Electrophysiological correlates of attraction in Trichoplusia ni

J. Insect t’hysiol., 1973, Vol. 19, pp, 1191 to 1198. Pergamon Press. Printed in Great Britain

ELECTROPHYSIOLOGICAL CORRELATES OF ATTRACTION IN TRICHOPLUSIA NI M. S. MAYER Insect

Attractants,

Behavior, and Basic Biology U.S.D.A., Gainesville, (Received

Research Laboratory, Florida 32601

27 September

Agr. Res.

Serv.,

1972)

Abstract-A

quantitative comparison was made between pheromone-induced electroantennogram (EAG) potentials and the attraction of male cabbage loopers, Tr:!chopZusiu ni. For this comparison, duplicate pheromone dispensers were used in both assays. The slope of the function of evoked EAG potentials was the same as the slope of the function of percentage attraction to different amounts of pheromone, but the EAG was calculated to be about 3 x lo* times less sensitive than the attraction response. Thus, the EAG of T. ni was not a reliable indicator of relative attraction to various batches of synthetic pheromone, and no dif?erence in the evoked EAG was observed between an inhibitor of the behavioural response to the pheromone and the pheromone itself. INTRODUCTION

ALTHOUGH the electroantennogram (EAG) is increasingly being employed for determination of compounds that an insect may perceive as ‘odours’ and as a tool in pheromone identification (ROELOFSet al., 1971a, b); more information about the EAG from greater numbers of insects is required to establish the usefulness of this tool in such studies. In addition, though the EAG is generally believed to represent the algebraic sum of antenna1 receptor potentials (SCHNEIDER,1969), these slow potentials per se are unlikely to convey information to the central nervous system. Consequently, behaviour may not always be predictable from the evoked RAG. The lepidopterous antenna has a large number of specialized receptors for perception of a specific pheromone(s). Thus, the EAG of this order might provide a more realistic prediction of behaviour than that of other orders of insects (SCHNEIDERet al., 1967; PRIESNER, 1968). Also, with male Lepidoptera, it is assumed that a sex pheromone will evoke greater EAG potentials because of this great number of olfactory receptors specific for the sex pheromone. Such, indeed, has been demonstrated with pheromone isomers and analogues for a number of moths including the cabbage looper, Trichoplusia ni (Hi.ibn) (SCHNEIDERet al., 1967; GBANT, 1970; ROELOFS and COMEAU, 1971; ROELOFS et al., 1971a, b). However, some inherent difficulties do exist in using the EAG in odour quality determination, some of which were discussed by ST~~RCKOW (1970) who considers * Mention of a proprietary product of this product by the U.S. Department

in this paper does not constitute of Agriculture. 1191

an endorsement

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M. S. MAYER

the EAG to be unreliable for this use. Other possible exceptions to the assumption that the pheromone evokes the greatest EAG have been noted (ST~~RCKOW, 1965 ; ABUSHAMA, 1966) and this report demonstrates another exception with the behaviourally inhibitory alcohol (cis-7-dodecen-l-01) identified by TUMLINSON et al. (1972). For example, the quantitative correlation between the EAG and behaviour is frequently difficult to determine because different methods are usually used to present the stimulus in the two types of tests. Another and more difficult problem involves the amount of odour released from the dispenser and the question of how much of this odour actually impinges on the chemosensory transducers of the antenna1 receptors. The present paper, therefore, reports an examination of the quantitative correlation between the attraction of the male cabbage looper moth to the synthetic female sex pheromone, cis-7-dodecen-l-01 acetate, and to the EAG evoked by the same dispensing technique and over the same ranges of concentration of the pheromone used in assays of attraction (MAYER, 1973). MATERIALS

AND METHODS

For the present test, moths were taken from a laboratory colony modified pinto bean diet (BURTON, 1969). Th e results are compared obtained in the behavioural assay of attraction made in an olfactometer, tunnel O-3 x O-3 x 3.5 m long (MAYER, 1973). In this earlier test, only flew 2.7 m upwind within 3 min in response to the pheromone were to be attracted.

reared on a with those a Plexiglas@ moths that considered

Dispenser and output quant$ication The dispensers of pheromone used in the present test were the same glass tubes (described by ACREE et al., 1968) that were used in the behavioural studies (MAYER 1973). These tubes were assembled male and female 24/40 ground glass joints (2 cm i.d. x 14 cm long) with the o.d. of the unground ends reduced to 0.9 cm (i.d. 5 mm). Known amounts of pherohone were coated evenly over the inside of the tubes in 0.5 ml of acetone, and the acetone was evaporated by rolling the tube in a low velocity air stream until the odour was no longer noticeable to the human nose (approx. l-2 min). The pheromone released from the tubes over a 3 min interval (25 ml/min rate of airflow) was trapped in a glass capillary tube chilled in a dry-ice-acetone bath, and aliquots were subjected to quantitative gas-liquid chromatography. Electrophysiology and stimulation For the electrophysiological tests, the male moths were restrained by embedding them in Plasticine, and two small wire hooks were used to immobilize the antenna1 flagellum. The evoked potentials were recorded with glass capillary Ag-AgCl electrodes filled with 3 M NaCl. The recording electrode was positioned over the intact distal tip of the flagellum. The reference electrode was inserted into the bloodspace in the basal quarter of the flagellum. The evoked potentials were

ITLECTROPHYSIOLOGICAL

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differentially amplified, monitored visually on an oscilloscope, and recorded on an X-Y recorder. Compressed air (commercial quality) was dried and filtered through activated charcoal in a system terminating in a flexible Teflon@ tube into which were inserted the glass tubes containing the pheromone. The outlet end of the dispenser was located about 1 cm from the centre of the antenna. The air speed at the outlet end of the tube varied linearly with the volume of air: at 50 ml/min, it was 3.7 cm/set, and at 1000 ml/min it was 38 cm/set. A variable time relay controlled a solenoid-operated 3-way valve so stimuli could easily be introduced over various time intervals. The EAG’s recorded with this technique were comparable in form to those obtained by GRANT (1970); also, a similar positivegoing potential was obtained in response to dry air alone (control). Otherwise, most other observations also agree with those of PAYNE et al. (1970). The pheromone standard used in the tests was a large batch (H) of synthetic pheromone that had proved highly attractive in both field and laboratory tests (MAYER, 1973). This sample of pheromone contained about 89% cis-7-dodecenl-01 acetate, 6% trans-7-dodecen-l-01 acetate, and 5% unknown impurities, none of which was cis-7-dodecen-l-01 (TUMLINSON et al., 1972).

RESULTS EAG alrd behavioural threshold responses The comparison between percentage attraction and the EAG in response to various concentrations of pheromone is illustrated in Fig. 1. The line describing the attraction evoked by various amounts of the pheromone (- - - - -) was redrawn from MAYER (1973). No significant statistical difference was evident in the slop~es of the lines measured over at least 4 log units of concentration. Thus, a direct quantitative comparison can be drawn between the behavioural responses and the EAG’s because the airflow through the dispenser was 50 ml/min in both cases. However, this rate of airflow, though appropriate for the 3 min duration of the behavioural experiments, created some rather severe problems for the electrophysiological experiments in which a flow rate of about 1000 ml/min is desirable. The primary purpose in using the low flow rates was to avoid, if possible, the exponential dissipation of the cabbage looper pheromone observed by several investiga.tors (TOBA et al., 1968; SOWER et al., 1971). In fact, it did probably result in a reasonably linear rate of dissipation over a period of at least 10 min (see following), though it created problems in calculating threshold concentrations of pheromone. The following calculations are, therefore, considered to result in maximum divergence between the attraction and EAG thresholds. The ‘behavioural threshold in the olfactometer tests was calculated to be 0.33 ng of pheromone in the dispenser. Emission of 10 per cent of this quantity over a 3 min irrterval would release 3.3 x 1O-2 ng (MAYER, 1973). The amount of air flowing through the olfactometer in 3 min was 4.25 x lo6 cm3. Therefore, full 38

M. S. MAYER

1194

dilution with air would result in a behavioural threshold at 7.8 x 1O-g ng/cm3 air (or 2.1 x lo4 molecules/cm3). For the electrophysiological measurements, however, the outlet end of the dispenser was close enough to the antenna so the dilution factor could be neglected. Then, if 50 msec is taken as the minimum length of stimulus required to evoke a maximum EAG (see following) the amount of air of the data in Fig. diluting each stimulus would be 4.2 x 10m2 cm 3. Extrapolation 1, therefore, gives 0.33 ng of pheromone in the dispenser as the threshold for the EAG (Fig. 1); conversely, from the assumption that the emission characteristics

log

pheromone

cont.,

ng

FIG. 1. Comparison of pheromone-induced attraction (dotted line, from MAYER, 1973) and amplitude of the EAG (solid line, data points). Numbers above points refer to number of EAG’s average (greatest S.E.M. = 0.15 Mv); numbers in parentheses refer to the number of insects tested. Tracings of typical EAG’s at three log concentrations of pheromone. Attraction, 17.9 + 12.1 (log X ng); EAG, O-16 + 0.4 (log X ng). of the pheromone are the same for a 50 msec interval as for a 3 min interval, 9.2 x lo6 ng of pheromone would be dispensed in 50 msec, which would provide 2.2 x lop4 ng/cm3 of air (5.5 x lo* molecules/cm3). This calculated threshold for the EAG is 2.6 x lo4 times greater than that required for attraction. Obviously the factor that could contribute the greatest error to the determination of the behaviour and EAG thresholds is that of dilution. E#ect of duration of stimulus on the EAG At a flow rate of durations of stimulus min of air through decreasing durations

50 ml/min, the magnitude of the EAG was little affected by ranging from 0.05 to 5 sec. However, at flow rates of 1000 ml/ the dispenser, the magnitude of response decreased with (Fig. 2) and the positive-going deflexion in response to air

ELECTROPHYSIOLOGICAL

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increased with increasing duration. Therefore, at least part of the measured EAG a.t the higher flow rate may result from stimulation of mechanosensory receptors that are unaffected at the low flow rate. Thus, the effect of flow rate on behavioural response and the EAG warrants further investigation. lmsec

IOmsec

3Cmsec

60msec

O,isec

2.2 mV

O.SSt?C

Y 24mV

isec

Ij I 26mV

FlG. 2. Change in magnitude of EAG with duration of stimulus. C&-7dodecen-l-01 acetate, 0.5 pg in dispenser; airflow, 1 l./min; Cal., 0.5 Mv, 1 sec. Numbers below each representative tracing are average of five or more EAG’s. Adaptive-type phenomena

At a fiow rate of 50 mlfmin, little adaptation was observed. Generally, 40 to 50 stimuli could be delivered with 10 to 15 set between stimuli without a measurable decrease in the EAG. Also, several moths were subjected to repetitive stimulation of 200 msec duration every 0.5 set: in 21 min (more than 1800 stimuli), there was virtualby no adaptation. However, when airflow was as great as, or greater than, 800 ml/min, an apparent adaptation began after only 7 to 10 stimuli. Obviously, repetitive stimuli at the high Aow rate was occurring before full recovery and resulting in a continuing negative depression with small potential changes superimposed (Fig. 3). Such an effect was also observed by SCHNEIDER(1957) when he used increasingly forceful air streams. The phenomenon occurs in most sensory

Fro. 3. Effect of repetitive pheromone stimuli. Pheromone cont., 1.0 pg; airflow, 800 ml/min.

Upward deflexion of upper line indicates stimulus on.

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M. S. MAYER

systems and is associated with prolonged depolarization of the receptor was also observed with the low rate of air flow and with concentrations mone greater than those tested at 1000 ml/min.

units. It of phero-

EAG response to various odorants Three commercially prepared samples of cis-7-dodecen-l-01 exposed to both the behavioural and the EAG assays. The results in Table 1.

acetate were are compared

TABLE 1

Batch

Relative attraction in laboratory tests

H

Relative attraction in field tests *

++ + S-l-

:;

++ 0 +

EAG

++ ++ +

* E. R. MITCHELL of this laboratory (personal communication) and TUMLINSONet al. (1972). t Contained about 5% cis-7-dodecen-l-01, an inhibitor of pheromonally induced attraction; 89% cis-7-dodecen-l-01 acetate; and 6% trans-7-dodecenl-01 acetate (TUMLINSONet al., 1972). 1 Contained about 15 per cent impurities most of which was the trans-isomer.

The form of the EAG evoked by these three samples was similar (Fig. 4), but batch L evoked an EAG at flow rates of 50 and 700 ml/min that was about Ii per cent smaller than those evoked by the other two batches. This result correlated with the diminished attraction relative to batch H, demonstrated in the field tests, but did not explain the results of the laboratory test. Also, batch K obviously had an inhibitory effect in the field and the laboratory tests, but this effect could H

w.

L

DA

OH

FIG. 4. Typical EAG’s obtained from male cabbage looper antenna with three batches (H, K, L) of pheromone, dodecan-l-01 acetate (DA), and k-7dodecen-l-01 (OH). Airflow, 50 ml/min; quantity, 1.0 pg; DA, 500 pg; duration of stimulus, 0.1 set; Cal., O-5 mV, 1 sec.

ELECTROPHYSIOLOGICAL CORRELATESOF ATTRACTION IN TRICHOPLUSIA

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not be distinguished with the EAG. Also, when the insects were exposed to pure (99+ per cent) inhibitor, cis-7-dodecen-l-01, or to the pure pheromone, no statistical dfferences were apparent in the magnitude or shape of the EAG evoked. Indeed, the EAG evoked in response to stimulation with the saturated analogue of the pheromone, dodecan-l-01 acetate, indicated a sensitivity roughly 500 times less than that to the pheromone (Fig. 4). DISCUSSION

AND CONCLUSIONS

A direct correlation was demonstrated in the present test between the EAG and the behavioural responses to a sex pheromone. However, the EAG was a much less sensitive measure of pheromone quality and quantity than the behavioural assay, as expected (BOECKH et al., 1965; SCHNEIDER, 1969; ST~~RCKOW, 1970). The greatest difference in the two measurements can be explained from the calculation of the amount of air passing the receptors. Smoke generated in the olfactometer from the dispenser was observed to preserve a filamentous character 300 cm downwind from the point of origin at an airflow of 1525 cm/min; thus, complete dilution of the pheromone into the total available air probably did not occur in the behavioural tests, and the actual difference between the behavioural and th’e electrophysiological assessments of the threshold may be less than the calculated difference. SOWER et al. (1971) arrived at a quantity of pheromone for the behavioural threshold differing by only 0.4 per cent from that obtained by MAYER (1973); h owever, the same problems in gas dispersion could have been a factor in their tests. The EAG technique also involves other potential errors. For example, it is sensitiv,e to airflow and temperature changes ; air speed governs the quantity as well as the velocity of the stimulus (ST~~RCKOW, 1970); and the duration of the stimulus may also cause variation. More important, the form of the EAG response did not differentiate various batches of synthetic pheromone that evoked widely different results in behavioural assays. Even the inhibitor of behavioural response evoked an EAG no different in form or amplitude from that evoked by the pheromone itself. Thus, the amplitude or shape of the EAG of T.nicannot be used as an unambiguous assay of its most biologically active odorants though that of some other species may be adequate (ROELOFS et al., 1971a, b). Ack~~oeuledgement-I thank J. H. of pherc’mone

TUMLINSON of this laboratory

for analysing

the amount

dispensed.

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CoZd

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M. S. MAYER

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