Plant volatiles activating specific olfactory receptor neurons of the cabbage moth Mamestra brassicae L. (Lepidoptera, Noctuidae).

Herbivore insects are suitable model organisms for studying how plant odor information is encoded in olfactory receptor neurons (RNs). By the use of gas chromatography linked to electrophysiological recordings from single RNs, screening for sensitivity to naturally produced plant odorants is possible in order to determine the molecular receptive ranges of the neurons. Using this method, we have in this study of the cabbage moth, Mamestra brassicae, classified 21 types of olfactory RNs according to their responses to odorants present in the host plants of Brassicae, in the related species of Arabidopsis, as well as in essential oils of nonhost plants like ylang-ylang. Most of the RNs were tuned to one or a few structurally similar compounds, showing minimal overlap of their molecular receptive ranges. Whereas some RNs displayed a novel tuning, others were tuned to the same compounds as neurons in other insect species. We also found colocation in the same sensillum of 3 RN types with the same response characteristics and tuning as 3 colocated types described in heliothine moths living on different host plants. The presence of similar RN types across different insect species implies conservation or reappearance of the RN types, independent of the evolution of host plant ranges.

Methyl salicylate, identified as primary odorant of a specific receptor neuron type, inhibits oviposition by the moth Mamestra brassicae L. (Lepidoptera, noctuidae).

The cabbage moth, Mamestra brassicae L. (Lepidoptera, Noctuidae), is a polyphagous species that is often choosing plants of Brassica as hosts for oviposition. In the search for biologically relevant odorants used by these moths, gas chromatography linked to electrophysiological recordings from single receptor neurons (RNs) has been employed, resulting in classification of distinct types of neurons. This study presents specific olfactory RNs responding to methyl salicylate (MeS) as primary odorant and showing a weak response to methyl benzoate, the 2 aromatic compounds occurring together in several plant species. In 2 cases, the neuron was colocated with another RN type responding to 6 green leaf volatiles: 1-hexanol, (3Z)-hexen-1-ol, (2E)-hexen-1-ol, (3Z)-hexenyl acetate, (2Z)-hexen-1-ol, and an unidentified compound. Whereas the specific RNs detected the minor amounts of MeS in some plants, the compound was not found by gas chromatography linked to mass spectrometry in intact plants, but it was found after herbivore attack. The behavioral effect of MeS was studied in outdoor test arenas with Brassica napus and artificial plants. These experiments indicated that mated M. brassicae females avoid plants with dispensers emitting MeS. As it is induced by caterpillar feeding, this compound may mediate a message to mated M. brassicae females that the plant is already occupied.

Discrimination between enantiomers of linalool by olfactory receptor neurons in the cabbage moth Mamestra brassicae (L.).

Plants emit complex blends of volatiles, including chiral compounds that might be detected by vertebrates and invertebrates. Insects are ideal model organisms for studying the underlying receptor neuron mechanisms involved in olfactory discrimination of enantiomers. In the present study, we have employed two-column gas chromatography linked to recordings from single olfactory receptor neurons of Mamestra brassicae, in which separation of volatiles in a polar and a chiral column was performed. We here present the response properties of olfactory receptor neurons tuned to linalool. The narrow tuning of these receptor neurons was demonstrated by their strong responses to (R)-(-)-linalool, the weaker responses to the (+)-enantiomer as well as a few structurally related compounds, and no responses to the other numerous plant released volatiles. The enantioselectivity was verified by parallel dose-response curves, that of (R)-(-)-linalool shifted 1 log unit to the left of the (S)-(+)-linalool curve. A complete overlap of the temporal response pattern was found when comparing the responses of the same strength. Analysis of the spike amplitude and waveform indicated that the responses to the two enantiomers originated from the same neuron.

Olfactory receptor neurons in two Heliothine moth species responding selectively to aliphatic green leaf volatiles, aromatic compounds, monoterpenes and sesquiterpenes of plant origin.

Moths of the subfamily Heliothinae are suitable models for comparative studies of plant odour information encoded by the olfactory system. Here we identify and functionally classify types of olfactory receptor neurons by means of electrophysiological recordings from single receptor neurons linked to gas chromatography and to mass spectrometry. The molecular receptive ranges of 14 types in the two polyphagous species Heliothis virescens and Helicoverpa armigera are presented. The receptor neurons are characterized by a narrow tuning, showing the best response to one primary odorant and weak responses to a few chemically related compounds. The most frequently occurring of the 14 types constituted the receptor neurons tuned to (+)-linalool, the enantioselectivity of which was shown by testing two samples with opposite enantiomeric ratios. These neurons, also responding to dihydrolinalool, were found to be functionally similar in the two related species. The primary odorants for 10 other receptor neuron types were identified as (3Z)-hexenyl acetate, (+)-3-carene, trans-pinocarveol, trans-verbenol, vinylbenzaldehyde, 2-phenylethanol, methyl benzoate, alpha-caryophyllene and caryophyllene oxide, respectively. Most odorants were present in several host and non-host plant species, often in trace amounts. The specificity as well as the co-localization of particular neuron types so far recorded in both species showed similarities of the olfactory systems receiving plant odour information in these two species of heliothine moths.

Associative learning of plant odorants activating the same or different receptor neurones in the moth Heliothis virescens.

The importance of olfactory learning in host plant selection is well demonstrated in insects, including the heliothine moths. In the present study olfactory conditioning of the proboscis extension response was performed to determine the moths' ability to learn and discriminate three plant odorants: beta-ocimene and beta-myrcene (activating the same receptor neurone type), and racemic linalool (activating two different types). The conditioned stimulus (CS) was an air puff with each odorant blown into a constant air stream and over the antennae, and the unconditioned stimulus (US) was sucrose solution applied first to the antennal taste sensilla, then to the proboscis. Conditioning with increasing odorant concentrations induced increased learning performance. The concentration threshold for learning was 100 times lower for racemic linalool than for the two other odorants, a fact that can be correlated with a higher sensitivity of the moths' antennae to racemic linalool as shown in electroantennogram recordings. After correcting for the different odour sensitivities, the moths' ability to discriminate the odorants was studied. Differential conditioning experiments were carried out, in which moths had to distinguish between a rewarded (CS+) odorant and an explicitly unrewarded odorant (CS-), choosing odour concentrations giving the same learning rate in previous experiments. The best discrimination was found with beta-myrcene as the rewarded odorant and racemic linalool as the unrewarded. The opposite combination gave lower discrimination, indicating a higher salience for beta-myrcene than for racemic linalool. The moths could also discriminate between beta-ocimene and beta-myrcene, which was surprising, since they activate the same receptor neurone type. No difference in salience was found between these two odorants.

Representation of primary plant odorants in the antennal lobe of the moth Heliothis virescens using calcium imaging.

The primary olfactory centre, the antennal lobe of Heliothis virescens moths, contains 62 glomeruli which process plant odour information and four male-specific glomeruli which form the macroglomerular complex, involved in processing information about pheromone and interspecific signals. Using calcium imaging, we recorded the spatio-temporal activity pattern of the glomeruli in the anterior antennal lobe during stimulation with odorants produced by plants or insects. Each odorant elicited specific excitatory responses in one or a few glomeruli: the major pheromone component did so exclusively in the large glomerulus of the macroglomerular complex and the plant odours exclusively in the ordinary glomeruli. Eight glomeruli, with corresponding plant odour responses and positions, were identified within each sex. Glomeruli responded specifically to linalool, beta-ocimene/beta-myrcene or germacrene D/alpha-farnesene. Responses to two essential plant oils covered the response areas of their major constituents, as well as activating additional glomeruli. Stronger activation in the AL due to increased odour concentration was expressed as increased response strength within the odorant-specific glomeruli as well as recruitment of less sensitive glomeruli.

(-)-Germacrene D receptor neurones in three species of heliothine moths: structure-activity relationships.

Specificity of olfactory receptor neurones plays an important role in food and host preferences of a species, and may have become conserved or changed in the evolution of polyphagy and oligophagy. We have identified a major type of plant odour receptor neurones responding to the sesquiterpene germacrene D in three species of heliothine moths, the polyphagous Heliothis virescens and Helicoverpa armigera and the oligophagous Helicoverpa assulta. The neurones respond with high sensitivity and selectivity to (-)-germacrene D, as demonstrated by screening via gas chromatography with numerous mixtures of plant volatiles. Germacrene D was present in both host and non-host plants, but only in half of the tested species. The specificity of the neurones was similar in the three species, as shown by the "secondary" responses to a few other sesquiterpenes. The effect of (-)-germacrene D was about ten times stronger than that of the (+)-enantiomer, which again was about ten times stronger than that of (-)-alpha-ylangene. Weaker effects were obtained for (+)-beta-ylangene, (+)-alpha-copaene, beta-copaene and two unidentified sesquiterpenes. The structure-activity relationship shows that the important properties of (-)-germacrene D in activating the neurones are the ten-membered ring system and the three double bonds acting as electron-rich centres, in addition to the direction of the isopropyl-group responsible for the different effects of the germacrene D enantiomers.

Identification of plant odours activating receptor neurones in the weevil Pissodes notatus F. (Coleoptera, Curculionidae).

Plants release complex mixtures of volatiles important in the interaction with insects and other organisms. In the search for compounds that contribute to the perception of odour quality in the weevil Pissodes notatus, single olfactory receptor neurones on the antennae were screened for sensitivity to naturally produced plant volatiles by the use of gas chromatography linked to single cell recordings. We here present 60 olfactory neurones responding to 25 of the numerous compounds released by host and non-host plants. All the neurones show high selectivity and are classified into 12 distinct types. The two most abundant types respond to alpha-pinene, beta-pinene, and 3-carene ( n=17), and to isopinocamphone and pinocamphone ( n=17), respectively. Other neurone types respond to limonene ( n=9), beta-phellandrene ( n=3), and fenchone ( n=4). Responses to beta-caryophyllene ( n=1) and to ethanol ( n=4) are also shown. Except for two pairs, the neurone types do not show overlap of the molecular receptive range. The active compounds are present in the host, Pinus pinaster, as well as in non-hosts, supporting the idea that plant odour quality is mediated by the ratio of the compounds rather than specific odorants.

(-)-Germacrene D increases attraction and oviposition by the tobacco budworm moth Heliothis virescens.

The sesquiterpene germacrene D (GD) activates a major type of olfactory receptor neuron on the antennae of the heliothine moths. In Heliothis virescens females, 80% of the recordings have shown activity of one neuron type responding with high sensitivity and selectivity to GD. With the aim of determining the behavioural significance of this sesquiterpene, we have used a two-choice wind-tunnel to study the preference of mated H. virescens females for host plants with and without (-)-GD added. Tobacco plants containing dispensers with low release rate of (-)-GD had a greater attractiveness than tobacco plants without this substance. In addition, a significant increase of oviposition was found on the plants with (-)-GD.

Receptor neuron discrimination of the germacrene D enantiomers in the moth Helicoverpa armigera.

Plants release complex mixtures of volatiles, including chiral constituents. In the search for the biologically relevant plant odorants, gas chromatography linked to electrophysiological recordings from single receptor neurons has been employed. In heliothine moths, including the females of the Eurasian cotton bollworm moth Helicoverpa armigera, a major type of receptor neurons is identified, showing high sensitivity and selectivity for the sesquiterpene germacrene D. In the present study, gas chromatography with a chiral column linked to single cell recordings were performed. It was found that all germacrene D neurons belonged to one type; all responded to both enantiomers, but (-)-germacrene D had approximately 10 times stronger effect than (+)-germacrene D. Parallel dose-response curves for the two enantiomers were obtained by direct stimulations. The enantiomeric composition of germacrene D, which differed in six plant species and in different individuals of one species, was determined on the basis of the neuron responses. The results, showing the presence of one neuron type for receiving the information about germacrene D in the various plants, suggests that the two enantiomers mediate the same kind of information to the moth, but with different intensity.

Selective receptor neurone responses to E-beta-ocimene, beta-myrcene, E,E-alpha-farnesene and homo-farnesene in the moth Heliothis virescens, identified by gas chromatography linked to electrophysiology.

An important question in olfaction is for which odorants receptor neurones have evolved. In the present study, olfactory receptor neurones on the antennae of the tobacco budworm moth Heliothis virescens were screened for sensitivity to naturally occurring plant-produced volatiles by the use of gas chromatography linked to electrophysiology. Volatiles of host as well as non-host plants collected by headspace techniques were used for stimulating the neurones, sequentially via two columns, one polar and one nonpolar installed in parallel in the gas chromatograph. Three types of neurones presented in this paper responded to one, two or three compounds for which the retention times were determined in both column types. The chemical structures of the active components were determined on the basis of mass spectrometry linked to gas chromatography, indicating E-beta-ocimene and beta-myrcene as stimulants for neurone type 1, E,E-alpha-farnesene for neurone type 2 and homo-farnesene for neurone type 3. Re-testing authentic materials verified the identifications for the type 1 neurones. The results demonstrate a high specificity for the three types of neurones by strong responses to one or two structurally similar compounds out of hundreds present in a large variety of plants. The study exemplifies plant odour detection by narrowly tuned receptor neurones in a polyphagous moth species.

The plant sesquiterpene germacrene D specifically activates a major type of antennal receptor neuron of the tobacco budworm moth Heliothis virescens.

Plants release hundreds of volatiles that are important in interactions with insects or other organisms. However, knowledge is scarce as to which of the compounds are detected by the organism's olfactory receptor neurons. In the present study, single receptor neurons on the antennae of the tobacco budworm moth, Heliothis virescens, were screened for their sensitivities to naturally produced plant volatiles by the use of gas chromatography linked to electrophysiological recordings from single cells (GC-SCR). Plant volatiles, collected by aeration of host and non-host plants, were tested on each receptor neuron via parallel GC-columns. Thus, simultaneous recordings of the gas chromatogram and the neuron responses to each component were obtained. One type of receptor neuron, appearing in 80% of all experiments, responded with high sensitivity and selectivity to one particular component, present in host as well as non-host mixtures. The component, identified as a sesquiterpene hydrocarbon by linked gas chromatography-mass spectrometry, was isolated from a sesquiterpene fraction of cubebe oil and identified by NMR as germacrene D. The purified compound was then re-tested via gas chromatography on the same receptor neuron type, verifying the identification. A weaker response to another sesquiterpene hydrocarbon was also recorded.

Calcium responses to pheromones and plant odours in the antennal lobe of the male and female moth Heliothis virescens.

In male moths, the primary olfactory integration centre, the antennal lobe, consists of two systems. The macroglomerular complex processes pheromone information, while the ordinary glomeruli process plant odour information. Females lack a macroglomerular complex. We measured the spatial representation of odours using in-vivo optical recording. We found that: (1) pheromone substances elicited activity exclusively in the MGC. No response was found in female antennal lobes. (2) Plant odours elicited combinatorial activity patterns in the ordinary glomeruli in both males and females. No response was found in the MGC of male moths. (3) A clean air puff often led to activity, in both males and females, suggesting that mechano-sensory information is also processed in the antennal lobe. (4) With an interstimulus interval of 5 or 10 s, strongly activated glomeruli were able to follow the temporal structure of the stimulus, while others lost their phase-locking. Some glomeruli showed "off" responses. These properties were odour dependent. This confirms and extends previous studies, showing the functional significance of the two subsystems for processing olfactory information. Pheromones are coded in a combinatorial manner within the macroglomerular complex, with each glomerulus corresponding to one information channel. Plant odours are coded in an across-glomeruli code in the ordinary glomeruli.

Central mechanisms of pheromone information processing.

An advantage of using pheromones in olfactory studies is that they are chemical signals for which receptor neurons are evolved and thus elicit biologically relevant odour-information to be processed in the brain. In many vertebrate and insect species, the olfactory system is separated into a 'main' and an 'accessory' division, the latter mediating pheromone information. In moths, the pheromone information is first processed in the brain in a large and sexually dimorphic structure, the macroglomerular complex (MGC) of the antennal lobe (AL). Also in vertebrates the pheromone information is processed in specific or modified glomerular complexes. One principle question is whether individual olfactory glomeruli are functional units, processing specific information concerning both the chemical quality and spatiotemporal features of the stimulus, like the pheromone plume. Indeed it has been shown that the axons of different pheromone-selective receptor neurons project into different MGC-glomeruli. Intracellular recordings from the AL projection (output) neurons also show that information about single components of the pheromone blend is preserved in some output pathways, whereas other output neurons respond in a unique fashion to the blend. The information about interspecific signals, which interrupts pheromone attraction, is processed in a specific MGC-glomerulus and is to a large extent kept separate from the pheromone information throughout the AL. Many of the output neurons accurately encode changes in the temporal characteristics of the stimulus.

Heliothis virescens: Response characteristics of receptor neurons in Sensilla Trichodea type 1 and type 2.

Partial electroantennograms (EAGs) and single cell recordings fromHeliothis virescens males have demonstrated the presence of pheromones receptor neurons in sensilla trichodea type 2 as well as in type 1. This is supported by cobalt tracing experiments, showing that primary axons from the distal flagellum, containing only s. trichodea type 2, project into the macrogiomerulus complex in the male antennal lobes. Four types of finely tuned pheromone receptor neurons were found in males, whereas in females the corresponding neurons responded mainly to host odors. In males the majority (75 and 18%, respectively) were tuned to the majorHeliothis virescens pheromone components (Z)-11-hexadecenal (Z11-16∶A1) and (Z)-9-tetradecenal (Z9-14∶A1). The others (5 and 2%, respectively) responded specifically to (Z)-1 1-hexadecen-1-ol (Z1 1-16∶OH) and (Z)-1 1-hexadecen-1-ol acetate (Z1 1-16∶Ac). No neurons responding selectively to the minor pheromone components were found. The Z11-16∶A1 neurons of both sensilla types possessed similar specificity. However, the sensitivity decreased toward the medial and distal part of the flagellum, where s. trichodea type 2 are located. This suggests that the pheromone concentrations can be detected peripherally by a spatial as well as a temporal mechanism. Differences in temporal response patterns (pronounced phasic vs. tonic component) were found within the same type of neurons, suggesting different ability to encode intermittency of the pheromone plume as well as to mediate maintenance of flight.

Pheromone reception in tobacco budworm moth,heliothis virescens.

Eleclrophysiological recordings from single olfactory receptor cells were carried out in the male tobacco budworm moth,Heliothis virescens. Recordings were made primarily from the sensilla trichodea type 1, which are located in the characteristic circumferential rows on the antennae. They possess the longest sensilla hairs as revealed by scanning electron microscopy (SEM). The sensory cells of these sensilla responded specifically to pheromones. Only three types of receptor neurons were found, each tuned to one of the female-produced components. The majority (58%) of the neurons were tuned to the major component (Z)-11-hexadecenal (Z11-16:A1). Another large group (27%) responded specifically to stimulation with (Z)-9-tetradecenal (Z9-14: Al). These two compounds are the most important components of the pheromones as judged by their influence on the behavioral responses of the males. The third type of neurons responded specifically to (Z)-11-hexadecen-1-ol (Z11-16: OH), which may act either as a pheromone component or as an interspecific cue. None of the receptor neurons in the long sensilla trichodea responded specifically to the minor aldehyde components of the pheromone, which have subtle effects on behavior. Mixture experiments provided no evidence that minor components influence the receptor responses to the major components. Olfactory sensilla outside the crosswise rows were also characterized morphologically by SEM. Included in these were sensilla of different lengths, corresponding to a classification as s. basiconica and s. trichodea type 2. Electrophysiological recordings from these sensilla showed that they are involved primarily in host odor reception. However, a few of these neurons responded to pheromones.

Pheromone receptor cell specificity in interpopulational hybrids ofIps pini (Coleoptera: Scolytidae).

Electrophysiological studies of pheromone receptor cells keyed to ispdienol were performed in laboratory-raised hybrids of the eastern and western populations of the pine engraver,Ips pini. As previously shown in the parental beetles, the receptor cells keyed to ipsdienol could be classified as two distinct types: one keyed to (+)- and one to (-)-ipsdienol. None of the 20 ipsdienol cells recorded from F1 hybrids were of an intermediate type. Recordings of the summated receptor responses (EAGs) showed no significant difference between parental beetles and hybrids. Similar results were obtained in reciprocal crosses, eastern females with western males and the reverse. Thus, there was no indication that sex-linked alleles determined the specificity of the ipsdienol receptor cell. The ratio between (+) and (-) cells was 14∶6 in the hybrids compared to 1∶12 in the western and 9∶12 in the eastern populations.

Receptor cells inIps typographus andDendroctonus micans specific to pheromones of the reciprocal genus.

Olfactory receptor cells were studied electrophysiologically inIps typographus andDendroctonus micans. The investigation revealed cells which were keyed to pheromone compounds characteristic of the reciprocal genus. Thus, cells keyed toexo-brevicomin were found inI. typographus, whereas cells keyed to (+)-ipsdienol were present inD. micans. Laboratory behavioral tests indicated an attractive effect of the two compounds on beetles of the reciprocal genus. InI. typographus the effect ofexo-brevicomin predominantly concerned males and enhanced their response to the pheromone "ipslure." It is suggested thatexo-brevicomin serves as an interspecific attractant forI. typographus, which may be guided by pheromone compounds of the reciprocal genus in finding suitable breeding material. The function of (+)-ipsdienol inD. micans is more uncertain. It may be either a pheromone or an interspecific messenger.