Journal of Insect Physiology 48 (2002) 825–834
www.elsevier.com/locate/jinsphys
Repetitive stimulation of olfactory receptor cells in female
silkmoths Bombyx mori L.
Romina B. Barrozo a, Karl-Ernst Kaissling b,∗
a
Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria,
C1428EHA Buenos Aires, Argentina
b
Max-Planck-Institut für Verhaltensphysiologie Seewiesen D 82319 Starnberg, Germany
Received 10 June 2002; accepted 11 June 2002
Abstract
The pheromone-sensitive receptor cells of male moth antennae are capable of detecting the rapid changes in stimulus intensity
encountered in natural pheromone odour plumes. We investigated temporal response characteristics of the two receptor cell types
of the sensillum trichodeum of female Bombyx mori, which are most sensitive to benzoic acid and 2,6-dimethyl-5-heptene-2-ol
(DMH), respectively. The cells were repetitively stimulated with 50-ms pulses of benzoic acid and (±)-linalool, an effective mimic
of DMH, at various pulse rates and different stimulus intensities. By recording receptor potentials and nerve impulses we demonstrated that both receptor cell types were able to follow stimulus pulses at least up to eight pulses per sec, with a more pronounced
modulation of the responses in the DMH cell. The resolution capability of the two cell types showed little dependence on stimulus
intensity. In their ability to resolve pulsed odour stimuli, the receptor cells for benzoic acid and DMH were as good as pheromone
receptor cells.  2002 Elsevier Science Ltd. All rights reserved.
Keywords: Resolution of odour pulses; Single-cell recordings; Benzoic acid; 2,6-dimethyl-5-heptene-2-ol; (±)-linalool; Sensory adaptation
1. Introduction
Turbulence in the air causes the odours liberated from
small odour sources, such as insect pheromone glands,
to pass a detector downwind intermittently, with frequent
and rapid changes of odour concentration (Murlis and
Jones, 1981; Murlis et al., 1992). Insects orienting within
turbulent pheromone plumes are well adapted to such
patterns and have olfactory receptor cells that are able
to encode the temporal characteristics of the stimuli
(reviewed by Kaissling (1997). This has been shown for
pheromone receptor cells of male moths by studying
responses to pulsed odour stimuli. In Antheraea pernyi,
Kaissling (1986) found sensory neurons following pulses
of pheromones delivered at 4 Hz. Pheromone receptor
cells of Antheraea polyphemus responded in synchrony
to stimuli at rates as high as five or more odour pulses
per s (Rumbo and Kaissling, 1989; Kodadová, 1996).
The pheromone receptor cells in Manduca sexta also fol∗
Corresponding author.
lowed pheromone pulses up to at least five per s
(Marion-Poll and Tobin, 1992). Not only receptor cells
but also projection neurons of the antennal lobe of this
species responded to pheromone pulses up to ten per s
with discrete bursts of nerve impulses (Christensen and
Hildebrand, 1988). The resolution of repetitive stimulus
pulses has also been studied in insect carbon dioxide
receptors (Stange, 1992).
Our study is directed to receptor cells tuned to odours
other than pheromones, such as are found on the
antennae of female silkmoths. The sensilla trichodea of
male and female silkmoths Bombyx mori are 100-µmlong olfactory hairs distributed all over the antenna. In
the male moth the two cells of each sensillum trichodeum detect extremely low concentrations of the two
pheromone components bombykol and bombykal
(Kaissling and Priesner, 1970; Kaissling et al., 1978).
Like the male, the female has two olfactory receptor
cells of different specificity inside each sensillum trichodeum. In the female one cell responds best to benzoic
acid (benzoic acid cell), whereas the other reacts most
sensitively to 2,6-dimethyl-5-heptene-2-ol (DMH cell)
0022-1910/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 2 2 - 1 9 1 0 ( 0 2 ) 0 0 1 0 9 - 9
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R.B. Barrozo, K.-E. Kaissling / Journal of Insect Physiology 48 (2002) 825–834
and somewhat less sensitively to (±)-linalool (Priesner,
1979). To our knowledge, DMH is so far not known as
a natural product; it is used as an internal standard in
the analysis of components of wine aroma (Rapp et
al., 1994).
Both of these cell types are odour specialists since the
side spectra of the cells of each type are identical (De
Brito Sanchez, 1996, 2000; Van der Goes van Naters,
in prep.). Little is known about their role for the female
moth (see Discussion); it must, however, be important
in view of the high sensitivity to the respective key compounds. A single benzoic acid cell responds to a concentration of 7×108 molecules of benzoic acid per ml of air
(Ziesmann et al., 2000). Single DMH cells responded to
(±)-linalool roughly at concentrations just detected by
human noses (in the range of 2×1011 molecules/ml of
air, Ohloff and Klein (1962); Buttery et al. (1968)). The
threshold concentration of DMH was estimated by Van
der Goes van Naters (in prep.) from gas-chromatographic measurements and linear extrapolation over
three decades of stimulus loads as 1010 molecules per ml
of air. Because of the large number of the cells (⬎10,000
sensilla trichodea per antenna, Schneider and Kaissling
(1957); Steinbrecht (1970) the behavioural threshold
could be even lower.
Studying the temporal characteristics of these cells
seemed to us promising not only for a better understanding of their biological significance but also because of
the strikingly different temporal response patterns of the
two cell types. While both types of cells show tonic
receptor potentials, they differ widely in their temporal
pattern of nerve impulse firing upon olfactory stimulation. The DMH receptor cells respond phasically, i.e.
with a burst of nerve impulses after stimulus onset, and
without background firing between stimuli. The benzoic
acid receptor cells show a phasic–tonic response and a
background firing of 3–6 nerve impulses per s
(Heinbockel and Kaissling, 1996). We expected that the
two types of receptor cells would differ with respect to
the pulse rates resolved.
2. Materials and methods
Pupae of Bombyx mori were obtained from commercial breeders and kept at room temperature. After emergence the adults were stored at 12 °C before the experiments started. All the experiments were carried out at
room temperature (22–24 °C).
Transepithelial recordings were made from the cut tips
of single sensilla trichodea located on side branches from
the middle portion of isolated antennae, using glass
capillaries with Ag-AgCl electrodes. Recording electrodes had a diameter at the tip of 10–15 µm. The reference electrode was filled with hemolymph ringer solution and the recording electrode with sensillum lymph
ringer solution (technique and solutions described by
Kaissling (1995).
After amplification low-pass (2 kHz) filtered signals
were stored by a tape recorder. To analyze the nerve
impulse data, the signals from the tape recorder were
digitized (sample rate 2.5/ms) and stored in the computer. The nerve impulses were detected by routine
software (Superscope II2.31 for Macintosh) extended by
means of included functions.
The antenna was stimulated with benzoic acid
(Henkel, purity 99%) and (±)-linalool (Aldrich, purity
97%) released from 1-cm2 filter papers placed in a glass
tube of 7 mm inside diameter, with a lumen volume of
2.5 ml. The antenna was positioned 2 cm from the outlet
of the glass tube. The stimulus loads per filter paper were
as follows: 1, 3, 10, 30 µg of benzoic acid and 5, 50,
500 µg, and in some cases 5000 µg of (±)-linalool, the
latter compound dissolved in 50 µl of paraffin oil. The
solvent for benzoic acid was acetone, which was evaporated from the filter paper before using it as a stimulus
source. Pulsed stimuli 50 ms in duration (half width)
with an air stream of 100 ml/s were presented at increasing pulse rates , starting with the smallest stimulus load.
The interval durations (times between stimulus-pulse
onsets) used were (in this sequence) 1.92, 0.96, 0.48,
0.24, 0.12 s and in some cases 0.06 s (where consecutive
stimulus pulses partially overlap). Series of ten pulses
were applied for each pulse rate. An electrical valve controlled the rate and duration of the stimuli; the airstream
velocity was monitored with a thermistor located 2 mm
behind the antenna. After each series of ten stimulus pulses, the antenna was allowed to recover for 30 to 120 s,
with longer recovery times for higher stimulus intensities. The responses of five and six sensilla from different animals were evaluated for benzoic acid and for (±)linalool, respectively.
After the numbers of nerve impulses within 40 ms
bins had been counted, the correlation of nerve impulse
responses with the stimulus pulses was tested using peristimulus time histograms (StatView 5.0). Averaged histograms were constructed by using the cell responses to
eight consecutive pulses, discarding the responses to the
first two of each series of ten pulses. In order to find out
if a cell is capable of following stimulus pulses at a certain pulse rate we carried out time series Fourier analyses
of the nerve impulse responses. For testing the statistical
significance of the identified periodical components of
the responses, we used the Lomb–Scargle periodogram
(Lomb, 1976; Scargle, 1982).
To find out whether the odour source was constant
during a stimulation series of ten pulses the first few
stimulus pulses of a series of ten pulses were directed
away from the antenna by an interfering airstream. For
the remaining pulses, the airstream coming from the
odour source was reoriented towards the sensillum. In
the example shown in Fig. 1 we delivered from the same
R.B. Barrozo, K.-E. Kaissling / Journal of Insect Physiology 48 (2002) 825–834
827
pulse. Since the responses to the first pulses of the first
and last stimulus series (traces a, e) were similar, the
odour source must have ‘recovered’ from the small
fatigue within the series during the one min between two
successive stimulus series. Thus, stimulus pulses at the
same positions within different series ought to have the
same degree of (small) fatigue. Consequently, fatigue
cannot be the reason if responses to pulses at the same
position within different series differ in size. This
applies, e.g., to the first responses in the traces b, c, or
d as compared with the responses to the pulses at the
same positions in the respective preceding series.
3. Results
Fig. 1. Receptor potentials and nerve impulse responses of a single
DMH cell to an odour source (filter paper) loaded with 500 µg of (±)linalool. Responses to stimulus pulses of 50 ms duration at intervals
of 0.24 s. a–e: responses to 1st, 5th, 6th, 8th and 9th series of stimulus
pulses delivered from the same filter paper. The numbers of nerve
impulses within the first responses shown in a –e were 16, 16, 15, 11,
and 15, respectively; the corresponding mV amplitudes were 8.6, 6.0,
6.0, 5.3, and 8.3. The intervals between successive series lasted one
min. Lower traces show the velocity of the stimulating airstream registered by a thermistor. The air from the odour-laden airstream was
directed away from the antenna for the first three, four or five pulses
(b, c, and d). Extra trace in e: Calibration 3 mV, 0.2 s. Note a transient
inhibitory effect of linalool on the background activity of the benzoic
acid cell (Pophof and Van der Goes van Naters, 2002).
filter paper, loaded with 500 µg of (±)-linalool, 9 series
of ten stimulus pulses with inter-pulse intervals of 0.24
s. The successive series were separated by 1-min pauses.
The figure shows the responses of the same receptor cell
to series 1 (trace a), 5 (trace b), 6 (trace c), 8 (trace d)
and 9 (trace e). In the traces b, c, and d the 4th, 5th and
6th stimulus pulses, respectively, were the first pulses
directed to the antenna. In the control series 1–4, 7 and
9 all ten pulses were directed to the antenna. Comparing
the response to the first stimulus pulse in a control series
(traces a, e) with the first response to a test series (traces
b–d), a small reduction in the receptor potential and in
the number of nerve impulses can be seen. This means
that from the 1st to the 4th stimulus of a series the odour
source showed a slight ‘fatigue’. The first responses in
b, c, and d were almost equal, indicating comparatively
little further fatigue of the source from the 4th to the 6th
The two types of receptor cells could be discriminated
from each other by the amplitude of the nerve impulses
(about twice as large for the DMH cell as for the benzoic
acid cell). Figs. 2 and 3 show examples of receptor
potentials (i.e. changes of transepithelial potentials) and
nerve impulses recorded from a sensillum trichodeum
upon stimulation with 50-ms pulses. These responses
were produced by individual cells of the two types, when
stimulated with 500 µg of linalool and 10 µg of benzoic
acid, respectively. The receptor potentials elicited in
both cells reached maximum amplitude within less than
100 ms. The subsequent decline was faster in the DMH
cell than in the benzoic acid cell. The receptor potential
Fig. 2. Receptor potentials and nerve impulses of an individual DMH
cell stimulated with 50-ms pulses of 500 µg of (±)-linalool per filter
paper given at intervals ranging from 1.92 to 0.06 s. Lower traces:
airstream velocity as measured by a thermistor.
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R.B. Barrozo, K.-E. Kaissling / Journal of Insect Physiology 48 (2002) 825–834
Fig. 3. Receptor potentials and nerve impulses of a benzoic acid cell
stimulated with 50-ms pulses of 10 µg of benzoic acid per filter paper
presented at intervals ranging from 1.92 to 0.12 s. Lower trace: airstream velocity.
fully returned to the baseline when the inter-pulse intervals were 0.96 s and 1.92 s (Fig. 2 e–f and Fig. 3 d–e).
Consecutive responses were similar to each other for
pulse intervals of 1.92 s. For pulse intervals of 0.48 s
and for smaller intervals the potential changes elicited
by the 2nd to 10th stimulus pulse became smaller than
those elicited by the first pulse. Correspondingly, the
number and frequency of nerve impulses decreased after
the first stimulus pulse. At pulse intervals down to 0.12
s both cells responded with pulse-correlated discharge of
nerve impulses. However, the correlation at intervals of
0.12 s appeared less clear for the benzoic acid cell; the
stimulus-induced modulation disappeared after the first
few stimulus pulses (Fig. 3 a). When stimulated at 0.06
s intervals both cell types—the DMH cell (Fig. 2 a) and
also the benzoic acid cell (not shown)—discharged nerve
impulses uncorrelated with the stimulus pulses.
The nerve-impulse responses of all cells for different
pulse intervals and intensities were evaluated by direct
inspection of recordings (such as shown in Figs. 2 and
3) or of peristimulus time histograms, and by means of
periodograms. Tables 1 and 2 summarize the analyses
for the DMH cells and the benzoic acid cells tested,
respectively. Single asterisks denote the cases of pulsecorrelated responses judged by direct inspection. Double
asterisks indicate that, according to the periodogram
analysis, the periodicity of the nerve impulse responses
was statistically significant and similar to the pattern of
the stimulus pulses. These analyses confirmed that cells
of both types followed pulses with intervals down to
0.12 s. At small intervals or at low stimulus intensities,
both cells often showed responses that were clearly correlated with the stimulus pulses (single asterisks)
although not every pulse elicited a nerve impulse. Under
these conditions, the benzoic acid cell gave uncorrelated
responses more often than the DMH cell. No pulse-correlated responses were found in either cell type with
0.06-s intervals between stimulus pulses (evaluated with
30-ms bins). In conclusion, these data show that the resolution frequency of most cells of each type reached
values of at least eight stimulus pulses per s, while the
correlation of responses and stimuli generally appeared
to be weaker in the benzoic acid cells than in the DMH
cells. There was little dependence of the resolution capability on stimulus intensity.
The time course of the averaged responses (discarding
the first two responses of each series of ten pulses) are
shown by histograms for the three cells of each type
showing the largest responses (Figs. 4 and 5). For both
cell types we selected responses to stimulus intensities
producing large but not maximal peak impulse frequencies. Although with shorter stimulus pulse intervals
the peak frequencies of both cell types became smaller,
the time course of the responses remained similar, at
least down to intervals of 0.48 s.
Typical differences between the two cell types are
made more distinct by combining semilog histograms
(Fig. 6) of the averaged responses at stimulus pulse intervals of 1.92, 0.96, and 0.48 s. The initial burst of nerve
impulses lasted for two bins in the DMH cell; the
impulse numbers per bin declined with a half time of
about 40 ms and reached zero usually after 200–400 ms.
In the benzoic acid cell the impulse burst lasted a shorter
time and the initial decline of the response was as fast
as in the DMH cell. However, the later response
decreased more slowly, with a half time of about 220
ms. The average background level of the three benzoic
acid cells was 2.7 nerve impulses per s.
For studying short-term adaptation and recovery (in
the range of seconds, Kodadová (1996)) the reduction
of nerve impulse responses after the first stimulus pulse
of a series of pulses was evaluated. The reduction of
responses was usually strongest after the first stimulus
pulse in a series. At pulse intervals of 0.12 s the
responses continued to decrease after the second stimulus pulse (Fig. 2 b and 3 a). Since an almost constant
level of reduction was reached with the third response,
we plotted the height of the third response relative to
that of the first response in the same series, as a function
of the stimulus pulse interval (Fig. 7). In the DMH cell
the initial response reduction was 80, or 100% at higher
stimulus strength. Recovery by 50% was reached after
about 0.63 s. With the higher stimulus load (5000 µg)
50% recovery was not yet reached after 1.92 s. In the
benzoic acid cell the initial relative response reduction
was smaller, about 50%. The time for half-restoration of
the response of the benzoic acid cell was 0.35 s; it
increased to about 1.3 s at the higher stimulus load of
30 µg per filter paper.
Since the experiments shown in Fig. 7 include the
R.B. Barrozo, K.-E. Kaissling / Journal of Insect Physiology 48 (2002) 825–834
829
Table 1
Summary showing the periods obtained for benzoic acid cells, the statistical significance (Lomb–Scargle periodogram, p ⬍ 0.05) denoted by ‘∗∗’,
and ‘–’ for no period identified. ‘∗’ for pulse-correlated responses to ⬍ 10 pulses, and blank for not tested
stim. interval
50 µg
500 µg
cell
1.92s
1.9∗
1.943∗∗
1.973∗∗
1.937∗∗
1.964∗∗
1.928∗∗
1.937∗∗
1.928∗∗
0.977∗∗
0.977∗∗
–
0.968∗∗
0.977∗∗
0.980∗∗
0.497∗∗
0.497∗∗
–
0.488∗∗
0.488∗∗
0.491∗∗
0.23∗
0.248∗∗
0.211
0.24∗
0.248∗∗
0.283∗
–∗
0.126∗
0.144
0.114∗
0.126∗
0.117∗
DMH1
DMH2
DMH3
DMH4
DMH5
DMH6
DMH1
DMH2
DMH3
DMH4
DMH5
DMH6
DMH1
DMH2
DMH3
DMH4
DMH5
DMH6
DMH1
DMH2
DMH3
DMH4
DMH5
DMH6
DMH1
DMH2
DMH3
DMH4
DMH5
DMH6
1.937∗∗
0.96s
0.48s
0.967∗∗
0.972∗∗
0.969∗∗
–
0.496∗
0.49∗∗
0.50∗
0.24s
0.288∗
0.276∗
0.261∗
0.12s
–
0.09∗
0.138∗
slight fatigue of the odour sources within a series of
stimulus pulses (see Methods), the true reduction of the
nerve impulse response must have been slightly smaller
than shown in the curves, and the true recovery must
have been slightly faster. A stimulus reduction after the
first stimulus pulse of a series without the influence of
fatigue can be seen in Fig. 1 if one compares the first
response in track b with the response to the fourth stimulus in track a. In addition, the first responses in c or d
can be compared with those to the fifth and sixth stimuli
in the respective preceding tracks b or c.
4. Discussion
Temporal response characteristics of the two types of
receptor cells have been investigated by Heinbockel and
Kaissling (1996) using a quite different stimulus and
evaluation regime than used in our analysis: The stimuli
lasted for 10 s and the responses were evaluated by
means of histograms with 1-s bin width. In spite of a
large variability among the cells of each type, the
response patterns showed characteristic differences
between the two cell types. The DMH cell
(inappropriately termed terpene cell) responded phasically, with an initial burst of nerve impulses and practically no firing during the 2nd–10th s. In response to very
strong stimuli (500 and 5000 µg per filter paper) up to
30 impulses were fired within the first s, after which a
low tonic level of about three nerve impulses per s
appeared and lasted for the stimulus duration of 10 s.
The benzoic acid receptor cell showed tonic responses
of up to about 20 nerve impulses per s to stimuli up to 10
µg of benzoic acid per filter paper. Only at the strongest
stimulus load (100 µg per filter paper) did a phasic
component appear, with about 35 impulses within the
first s of stimulation. The decline of the impulse frequency after stimulus offset occurred within one s for
the DMH cell (at strong stimulation) and proceeded
much more slowly in the benzoic acid cell. The half-life
of the impulse frequency went from about 1 s at 1 µg
of benzoic acid per filter paper to 3 s at 10 µg and more
than 10 s at 100 µg.
In our analysis the bin width of 40 ms in the histograms allowed a much finer determination of the transients of the nerve impulse responses. Upon a single
stimulus pulse of 50 ms duration and a stimulus intensity
producing almost maximal impulse frequencies, both
830
R.B. Barrozo, K.-E. Kaissling / Journal of Insect Physiology 48 (2002) 825–834
Table 2
Summary showing the periods obtained for benzoic acid cells, the statistical significance (Lomb–Scargle periodogram, p⬍0.05) denoted by ‘∗∗’,
and ‘–’” for no period identified. ‘∗’ for pulse-correlated responses to ⬍10 pulses, and blank for not tested
stim. interval
1 µg
3 µg
10 µg
30 µg
cell
1.92 s
1.915∗∗
1.968∗∗
1.956∗∗
1.926∗∗
–
0.963∗∗
–
0.961∗∗
0.967∗∗
0.96∗∗
0.477∗∗
0.429
0.465
0.475∗∗
–
0.24∗∗
–
0.202
1.933∗∗
1.932∗∗
1.926∗∗
1.926∗∗
1.92∗∗
0.962∗∗
0.966∗∗
–
0.967∗∗
0.48∗∗
0.483∗∗
–
1.932∗∗
1.932∗∗
1.934∗∗
1.926∗∗
1.92∗∗
0.969∗∗
0.954∗∗
0.966∗∗
0.963∗∗
0.967∗∗
0.48∗∗
0.477∗∗
0.483∗∗
0.465
0.24∗∗
0.24
–
0.24∗∗
0.24
0.121∗∗
0.121
–
0.124∗
0.09
0.483∗
0.24∗∗
0.24∗∗
0.211∗
0.24∗∗
0.22
0.14
0.127∗∗
0.115
0.12∗∗
0.102∗
1.902∗∗
1.938∗∗
1.938∗∗
1.926∗∗
1.968∗
0.984∗∗
0.955∗∗
0.957∗∗
0.967∗∗
0.99
0.486∗
0.483∗∗
0.48∗∗
0.489∗
0.444
0.234
0.244∗
0.238∗∗
–
0.22
–
0.115∗
0.17
0.09
0.114∗
BA1
BA2
BA3
BA4
BA5
BA1
BA2
BA3
BA4
BA5
BA1
BA2
BA3
BA4
BA5
BA1
BA2
BA3
BA4
BA5
BA1
BA2
BA3
BA4
BA5
0.96 s
0.48 s
0.24 s
0.12 s
0.227
0.12∗
0.103
–
0.114∗
0.12∗
cells responded with a peak frequency which lasted for
two bins in the DMH cell and for only one bin in the
benzoic acid cell. The two cell types differed mainly
with respect to the following decline, which was fast in
the DMH cell whereas in the benzoic acid cell it showed
a fast first phase and a much slower second phase.
This difference in time course of the response to a
single stimulus pulse correlates with the differences
between the two cell types in the resolution of repetitive
pulses. While both cells were able to resolve stimulus
pulses up to pulse rates of at least eight pulses per s,
the DMH cell shows a clearer correlation of stimuli and
responses. With decreasing intervals between the stimulus pulses the time courses remained similar while the
response amplitude was reduced within a series of pulses.
This response reduction, most impressive after the first
of a series of stimulus pulses, cannot be explained by
the slight fatigue of the odour source during a series of
stimuli as demonstrated for the DMH cell and for pulse
intervals of 0.24 s (see Methods). The drop of response
amplitude must mainly be due to response properties of
the receptor cell. The reduction of the response after a
shortly preceding stimulus pulse was called short-term
adaptation by Kodadová (1996). As stated by this author,
this effect does not necessarily represent ‘true’ adaptation, i. e. a stimulus-induced decrease of responsiveness involving an alteration in the transduction pathway.
It could just reflect the dynamic properties of the
responses (Burkhardt, 1960). With smaller intervals
between pulses the response amplitude could be reduced
simply because there is insufficient time for the response
to return to the prestimulus level. The latter was zero in
the DMH cell and about 2.7 nerve impulses per s in the
benzoic acid cell.
If the response reduction merely reflects the response
transient, one would expect the response of the DMH
cell to recover completely about 400 ms after stimulation. Since full recovery took a much longer time,
‘true’ adaptation (reversible reduction of sensitivity) of
the response must have occurred in the DMH cell. This
suggests that the phasic character of the nerve impulse
response discussed above is a result of ‘true’ short-term
adaptation. In the benzoic acid cell the response
reduction was smaller and the recovery faster, while the
decline of the response proceeded more slowly than in
the DMH cell. Here the response reduction mainly
resulted from the dynamic properties of the response.
The weak adaptation in the benzoic acid cell type is consistent with its tonic response character. It should be
noted that in both cell types the recovery from response
reduction after a single stimulus pulse proceeded more
slowly after very strong stimuli, which suggests an
impaired resolution of the individual pulses in such stimuli.
The temporal pattern of response reduction and recov-
R.B. Barrozo, K.-E. Kaissling / Journal of Insect Physiology 48 (2002) 825–834
Fig. 4. Peristimulus time histograms of nerve impulse responses of
three DMH cells (DMH1–DMH3) stimulated with 50-ms pulses of 500
µg of (±)-linalool, at inter-pulse intervals ranging from 1.92 to 0.12 s.
The responses to eight consecutive pulses of each series were summed;
the responses to the first two of the ten pulses of each series were not
included. Ordinate: summed nerve impulses per 40-ms bin. An ordinate value of 10 corresponds to a frequency of 31.5 nerve impulses
per s.
Fig. 5. Peristimulus time histograms of nerve impulse responses of
three benzoic acid receptor cells (BA1–BA3) stimulated with 50-ms
pulses of 10 µg of benzoic acid. For further explanations see Fig. 4.
831
ery for the benzoic acid and the DMH receptor cells was
similar to that found in pheromone receptor cells of the
moth Antheraea polyphemus (Kodadová, 1996). The
recovery of the nerve impulse responses observed in this
species proceeded more slowly than in our study. While
50% recovery in Bombyx females took ⬍1.00 s, in A.
polyphemus males it was reached within 1–2 s (at 18–
28 °C). Correspondingly, the stimulus pulses were
resolved by the Bombyx female cells up to higher rates
than in the pheromone receptor cells of A. polyphemus
(2.5 pulses/s at 18 °C and 5 pulses/s at 28 °C, Kodadová
(1996). Pheromone receptor cells of the moth Manduca
sexta must be able to resolve up to ten stimulus pulses
per s, since projection neurons followed such rates
(Christensen and Hildebrand, 1988). In conclusion, the
cells of the Bombyx female tuned to DMH and benzoic
acid resolved stimulus pulses as well as pheromone
receptor cells.
The value of eight pulses per s can be considered as
a minimum. Firstly, the stimulus duration of 50 ms is
too long to study higher repetition rates. Secondly, at
small stimulus pulse intervals (and also at low stimulus
intensities) not every pulse elicited a response, especially
in the DMH cell, but the responses were well correlated
with the stimuli. The large number of cells per antenna
of both types might allow the moth to resolve repetitive
stimuli at rates even above eight per s, especially for the
DMH cell.
The biological role of the two cell types remains to be
elucidated. Neither cell responds to mulberry, the food of
Bombyx larvae (Van der Goes van Naters, in prep.).
Cells responding to mulberry leaves have been found in
sensilla coeloconica (Pophof, 1997). The benzoic acid
cell responds to the scent of the meconium, the intestinal
waste product of the adults containing benzoic acid, collected during the pupal stage and released upon disturbance of the moths (Heinbockel and Kaissling, 1990).
This cell type could signal the presence of other moths
to the female, even of moths of other species. So far, no
behavioural responses of females to meconium or benzoic acid have been observed. The benzoic acid cell
responds to many other compounds, although with at
least tenfold lower sensitivity (De Brito Sanchez, 1996,
2000; Ziesmann et al., 2000; Van der Goes van Naters,
in prep.).
In addition, the DMH cell responds to other compounds at relatively high concentrations (Ziesmann et
al., 2000; Van der Goes van Naters, in prep.). The third
most effective compound (after (±)-linalool) was 6methyl-5-heptene-2-ol (sulcatol), known as an insect
pheromone (Borden et al., 1976). Although DMH was
most effective among numerous tested compounds the
key compound for this cell might not yet have been
found. Priesner (1979) noted that DMH elicits a characteristic wing fluttering response, which might indicate a
832
R.B. Barrozo, K.-E. Kaissling / Journal of Insect Physiology 48 (2002) 825–834
Fig. 6. Peristimulus time histograms of Figs. 4 and 5 as semilog plots. For each cell type the nine histograms for the pulse intervals between
1.92 and 0.48 s were combined in one diagram. The lines were drawn according to the values for 1.92 s intervals (filled symbols). The hatched
line in the diagram for the benzoic acid cell represents the average background activity of the three cells (2.7 nerve impulses per s) as determined
for the second s after the responses at 1.92 s intervals.
pheromonal effect of this compound (Van der Goes van
Naters, in prep.).
The question remains to be discussed whether the temporal characteristics of the olfactory receptor cells of
female silkmoths Bombyx mori may help to understand
their biological function. Den Otter and Van der Goes
van Naters (1992) stated that the different temporal
response patterns might provide a good adaptation to the
natural stimulus situation. It is well known that males of
several insect species follow turbulent pheromone
plumes by flying faster and straighter upwind, and locating sources more efficiently, than do males following
continuous plumes (Baker et al., 1985; Mafra-Neto and
Cardé, 1994; for review see Todd and Baker (1999).
Bombyx mori males perform upwind turns in response
to each odour pulse during repetitive stimulation up to
a rate of about one pulse per s, and they showed optimal
anemotaxis at three pulses per s, the highest rate tested
(Kramer, 1986). However, they did not orient during
truly constant excitation of their pheromone receptor
cells (Kramer, 1992) as produced by a pheromone analogue (Kaissling et al., 1989). Interestingly, Geier et al.
(1999) found that Aedes aegypti is able to orient to both
continuous and intermittent stimuli, depending on the
odour quality. Intermittence was necessary for CO2 stimuli, whereas orientation to body odour was possible in
homogeneous concentrations.
Our results suggest that insects may resolve rapid
fluctuations in odour concentration not only for pheromones or CO2 but also for other odours. Although the
ability to resolve stimulus pulses does not differ much
between the two cell types investigated, it seems clear
that the DMH cell is better suited to locate punctate
odour sources. The benzoic acid cell is more able to
detect slow changes and constant levels of odour concentrations. These are produced by large-scale odour
sources, for instance the meconium fluid, which covers
large areas on substrates on which many moths are
present.
Acknowledgements
We are very grateful to B. Pophof for invaluable help
in the digitization of the data, to S. A. Minoli and C. R.
R.B. Barrozo, K.-E. Kaissling / Journal of Insect Physiology 48 (2002) 825–834
Fig. 7. Stimulus-induced reduction and recovery of the nerve impulse
responses of benzoic acid and DMH cells at different stimulus loads
(µg per filter paper) of benzoic acid and (±)-linalool. The reduction is
expressed by the number of nerve impulses produced by the third
stimulus pulse of each series of ten pulses relative to the response to
the first stimulus pulse of the series (=1). Ordinate: Averages of relative numbers of nerve impulses per burst for three cells. Abscissa:
inter-pulse intervals in each series evaluated, representing the recovery
time. The curves show the time course of recovery. For the responses
to 5 mg of (±)-linalool (from a single cell) the recovery was incomplete
after 1.92 s.
Lazzari for their help in the time series analysis, to A.
Thorson, A. V. Minor, and M. W. van der Goes van
Naters for critical reading of the manuscript. RBB also
expresses thanks to the DAAD-CONICET for a scholarship. We thank two anonymous referees for valuable
suggestions.
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