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On the relationship between the electroantennogram and simultaneously recorded single sensillum response of the european corn borer Ostrinia nubilalis.

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Archives of Insect Biochemistry and Physiology 85-91 (1983)
O n the Relationship Between the
Electroantennogram and Simultaneously
Recorded Single Sensillum Response of the
European Corn Borer, Ostrinia nubi/alis
Toshio Nagai
Agriculture Canada, Research Centre, University Sub P. O., London, Ontario, Canada
Electrical responses of the whole antenna (electroantennogram) and that of
the single sensillum trichodeum (electrosensillogram) of male moth of the
European corn borer, Ostrinia nubilalis, to their two pheromone components,
(Z)- and (E)-11-tetradecenyl acetates, were recorded simultaneously. The
configuration characteristics of both responses resemble each other, and
demonstrate an interaction between sensilla trichodea. The typical difference
in the response pattern between London (Ontario) and New York strains of
this moth seems t o be a property of the sensillum trichodeum.
Key words: electroantennogram, pheromone, sensillum, European corn borer, olfaction,
sensilla trichodea, Osfrina nubilalis, electrosensillogram, antenna
INTRODUCTION
Electroantennograms have been widely used to study antennal receptor
function of moths. The EAG* can be recorded as a potential difference between the distal end and the proximal base of the antenna. The EAG response is believed to be the summated recording of electrical potentials of
many antennal olfactory receptors excited almost simultaneously by odor
stimulants.
A gradient in the EAG response has been found along the axis of the
antenna of the European corn borer; the response amplitude becomes larger
*Abbreviations: electroantennogram = EAC; electrosensillogram = ESC; sensillum trichodeum = st; tetradecenyl acetates = tda.
I thank Dr D.G.R. McLeod for reading the manuscript and helpful criticism. I am also
indebted to Dr A.N. Starratt for the supply of the chemicals, and to M r G.R. Driscoll and Mrs
M.E. Stevens for their assistance.
Received January 3, 1983; accepted April 15,1983.
Address reprint requests to Toshio Nagai, Agriculture Canada, Research Centre, University
Sub P.O., London, Ontario, N6A 5B7, Canada.
0 1983 Alan R. Liss, Inc.
86
Nagai
toward the distal tip when the whole length was stimulated [l], and the
electrical potentials are summated more effectively in the proximal direction
than in the opposite direction. Moreover, it has been observed recently [2]
that the locally elicited response spreads from the stimulated point more
effectively in the proximal direction.
Considerable research has been conducted on the bioelectric activities of
the receptor unit in a sensillum [eg, 3-91; however, very few of these studies
involved investigation of the summation phenomenon in relation to a single
sensillum response, The present preliminary communication reports the first
simultaneously recorded response of the EAG and that of the single sensillum trichodeum, ESG. Recording from the cut tip of a single sensillum as
studied in this paper was introduced by Kaissling [7], and has also been
shown to be a useful technique for rather short sensilla [lo].
The EAG responses of male European corn borer moths to their two
pheromone components, (Z)- and (E)-11-tetradecenylacetates, were reported
previously [l, 11, 121, and the response characteristics were found to be
different in London (Ontario) and New York strains of the moth. Some of
the following experiments were designed to show whether the characteristic
strain difference resided at the level of the single sensillum.
MATERIALS AND METHODS
Three-day-old adult male European corn borer moths, Ostrinia nubilalis
(Hubn), from two cultures (London (Ontario) and New York strains) at this
laboratory were used. EAG recordings were made by a modification of the
method described before [l]. The excised antenna was bridged horizontally
between two small distal and proximal pools filled with saline. The distal
two or three segments were removed. An Ag-AgC1 electrode was placed in
both pools, and the distal electrode was connected to a Tektronix 502 oscilloscope through a neutralized input capacity amplifier (Bioelectric NF1). The
ground lead was attached to another Ag-AgC1 electrode placed in the proximal pool.
In order to record the ESG response from a single sensillum, the tip of a
randomly selected st was cut off by using two glass capillaries. Two salinefilled glass pipettes, routine glass microelectrode-type [eg,13], with obliquely
broken tips (C and RE in Fig. 1)were operated by micromanipulators, and
the tip of the st of about 5 pm was severed off by the smaller capillary edge
C (external diameter about 25 pm) on the internal surface of the capillary RE
(internal diameter about 30 pm), which is served as a cutting board as shown
in Figure 1.An Ag-AgC1 electrode is placed in RE capillary and is connected
to the same oscilloscope as the one for the EAG recording through another
identical amplifier. Therefore, the cutting board RE also served as a recording
electrode; ie, the bioelectrical activity of the sensillum can be registered from
the instant of cutting off without letting the severed tip become exposed to
the air. The proximal electrode for the EAG recording was also used for the
ESG as the indifferent electrode. The response amplitude obtained from an
st could be changed according to the amount of the sensillum length covered
Electroantennogram of the European Corn Borer
87
Fig. 1. Cutting device of sensillurn tip. S, single sensillurn; RE, recording electrode capillary
which i s also served as a cutting board; C, cutter capillary.
by RE (see Results and Discussion). The response was largest when the RE
was slipped over 50-60% of the sensillum length from the tip; ie, 50-40%
from the base was exposed to the stimulant. Therefore, all the recordings of
the single st response were conducted with 50-60% of the length covered
unless stated otherwise.
The device for application of the stimulant pheromones, (Z)- and (E)-11tetradecenyl acetates was described elsewhere [I, 121. The antenna was held
so as the ventral side faced the stimulant, and about 70% of the whole length
excluding both ends was stimulated for a measured time with a rotary valve
controlled by a solenoid. An interval of at least 2 min between stimuli was
given to avoid adaptation to odor.
The amplitude of EAG and ESG responses decreases with time (see Results and Discussion). For a comparison of the amplitude of response under
different experimental conditions such as in Table 1, amplitude-time curves
were prepared for each experimental condition over a period of 90 min, then
the response amplitude at a certain instant after excision was extrapolated
from the curves. The present communication did not focus on the nerve
impulse which could be obtained with ESG response by AC-amplified recording. All recordings were at room temperature (20-22°C).
RESULTS A ND DISCUSSION
Similarity of EAG and ESG Configurations
The EAG and ESG recordings obtained were consistent for any given
antenna1 preparation, but there was variabililty in response amplitudes between preparations. The EAG configurations evoked by the two pheromones, (Z)- and (E)-11-tda,have been reported previously [l];ie, a relatively
fast initial negative potential fall is followed by a slow plateau during the
time of stimulation, and then the response gradually returns to the initial
88
Nagai
TABLE 1. ESG Responses of European Corn Borer (London, Ontario Strain) to (Z)- and
(E)-11-tetradecenyl Acetates (20 pg)
Exposed length (YO)
Response height elicited
by (Z)-tda (mV)
Response height elicited
by (E)-tda (mV)
Response ratio, (E)/(Z)
70
3.7
50
4.4
40
5.7
20
4.1
0
3.8
2.6
3.5
4.1
3.2
2.9
0.70
0.80
0.72
0.78
0.76
Various lengths of a single sensillum trichodeum were exposed to the stimulant. Time after
excision of the antenna: 60 min.
Fig. 2. Simultaneously recorded EAC (upper traces) and ESG (lower traces) responses of
European corn borer. A and B were from same preparation of London (Ontario) strain; C and
D were from same preparation of New York strain. A, C) responses t o (Z)-11-tda, 20 pg; B, D)
responses t o (E)-11-tda, 20 kg. Forty percent of the sensillum length was exposed to stimulant
for ESG. Parallel lines show the time of stimulation: 0.5 sec. Calibration: 5mV. Time after
excision of the antenna: 37, 34, 23, and 26 m i n for A, B, C, and D, respectively.
level with a half decay time of 1-2 sec. Typical examples of the EAG response
and the simultaneously recorded response from a single sensillum, ESG, are
shown in Figure 2. The configurations of the ESG were found to be remarkably similar to those of the EAG. However, it is rather difficult to draw out
any quantitative relationship from simple comparison of the response configurations of the EAG and ESG. The EAG recordings with the cut tip preparation may have a reduced elicited potential amplitude because of a short
circuit through the antenna1 hemolymph at the cut distal edge. Moreover,
the underlying equivalent electrical system has not yet been clearly deter-
Electroantennogramof the European Corn Borer
89
mined, although spread phenomenon of the locally elicited response potential has been studied and discussed recently using a hypothetical circuit
model [2].
It is evident in Figure 3 that the amplitude of EAG and ESG responses
both decreased throughout the life-span of the preparation. Their decay
patterns obtained from 32 preparations were observed to be closely parallel.
An antenna1 preparation with rather short, or long, life-span of the EAG
generation also showed a similar life-span of ESG, suggesting the EAG and
ESG are physiologically related to each other. Figure 3 shows a typical
example. The present observations on the similarity of the response shape of
EAG and ESG, and on the correlation of their amplitude-time curves suggest
that the two responses are generated from excited olfactory cells and the
ESG is an unit component of the EAG.
Di f f e r e nc e
in Responses to (Z)-
a n d (El-11-tda
It has been reported 1121 that although the responses to both (Z)- and (E)11-isomers decreased with time, the ratio of these response amplitudes at a
particular time after excision remained fairly constant, and that the average
ratio was significantly different between two strains. The response ratio (El
Z) was 0.70 and 0.97 for the London (Ontario) and New York strains respectively. In the present study the response difference between strains was also
observed in the ESG, and the ratio was found to be coincide with that of
EAG-ie, 0.73
0.04
SE) (n = 18) and 0.96 f 0.04 (n = 9) for the
London (Ontario) and New York strains respectively.
(x
i;
6.0
0
Q
0
5 .O
w
U
3
Q
O
Q
0
-4.0
._
-c
a
O
0
0
-3.0
mIn
5
80
-2.0
I0
n
0
a
t
I
Q Q
000
0
'.O
50
100
200
150
1
Time
min
Fig. 3. EAG and ESC amplitude-time curves for European corn borer (London, Ontario
strain). Fifty percent of the sensillum length was exposed to the stimulant for ESC. C), EAC
response to (Z)-11-tda,20 pg; 0 , EAG response to (E)-11-tda,20 pg; Q, ESG response to (Z)-lltda, 20 pg; (3, ESG response to (E)-11-tda, 20 pg.
90
Nagai
The sensilla trichodea are distributed uniformly on the ventral side of the
antenna [12]. A random selection of 103 st from 70% of the whole length of
the antenna excluding both ends were examined, and none was found to be
differently specialized in the sensitivity. Therefore, the response pattern
typical of each strain seems to be a property of all st. It is unlikely that other
st having a different response characteristic exist near the unexamined tip or
the base of the antenna.
Responses of a Sensillurn With Different Length Exposed to the Pheromone
The sensillum tichodeum type A, believed to be specialized to detect the
female sex pheromone, is about 40 pm long [14]. When the st was gradually
covered by the recording capillary electrode (RE in Fig. 1)from the distal cut
end, the elicited response amplitude changed. it was thought that as more
of the st surface was covered, less area would be exposed to the stimulant,
and hense fewer pores on the st through which the stimulant odor molecules
presumably penetrate would be reduced, resulting in a reduction of the
response. However, the recorded ESG amplitude reached a maximum when
40-50% of the st length was exposed as shown in Table 1. If the recording
electrode was slipped toward the tip or the base of the st, the ESG response
became significantly smaller.
It is difficult to understand why the sensillum exposed more than 40-50%
of its length elicits a smaller response. Perhaps the high electrical resistance
through the sensillum structure near the tip accounts for the potential drop
between the recording electrode and the indifferent electrode. Response
reduction with a length shorter than 40-509'0 could be attributed to the
decreased number of pores available for stimulant penetration. It should be
noted here that a response could be recorded even with 0% exposure (Table
1);ie, the whole length was covered by RE, and as a result the sensillum
could not receive stimulant. This suggests an interaction of the response
from another source, possibly the surrounding sensilla.
it has been established previously [l]that the EAG response spreads from
the stimulated antennal region to neighboring unstimulated locations. This
EAG spread phenomenon may explain the ESG response to 0% length
exposure. On the other hand, this result indicates that the response of a
single sensillum without any neighboring activity interference cannot be
recorded, at least by the present device. In relation to the influence from the
adjacent sensilla, it is noteworthy that the elementary responses from one
sensillum of the silkworm moth antenna has been successfully studied by
nerve-blocking chemicals applied to the cut tip of the neighboring sensillum
next to the sensillum under investigation [15].
The present results indicate that the EAG response is related to the ESG.
The ESG potentials are assumed to contribute to the generation of the EAG,
but probably not by a simple summation. it has been frequently pointed out
[eg, 16, 171 that the EAG can only give information on the antennal sensitivity
to test chemicals, because of its summated nature of responses from many
receptor cells. The ESG method has been found to be adequate for the study
of the unit receptor cell response [6, 10, 181. Kaissling [7] has stated that the
attractiveness of the sex pheromone can be determined by the central nervous system on the basis of the relative level of responses in the different
types of olfactory cells, and that even a small region of antenna-in principle,
Electroantennogram of the European Corn Borer
91
a single sensillum-can provide the specific activity ratio needed. However,
it seems to be difficult not only to clearly separate the responses of more than
one receptor cell in the same sensillum, but also to isolate the single response
of a sensillum from the interaction of the neighboring sensilla. The observations in the present and the previous studies [I, 21 indicate the existence of
an activity interaction among sensilla, although the means of their communication is still unknown. Also remaining to be explained is how an individual ESG response can contribute to the overall response of the EAG. An
understanding of the equivalent electrical circuit network involved in the
ESG and EAG recordings will provide clarification.
LITERATURE CITED
1. Nagai T: Electroantennogram response gradient on the antenna of the European corn
borer, Ostriniu nubilulis. J Insect Physiol 27, 889 (‘1981).
2. Nagai T: Spread of local electroantennogram response of the European corn borer, Osfvinia
rmbilalis. Pestic Biochem Physiol 19, 291 (1983).
3. Schneider D: Electrophysiological investigation on the olfactory specificity of sexual attracting substances in different species of moths. J Insect Physiol 8, 15 (1962).
4. Schneider D: Insect olfaction: deciphering system for chemical messages. Science, Wash.
263, 1031 (1969).
5. Thurm U: Basics of the generation of receptor potentials in epidermal mechanoreceptors
of insects. In: Mechanoreception. Schwartzkopff J, ed. Abh Rhein Westf Akad Wiss,
Opladen, Westdeutscher Verlag, p 355 (1974).
6 Kaissling KE: Sensory transduction in insect olfactory receptors. In: Biochemistry of
Sensory Function. Jenicke L, ed. Springer, Berlin, New York, p 243 (1974).
7 Kaissling KE: Recognition of pheromones by moths, especially in Satiirniids and Borribyx
rriori. In: Chemical Ecology: Odour Communication in Animals. Ritter FJ, ed. Elsevier/
North-Holland Biochemical Press, Amsterdam, p 43 (1979).
8. Kaissling KE, Thorson J: Insect olfactory sensilla: Structural, chemical and electrical aspects of the functional organization. In: Receptors for Neurotransmitters, Hormones and
Pheromones in Insects. Sattelle DB, et al, eds. ElsevieriNorth-Holland Biochemical Press,
Amsterdam, p 261 (1980).
9 Erler C, Thurm U: Dendritic impulse initiation in a n epithelial sensory neuron. J Comp
Physiol 142, 237 (1981).
10 Den Otter CJ: Single sensillum responses in the male moth Adoxoph!/es o r m o (FvR) to
female sex pheromone components and their geometrical isomers. J Comp Physiol 121,
205 (1977).
11. Kochansky J, Carde RT, Liebherr J, Roelofs WL: Sex pheromones of the European corn
borer (Ostririia riubilnlis) in New York. J Chem Ecol 2 , 225 (1975).
12. Nagai T, Starratt AN, McLeod DCR, Driscoll GR: Electroantennogram responses of the
European corn borer, Osfriniu rnrhilrrlis, to (Z)- and (E)-11-tetradecenyl acetates. J lnsect
Physiol, 23, 591 (1977).
13. Nagai T: Insect visceral muscle. Responses of the proctodeal muscles to mechanical
stretch. J Insect Physiol 76, 437 (1970).
14. Cornford ME, Rowley WA, Klun JA: Scanning electron microscopy of antenna1 sensilla of
the European corn borer, Ostriniu nuhilalis. Ann Entomol Soc Am 66, 1079 (1973).
15. Kaissling KE: Action of chemicals, including ( + ) trans-Permethrin and DDT, o n insect
olfactory receptors. In: Insect Neurohiology and Pesticide Action. Rickett, FE, ed. Simp
Soc Chem Industry, London, p 351 (1980).
16. Kramer E: Insect Pheromones. In: Taxis a n d Bahavior. GL Hazelbauer, ed. Chapman and
Hall, London, p 205 (1978).
17. O’Connel RJ: Olfactory receptor responses to sex pheromone components in the red
banded leafroller moth. J Gen Physiol 65, 179. (1975).
18. Van der Pars JNC, Den Otter CJ: Single cell responses from olfactory receptors of small
ermine moths to sex-attractants. J Insect Physiol24, 337 (1978).
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