Discrimination of sex pheromone blends in the olfactory system of the moth

Intracellular analysis of olfactory neurons in the internallobes of several speicies of months has revealed a psysiologisallydivorse population of projection neurons connecting the pheromone-processingcenter (the male-specific macroglormerular complex) with severalarea of the protocerebrum. Some of these output elements carryinformation about only a single pheromone in the female's complexblend, which is consistent with the ‘component hypothesis’of behavioral excitation. Other output neurons, which receivemore complex synaptic input, can distinguish the complete blendfrom the individual pheromones, thereby lending support to the‘blend hypothesis’ of behavioral excitation. Theresults suggest that even within the pheromone-processing subsystermin male insects, which is largely distinct from the rest ofthe olfactory system, there exist different but parallel linesof pheromonal information coding that ultimately govern thesteteotyped mate-seeking behaviors.     

Unusual pheromone receptor neuron responses in heliothine moth antennae derived from inter-species imaginal disc transplantation

Single-cell electrophysiological recordings were obtained from olfactory receptor neurons housed in sensilla trichodea along the adult antennae arising from transplantation of the antennal imaginal discs between larval male Helicoverpa zea and Heliothis virescens. The olfactory receptor neurons from the majority of type C sensilla sampled on transplanted antennae displayed response characteristics consistent with those of the species that donated the antennae. However, some of the sensilla type C sampled in either transplant type contained olfactory receptor neurons that responded in a manner typical of the recipient species or other neurons that have not previously been found in the type C sensilla of either species. The single-cell data help to explain behavioral results showing that some transplant males do fly upwind to both species' pheromone blends, an outcome not expected based on known antennal sensory phenotypes. Our results suggest that host tissue can influence antennal olfactory receptor neuron development, and further that because of a common phylogenetic ancestry the donor tissue has the genetic capability to produce a variety of sensillar and receptor types.     

A general odorant background affects the coding of pheromone stimulus intermittency in specialist olfactory receptor neurones

In nature the aerial trace of pheromone used by male moths to find a female appears as a train of discontinuous pulses separated by gaps among a complex odorant background constituted of plant volatiles. We investigated the effect of such background odor on behavior and coding of temporal parameters of pheromone pulse trains in the pheromone olfactory receptor neurons of Spodoptera littoralis. Effects of linalool background were tested by measuring walking behavior towards a source of pheromone. While velocity and orientation index did drop when linalool was turned on, both parameters recovered back to pre-background values after 40 s with linalool still present. Photo-ionization detector was used to characterize pulse delivery by our stimulator. The photo-ionization detector signal reached 71% of maximum amplitude at 50 ms pulses and followed the stimulus period at repetition rates up to 10 pulses/s. However, at high pulse rates the concentration of the odorant did not return to base level during inter-pulse intervals. Linalool decreased the intensity and shortened the response of receptor neurons to pulses. High contrast (>10 dB) in firing rate between pulses and inter-pulse intervals was observed for 1 and 4 pulses/s, both with and without background. Significantly more neurons followed the 4 pulses/s pattern when delivered over linalool; at the same time the information content was preserved almost to the control values. Rapid recovery of behavior shows that change of perceived intensity is more important than absolute stimulus intensity. While decreasing the response intensity, background odor preserved the temporal parameters of the specific signal.     

Altered olfactory receptor neuron responsiveness in rare Ostrinia nubilalis males attracted to the O. furnacalis pheromone blend

Three percent of E-strain Ostrinia nubilalis males fly upwind in response to the Ostrinia furnacalis pheromone blend [a 40:60 ratio of (E)-12-tetradecenyl acetate to (Z)-12-tetradecenyl acetate (E12-14:OAc to Z12-14:OAc)], in addition to their own pheromone blend [a 99:1 ratio of (E)-11-tetradecenyl acetate to (Z)-11-tetradecenyl acetate) (E11-14:OAc to Z11-14:OAc)]. We assessed the olfactory receptor neuron (ORN) responses of these behaviorally "rare" males versus those of normal males. For the three ORNs housed within each sensillum, we tested responsiveness to Z12-14:OAc, E12-14:OAc, Z11-14:OAc, E11-14:OAc, and the behavioral antagonist (Z)-9-tetradecenyl acetate (Z9-14:OAc). Z11-14:OAc, E11-14:OAc, and Z9-14:OAc stimulated ORNs exhibiting distinct small, large, and medium spike sizes, respectively. For rare and normal males, both Z12-14:OAc and E12-14:OAc usually elicited responses from the largest-spiking ORN. In many ORNs of normal males, Z12-14:OAc or E12-14:OAc stimulated the smaller-spiking ORN that is responsive to Z11-14:OAc. In rare males, detectable ORN responses from the smaller-spiking ORN in response to Z12- and E12-14:OAc were virtually non-existent. These differences in ORN tuning in rare males will tend to create an ORN firing ratio between the large- and small-spiking ORNs in response to the O. furnacalis blend that is similar to that elicited by the O. nubilalis blend.     

Differential interactions of sex pheromone and plant odour in the olfactory pathway of a male moth

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.     

Guanosine 3',5'-cyclic monophosphate reduces the response of the Moth's olfactory receptor neuron to pheromone

The effects of the membrane-permeable dibutyryl guanosine 3', 5'-cyclic monophosphate (db-cGMP) on the bombykol-elicited receptor current and nerve impulse activity were studied using the open sensillum recording technique. db-cGMP was applied to the outer dendritic membrane of the olfactory receptor neuron of the moth Bombyx mori. db-cGMP reduced the amplitude of the overall receptor current activated by a pulse of strong pheromone stimuli as well as diminished the nerve impulse frequency elicited by continuously applied weak pheromone stimuli. The observed inhibition of the response to pheromone was due to size reduction of an elementary receptor current that elicits the nerve impulses and underlies the overall receptor current. It is suggested that cGMP is a factor which may adjust cell sensitivity to odour and play a role in olfactory adaptation.     

Frequency coding by central olfactory neurons in the sphinx moth Manduca sexta

Sexually receptive female moths and many other insects releasechemical attractants (sex pheromones) to lure conspecific mates.Recent evidence indicates, moreover, that the odor plume formeddownwind from the female possesses a discontinuous structurethat appears to provide the searching male with orientationcues.Using intracellular methods, we find that many central olfactoryneurons in male moths (Manduca sexta) can track pulsed pheromonalstimuli precisely. The cells respond to each brief odor pulsewith a similarly brief burst of action potentials, and the separationbetween response bursts is aided by inhibitory synaptic input.Furthermore, these neurons appear to participate in at leasttwo levels of ‘feature detection’: they respondselectively to pheromonal stimuli, and they follow pulsed stimulationonly in a limited range of frequencies Above the frequency limit,the cells respond as if the male is stimulated by a prolonged,uniform concentration of pheromone. The ability of these neuronsto encode changes in the temporal characteristics of pheromonalstimuli may provide the male with positional cues to help himlocate the pheromone source over long distances.     

Temporal coding in the auditory receptor of the moth ear

Temporal coding in the moth ear was inferred from the response of the auditory receptor to acoustic stimuli with different temporal characteristics.

Androgen increases olfactory receptor response to a vertebrate sex pheromone

Although it is well known that responses to ethologically-relevant odors are influenced by endocrine factors, it has not been clear whether these hormonal effects might be mediated at the level of the peripheral sensory neurons. During an investigation of hormonal pheromones in South-East Asian Cyprinids, we observed that in adult male Puntius schwanenfeldi, an androgen-dependent sex character was correlated with electro-olfactogram response to a putative sex pheromone (15-keto-prostaglandin-F₂ ). As secondary sex characteristics are androgen-dependent in male teleosts, this observation suggested a functional relationship between androgen and peripheral olfactory receptor response. We therefore investigated this possibility using androgen implants.In laboratory-raised juveniles, androgen treatment increased the magnitude and sensitivity of electro-olfactogram response to prostaglandin without affecting responses to other odors. Furthermore, androgen-treated juveniles performed pheromone-dependent sex behavior in the presence of a prostaglandin-injected stimulus fish. For the first time in vertebrates, the present data demonstrate hormone-induced plasticity of primary chemosensory neuronal responsiveness to an ethologically relevant compound.Abbreviations EOG electro-olfactogram - PGF Prostaglandin-F₂ - 15KPGF 15-keto-prostaglandin-F₂ - 17,2 1P 17,21 -dihydroxy-4-pregnen-3-one - MT 17-methyltestosterone - 11KA 11-ketoandrostenedione - 11KT 11-ketotestosterone - DHT 5-dihydrotestosterone     

Floral to green: mating switches moth olfactory coding and preference

Mating induces profound physiological changes in a wide range of insects, leading to behavioural adjustments to match the internal state of the animal. Here, we show for the first time, to our knowledge, that a noctuid moth switches its olfactory response from food to egg-laying cues following mating. Unmated females of the cotton leafworm (Spodoptera littoralis) are strongly attracted to lilac flowers (Syringa vulgaris). After mating, attraction to floral odour is abolished and the females fly instead to green-leaf odour of the larval host plant cotton, Gossypium hirsutum. This behavioural switch is owing to a marked change in the olfactory representation of floral and green odours in the primary olfactory centre, the antennal lobe (AL). Calcium imaging, using authentic and synthetic odours, shows that the ensemble of AL glomeruli dedicated to either lilac or cotton odour is selectively up- and downregulated in response to mating. A clear-cut behavioural modulation as a function of mating is a useful substrate for studies of the neural mechanisms underlying behavioural decisions. Modulation of odour-driven behaviour through concerted regulation of odour maps contributes to our understanding of state-dependent choice and host shifts in insect herbivores.     

Immunolocalization of a candidate pheromone receptor in the antenna of the male moth, Heliothis virescens

Pheromone recognition in insects is thought to involve distinct receptor proteins in the dendritic membrane of antennal sensory neurons. We have generated antibodies directed against a peptide derived from the sequence of the candidate pheromone receptor HR13 from Heliothis virescens. The antibodies specifically labelled the cell bodies of a distinct neuron population housed in male-specific pheromone-sensitive sensilla. Combining antibody staining with in situ hybridization the reactive cells were found to express the HR13 gene. In addition, dendrites projecting into sensilla hairs as well as the axonal processes of immunoreactive cells were labelled. Labelling of axons has allowed visualization of their fasciculation within antennal segments and permits tracking of axons as they merge into the antennal nerve. The HR13 protein was first detected 1 day before eclosion. Thus, the distribution of HR13 protein in the antennal neurons of the male moth strongly suggests a role of the HR13 receptor in recognition of pheromones.     

Olfactory receptor responses to sex pheromone components in the redbanded leafroller moth

Electrical responses of single olfactory receptor neurons of the male redbanded leafroller moth were elicited by each of the principle components of the sex pheromone and six other behaviorally active compounds. Response frequencies to equal intensities of each of these compounds and changes in response frequency with increasing amounts of any one compound, varied from receptor to receptor. These differences in response characteristics appear to be due to factors intrinsic to the olfactory recptor neuron and not to factors external to it. The encoding of odor quality by these receptor neurons cannot be in the simple presence or absence of activity in any one of them. Rather, odor quality may be encoded by the pattern of activity which invariably arises across an ensemble of receptor neurons, each having its own distribution of sensitivities.     

Stage-specific induction of DNA methyltransferases in olfactory receptor neuron development

DNA methylation-dependent gene silencing, mediated by DNA methyltransferases (DNMTs), is essential for normal mammalian development and its dysregulation has been implicated in neurodevelopmental disorders. Despite this, little is known about DNMTs in the developing or mature nervous system. Here, we show that DNMT1, 3a and 3b are expressed at discrete developmental stages in the olfactory neuron lineage, coincident with key shifts in developmental gene expression. DNMT1 is induced in cycling progenitors and is retained in post-mitotic olfactory receptor neurons (ORNs). DNMT3b is restricted to mitotic olfactory progenitors, whereas DNMT3a is expressed only in post-mitotic immature neurons prior to ORN terminal maturation, coincident with histone deacetylase 2 (HDAC2), a key downstream effector of methylation-dependent chromatin condensation. Similar stage-specific expression of DNMT3b and 3a was also found in other developing sensory and CNS neurons. This suggests that progressive lineage restriction regulated by methylation-dependent silencing could be a highly conserved mechanism shared by multiple lineages in the developing nervous system.     

Coding of odor intensity in a steady-state deterministic model of an olfactory receptor neuron

The coding of odor intensity by an olfactory receptor neuron model was studied under steady-state stimulation. Our model neuron is an elongated cylinder consisting of the following three components: a sensory dendritic region bearing odorant receptors, a passive region consisting of proximal dendrite and cell body, and an axon. First, analytical solutions are given for the three main physiological responses: (1) odorant-dependent conductance change at the sensory dendrite based on the Michaelis-Menten model, (2) generation and spreading of the receptor potential based on a new solution of the cable equation, and (3) firing frequency based on a Lapicque model. Second, the magnitudes of these responses are analyzed as a function of odorant concentration. Their dependence on chemical, electrical, and geometrical parameters is examined. The only evident gain in magnitude results from the activation-to-conductance conversion. An optimal encoder neuron is presented that suggests that increasing the length of the sensory dendrite beyond about 0.3 space constant does not increase the magnitude of the receptor potential. Third, the sensivities of the responses are examined as functions of (1) the concentration at half-maximum response, (2) the lower and upper concentrations actually discriminated, and (3) the width of the dynamic range. The overall gain in sensitivity results entirely from the conductance-to-voltage conversion. The maximum conductance at the sensory dendrite appears to be the main tuning constant of the neuron because it determines the shift toward low concentrations and the increase in dynamic range. The dynamic range of the model cannot exceed 5.7 log units, for a sensitivity increase at low odor concentration is compensated by a sensitivity decrease at high odor concentration.     

Inhibitors of sensillar esterase reversibly block the responses of moth pheromone receptor cells

Three volatile alkyl-thio-trifluoro propanones inhibiting the esterase in olfactory sensilla of the silkmoths Antheraea polyphemus and A. pernyi were used to test the hypothesis that enzymatic pheromone degradation is responsible for the decline of the receptor potential after pheromone stimulation. Test stimuli were the pheromone components (E,Z)-6,11-hexadecadienyl acetate, a substrate for the sensillar esterase, and (E,Z)-6,11-hexadecadienal, not degraded by the esterase. Each compound acts on a separate type of receptor cell. In both receptor cell types the trifluoro propanones caused a partially reversible reduction of sensitivity as indicated by smaller receptor potential amplitudes and lower nerve impulse frequencies. Since application of the esterase inhibitors did not prolong the decline of the receptor potential of the acetate cell, the esterase is not responsible for the rapid pheromone deactivation. When the trifluoro propanones were applied after the pheromone at high concentrations, they rapidly inhibited (repolarized) both receptor cell types. Experiments with local application of trifluoro propanones revealed that the inhibitory effect spreads within seconds along the length of the sensillum. The inhibition of the electrophysiological responses might be due to an antagonistic action of the trifluoro propanones at the pheromone-binding sites, either at the receptor molecules or at the pheromone-binding protein. Accepted: 4 February 1998     

Opposing effects of bone morphogenetic proteins on neuron production and survival in the olfactory receptor neuron lineage

In olfactory epithelium (OE) cultures, bone morphogenetic proteins (BMPs) can strongly inhibit neurogenesis. Here we provide evidence that BMPs also promote, and indeed are required, for OE neurogenesis. Addition of the BMP antagonist noggin inhibited neurogenesis in OE-stromal cell co-cultures. Bmp2, Bmp4 and Bmp7 were expressed by OE stroma, and low concentrations of BMP4 (below the threshold for inhibition of neurogenesis) stimulated neurogenesis; BMP7 did not exhibit a stimulatory effect at any concentration tested. Stromal cell conditioned medium also stimulated neurogenesis; part of this effect was due to the presence within it of a noggin-binding factor or factors. Studies of the pro-neurogenic effect of BMP4 indicated that it did not increase progenitor cell proliferation, but rather promoted survival of newly generated olfactory receptor neurons. These findings indicate that BMPs exert both positive and negative effects on neurogenesis, depending on ligand identity, ligand concentration and the particular cell in the lineage that is responding. In addition, they reveal the presence of a factor or factors, produced by OE stroma, that can synergize with BMP4 to stimulate OE neurogenesis.