Olfaction in the American roach as studied by nerve potentials

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QWACTIOH IN THE AMERICAN ROACH AS STUDIED BY 3= = NERVE POTENTIALS

by Chester Crosby Roys

A thesis submitted In partial fulfillment of the requirements for the degree of Doctor of Philosophy In the Department of Zoology in the Graduate College of the State University of Iowa June 1950

ProQuest Number: 10583788

All rights reserved INFORMATION TO ALL USERS The quality o f this reproduction is d e p e n d e n t upon th e quality o f th e copy subm itted. In th e unlikely e v e n t th a t th e author did not send a c o m p le te manuscript and there a re missing pages, these will b e n o ted . Also, if m aterial had to b e re m o ve d , a n o te will indicate th e deletion.

uest ProQuest 10583788 Published by ProQuest LLC (2017). Copyright o f th e Dissertation is held by th e Author. All rights reserved. This work is p ro te c te d against unauthorized copying under Title 17, United States C o d e Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

AOKHOWLBDGMEOT I wish to express my sincere appreciation of the assistance and encouragement given me in this research by Dr, Gordon Marsh and Dr, Theodore Jahn*

11

TABLE OF CONTENTS Pag© Intro&uet ion

......

1

Materials

4

Methods

5

Application of Olfactory Stimuli «•*•»••.«•••••«*« Recording of Olfactory Stimuli «•••«»•#•••*•.*•»•• Application and Recording of Other Types of Stimuli

5 11 IS

Results « * • « * » . • * * « » * « » • • « » • • • « « . • * *, **«* *

SO

Antenna® Maxillary Palpi t»gaaaj3««. Tarsi ** ... •, •. •.» ,•... os... o•o. ».*

BO 23 25 26 26

Ol SCUS 8 1On

2*7

Summary

29

Bibliography

30

iii

TABLE OF FIGURES pa 09 FigUr© i

Apparatus for Applying Olfactory Stimuli ***9

6

Fi glia?© 2

QctOr OtiamUer

8

Figure 5

FI oating Balance »• ®o»

Figure 4

Method of Confining the Roach •

12

Figure 5

Action Potential Records *«s»»

22

iv

»

«

s»« 10

TAB&E CF TABXES page Table

I

Table 11

Olfactory Thresholds of the Antennae »«,•** 21 Olfactory Thresholds of the Maxillary

Palpi

.***,.....

r

24

1

1HTR0DTOTI0H Investigation of insect olfaction has in the past been dependent on the observation of behavior responses. This has told us of the olfactory ability of the insect only in so far as these could be directly correlated with behav­ ior.

The early naturalist observed Insects coming to odorous

foods* and made the simple and apparently correct assumption that the Insect could smell that particular food odor*

In

the natural habitat the Investigator found it difficult to determine in what dilution th© Insect could detect the odor* so insects were brought into the laboratory and placed in various sorts of containers or olfactometers.

Air* bearing

odors of known concentration* was blown on them and their response observed.

If they moved toward or away from the

source of odor of a given strength* if they waved their an­ tennae or showed other unusual activity* the Investigator inferred that they could sense that odor at that concentra­ tion.

Whether concentrations which produced these responses

were true olfactory thresholds or merely the concentration at which the insect became Irritated enough to make some ob­ vious movement was not demonstrated.

Whether the failure to

respond indicated that the odor was not sensed or only that it produced no specific activity on th© part of th© Insect

2

again could not be proven by this method,

(Mclndoo 1915j

Glaser 19271 Abbot 1937)* In the Y-tub© olfactometer odorous and non**odorous air Is blown into the two branches while the insect enters at th© has® of th© v and advances into th© most attractive branch*

A series of tests comparing various odors with

pur© air and with each other shows which ones r©pel* attract* or produca no response*

Olfactometers of this type have been

greatly refined in recent years and have given excellent re­ sults as used* but they still have the limitation that they show only what the Insect reacts to and not what it senses, (Barrows 1907$ Hoskins and Craig 1939| Dethier 1941* *45)* This criticism cannot be applied to the work on conditioned reflexes for there th© insect is rewarded by food for demonstrating its olfactory acuity*

The insect is

trained to com© to food artificially scented with a given odor and then th© food is removed and the Insect tested with the odor alone.

Thus It has an Incentive to come as quickly

as possible to an odor which it might normally find without attraction or repellent.

Unfortunately this method is not

as well adapted to all types of Insects as to th© bees on which th© method has been used so successfully,

(v, Frisch

1926), With these limitations in mind work was undertaken

3

to record action potential® from nerves leading to various structures suspected of bearing olfactory receptors*

This

approach* in so far as th© method is successful* gives posi­ tive thresholds for any odor* whether th© animal be repelled* attracted* or indifferent to it* separates receptor response from general activity response* and is applicable to all in­ sects large enough to allow placement of the electrodes. Since olfactory receptors ar© more widely separated in in­ sects than in vertebrates* th© method also offers a means for further study of th© general nature of olfactory perception.

MATERIALS Th® American roach, Poriplaneta americana, was chosen as the insect for investigation because of the large sis© and the ease with which a laboratory supply could be secured and maintained.

Only adult males and females were

used* The test materials used were, with on© exception, taken from the Crocker-Henderson Odor Classification Set prepared and sold by Cargille Scientific Inc, of New York, This set was prepared primarily for testing food, perfume, and industrial odors, and v#as chosen because it is stand­ ardized as to quality and concentration of ingredients and can be duplicated for future work.

Although the set as

supplied contains 32 vials of material only 19 of these odors were used*

In addition, oil of cloves (Hutchinson USP)

was taken from chemical supplies available*

Th© odors sel­

ected ar© of a wide variety and include examples of th© flowery, fruity, spicy, burnt, resinous, acid, and eaprilic odors of Zwaar&emaker (1895), Henning (1916), Crocker and Henders on (1927)•

5

METHODS Application. Of. Olfactory Stimuli Olfactory stimuli wer© applied by a stream of air which carried the odorous material in carefully regulated concentration*

The apparatus is shown in Fig* 1*

Compressed

air at a constant velocity of 1*075 liters per minute and 25 per cent average relative humidity entered at a*

With

valves A and 0 closed It passed down tube abefk through charcoal f i It or V ' and out at 1*

Th© upper loop bode con­

trolled relative humidity between 25 per cent and 85 per cent. With valve A opened and valve B closed all the incoming air passed through Drechsel low form gas washing bottles 1 and II filled with strips of wet filter paper. was then measured by flow meter VI.

Th© total air flow

This was a capillary

tube orifice flow meter made to order*

The capillary was a

short tube with a constriction in it Inserted into two rub­ ber stoppers*

It could be replaced by one with a constric­

tion of a different diameter to change the range of the flow meter.

Headings wore In millimeters of water and were

# Since It was difficult to have an all glass sealed system with the type of flow motors used, only section ghi.jk with branch jl, was glass sealed, and filters of pea sized*activated charcoal in 8 inch test tubes were installed at g and k to remove any odor from rubber connections in preceding parts of the system or from the compressor.

6

Eig* 1 Apparatus for Applying Olfactory stimuli. Explanation: I andII > wash bottles filled with strips of wet filter paper; III, odor chamber; IV and V, charcoal filters; VI and VII, flow meters; A, B s G, and D, two-way glass stopcocks; E, three-way glass stopcock; a b c d e f g h i j k l , glass tubing with rubber connections*

7

calibrated to liters of air per minute experimentally*

By

controlling valves A and B th© proportion of air through the wash bottles could be decreased to any desired frac­ tion of the total flow*

Thus if the reading on flow meter

71 was 10 mm* for the total air flow, a reading of 5 mm* indicated that half th© air was being humidified in th© wash bottles and half was not* The lower loop fghi.1 controlled odorous air introduced

the proportion of

into th© total air flow*

This was

measured by flow meter¥11 and controlled by valves G and D in th© same way as for

humidity*

Valve E was a three way

stopcock with a discharge to th© outside so that th© odor chamber III and connecting tubing could be nrlnsedn by air discharged from the system through E without disturbing th© insect at 1®

Corks used to close the odor chamber were

changed after each test and exposed to the air in a warm place for at least five hours before being used again* Th© samples of test material in glass vials of one centimeter inside diameter fitted snugly into the odor chamber III*

Incoming air was directed down onto the sur­

face of th© material and then ©ddied up and out the discharge opening (sea Fi,g« 2)*

The concentration of test material

in grams per liter of air was determined under conditions Identical to those obtaining during th© olfaction tests*

8

SI

r vVl

Fig. 2 Odor Chamber Explanation: Vial of odorous material is shown in posi­ tion for olfaction test.

9

For the more volatile materials this was done by weighing the tube of test material on an analytical balance accurate to G«1 mg*, leaving it in the odor chamber for several hours with a fixed rat© of air flow, and reweighing*

The weight

loss divided by the total liters of air passed over the sample gave grams of odorous material per liter of air* For the less volatile materials a floating balance wad devised which registered small weight losses within a reasonable length of time*

This Is shown in Fig* 3*

Th©

sealed glass float £ has a piece of No* 29 wire n passing through it which supports a glass cup ra of 1 cm* inside dia­ meter counterbalanced by weight &• the float centered* supported below

A wire guide £ keeps

Th© float is in avessel

of water VIII

the odor chamber III, and th© cup m is

within the odor chamber at approximately the same height as a test material

in a via3.*

Th© bottom of the chamber is

closed by water

in vessel VIII*

In us© the cup was filled with test material until the float just sank*

As the material evaporated, .the

float rose, and because of the small diameter of wire n, each millimeter of rise represented a loss of 62*5 micro­ grams*

When th© top edge of th© cup was observed through

a horizontal microscope ©quipped with an ocular micrometer, a rise equivalent to a weight loss of 1*0 microgram was

10

IE

w

Fig* 3 Floating Balance explanation: I H * odor chamber; VIII, glass vessel; m / glass cup; n, wire; o, guide; p, float; q* weight*

11

easily observed#

The roach was anesthetized with carbon dioxide and confined as shown in Fig* 4*

It is essential that the

part tested be completely immobilized, since any movement of th© electrodes causes electrical disturbances which resemble action potentials.

The bases of the palpi were

sometimes embedded in wax of very low melting point to hold them rigid* Us© of th© whole animal with normal blood and air supply to the sense organs was considered preferable, but any general activity of the animal gave the appear­ ance of a sensory nerve response*

Therefore all tests were

repeated on isolated parts to insure that the electrical activity noted was due to response of that part to th© stimulus and not to reception elsewhere and secondary activity in th© part studied*

Such preparations were usu­

ally short lived, but some gave good responses for several hours when kept moist with a cotton wick leading from a drop of insect Ring©r*s solution (Blifer 1934) to th© cut end of the appendage. The electrodes were of Mo* 54 tungsten wire one centimeter long soldered to Mo. 28 enameled copper wire and mounted in small lumps of soft dental wax*

The tip of th©

X' '

*

rr

Method of Confining the Roaoh Explanation : A, Incite block hollowed to receive roach, and yoke to secure head; B, roach confined by yoke and scotch tape with wax supported electrodes in place.

13

electrode was ground to as fine a point as possible.

A small

hole was mad© in the appendage with a minute nadel or specially sharpened steel needle and th© electrode inserted into this hole just far enough to insure good contact with the tissue. Th* placing of th© electrodes is th© vital part of th© tech­ nique, and even after months of practice it was considered good if 50 per cent of th© preparations gave recognizable responses*

Electrodes were located in one of three ways.

On th© intact animal they were placed one to two millimeters apart at the joints of the appendage, or on© electrode was stuck into th© head or body at th© base of the appendage and one placed at a joint on© to two millimeters from th© base. On th© isolated appendage on© electrode was put into the cut basal end and the other inserted at a joint on© to two milli­ meters away*

In such small appendages it seemed that th©

electrode must always contact some nerve fibers, but which ones it contacted and how badly it damaged them seemed en­ tirely a matter of chance* Th© Insect or appendage with the electrodes in place was put into an aluminum box 16 centimeters long, 14 centimeters wide, and 8.5 centimeters deep Inside and covered with a removable glass plat© sealed In place with vaseline.

Tube 1 (Fig* 1) entered th© box in the center

of one end, and th© insect was so placed that the part to

14

be tested was one to three centimeters from the end of the tube and directly in the air stream from it#

Therefore no

allowance was made for dilution of the odorous material in the box sine© most of the dilution there took place after th© insect had been stimulated* not before#

Th© leads from

th© electrodes w©r© attached to binding posts in th© box and these in turn connected to th© amplifier*

The metal

box was grounded and all leads outside the box were shielded and grounded#

It was impossible to keep th© box entirely

free of extraneous odors* since of necessity It contained materials for mounting th© insect* the electrodes, and various wiring*

It seems reasonable to assume, however,

that th© recorded responses of th© Insect are responses to odor or lack of odor In th© air stream from 1 and not re­ sponses to general conditions in th© box# Th© experiments were performed at room temperature maintained at 23 °

±

1° C»

Th© amplifier was an Offner Type A Electroenceph­ alograph unit.

Results were recorded on an Offner Crysto-

graph ink writer*

Frequent checking on an oscilloscope

showed no significant oscillations of higher frequency than could b© recorded by the Cryptograph#

Responses of

10 microvolts could be distinguished above the has© line under average conditions,

15

The antennae of raal© and female adult roaches were tested for responses to twenty odors*

All thresholds

war© recorded from complete animals with on© electrode placed In the head at the has© of the antenna and the other at some joint between th© tenth and fifteenth*

The vial of

test material was placed In th© odor chamber III* the cham­ ber closed* and valve C opened to pass 0*25 per cent of th© total air through th© chamber*

If no response was noted in

40 seconds* the flow through th® odor chamber was raised by closing valve D to 0*50 per cent and then to 1*00 per cent* 2*50 per cent* 5*00 per cent and 10*00 per cent successively at 40 second Intervals until a response was noted*

After a

test th© roach was allowed to rest for an hour with odorless air from 1 flowing over it*

Meanwhile the loop of apparatus

f&hi was rinsed with 5 per cent of the total air flow which was discharged from the system through valve E*

Each hour

the procedure was repeated with another odor until the series of 20 odors was completed or the roach ceased to respond* When tests were interrupted for eight hours overnightp the threshold taken just after th© rest was usually lower than on© taken just before it for the same odor*

This Indicated

that a rest longer than one hour between tests would be \

desirable* but It was felt that confining the roaches for more than th© two days already required would bring in more

16

undesirable factors than It would eliminate*

If riven a

little water with a pipette the roaches usually survived the tests for 20 odors, but the same animal was never used for a second complete set of tests, and with few exceptions each threshold for a given odor Is from a different animal® Odors were tested in different sequence on different animals to guard against one odor consistently being affected by the one which proceeded It in the series®

Blanks were run

frequently with chamber III empty to Insure that no stimulate ing odor was retained In the apparatus from one test to the next* Application .and Be cording of Other Type a of Stimuli Sine© all nerv© responses and many olectrical disturbances from other sources look very much alike on the record, it became necessary to eliminate as many undosirod responses as possible and to set up some criterion for de­ termining acceptable ones®

To this end tests were made

with other types of stimuli to determine their effect on receptors in the appendages studied® Touching th© animal anywhere with a rod or needle often Induced a static disch&rg© which appeared as a spike on the record, so appendages were tested for movement by blowing air on them through a pipette*

The nerve response

to a single movement was a single strong spike followed by

17

two or three much smaller ones*

Antennae, palpi, cerci*

and tarsi both on th© animal and as separate preparations, all responded in substantially th© same way.

Antenna©,

palpi, and particularly cerci were found to be especially sensitive to slight movements of air.

Experiments which

might cause a change in rate of air flow were always checked against the effect of changes in air flow alone.

It

was

necessary to substitute strips of wet filter paper for water In th© wash bottles because bubbling through th© water produced a fluctuating air flow detected by the cerci. Antennae, palpi, tarsi, and cerci were tested for response to sounds In the room and close to the box containing the insect with the box covered and open.

The

only responses noted were such as could be attributed to gross vibrations such as tapping the box or holding a low frequency tuning fork very close to the appendage.

Ho attempt

was mad© to duplicate the work of Pumphroy and H awd on-Smi th (1936) on th© cerci as organs of tearing.

The work with th©

other appendages did not indicate that any of them are organs of hearing as we understand th© sens©. All th© appendages studied were checked for re­ sponse to light by darkening th© box with an opaque cover and then quickly removing it and vice versa, to changes in light was noted in any case.

Ho response

18

Each of th© appendages was also tested for response to increase in temperature by bringing a hot needle to within two millimeters of it#

This was repeated many times with

the needle ranging from warm to red hot.

The antenna© and

maxillary palpi were also tested for response to a decrease in temperature by bringing a chilled pioce of Iron near them* No satisfactory evidence for th© presence of temperature receptors was found for any of the appendages tested* This informat ion made it possible either to eli­ minate thes© stimuli or to discount responses obviously du© to one of th©m*

But in addition to the effects of these

stimuli* the insect sometimes showed a response when no known stimulus had been applied*

Therefore it was neces«

sary to rely on timing as the final criterion for selecting true responses to the applied stimulus*

A wav© or series

of waves was considered a valid response only ?fh©n no such disturbance had occurred prior to th© application of th© test stimulus* when the disturbance closely followed th© application of the stimulus* and \¥hen the disturbance was not repeated for some time after th© obvious effect of th© stimulus had disappeared,

In tests of stimuli with unknown

effect* a record was mad© for two or three minutes preceding stimulation and for two or throe minutes after the test was apparently complete.

Tests repeating stimuli previously

19

round effective were run for only about one half minute before and after th© test.

Any tests which shewed a response

less than two seconds or more than thirty seconds after stimu­ lation were considered questionable and carefully checked*

20

RESULTS Antennae Boaeh antennae wore first tested for response to a high concentration of odorous material in the air until responses were obtained•

Then th© next group of animals

was tested at a lower coneentration.

This was repeated

with five successive groups of insects as techniques were improved^ the results of the final tests being recorded in Table X*

Ho difference was noted between males and females*

Responses at the lowest concentration used were considered too weak to warrant further dilution of the odorous material. In addition th© antenna© were tested with water vapor and were found to respond to a change from 25 par cent to 40 per cent and from 40 per cent to 25 per cent relative humidity. A typical record of antennal nerve response to stimulation is shown in Fig* 5, A«

It Is diphasic as might

be expected from the position of the electrodes in the antenna.

The response may be presumed to com© from a num­

ber of nerve fibers and shows no Individual spikes but a composite wavo pattern with increase in height as the stimulus was increased*

Fig. 5^ B shows a pattern of

response similar to that of a single nerve fiber.

Such

results were extremely rare with th© technique used.

Since

21

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24

25

Isolated palpi were also tested*

No consistent differences

between palpi of male and female roaches were noted although the variation between individuals was as great as in the antennae*

It was seldom possible to test both th© antennae

and palpi of the same insect, but when it could be don© th© thresholds were found to be approximately th© same for both* The records of responses resemble those mad© with antennae, but are of slightly lower magnitude*

The responses to water

vapor were also similar* X*abial Palol Because of th© difficulty in immobilising them, thresholds from the labial palpi on th© intact Insect were not considered reliable*

Therefor© only responses from

excised palpi were considered,.

These war© of much lower

magnitude than the responses from th© antenna© and maxil­ lary palpi, and no thresholds could be established for them which were considered satisfactory*

However, out of some

25 trials with different palpi positive responses to high concentrations of all 20 odors were obtained* Responses to a 15 per c©nt chang© in relative humidity were obtained as for the antenna©*

An exceptionally

good response to water vapor in a labial palp Is shown In Fig. 5, C*

This preparation did not respond to any of the

odors tested, clearly indicating that there are roceptors

26

tov

water vapor independent of the olfactory receptors.

This

was supported by more general observations on antennae and palpi some of which responded to water and not to odor and vice versa* Corel Th© eerci were tested for odor perception both on th© intact animal and separately.

No response to any of

the test odors was noted in either males or females * al­ though a drop of the test material was brought to within a few millimeters of th© corcl when they failed to respond to lower concentrations * The cerci responded to a 5 per cent change in relative humidity between 25 per cent and 30 per cent* Tarsi Th© front* middle* and hind tarsi vrere clipped from decapitated roaches and tested for response to odors and water vapor in the same ways as the cerci. to either was noted*

No response

27

DISCTJSSX OW Th© results indicate that th© roach senses a wide variety of odors and has no obvious gaps in its rang© of odor perception*

They also show that receptors for all

these odors ar© found along antennae and on both sets of palpi , and it seems reasonable to assume that the decrease in magnitude of response from antenna© to maxillary palpi to labial palpi is duo to a decrease in the number of recep­ tors rather than to any qualitative difference between re­ ceptors on the different parts* The thresholds noted are th© lowest which could b© obtained under th© conditions of th© experiments and with th© techniques used*

There are many variable factors

in thes© conditions such as temperature, humidity, rat© of air flow, ag© of animal, etc, which might be altered to give lower valuess

Therefore, it can only be said that th©

true thresholds are at least as low as those given*

It may

be of Interest to note that these threshold concentrations wore on© tenth to on© hundredth of th© concentrations which could be perceived by the experimenter in a crude test* Since most of the responses represent the sum of th© action potentials from many norv© fibers, the resulting wave forms are very complex to analyse*

Hot only do they

represent a mixture of many waves of different magnitude,

28

frequency and phase, each with a positive and a negative componentp but thoy also represent the algebraic addition of these waves so that some components add to the total magnitude while others subtract from it.

Therefore, the

form of the total response may be unlike that of any of its components#

Since the number of fibers from each type of

receptor varies by chance from one preparation to another it seems quite unlikely that any typical response for a parti­ cular odor would re suit , and none was observed#

It was

noted that the electrical response increased as the concen­ tration of th© stimulating material increased, indicating that more receptors were stimulated as th© odor increased. Also the response delay following initial stimulation sug­ gested that a definite time-duration relationship exists between stimulation and response,

Unfortunately not enough

comparative data were secured to make any conclusive state­ ment on this.

Little could be learned of th© number of

types of receptors because so few single nerve fiber prepara­ tions could b® obtained, but it Is hoped that further work will produce more of these with subsequent clues to the nature of olfactory perception.

29

SUMMARY 1*

Olfactory perception of the American roach

(Feriplaneta americ&na) was studied through action poten­ tials in th© olfactory nerves*

Thresholds of the antennae

and maxillary palpi for 20 odors were determined*

Labial

palpi war© shown to respond to these odors but no thresholds were determined* whil© the cerci and tarsi showed no olfac­ tory responses® 2® to respond to

Antennae* palpi* and ceroi were also found movement, air currents* loud sounds, and

relative humidity changes* but showed no response to changes in temperature or light*

Tarsi responded only to

movement and air currents* 3®

Most records were taken from bundles of nerve

fibers and the responses were very complex waves.

An in­

crease in stimulus increased response magnitude and decreased response delay* but no further information on th© nature of the olfactory receptors was obtained*

30

BIBLIOGRAPHY Abbott t C® E* 1937 The Physiology of Insect Senses« Entomolop;!ca Americana, 16 {4), 225-280* Barrows, W* l*L 1907 The Reactions of the Pomace Fly, Drosophila ampelophila Loew, to Odorous Substances* Journal of Experimental Zoology 4(4), 515-537* Crocker, E* C* 9 and Henderson, L* P* 1927 Analysis and Classification of Odors, American Perfumer, 22, 325-356, Dethier, V, 0, 1941 The function of the Antennal Receptors in Lepidopterous Larvae, Biological Bulletin, 80(3), 402-414* ~~ 1943 Testing Attractants and Repellents, Laboratory Procedures in Studies of the Chemical Control of In­ sects ® Washington, 0* 0*, American Association for""* Advancement of Science, pp* 167-172* v* Frisch, K® 1920 Tiber der Geruchslnn d©r Biene and seine blutenbiologische Be&eutung, Zooloffdsch© Jahrbuch fur Physiolop:ie und Zoologde 37, 1-238, Glaser, R* W* 1927 Evidence in Support of the Olfactory Function of the Antennae of Insects, Psyche, 34, 209-215* Henning, H* 1916

Per Geruch*

Leipzig*

Hoskins, W, M * 9 and Craig, R, 1939 'The Olfactory Responses of Flies in a Hew Type of Insect Olfactometor. Journal of Economic Entomologya 27(5), 1029-1036* Mclndoo, N* E* 1915 The Olfactory Sens© of Cole opt© ra Biological Bulletin, 28, 407-455® Pumphrey, R® J«, and Rawdon—Smith, A* P® 1936 Synchronized Action Potentials In the Cereal Nerve of the Cock­ roach (Perlplaneta americana) in Response to Auditory Stimuli, Journal of Physiology 87 (Proceedings of the Physiological Society March 14, 1958)®