Chem. Senses 26: 1175-1186,
2001
© Oxford University Press 2001
Detection of Sex Pheromone Components in Manduca sexta (L.)
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, CZ-166 10 Prague 6, Czech Republic 1 Ecological Chemistry Group, Organic Chemistry, KTH, SE-100 44 Stockholm, Sweden 2 Department of Crop Science, Swedish University of Agricultural Sciences, SE-2230 53 Alnarp, Sweden
Correspondence to be sent to: Dr Blanka Kalinová, Institute of Organic Chemistry and Biochemistry ASCR, Flemingovo nám. 2, CZ-166 10 Prague 6, Czech Republic. e-mail: blanka{at}uochb.cas.cz
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Abstract |
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The ability of olfactory receptor neurons to detect female-produced sex pheromone components and a limited sample of potential host plant odours was studied by single-sensillum recordings from olfactory sensilla present on male and female antennae in Manduca sexta. The majority of pheromone-sensitive receptor neurons examined in males was specialized for detection of the two major pheromone components, E10,Z12-hexadecadienal and E10,E12,Z14-hexadecatrienal or E10,E12,E14-hexadecatrienal. New olfactory receptor neurons tuned to the minor components E10,E12-hexadecadienal and Z11-hexadecenal were found. In females, olfactory receptor neurons specific to Z11-hexadecanal were discovered. Pheromone components and host volatiles were detected by separate sets of receptor neurons.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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The tobacco hawk moth, Manduca sexta (Linnaeus) (Lepidoptera, Sphingidae), is one of the most thoroughly studied insect models used in olfactory research. The main interest has been directed towards the central nervous system, especially to processing of the two main sex pheromone components in males [for review see (Hildebrand, 1995
,
1996;
Hildebrand and Shepherd,
1997)]. In comparison, the peripheral aspects of pheromone
detection have not been studied so intensely
(Kaissling et al.,
1989).
Twelve pheromone-like compounds have been identified in solvent rinses of
the female sex pheromone gland (Tumlinson
et al., 1989). Behavioural observations in the wind
tunnel and in the field revealed that eight of them, namely hexadecanal
(16:Ald), three isomeric hexadecenals [Z9-16:Ald, E11-16:Ald
and Z11-16:Ald (Z9, E11 and Z11, respectively)], two isomeric
hexadecadienals [E10,E12-16:Ald (EE) and
E10,Z12-16:Ald (EZ; bombykal)] and two isomeric
hexadecatrienals [E10,E12,E14-16:Ald (EEE) and
E10,E12,Z14-16:Ald (EEZ)], play a role in the
attraction of males to conspecific females
(Starrat et al.,
1979; Tumlinson et al.,
1989,
1994).
Males detect pheromone by olfactory receptor neurons (ORNs) within
male-specific sensilla trichodea (Sanes
and Hildebrand, 1976;
Schweitzer et al.,
1976; Keil, 1989;
Lee and Strausfeld, 1990;
Shields and Hildebrand,
1999a,b).
Two morphological classes of sensilla trichodea—type I and II—have
been found on the male antennal flagellum
(Sanes and Hildebrand, 1976;
Keil 1989;
Lee and Strausfeld, 1990).
Type I trichoid sensilla form typical arch-like rows on the dorsal and ventral
surfaces of each annulus. Slender type II trichoid sensilla are located,
together with sensilla basiconica and other sensillar types, in areas of the
annulus not occupied by type I elements
(Lee and Strausfeld,
1990).
Though females lack type I trichoid hairs, they possess trichoid elements
too (Lee and Strausfeld, 1990;
Shields and Hildebrand,
1999a,b).
Similarly as in males, slender A and stout B elements have been recognized
(Shields and Hildebrand,
1999a,b).
The female-specific trichoid types do not exceed 50 nm in length and are
distributed over the annular surfaces among the population of much shorter
sensilla basiconica. Females are considered to be pheromone anosmic
(Schweitzer et al.,
1976; Hildebrand,
1996).
Previous electrophysiological recordings from male-specific type I trichoid
sensilla of M. sexta showed that one of the two ORN types present is
specific to EZ (the most prominent component in the pheromone blend) and the
second, to either EEZ or EEE (Kaissling
et al., 1989). No other male-specific ORNs have been
characterized. But it has been shown that minor pheromone components have
physiological effects in the brain (Christensen et al., 1989b). In
females, no ORNs tuned to EZ and/or E11,Z13-penta-decadienal
(a mimic of EEZ) were observed when single sensillum responses were recorded
from type A sensilla trichodea (Shields
and Hildebrand, 2001). But expression of the `male-specific'
pheromone binding protein in female antennae has been reported
(Györgyi
et al., 1988; Vogt
et al., 1991). The aim of our study was to find out how
males detect minor pheromone components and if there are any
pheromone-sensitive ORNs on female antennae.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Insects and electrophysiological recordings
Manduca sexta moths were reared on an artificial diet [modified
from that of Bell and Joachim (Bell and
Joachim, 1976)] under a L:D 16:8 photoperiod regime (23-25°C,
40-50% relative humidity). Males and females 1-2 days old were used for
experiments. Moths were restrained in a tightly fitting plastic tube. The head
was encased in wax with the antennae firmly fixed at their bases.
The neuronal activity was recorded extracellularly by means of an
electrolytically sharpened tungsten electrode penetrating the antennal cuticle
at the base of a sensillum (Hubel,
1957; Boeckh, 1962). The position of each electrode contact was
recorded to achieve information about the distribution of different
physiological types of ORNs on antennal annuli. The recording electrode was
connected to a high impedance AC amplifier (Syntech, Hilversum, The
Netherlands) operating at 1000 times amplification and with a 500 Hz bandpass
filter. The indifferent electrode (Ag/AgCl wire) was inserted into the moth's
abdomen. An audio-monitor was used to indicate contact quality. The signals
were observed on a Phillips oscilloscope and recorded on a Vetter
videocassette recorder, SLF-750HF (Vetter, NJ) for later processing. Responses
were then digitized (sampling rate 10 416 samples/s) and PC analysed using
Syntech Auto-spike software version 3.0 and 4.0 (Syntech). Experiments were
performed on 20 males and 25 females.
Chemicals
The commercially available host plant-related volatiles were used as
representatives of non-pheromonal stimuli. Among the selected compounds were
those affecting oviposition in M. sexta
(Tichenor and Seigler, 1980)
or compounds identified in emanations of tomato and tobacco, the preferred
host plants in M. sexta (Andersen et al.,
1986,1988;
Buttery et al.,
1987a,b;
Loughrin et al.,
1990). The selected volatiles and their purities, determined by
gas chromatography, are listed in Table
1.
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Pheromone components were synthesized in the laboratory. Monounsaturated
aldehydes were prepared from corresponding alkenols by a simple oxidation
procedure with pyridinium chlorochromate (PCC)
(Corey and Suggs, 1975). The
starting alkenols were synthesized by an alkyne chain elongation (from
-bromo alkanols and corresponding 1-alkynes) and subsequent
reduction/hydrogenation of the triple bond.
The key intermediate for synthesis of 10,12-hexadecadienals, O-protected
1-iodo-E1-undecen-11-ol, was prepared from O-protected
10-undecyn-1-ol by a standard hydro-alumination/iodation procedure
(Tellier and Descoins, 1991).
The palladium catalysed cross-coupling reaction of this iodocompound with
1-diisobutylaluminium-E1-pentene
(Negishi et al.,
1988) directly provided the required E10,
E12-dienic system. The product of this coupling was deprotected and
oxidized (PCC) to a desired E10,E12-hexadecadienal (EE).
Isomeric E10,Z12-hexadecadienal (EZ; bombykal) was prepared
in a similar way. The palladium catalysed cross-coupling of the key
intermediate with 1-pentyne
(Ratovelomanana and Linstrumelle,
1981) was followed by a hydroboration with dicyclohexylborane,
which gave the corresponding O-protected E10,Z12-dienic
alcohol. The last steps of the synthesis were the same as in the case of the
above-mentioned E10,E12-hexadecadienal.
E10,E12,E14-hexadecatrienal was prepared according to the following procedure: the triphenylphosphosphonium salt prepared from 10-bromo-1-decanol was converted to the corresponding ylide which was reacted with sorbinal in the presence of LiBr and excess of base. From the obtained mixture of Z10,E12,E14-hexadecatrienol (major product) and the E10,E12,E14-isomer, the latter isomer was isolated by an urea complex inclusion procedure, then oxidized to the desired product, E10,E12,E14-16:Ald (EEE) by Swern oxidation.
The synthesis of E10,E12,Z14-hexadecatrienal started with oxidation of 1-(2-tetrahydropyranyloxy)-10-bromodecane to the corresponding O-protected decanal by N-methylmorpholine N-oxide. This aldehyde was reacted with the anion of methyl 4-dimethylphosphonate-E2-butenoate (Wadsworth—Horner—Emmons reaction). The hydroxy function in the resulting dienoate was deprotected before the ester functionality was converted to an aldehyde by use of diisobutylaluminium hydride and manganese dioxide in two subsequent reactions. The product, 14-hydroxy-E2,E4-tetradecadienal, was converted to the desired E10,E12,Z14-hexadecatrienol by a reaction with a corresponding ylide. The trienol was oxidized (Swern oxidation) to 10E,12E,14Z-16:Ald (EEZ) in the last step of the synthesis.
The purity of all synthetic pheromone components used was between 95 and 99% (as determined by GC—MS and HPLC—MS).
Stocks of test compounds were prepared by diluting the neat compound in
hexane in decadic steps. From each stock concentration, 10 µl were pipetted
onto a strip of filter paper (
10 x 15 mm) placed in a Pasteur
pipette, where the solvent was allowed to evaporate. The amount of substance
in pipettes ranged from 100 pg to 1 µg in decadic steps for the
dose—response trials. For screening, 100 ng were used in each pipette.
Blank stimulations were performed with a cartridge containing a filter paper
onto which only solvent had been applied. The test cartridges were kept at
-20°C when they were not used to prevent degradation of the compounds. New
pheromone cartridges were prepared every second day, cartridges loaded with
plant volatiles were prepared prior to every experiment.
Odour delivery system
The antenna was continuously ventilated with a stream of purified, humidified air (0.5 m/s) that passed through a glass tube (8 mm i.d.) with the outlet (3 mm i.d.) positioned 0.5 cm from the antenna. Neurons were stimulated with 0.5 s puffs of each component by injecting 1 ml of air from the odour cartridge into a continuous air-stream through a hole (i.d. 0.4 cm) in the glass tube located 15 cm from the outlet. Odour stimulations were controlled by a Syntech stimulus controller operated by a foot switch. The time of closed switch was indicated on the computer screen as a stimulus bar. In selective blocking experiments, two stimulation channels were synchronized to deliver the blocking and test stimuli (duration 0.3 s) with a 0.1 s interval.
Each time a contact with a sensillum was established, the spontaneous activity of associated ORNs was recorded for 30 s and the number of neurons within a sensillum was determined. Then, pheromone and host plant-related compounds at the screening dose, and a blank, were used to test whether any ORN of the contacted sensillum gave a response stronger than the blank. If an ORN responded to any of the test substances, dose—response trials were performed. The test substances were presented to the antenna at all dose levels, starting with the lowest doses. At lower doses (<100 ng) the stimuli were presented with an interval of 60 s, at higher doses the ORNs were allowed to recover for longer periods up to 5 min. Spikes were counted during the period of stimulation. When a dose—response curve for a key compound was established, the lowest dose that gave responses significantly higher than the spontaneous activity was determined by a Wilcoxon rank test (one-sided, P < 0.05). When all tests were done, the antenna was fixed in a new position that made it possible to contact previously un-stimulated sensilla.
Selective blocking technique
In sensilla where spike amplitudes of individual ORNs could not be
discriminated, separation of individual ORNs within the sensillum was
performed using a technique modified from differential adaptation as described
elsewhere (Payne and Dickens,
1976; Kaissling et
al., 1989). Initially, the sensillum was exposed to 0.3 s
stimulation with one compound active in the screening procedure (blocking
compound, 500 ng) and then, within 0.1 s interval, with another compound
active in the screening procedure (test compound, 100 ng). If blocking and
test compounds were detected by the same ORN, no response or a weak response
was supposed to be elicited by the test compound. If the test compound was
detected by a different ORN within a sensillum, the response to the test
compound was considered to be more or less unaffected by blocking.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Sensillar classification
The male-specific type I sensilla trichodea (long trichoids) of the phallanxes were determined unambiguously due to their anatomical separation and length. Outside phallanxes however, morphological characteristics visible in the stereomicroscope of the recording set-up did not allow clear discrimination among other, much shorter, morphological types (e.g. shortest type I sensilla trichodea, type II sensilla trichodea and/or sensilla basiconica). All sensilla outside phalanxes were therefore assigned as short ones. Similarly, morphological types were not distinguished in females.
Sensillar physiology
Out of 431 sensilla investigated in males, 170 sensilla were type I trichoids (long sensilla) and 261 were contacted in areas outside phallanxes (short sensilla). All long trichoids examined contained pheromone-sensitive ORNs (the representation of all ORN types found on male and female antennae is summarized in Table 2). Out of the 261 short sensilla, 86 contained pheromone-sensitive ORNs. In 81 short hairs, ORNs sensitive to one or more host plant odours were found (detailed physiological results will be reported elsewhere). In seven contacts, ORNs responded equally to pure air and to all applied stimuli. In 87 sensilla, associated ORNs did not respond to any compound tested.
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In females, 200 sensilla were studied. Out of all impaled sensilla, 121 contained ORNs sensitive to one or more host plant odours, 71 sensilla did not respond to any odour tested, eight ORNs were found to be specific to Z11.
In most contacts in both sexes, the spontaneous activity showed more than one class of spike amplitudes, indicating the presence of two or three ORNs.
Type I sensilla trichodea
In agreement with previously published data, male-specific sensilla trichodea type I contained two cells. In the majority of them, an EZ-specific neuron was paired with an EEZ-specific one (Figure 1A). In only four sensilla, the trienal-specific cell showed higher sensitivity to EEE than to EEZ (Figure 1B). In 16 long hairs, the EZ cell was associated with a so far unknown ORN type tuned to EE. In some cases, the EE cell responded selectively to EE (Figure 1C). In others, however, the EE cell responded also to EEE but at a somewhat lower sensitivity (Figure 1D). In few contacts, only one ORN—tuned either to EZ or EEZ—was found. Due to very high cross-reactivity and/or contact deterioration, the specificity of associated ORNs was not determined in 12 contacts.
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The ORNs present in type I sensilla trichodea displayed spikes of very
similar shape and amplitude. In most of the naive (un-stimulated) sensilla, EZ
spikes were slightly higher than spikes of the EEZ cell, but sensilla with
both cells spiking similarly were also found. The overall spontaneous activity
recorded in long trichoids was 0.93 ± 0.69 imp./s. Spontaneous spikes
quite often occurred in bursts of three to five. The dose—response
curves for EZ, EEZ, EE and EEE (Figure
3) show the response threshold of pheromone-sensitive ORNs at a
stimulus load 1 ng (Wilcoxon rank test, one-sided, P < 0.05).
Saturation was observed at doses 
1 µg. At doses 
10 ng, responses
tended to be organized in an initial phasic burst of action potentials
followed by a tonic rate of firing, which diminished after the end of
stimulation. The frequency of spikes within a burst gradually increased (up to
200-250 Hz) with increased stimulation doses. Close to saturation (and or
after repeated stimulation), the tonic phase disappeared, bursts shortened,
spike amplitudes within burst rapidly declined, number of spikes decreased and
action potential firing was eventually blocked
(Figure 2). The increased
stimulation dose reduced the latency of the neuronal response until ORN
adapted (Marion-Poll and Tobin,
1992).
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, n = 3) and female (
,
n = 3) antennae. The y-axis represents the number of spikes
elicited during 500 ms stimulation by a given dose, the x-axis
delineates the stimulation intensity given by an amount of the stimulus
compound loaded onto a filter paper disc in the stimulation pipette. Vertical
lines indicate the standard error of the mean.
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The ORNs associated with type I sensilla trichodea responded to non-key pheromone components only when stimulus doses were elevated substantially (>100 times). Figure 3A displays the dose—response characteristics of ORNs present in the most abundant type I sensilla trichodea to key and non-key pheromone components. In these sensilla, EE was the second most effective compound, followed by Z11 and E11. Compounds EEE, Z9 and 16:Ald elicited weak or no responses. Selective blocking proved that the EE was the second best stimulus for EZ-specific neurons. The EE cells responded second best to EEE.
A relatively large variation in the specificity and sensitivity of ORNs in male-specific sensilla trichoidea was observed. Some ORNs responded very specifically only to their key compound even at elevated doses (Figure 1A,B,1C,1E), while others were significantly sensitive also to other stimuli (Figure 1D). No ORNs within type I sensilla trichodea responded to any plant odour tested.
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Selective blocking technique
The principle of how the selective blocking technique was used in our study to discriminate between neurons in a contacted sensillum is demonstrated in Figure 4. Two columns (A, B) represent typical examples of physiological responses recorded from type I sensillum trichodeum. Responses displayed in column A were obtained from the most abundant sensillum type containing EZ and EEZ neurons. The first trace displays neuronal responses to two successive EZ stimuli. The EZ-specific ORN responded to the first EZ stimulus with a strong phasic burst of spikes. By the time of the second EZ stimulus onset, the EZ cell remained blocked and EZ re-stimulation did not elicit any further spike activity (similarly, EEZ block eliminated the response of the EEZ neuron to the second EEZ stimulus—not shown). On the other hand, the EZ block did not eliminate the responses of the EEZ neuron to EEZ stimulation and vice versa (Figure 4A, the second and the third trace). A clear response to EEZ after EZ block (and the other way round) was considered as a proof that EZ and EEZ were detected by two discrete ORNs. Column B displays recordings from a sensillum where an EZ-specific neuron was located together with an EE-specific one. The first and second traces show how the EZ cell responded to EZ stimulation after EE or EEE blocking (large spikes detected in the background of rapidly declining spikes of the first burst represent activity of the EZ-specific ORN). The selective blocking by EE abolished the response to EEE (Figure 4B, third trace) and vice versa (not shown). The third trace thus demonstrates that EE and EEE were detected by the same ORN not identical to the EZ one. As could be seen from averaged frequency histograms displayed in Figure 5, the selective blocking was quite efficient and reduced the spiking activity of the blocked cell considerably, while the response of other cell within the sensillum to the key stimulus remained unaffected.
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Short sensilla
Extracellular recordings from short sensilla on male antennae revealed activity of one, two or three ORNs. Based on the specificity of ORNs present within the contacted sensillum, three discrete physiological subtypes of short sensilla were identified: (i) sensilla with two ORNs sensitive to the major pheromone components and/or to their isomers, the physiology of which (spontaneous activity, spike patterns, amplitudes, sensitivity and specificity) was very similar to that found in trichoid sensilla within phallanxes, (ii) sensilla with an ORN sensitive to Z11, and (iii) sensilla with one up to three ORNs sensitive to plant volatiles. The respective physiological types were found in different areas on antennal anulli (Figure 6D). The ORNs specific to major pheromone components were occasionally found on the leading edge of the annuli, in areas along the phallanxes and more frequently in the U-shaped pocket of long trichoids at the trailing edge. On the other hand, 14 ORNs tuned to Z11 were interspersed among the host odour-sensitive sensilla on the free surface of the annuli.
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The spontaneous activity recorded from sensilla with the Z11 -specific ORN sometimes indicated the presence of more than one ORN. The associated cell(s), however, did not respond to any stimulus tested. A typical example of physiological responses recorded from sensillum with the Z11 -specific ORN is displayed in Figures 1E and 2. The responses of Z11 -specific ORNs were dose-dependent at doses above 10 ng. Saturation was observed at 10 µg (Figures 2 and 3D).
Pheromone-sensitive ORNs in females
In females, eight ORNs tuned to Z11 were found. The sensilla associated with Z11 ORNs were distributed over much of the annular surface, among the population of host-odour sensitive sensilla. An accumulation in any specific area of the antennal annulus was not observed. The physiology of Z11 ORNs was similar in males and females (Figures 1E, 3D and 7).
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Discussion |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Our study provides evidence about the presence of two new, previously unknown, pheromone-sensitive ORN types on male antennae. The first one, EE-specific ORNs, was found in sensilla within phalanxes and between short sensilla medially along phallanxes and among inner hairs of U-shaped cul-de-sac formed by phallanxes at the trailing edge of each annulus. This distribution corresponds with the distribution of sensilla trichodea type I (Lee and Strausfeld, 1990
).
Previous electrophysiological study of type I sensilla trichodea reported the
presence of three types of ORNs: cells tuned to EZ (ORN type A), EEZ (type B)
and EEE (type C) (Kaissling et
al. 1989). In addition to EE-specific ORNs, we found all
already known receptor types. However, the EEE-specific ORNs were found in
relatively lower abundance [compare four EEE ORNs out of 431 contacts in our
study (0.9%) and three EEE ORNs out of 50 contacts (6%)
(Kaissling et al.,
1989)]. Such a discrepancy could be explained by the fact that
different laboratory colonies of M. sexta were used. However, since
in earlier investigation the EE was not tested, we can also speculate that
some previously identified EEE neurons might be identical to the EE ones. Some
EE neurons impaled in our experiments were indeed also sensitive to EEE and
selective blocking experiments showed undoubtedly that EE and EEE stimulated
the same ORN.
Surprisingly, a relatively large variation in specificity among ORNs
associated with male-specific type I sensilla trichodea was observed. Some
ORNs responded highly specifically only to the key compound, even at elevated
doses, while others were quite sensitive also to other stimuli of pheromonal
origin. The excitability of impaled cells could in some cases be affected by
the penetration of the electrode. However, the observed variability might
reflect real differences in ORN physiology. The wide range of lengths of type
I trichoids may represent the various physiological subclasses
(Lee and Strausfeld, 1990).
This suggestion was proposed by Kanaujia and Kaissling who studied sensillar
physiology in Antheraea polyphemus
(Kanaujia and Kaissling,
1985). Their study implies that different lengths of sensilla, and
hence of dendrites, may indicate specific functional roles amongst members of
the respective classes. The different trichoid lengths may confer different
biophysical properties relating to sensitivity and transduction
(Kanaujia and Kaissling,
1985). It would be interesting to know if there is any systematic
correlation between physiology of the ORNs and the sensillar position on
antennal annuli in M. sexta.
The selective blocking technique proved to be an efficient tool in discrimination of different ORNs responding with similar spike amplitudes in M. sexta. We have chosen to name the technique selective blocking, as we cannot be sure if the observed effect is a result of an adaptation process or of a depolarization block. Both mechanisms might contribute in this case. Adaptation could be argued to allow doubt regarding the specificity of the neuron adapted, as a second receptor type could theoretically be expressed in the dendritic membrane of the same neuron. Under such circumstances a single, adapted neuron could still respond to a second component. Depolarization block would provide an unambiguous result, as all responses of the affected neuron would be abolished.
The second new ORN type found in our study was specific to Z11. Neurons
sensitive to Z11 were discovered in short sensilla of the free space between
phalanxes spotted among sensilla sensitive to plant-related odour. In this
area, sensilla trichodea type II and sensilla basiconica are found. These
sensilla are supposed to carry information about non-pheromonal odours
(Christensen et al.,
1995), since axons of associated ORNs target glomeruli outside the
macroglomerular complex (MGC)—the structure where all pheromone-specific
ORNs have traditionally been considered to project
(Christensen and Hildebrand,
1987). Our finding that among these sensillar types do exist ORNs
sensitive to one of the pheromone components is noticeable and raises some
interesting questions. Could these sensilla be identical to those expressing
the pheromone-binding protein (PBP) in free space between phalanxes
(Vogt et al., 2002)?
Do neural circuits outside the MGC process some features of the pheromone
signal? Recent findings that pheromone responses can indeed be recorded from
antennal lobe neurons restricting their arbors to ordinary glomeruli
(Anton and Hansson, 1999)
support such a possibility. However, further studies are needed to answer
these questions and to understand entirely pheromone processing in M.
sexta.
We did not find ORNs specific to E11, Z9 or 16:Ald. Considering the number
of sensilla present on each antennal annulus and the number of sensilla
contacted in this study, we cannot conclude whether these receptor types exist
or not. If very few specific ORNs are present on each antennal annuli, the
possibility of contacting them is low. In the turnip moth, Agrotis
segetum, ORNs tuned to one of the major pheromone components occur only
in 0.1% of the sensilla (Hansson et
al., 1990). If a similar relationship is present in M.
sexta, a sample of 1000 sensilla would statistically be required to
encounter all types.
The processing of minor pheromone components in the male brain of M. sexta
has been investigated in the deutocerebrum
(Christensen et al.,
1989), where the activity of antennal lobe interneurons was
recorded intracellularly. These experiments proved that minor components have
some physiological effect in the male brain, however, the neural substrate for
their detection remains unknown. Our study shows that male-specific ORNs tuned
to major pheromone components responds to other pheromone components only when
elevated doses are used and with high certainty do not represent a relevant
channel to the brain regarding their detection. On the other hand, the newly
identified ORNs specifically tuned to EE and Z11 undoubtedly delineate the
previously unknown sensory pathway.
The role of minor pheromone components in sexual communication in M.
sexta is not yet fully understood. One of the difficulties in working
with the pheromone of this species is the instability and unavailability of
the triene aldehydes. In wind tunnel experiments it has been shown that from
all components produced by female sex pheromone glands, a blend of two
components, EZ and EEZ, is essential to elicit male precopulation behaviour
(Tumlinson et al.,
1989). Further tests in the wind tunnel suggested, but did not
clearly demonstrate, that other components of the gland rinse played a role in
mating communication in this species. These experiments also showed that a
four-component blend (EZ, EEZ, EE and EEE) was less effective than either the
two-component blend or the full component blend (Tumlinson et al.,
1989,
1994). Field experiments
showed that the synthetic full component blend is attractive for males in the
field. Addition of one or more of the saturated and monounsaturated components
to EZ and EEZ improved the male response. The authors of the study suggested
that all eight 16-carbon aldehydes are active
(Tumlinson et al.,
1994). In the male brain, all the 16-carbon aldehydes found in the
pheromone gland elicit some form of response in olfactory interneurons
(Christensen et al.,
1989), but EZ, EEZ and EEE evoke the greatest responses. Our
finding of two new ORN types tuned to EE and Z11 suggests that except EZ, EEZ
and EEE also EE and Z11 play an active role in sexual communication of M.
sexta. However, their exact roles must be further investigated.
Females of M. sexta have been consistently noted not to respond
physiologically or behaviourally to sex pheromone
(Schweitzer et al.,
1976; Hildebrand,
1996). In spite of this, the expression of `male-specific'
pheromone binding protein (PBP) in antennae of female M. sexta
(Györgyi
et al., 1988; Vogt et al.,
1991,
2002) and in some other
species, females of which have been previously considered as pheromone
anosmic, have been reported (Steinbrecht
et al., 1992;
Nagnan-Le Meillour et al.,
1996;
Maibeche-Coisné
et al., 1997; Callahan
et al., 2000). Immunological and histological studies
have shown that PBP is expressed at a low level compared with that in male
antennae and the expression is associated with a small number of specific, but
otherwise uncharacterized, group of olfactory sensilla (Steinbrecht et
al., 1992,
1995;
Laue and Steinbrecht, 1997;
Vogt et al., 2002).
Our data bring the first physiological evidence that M. sexta females
do respond to at least one pheromone component. Our sensilla associated with
ORNs specifically tuned to Z11, found in a small number on female antennae,
may be among those showing expression of PBP. The behavioural meaning of
female ability to detect Z11 is not known and should be further
investigated.
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Acknowledgments |
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Financial support from the Grant Agency of the Czech Republic (Grant No. 204/95/1029, research project Z40055905), the Royal Swedish Academy of Sciences and the Swedish Natural Science Research Council are ack- nowledged. We thank the anonymous referees for their critical comments and suggestions that improved the manuscript.
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Accepted August 6, 2001
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