Injury to one side of the face, whether on the frons or an antennal patch, affects the orientation of adult locusts to a lamp. For the first 5 days after injury most of them turn the injured side towards the lamp. This tendency is lost or reversed after the fifth day. The change is most evident with a lamp of relatively high power (100 W.); with weaker lamps (60 or 40 W.) the differences are not statistically significant after five days.
This phenomenon explains the finding of previous authors that the injured side is usually turned away from a lamp. When the results of numerous tests are added together, those obtained more than 5 days after injury can swamp the earlier ones.
Suggested interpretations include the possibility that injury makes the damaged part ‘numb’ until type II neurones are formed in the healed area.
Under natural conditions locusts spend much of their time basking in sunshine. This response to insolation (men-akinesis) characteristically involves orientation of the body at right-angles to the sun’s rays, thus achieving the maximum radiation load (Volkonsky, 1939). In the laboratory the same response can be elicited by an ordinary tungsten-filament lamp which emits both heat and light. If a locust is released facing the lamp, there is an equal chance of it turning the left or the right side towards the lamp, unless its behaviour has been affected by injury or experimental interference.
Slifer (1951) showed that damaging one of the specialized areas (‘Slifer’s patches’) on the head significantly affected the orientation of locusts to a lamp in a small arena, but Makings (1964) found that damaging a part of the normal cuticle had the same effect. He suggested that injury lowered the nociceptive threshold of the cuticle as if it were ‘sore’. This hypothesis appeared to explain all the results obtained at that time, for there was generally a tendency to turn the injured part away from the source of heat and light. At the same time, it was pointed out that the locusts’ behaviour might vary according to the precise combination of experimental conditions and according to the progress of healing.
Those experiments have now been extended, using a greater range of experimental conditions and correlating the results with time. For the first few days the injured part seems to be turned towards, rather than away from, the source of radiation, and this behaviour is more or less reversed as healing proceeds. The same results are obtained by damaging one of the Slifer’s patches as by damaging an unspecialized part of the frons, but they cannot be explained in the way previously suggested.
II. MATERIAL AND METHODS
Adults of the African migratory locust, Locusta migratoria migratorioides (R. & F.) were used for all the experiments. They were bred gregariously in small stock cages (15 × 15 × 18 in.) in an animal house maintained at approximately 26° C. The routine maintenance procedure was as advocated by Hunter-Jones (1961), but the locusts were often fed on trays of sprouting wheat as an alternative to fresh grass and bran. A minimum day length of 9 hr. was provided by the cage lights (40 W.). The locusts were normally 3 weeks old (from fledging) when experimentally injured, and were tested for up to 3 weeks afterwards. Occasionally some slightly younger animals were used.
The lamp and arena arrangement for these experiments was similar to that used by Slifer (1951) and by Makings (1964), but with lamps of various power ratings. Two dark rooms were used; one was a photographic dark room at 16–19° C. and the other was a controlled-temperature room at 16–17° C. with mechanical air circulation.
For all surgical operations on the locusts CO2 anaesthesia was employed. The part selected for injury was the lateral area of the frons near the base of the antenna, as described by Makings (1964), or the Slifer’s patch near the base of the antenna. In each case the cuticle was pricked repeatedly with a fine needle in the specified area, loosened pieces being removed with fine forceps. Care was taken to prevent the needle going deeply into the face. The damaged area of the frons amounted to approximately mm.2, about the same as the area of an antennal patch. Only one side of the face was injured, equal numbers of animals being treated on the left side and on the right side.
After the operation the locusts were kept in the stock cages in the animal house until required for testing. Tests on the same day as injury were performed 3 hr. after treatment, the locusts being returned to the stock cage during this time.
The behaviour of the locusts in the arena was as described by Slifer (1951) and Makings (1964). Their positions were scored when they had adopted a distinctive basking (men-akinetic) posture and maintained it for at least 5 min. Sometimes a locust would turn first one side then the other towards the lamp before settling down in its final scored position. As far as possible the preliminary orientations were noted as well as the final position, but they were often difficult to determine, as the movements involved varied from slight swaying and turning movements through the whole range to complete orientation. Also, the initial position was often changed rapidly, suggesting that the final position was the ‘preferred’ one. In fact the results obtained from the preliminary orientations were similar to the final ones, showing the same trends, but were less often statistically significant. The final position thus provided a better and more useful criterion and the results given in Tables 1-4 are the final positions.
The χ2 method was used to test the results for statistical significance.
III. THE EFFECT OF INJURING THE FRONS
The apparatus was tested by first running a control series of 15 male and 10 female uninjured locusts in the arena. For this control (Expt. 1) and for Expt. 2, the procedure described by Makings (1964) was followed. The locusts were put into large glass tubes and cooled in a refrigerator for 30–40 min. prior to testing. Makings used a 60 W. electric bulb, but for these two experiments a 40 W. bulb was used. The temperature of the dark room (16–19° C.) was similar to that used by Slifer, lower than that used by Makings, and it was intended to determine whether the results obtained by Makings (1964) would be the same if the lamp intensity, as well as the air temperature, had the lower values used by Slifer (1951).
In Expt. 1 (the control) the right and left sides were turned towards the lamp in approximately equal numbers (P > 0·1) confirming the absence of bias (Table 1).
Experiment 2 employed nearly equal numbers of male and female locusts, each injured on one side of the frons. They were tested every day for about a fortnight after injury, but in Table 1 the results are simply added together. The results of this experiment appear to contradict those of Makings (1964) because they show a significant tendency to turn the injured side towards the lamp, whereas in his experiments the injured side was more often turned away. Further investigation was obviously required.
The period of cooling in the refrigerator was considered to be a possible source of error, perhaps being excessive. Other potential sources of error were : the power of the lamp, the room temperature, other details of the experimental conditions, and changes in the locusts themselves with time following injury (see Makings, 1964).
Further tests, Expts. 3–5, were carried out taking some of these factors into account. The refrigeration treatment was omitted, but the locusts were left instead in the glass tubes in the cool dark-room for 1–2 hr. before being tested. The lamps used for Expts. 3–5 were 40, 60 and 100 W. respectively. A second control (Expt. 6) was carried out with a 100 W. bulb under the same conditions as Expt. 5 and confirmed the absence of bias (P > 0·5, Table 1).
If any of the locusts died before the series of tests was completed, additional tests were carried out by treating more animals and testing them at the appropriate time. Less than 10% died, so the results are not dependent on this procedure, but it explains the variation of daily totals which will appear in Table 2 for some experiments.
As Table 1 shows, the overall results of all the experiments showed a significant difference between the scores for injured and intact sides, but with a weaker lamp (40 W. or 60 W.), the injured side was mostly turned towards it, whereas with the stronger source (100 W.), the injured side was mostly turned away. An important clue to the interpretation of this behaviour was obtained by examining the results on a daily basis.
Table 2 gives separately the results obtained on each day after the injury and shows the striking variation associated with the passage of time. In all four injury experiments, including Expt. 2, the injured side was turned towards the lamp more often than the intact side for the first 5 days after injury. This tendency is significant in all four sets. From day 6 onwards, this preference disappeared except in Expt. 5. In Expt. 5, the tendency was not merely lost, but reversed, so that from day 6 onward there was a significant tendency to turn the injured side away from the lamp. The overall result of this experiment was, therefore, due to the results from days 6 to 19 outweighing those of days o to 5. The 6-day sequence of the control experiment, no. 6, is shown here for comparison. The results of the first control, Expt. 1, were not obtained in a continuous series.
For the sake of comparison the results in Table 2 are analysed in blocks each of 6 days, namely from o to 5 days inclusive, 6 to 11, and 12 to 17 days. For Expts. 2 and 3 tests were not carried out on the same day as injury (day o) or beyond the 15th day. These were added for the later experiments but made no difference to the results.
When indications of a reversal of behaviour after the fifth day were found in Expts. 4 and 5, it was anticipated that this in turn might disappear when the locusts had been allowed to recover for more than a week. This was another reason for analysing the results in blocks of 6 days at a time. For instance, it might be expected that the effects of injury would have disappeared by days 12–17, leaving no indication of a preference for one side or the other. This was not the case. In Expt. 4, on days 6–11, the intact side was turned towards the lamp only slightly more often than the injured side, the ‘preference’ not being significant, and later tests showed the same lack of preference. Conversely, in Expt. 5, where there was a strong reversal of behaviour with a significant tendency to turn the intact side towards the lamp from day 6 to 11, this tendency persisted unaltered to day 19 when the tests were discontinued.
In a complex situation like this it is obviously wrong simply to sum the results of several days’ observations as was done previously. It was therefore thought desirable at this stage to carry out a fresh analysis of the orientation behaviour following injury of the antennal patches. This was necessary to determine whether Makings’s conclusion, that damaging the frons has the same behavioural effect as damaging one of the patches, was correct in the light of this new information.
IV. THE EFFECT OF INJURING AN ANTENNAL PATCH
The conditions for these experiments were the same as for Expts. 1–6, so the controls were not repeated. The antennal patch was injured on one side of the head of each locust, as described in § 11. Two experiments were carried out; no. 7 with a 40 W. bulb and no. 8 with a 100 W. bulb. With a 60 W. bulb, the results for the frons (Expt. 4) seemed to be intermediate between the other two, so this was not repeated for the antennal patch. The results of these experiments, shown in Table 3, are virtually the same as those of Expts. 3 and 5 given in Table 1, which show the effects of frons injury at the same two lamp intensities.
Both for the damaged frons and for the damaged antennal patch the overall result with a 100 W. lamp was a significant tendency to turn the injured side away from the lamp (Expts. 5 and 8). With a 40 W. bulb the opposite effect, a tendency to turn the injured side towards it, was obtained for the frons (Expt. 3). The same applies to the antennal patch (Expt. 7) except that in this case the numbers were not statistically significant.
Analysis of Expts. 7 and 8 on a daily basis is shown in Table 4. This shows the same features as Table 2, in that there is at first a significant tendency to turn the injured side towards the lamp, whether of low or of high intensity, but this tendency is lost or reversed on about the fifth day after the operation.
These findings confirm Makings’s conclusion that damaging the antennal patch has the same effect as damaging the frons or presumably any other part of the ‘normal’ cuticle. However, they contrast markedly both with his conclusions and with those of Slifer, in that there is not a consistent tendency to turn the injured side away from a lamp or to turn the intact antennal patch towards it. Such tendencies can be found, but they change with time, presumably due to healing.
These changes are fairly clear-cut and striking but not immediately obvious from the detailed results given in Tables 2 and 4. They are best illustrated graphically in the form of histograms as in Fig. 1. Fig. 1,A shows the results obtained after damaging the frons (Expt. 5) and Fig. 1 B the results after damaging the antennal crescent (Expt. 8).
Some of the orientation experiments, namely those using high-wattage lamps, gave results like those of earlier work, but time is a vital factor. Neither Slifer (1951) nor Makings (1964) differentiated between results obtained at different times after injury, although the latter pointed out that changes might be expected as healing proceeds. It seems clear that the tendency to turn the injured side away from the heat, indicated by the overall totals of these authors, was due to the relevant number of tests before and after 5 days from injury. The method described by Slifer was to begin experiments after the injured locusts had been left to recover for 4 or 5 days. If the majority of results are obtained after this point, they can outweigh the rest. This effect was obtained in the present work in Expts. 5 and 8, with a 100 W. bulb, even though daily tests were begun on the same day as injury.
Since the results of orientation experiments in these arena tests are so strongly affected by time, it is perhaps of little significance to find that the overall result obtained by Makings was duplicated only when a 100 W. lamp was used, although he used one of 60 W. His results were not expressed on a daily basis and the experimental conditions were slightly different. Similarly, it may be noted that the results of Expt. 7, using locusts with one antennal patch damaged and a 40 W. bulb, differed from those obtained by Slifer in that the tendency to turn the intact side towards the lamp after the fifth day was not statistically significant in the present case (Table 4). With a 100 W. bulb the tendency was highly significant.
It is somewhat surprising to find that injured locusts at first seem to ‘prefer’ to turn the injured side towards a source of light and heat. The most likely explanation is that the injury eliminated receptors, including nociceptors, making the part at first ‘numb’ rather than ‘sore’ and resulting in the normally sensitive, intact side being turned away from the heat. The reversal of this tendency after 5 days may be brought about by abundant re-innervation following healing and resulting in enhanced sensitivity. It would be inappropriate to describe such sensitivity after healing as ‘soreness’. Of course this is an entirely speculative idea. It is simpler than the alternative hypothesis that the sensitivity of the injured side is at first reduced, then later increased, by chemical correlates of the injury and healing processes. An immediate and continuing sensitization might be masked by a clot until it is shed after healing, but this notion has not been supported by later experiments.
One cannot argue that this behaviour is due to the intact side being at first turned away from the heat and later turned towards it, unless a recent injury is preferentially turned towards the heat. This would demand the assumption that injury could increase the excitability of adjacent thermoreceptors without affecting nociceptors.
One more possibility may be mentioned. This is that during the first few days after injury the locusts were simply tending to circle towards the normal intact side when stimulated by the lamp, thus automatically presenting the opposite, damaged, side to the lamp. This would be analogous to the circus movements of unilaterally blinded insects. This idea is rejected because it does not agree with the fact that the locusts would often turn both sides towards the lamp before settling down in the final position, nor does it explain the reversal at about the sixth day. Makings (1964) found that locusts with extensive damage on the head and body tended to veer to one side when moving towards a lamp, but this is seldom seen when only the head is damaged.
Rinterknecht (1963) has shown that at 32° C. the healing processes bring about closure of the wound by the fourth day after injury, when new cuticle is laid down. It is not known to what extent new innervation may be developed, but Wigglesworth (1953) has shown that in Rhodnius new sensory neurones are differentiated from amongst the ordinary epidermal cells during postembryonic growth. He has also shown that if sensory nerves in fourth-instar larvae are interrupted by burning, new axons grow out from the peripheral end and eventually join up with an existing nerve. A further interesting observation was that no sensilla appeared in the regenerated area of a healed burn or wound at the first moult after injury, but at the subsequent moult new sensilla were regenerated. Regeneration of sensilla is unlikely to occur in adult Locusta, but epidermal type II neurons might be formed readily enough, as they need no cuticular specialization.
Study leave for C.A.H., granted by the Education Department, Government of West Pakistan, is gratefully acknowledged; also a travel grant for C.A.H. provided by the British Council. We should like to thank Professor E. W. Knight-Jones for the provision of laboratory facilities and the Anti-Locust Research Centre for the loan of reprints.