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