessary. This required a new male AL atlas, included in this paper. As demonstrated by the integration of three AL projection neurons of different preparations, the new SBA with the integrated glomruli is a helpful tool for determining the glomeruli innervated as well as the relative position of the axonal projections in the protocerebrum.
insect; olfaction; three dimensional reconstruction; mushroom body calyces; lateral protocerebrum
The molecular and cellular events mediating complex behaviors in animals are largely unknown. Elucidating the circuits underlying behaviors in simple model systems may shed light on how these circuits function. In Drosophila, courtship behavior provides a tractable model for studying the underlying basis of innate behavior. The male-specific pheromone 11-cis-vaccenyl acetate (cVA) modulates courtship behavior and is detected by T1 neurons, located on the antenna of male and female flies. The T1 neurons express the odorant receptor Or67d, and are exquisitely tuned to cVA pheromone. However, cVA-induced changes in mating behavior have also been reported upon manipulation of olfactory neurons expressing odorant receptor Or65a. These findings raise the issue of whether multiple olfactory-driven circuits underlie cVA-induced behavioral responses, and what role these circuits play in behavior. Here, we engineered flies in which the Or67d circuit is specifically activated in the absence of cVA in order to determine the role of this circuit in behavior. We created transgenic flies that express a dominant-active, pheromone-independent variant of the extracellular pheromone receptor, LUSH. We found that, similar to the behaviors elicited by cVA, engineered male flies have dramatically reduced courtship, while engineered females showed enhanced courtship. Furthermore, cVA exposure did not enhance the dominant LUSH-triggered effects on behavior in the engineered flies. Finally, we show the effects of both cVA and dominant LUSH on courtship are reversed by genetically removing Or67d. These findings demonstrate that the T1/Or67d circuit is necessary and sufficient to mediate sexually dimorphic courtship behaviors.
lush; Or67d; cVA; courtship; pheromone; olfaction
Odors are key sensory signals for social communication and food search in animals including insects. Drosophila melanogaster, is a powerful neurogenetic model commonly used to reveal molecular and cellular mechanisms involved in odorant detection. Males use olfaction together with other sensory modalities to find their mates. Here, we review known olfactory signals, their related olfactory receptors, and the corresponding neuronal architecture impacting courtship. OR67d receptor detects 11-cis-Vaccenyl Acetate (cVA), a male specific pheromone transferred to the female during copulation. Transferred cVA is able to reduce female attractiveness for other males after mating, and is also suspected to decrease male-male courtship. cVA can also serve as an aggregation signal, maybe through another OR. OR47b was shown to be activated by fly odors, and to enhance courtship depending on taste pheromones. IR84a detects phenylacetic acid (PAA) and phenylacetaldehyde (PA). These two odors are not pheromones produced by flies, but are present in various fly food sources. PAA enhances male courtship, acting as a food aphrodisiac. Drosophila males have thus developed complementary olfactory strategies to help them to select their mates.
courtship; Drosophila; olfaction; receptor; nervous system
Nocturnal insects such as moths are ideal models to study the molecular bases of olfaction that they use, among examples, for the detection of mating partners and host plants. Knowing how an odour generates a neuronal signal in insect antennae is crucial for understanding the physiological bases of olfaction, and also could lead to the identification of original targets for the development of olfactory-based control strategies against herbivorous moth pests. Here, we describe an Expressed Sequence Tag (EST) project to characterize the antennal transcriptome of the noctuid pest model, Spodoptera littoralis, and to identify candidate genes involved in odour/pheromone detection.
By targeting cDNAs from male antennae, we biased gene discovery towards genes potentially involved in male olfaction, including pheromone reception. A total of 20760 ESTs were obtained from a normalized library and were assembled in 9033 unigenes. 6530 were annotated based on BLAST analyses and gene prediction software identified 6738 ORFs. The unigenes were compared to the Bombyx mori proteome and to ESTs derived from Lepidoptera transcriptome projects. We identified a large number of candidate genes involved in odour and pheromone detection and turnover, including 31 candidate chemosensory receptor genes, but also genes potentially involved in olfactory modulation.
Our project has generated a large collection of antennal transcripts from a Lepidoptera. The normalization process, allowing enrichment in low abundant genes, proved to be particularly relevant to identify chemosensory receptors in a species for which no genomic data are available. Our results also suggest that olfactory modulation can take place at the level of the antennae itself. These EST resources will be invaluable for exploring the mechanisms of olfaction and pheromone detection in S. littoralis, and for ultimately identifying original targets to fight against moth herbivorous pests.
In insects, olfactory receptor neurons (ORNs), surrounded with auxiliary cells and protected by a cuticular wall, form small discrete sensory organs – the sensilla. The moth pheromone-sensitive sensillum is a well studied example of hair-like sensillum that is favorable to both experimental and modeling investigations. The model presented takes into account both the molecular processes of ORNs, i.e. the biochemical reactions and ionic currents giving rise to the receptor potential, and the cellular organization and compartmentalization of the organ represented by an electrical circuit. The number of isopotential compartments needed to describe the long dendrite bearing pheromone receptors was determined. The transduction parameters that must be modified when the number of compartments is increased were identified. This model reproduces the amplitude and time course of the experimentally recorded receptor potential. A first complete version of the model was analyzed in response to pheromone pulses of various strengths. It provided a quantitative description of the spatial and temporal evolution of the pheromone-dependent conductances, currents and potentials along the outer dendrite and served to determine the contribution of the various steps in the cascade to its global sensitivity. A second simplified version of the model, utilizing a single depolarizing conductance and leak conductances for repolarizing the ORN, was derived from the first version. It served to analyze the effects on the sensory properties of varying the electrical parameters and the size of the main sensillum parts. The consequences of the results obtained on the still uncertain mechanisms of olfactory transduction in moth ORNs – involvement or not of G-proteins, role of chloride and potassium currents – are discussed as well as the optimality of the sensillum organization, the dependence of biochemical parameters on the neuron spatial extension and the respective contributions of the biochemical and electrical parameters to the overall neuron response.
An essential part of sexual reproduction typically involves the
identification of an appropriate mating partner. Males of many moth species
utilize the scent of sex pheromones to track and locate conspecific females.
However, before males engage in flight, warm-up by shivering of the major flight
muscles is necessary to reach a thoracic temperature suitable to sustain flight.
Here we show that Helicoverpa zea males exposed to an
attractive pheromone blend (and in some instances to the primary pheromone
component alone) started shivering earlier and took off at a lower thoracic
temperature than moths subjected to other incomplete or unattractive blends.
This resulted in less time spent shivering and faster heating rates. Two
interesting results emerge from these experiments. First, the rate of heat
generation can be modulated by different olfactory cues. Second, males detecting
the pheromone blend take off at lower thoracic temperatures than males exposed
to other stimuli. The take-off temperature of these males was below that for
optimal power production in the flight muscles, thus generating a trade-off
between rapid departure and suboptimal flight performance. Our results shed
light on thermoregulatory behaviour of unrestrained moths associated with the
scramble competition for access to females and suggest ecological trade-offs
between rapid flight initiation and sub-optimal flight performance.
thermoregulation; shivering; flight; sex pheromone; Noctuidae; insect
Remarkably little is known about the molecular and cellular basis of mate recognition in Drosophila . We systematically examine one of the three major types of sensilla that house olfactory receptor neurons (ORNs) on the Drosophila antenna, the trichoid sensilla, by electrophysiological analysis. We find that none respond strongly to food odors, but all respond to fly odors. Two subtypes of trichoid sensilla contain ORNs that respond to cis-vaccenyl acetate (cVA), an anti-aphrodisiac pheromone present in males and transferred to females during mating [2–4]. All trichoid sensilla yield responses to a male extract; a subset yield responses to a virgin female extract as well. Thus males can be distinguished from virgin females by the activity they elicit among the trichoid ORN population. We then systematically test all members of the Odor receptor (Or) gene family [5–7] that are expressed in trichoid sensilla , using an in vivo expression system . Four receptors respond to fly odors in this system: two respond to extracts of both males and virgin females, and two respond to cVA. We propose a model for how these receptors might be used by a male to distinguish suitable from unsuitable mating partners through a simple logic.
Most animals including insects rely on olfaction to find their mating partners. In moths, males are attracted by female-produced sex pheromones inducing stereotyped sexual behavior. The behaviorally relevant olfactory information is processed in the primary olfactory centre, the antennal lobe (AL). Evidence is now accumulating that modulation of sex-linked behavioral output occurs through neuronal plasticity via the action of hormones and/or catecholamines. A G-protein-coupled receptor (GPCR) binding to 20-hydroxyecdysone, the main insect steroid hormone, and dopamine, has been identified in Drosophila (DmDopEcR), and was suggested to modulate neuronal signaling. In the male moth Agrotis ipsilon, the behavioral and central nervous responses to pheromone are age-dependent. To further unveil the mechanisms of this olfactory plasticity, we searched for DopEcR and tested its potential role in the behavioral response to sex pheromone in A. ipsilon males. Our results show that A. ipsilon DopEcR (named AipsDopEcR) is predominantly expressed in the nervous system. The corresponding protein was detected immunohistochemically in the ALs and higher brain centers including the mushroom bodies. Moreover, AipsDopEcR expression increased with age. Using a strategy of RNA interference, we also show that silencing of AipsDopEcR inhibited the behavioral response to sex pheromone in wind tunnel experiments. Altogether our results indicate that this GPCR is involved in the expression of sexual behavior in the male moth, probably by modulating the central nervous processing of sex pheromone through the action of one or both of its ligands.