On The Measurement and Modification of the Olfactory Responses of Blowflies

As they stand these experiments are unsatisfactory chiefly for the following reasons. First, in several cases the flies began to die off before the experiments were over. The cause of this mortality was apparently not old age, because the average longevity of flies kept in captivity under the same conditions was found to be over 6 weeks for Lucilia and over 4 weeks for Calliphora (cf. Mackerras, 1933), whereas in one case the experimental Lucilia practically all died between the thirteenth and fifteenth days of life (Table 5), and in two experiments almost all the Calliphora were dead by the tenth day of hfe (Tables 3,4). This suggests that the longevity of the flies was less than usual because of the experimental conditions to which they were subjected. Secondly, the example of ‘conditioning’ mentioned below needs further investigation. Finally, in the experiments described in § I, the minimum change in menthol concentration required to cause a change of response, at different intensities of both light and odour, has not been measured. This could be ascertained by performing more tests at intermediate concentrations of menthol vapour (Fig. 1 a). The whole purpose of these experiments would, however, have probably been more effectively achieved if, instead of measuring the percentage response, the technique had been used of measuring the different intensities of odour and light which balanced each other, leaving 50 % of the flies in each arm of the Y. But as it is likely that circumstances will prevent a return to the subject for the time being, it seems useful to publish the results as they are.

The responses of the blowflies were tested in an olfactometer which was essentially similar to that used by Thorpe & Jones (1937) and Thorpe (1938), except that the Y-tube was 2 in. in diameter to accommodate the larger insects used here (see Crombie, 1941). The chief defect of this apparatus lies in the difficulty, common to many investigations of the sense of smell (cf. Dethier, 1937 ; Elsberg, 1937), of measuring exactly the intensity of the olfactory stimulus. An attempt was made to overcome this.

Two species of blowflies were used, viz. Calliphora erythrocephala and Lucilia tericata. The experimental insects belonging to each species were respectively descended from a single pair of original parents. The flies used in the experiments described in the first section of this paper were one day old. They had been fed from emergence on a diet of crystalline sucrose, meat and water (all renewed daily), and then starved for 4 hr. before being tested in the olfactometer. In all the experiments described in both sections of the paper the adults were, when not being tested, kept in a constant-temperature room, open to the daylight, at 23°C. and 70% R.H. All the olfactometer tests were performed in a blacked-out constant-temperature room at 23°C. and 70% R.H. These insects gave clear-cut responses in the olfactometer. With Lucilia, for instance, 151=82% (S.E. = ± 3·7 %) out of 185 insects went to the arm of the olfactometer containing a bait of high meat. The actual number of insects tested was forty, and each individual was put through the olfactometer about four times. The symmetry of the Y-tube was 11 examined by placing the lamp on the axis of the stem and testing the responses of flies with no ‘bait It was found that approximately 50% of the flies went into each arm whether the air currents in them were equal or one was ten times as strong as the other, which showed both that there was no bias in the tube affecting the flies’ behaviour and that the latter did not respond to wind currents (Hoskins & Craig, 1934). The nature of the air flow in the Y-tube was investigated as follows. To one arm of the Y was attached a tube which led from two flasks, on containing hydrochloric acid and the other ammonia. The white vapour of ammonium chloride then entered the arm of the Y-tube. At the junction of the arms with the stem of the Y there was some turbulence, but in spite of this the streams from each arm remained clearly distinct all the way down to where the air was drawn out. The flies would therefore have the whole length of the stem in which to make their choice. In fact what they did if there was no ‘bait’ present was to pursue a zigzag course up the stem towards the light, while if there were a repellent or attractant odour coming down one arm they soon made their choice of sides and continued along it until the junction, when they entered the arm on the chosen side.

The strength of the olfactory stimulus, which in this case was provided by menthol vapour, was controlled as follows. Part of the air which entered the ‘bait’ arm of the olfactometer was passed through tubes containing solid menthol so that it was saturated with the vapour of this substance. This menthol-saturated air was then diluted to any desired degree by introducing a stream of pure air which, with the menthol stream, was passed through mixing tubes before entering the ‘bait’ arm (cf. Wirth,. 1928). The strength of the olfactory stimulus may thus be expressed in terms of the proportion of menthol-saturated air in the air entering the ‘bait’ arm. The volumes of these two streams of air entering the ‘bait’ arm were measured by means of flow-meters. Each of the latter had been calibrated by attaching an aspirator to it and measuring the time for 500 c.c. to run out. The pressure difference in the flow-meter was adjusted to each point in turn by a stop-screw on one of the rubber leads, and by the same means the meniscus of the liquid in the U-tube capillary was kept at the point concerned as the water ran out of the aspirator. The flow-meter readings were plotted against the rate of flow of the air, expressed in c.c. per sec., and a curve drawn from which the rate of flow of air corresponding to any reading on the flow-meter could be read. Thus the number of c.c. of air entering the ‘bait’ arm per second could be determined directly from the flowmeter readings, and the volume of menthol-saturated air expressed as a percentage of the total volume of air entering this arm per second. The rates of air-flow in each of the two arms were also equalized in the usual way by means of flow-meters.

The following experiment was performed to test this method of measuring and controlling the intensity of the olfactory stimulus. As described later, when the light was as usual in line with the axis of the stem, about 75 % of the flies were repelled by the vapour of menthol coming down the ‘bait’ arm (vide infra, Table 2). In this experiment, the attracting lamp, instead of being placed on the axis of the stem, was placed on a line making an angle of 45⁰ with it, so that one arm was nearer the lamp than the other. The latter will be called the ‘illuminated’ arm. The adults of both Lucilia and Calliphora are of course strongly photopositive, and with the lamp so placed about 90% of the flies go to the ‘illuminated’ arm when there is no menthol vapour present. By introducing menthol vapour into the ‘illuminated’ arm, therefore, the attraction to the light may be balanced against repulsion by the menthol vapour at different strengths of both light and odour. It should be noted that the ranges of intensity over which the change in response with change in stimulus are measured by this method will be well above the thresholds of response to both stimuli. The lamp was operated by a good, well-charged accumulator with a variable resistance in series. The strength of the current was measured with an ammeter, which was calibrated to give the values of the brightness of the lamp in metre-candles corresponding to each amperage. This calibration was performed by means of a photoelectric cell, itself calibrated with a standard lamp. The accurate measurement of absolute brightness is it seems a matter of some difficulty, and while this method cannot give accurate values for this variable the values of the relative brightness of the lamp with currents of different strengths will be reasonably accurate. Light and odour were now presented to the flies as opposing stimuli in the following way. The lamp was kept at a fixed distance from the junction of the two arms of the olfactometer, and for each series of observations its brightness was kept constant while the strength of the odour was increased by manipulating the flow-meters. The Strength of the light was expressed in terms of the intensity of illumination (metre-candles) at the junction of the arms, which was calculated from the brightness of the lamp by means of the inverse square law. The strength of the odour was expressed, as stated above, in terms of the percentage of menthol-saturated air in the air introduced into the ‘bait’ arm.

The experimental procedure adopted was to adjust the suction pump, which drew air from the stem of the Y, to a fixed point so that the volume of air entering each arm remained constant at 5 c.c. per sec. The amount of menthol vapour in the ‘bait’ arm was then varied by varying the relative amounts of menthol-saturated and pure air entering it, the sum of the menthol-saturated and pure air always remaining constant at 5 c.c. per sec. Four different intensities of illumination were used, at each of which observations were made with menthol of five different strengths and with no menthol. Sixty insects were used in each test and these were put through the olfactometer between two and four times. The results are shown in Fig. 1 and Table 1. The standard error of the proportion of flies entering the ‘blank’ arm never exceeded 4 %. There was no difference between the responses of samples of flies taken from the ‘menthol’ and ‘blank’ arms, showing that the population was homogeneous in its responses to these sensations (vide infra).

No theoretical deductions seem to be easily drawn from these results. The chief conclusions will therefore be baldly stated as follows. Fig. i a will be considered first, (a) With constant illumination the relationship between the proportion of flies repelled by the odour of menthol and the concentration of this substance seems (within the limits of the experimental error, which is fairly large), to be rectilinear between 10 and 75 % menthol for the first three intensities of illumination, and over the whole range for the highest illumination, (b) The flattening of the curves which tends to occur at high concentrations of menthol seems to be due to the fact that the limit of accuracy of the olfactometer has been reached. The flies made an error of choice of about 25% in their responses to this odour (cf. Table 2).

An error of similar magnitude was made in responding to the odour of meat (vide supra), (c) Likewise, the flies made an error of choice of about 10% in their responses to light. Even when there was no menthol vapour in the ‘illuminated’ arm never more than about 90% of them succeeded in getting into it. (</) The lowest illumination used is above the threshold of response to light, and higher intensities of illumination produced no greater response than lower intensities when there is no menthol vapour present (cf. Brown & Hall, 1936). (e) Between 10 and 75 % menthol concentrations all the lines seem to be parallel within the limits of the experimental error (S.E. x 2). (f) It appears from Fig. 1 b that with constant concentration of menthol vapour the proportion of flies going towards the light is not linearly related to the intensity of illumination. The range of intensities of illumination tested is, however, too small for the exact relationship to be ascertained here. It may be noted that when the light was placed on the axis of the stem of the Y-tube (Table 2), i.e. when there was no attraction by light towards either arm, the proportion of flies entering the ‘bait’ arm (with 2 % menthol) was 24 5 %. The five bottom curves in Fig. 1 b would probably tend to approach this point at the left-hand end. (g) These results give some indication of the degree of accuracy to which the olfactory stimulus has been controlled. The intensity of illumination can be relatively accurately controlled, and the control of the intensity of the olfactory stimulus is, like light, evidently of an order of accuracy at least equal to that of the apparatus in measuring the response. In different regions of intensity of light and odour, changes of 10, 15 and 25% produced statistically significant changes in response (see Table 1). Furthermore, the behaviour of the flies in the apparatus is what it would be expected to be: in each case a greater intensity of stimulus tended to produce a greater response, and the relationship between stimulus and response is more or less regular and orderly. The regular way in which the response is balanced between the two opposing stimuli at different intensities of the latter recalls the observations on responses to light and gravity of Crozier & Pincus (1927) with the rat and of Yagi (1928) with Dixippus morosus. No theoretical conclusions seem to be easily deducible from this behaviour, however, although it perhaps points to the operation of some exact physiological or psychological factor. It should be noted that the method of orientation to the two stimuli would be different: that to the odour of menthol is probably a combination of klino-taxis and tropo-taxis, and that to light telo-taxis (Fraenkel & Gunn, 1940).

The insects were reared in a sterile artificial medium (Lennox, 1939) which had been autoclaved. Before being introduced into it the eggs were sterilized by washing with 0·5 % Agral I solution and treating with 0·1 % mercuric chloride for 15 min. (Hobson, 1932). Five groups of flies of each species were reared: (A) Controls were reared on the artificial medium and their response to menthol tested on emergence (Table 2). (B) The second group was reared from hatching on artificial medium containing 0·2% by weight of menthol. When feeding had finished the larvae were washed in distilled water and removed to clean sawdust and the response of the adults to menthol tested on emergence (Table 3, Fig. 2). (C) The third group consisted of Callliphora only and was reared like the controls on the artificial medium, but when they had finished feeding the larvae were transferred to clean sawdust through which air containing 10% of menthol-saturated air was passed until pupation occurred. The pupae were then washed in distilled water and the response of the adults to menthol tested on emergence (Table 4, Fig. 3). (D) The fourth group was reared like the controls on artificial medium but the adults were allowed to emerge into an atmosphere containing 2% of menthol-saturated air. Their response to menthol was tested after 3 days in this atmosphere in the case of Calliphora, and after 11 days in the of case Lucilia (Table 5, Fig. 4). (E) The fifth group consisted of Calliphora only and was treated like the fourth except that the adults did not emerge into the atmosphere of menthol but were placed in it when they were several days old. Adult flies belonging to all groups were starved for 4 hr. before being tested in the olfactometer, but otherwise were fed as before on a diet of crystalline sucrose, meat and water. The latter were renewed daily. Freshly emerged flies belonging to the first three groups were fed, before being starved for the standard 4 hr. While the tests were being earned out the adults were in all cases kept in a menthol-free atmosphere and fed on the diet mentioned above. There is thus a series of flies which were exposed to a specific chemical sensation during progressively later periods of their life and, as described below, their behaviour towards it was modified according to the stage in which they first experienced this sensation. The response of each group as a whole was tested first. In all cases the flow-meters were adjusted so that the air entering the ‘bait’ arm contained 2 % of menthol-saturated air. The lamp was, of course, placed on the axis of the stem of the Y so that the flies were attracted towards the arms against the air stream. It was found that the best results were obtained when the lamp was very dim. The intensity of illumination was 0-095 m.c. at the junction of the arms of the Y. With a bright lamp the flies tended to become excited and attempt to fly, while when it was dim they crawled quietly along the Y-tube and responded well. When the whole group had been tested the insects going to eitherarmof the olfactometer were removed from the traps concerned and kept to form the ‘menthol’ and ‘blank’ samples. These were then tested separately every 2 or 4 days. The actual number of insects used in each test is shown in the last column of each table. The same insects were put through the olfactometer between about two and ten times in differeiit experiments. At the end of each experiment all the flies were sexed, as were any of them that succumbed before it was finished. The standard error of the proportion of flies entering the ‘blank’ arm never exceeded 4% except where it is indicated to the contrary in the tables.

The controls (A) of both species were strongly repelled by menthol (Table 2). The responses of flies of both species belonging to groups B, C and D are shown in Tables 3–5 and Figs. 2–4, respectively. In each case the adults achieved a tolerance of the odour of menthol which gradually disappeared after a few days in a menthol-free atmosphere. By ‘tolerance’ is meant, of course, that in the olfactometer 50 % of the insects went to each arm, i.e. the menthol did not affect the ‘chance distribution’, so that it may be concluded that the flies are not responding to it. The response of the flies to menthol was thus modified when they first experienced this normally repellent sensation in the larval stage or immediately upon emergence. If, however, as in group E, they were first exposed to the odour of menthol a week after emergence as adults, their behaviour towards this sensation was not modified. Thus ninety Calliphora adults, which had been exposed to the odour of menthol for 2 weeks beginning when they were a week old, were still repelled 230 = 73·5% (s.E. = ±2·83%) out of 313 times by this odour, and thirty Lucilia adults which had been similarly treated were repelled 107 = 75·4% (s.E. = ±4·19%) out of 142 times by the odour of menthol. It may therefore be concluded that immediately after emergence the adults are in a plastic state in which they are more easily influenced by the external situation than when they are older.

There is a possible objection to the conclusion that the change in response to the odour of menthol which occurred in groups B and C was due to the memory of a larval experience, for if the contents of the Malpighian tubes reached the ecdysial fluid, either with the faeces via the rectum as in the silkworm (Esperón, 1937) or via the perivisceral fluid as in Acidia (Keilin, 1921), and became spread out over the inner surface of the puparium, then the adult might experience the odour of this substance just before its emergence from the puparium. Eastham (1925), however, found that the contents of the Malpighian tubes of Drosophila funebris were passed to the gut about the sixth day of pupal life, and only expelled from the body on the emergence of the adult. The fate of the contents of the Malpighian tubes at metamorphosis of Lucilia sericata and CaUiphora erythrocephala was investigated by the following experiment, which was devised by Thorpe (1939)- The larvae were fed on minced meat to which sufficient carmine had been added to give a bright red colour. After they had ceased feeding the larvae were washed in distilled water and placed in clean sawdust to pupate. A few days before emergence some of the pupae were dissected out of the puparia and both examined for carmine. None was found either between the pupa and puparium, nor, after adults had emerged, inside the pupa case itself. These observations were made with carmine because it is easy to see, but it is probable that the fate of menthol would be the same. It may therefore be concluded that the above objection does not hold and that an alteration has been effected in the larval nervous system by their experience in this stage and has survived metamorphosis to influence the behaviour of the adults (cf. Thorpe & Jones, 1937 ; Thorpe, 1938-9; Cushing, 1941).

When the ‘menthol’ and ‘blank’ samples from groups B, C, D are compared it is seen that there is a marked difference in their responses to the odour of menthol, and that in fact the behaviour of the latter sample has scarcely been modified at all. Thus when a population of flies appears in the olfactometer to be ‘tolerant’ to an odour each individual fly is not necessarily indifferent to it, but some may be repelled and some attracted. A statistical method such as this obscures this result and may give a totally wrong impression of the situation. No such difference was observed in the two samples of the controls (A) (Table 2), both being equally repelled. The treatment with menthol has therefore brought about a division of the population into two sections differing in their responses to this odour, and it is conceivable that such behaviour may lead to genetic isolation and thus to the formation of ‘biological races’ (Thorpe, 1930). It should be pointed out, however, that the modification of response to the odour of menthol of flies which had been exposed to this odour as larvae had, in almost all cases, disappeared by the end of the pre-oviposition period (approximately 6 days for Lucilia and 10 days for Calliphora). After this it would therefore cease to be a factor of isolation. But it remains possible that the effects of larval experience of less repellent odours may not disappear so quickly. It would be interesting to know what causes the difference in behaviour of blowflies belonging to the two samples. There are cases in which variations in the behaviour of members of the same species are known to have a genetic origin (e.g. McEwen, 1918, 1925; Brown & Hall, 1936), although there is no evidence that this is so here.

The change in behaviour observed in Tables 3-5 may probably be described as ‘habituation’. Thorpe (1943a) defined habituation as ‘an activity of the central nervous system whereby innate responses to mild shock and warning stimuli wane as the stimuli continue for a long period without unfavourable result’. The insects became accustomed to the odour of menthol and ceased to respond to it (Humphrey, 1933). f^{n one} case, however, when Calliphora had been exposed to menthol as post-feeding larvae, the flies of the ‘menthol sample’ seem actually to have been attracted by the odour of this substance (Table 4). These flies thus seem to have become ‘conditioned’, their behaviour changing from negative to positive (see Crombie, 1941), although it must be admitted that the results fall under the shadow of a statistical doubt. Thorpe (1938–9) suggested in explaining his results with Nemeritis and Drosophila that the modification of behaviour is not as in Pavlov’s ‘conditioned reflex’ an association between a definite stimulus and a definite reaction, but rather between one constituent of the environment (e.g. the odour of menthol) and optimum conditions of life in general. The post-feeding larvae perform no functions such as feeding, oviposition, etc., but merely walk about until they pupate. The odour of menthol could therefore not have become associated with any specific function, and thus appears to have been associated with a favourable environment as a whole. Furthermore, during the period of their exposure to menthol this odour could have had no particular meaning for the animals, but must have been merely one of many other sensations which they experienced. It was only when the odour was experienced again out of its context, so to speak, in the olfactometer, that it acquired significance and became a signal for that pattern of comfortable sensations of which it was a part when first encountered. This sensation acquired significance (cf. Russell, 1938, 1941), then, only after it had been learnt ; when it was learnt it had no specific meaning. This is perhaps an example of ‘latent learning’, or ‘learning without patent reward’. According to Thorpe (1943a), ‘Latent learning experiments seem to involve the preliminary learning of a locality or environment as a whole together with the ability subsequently to select certain parts of that whole for use as guides or signals for the whole or for some other part thereof or in connexion with a new motivation’ (see also Thorpe, 1943b).

Post-feeding larvae (C) may be prevented from pupating by keeping them at 0°C. Under these conditions they were exposed to the odour of menthol for 30 days, but except that the habituation seemed to disappear more quickly the result was identical with that obtained after an exposure of 8 days at 23°C. (Table 4, Fig. 3).

The ‘habituated’ flies of both species were tolerant of the odour of menthol when the air entering the ‘bait’ arm of the olfactometer contained 2% of menthol-saturated air. When, however, the diluting flow-meter was shut off so that the air entering the ‘bait’ arm was 100% menthol-saturated, then the ‘habituated’ flies were just as strongly repelled as the controls, i.e. about 75 % of them went to the blank arm. The strength of the odour (above 2%) entering the ‘bait’ arm made no difference to the proportion of controls repelled. When the menthol was as strong as this it probably acted as an irritant, in which case one would expect the olfactory responses to be obscured or upset. No difference was observed between the responses of the two sexes in any of the experiments.

Thus although the evidence is against it being the cause of economic damage to sheep in Australia, the responses of blowflies may be modified by their past experience as larvae or as young adults, and Froggatt’s theory, although improbable, is possible.

The accuracy of the control of the olfactory stimulus was tested by balancing olfactory and visual stimuli of different intensity. This demonstrated that a reasonably accurate control of the intensity of the olfactory stimulus was achieved, and also disclosed some facts about the quantitative relationship of stimulus to response.

These flies are normally repelled by menthol but their response to the odour of this substance was modified when they first experienced it in the larval stage or immediately upon emergence. The memory of an experience in the larval stage thus survives metamorphosis and affects adult behaviour. Most of the flies then became ‘habituated’ to the odour, but in one case they appear to have become ‘conditioned’. The latter may be a case of ‘latent learning’. There was no modification of the response of adults which first experienced the odour when they were several days old. The populations of habituated or conditioned flies were not homogeneous. Different samples of populations which seemed at first to be indifferent to the odour of menthol often proved to respond differently, some being repelled by it, others indifferent or even attracted to this odour.

I should like to thank Dr A. D. Imms, F.R.S., for his interest in these experiments, Dr F. S. Dainton for kindly measuring the solid vapour pressure of menthol, and especially Dr W. H. Thorpe for helping me to clarify my ideas on the subject. This work was done while holding a research scholarship from the University of Melbourne.