Effects of temperature on the irritability caused by DDT and DDT-analogues in anopheline mosquitos*

Bull. Org. mond. Santo 1970, 42, 917-930 Bull. Wld Hlth Org. Effects of Temperature on the Irritability Caused by DDT and DDT-analogues in Anopheline Mosquitos* A. H. KASCHEF 1 The author reports a series ofinvestigations into the effects oftemperature on irritability, caused by DDT or its analogues methoxychlor and DDD, in Anopheles labranchiae atro- parvus and An. gambiae, species B; into the irritability of three other anophelines upon exposure to deposits of water-dispersible DDT powder; and into the relation between aging and DDT-irritation in anophelines. Irritability was measured by both the number of take-offs and the duration offlight over a 20-minute period at temperatures offrom 19- C to 320C. The flight activity of An. 1. atroparvus and An. gambiae B increased with increasing temperature to apeak at 23°Cfor theformer and 27°Cfor the latter; it then fell with further increase in temperature to 32°C. Over 20 minutes, the DDT-resistant An. pharoensis was less irritated than either An. gambiae A or An. punctulatus farauti (both susceptible) but over 30 minutes was mor e so than An. p. farauti. Among other findings were that methoxychlor and DDD were stronger irritants thai DDT and that irritability declined with increasing age of the mosquitos. DDT is known to affect the behaviour of mos- quitos, apparently by causing irritability in them which induces flight and sometimes enables them to escape from the toxic residue. This reaction to DDT has been reported and investigated in about 14 spe- cies of mosquitos, and there are numerous publica- tions on the subject. Despite the obvious importance of temperature on physiological processes, there seems to have been only one investigation of the effects of temperature on this irritant effect. The subject is of particular interest since the toxic effect ofDDT is well known to have a negative temperature correlation, whereas one would expect normal activity to be positively correlated with it. The present work represents a series of investiga- tions on the effects of temperature on the irritant effect of DDT and DDT-analogues on Anopheles * This study was supported by a research grant from the World Health Organization and carried out at the Medical Entomology Department, London School of Hygiene and Tropical Medicine, London, England. 1 Present address: Entomology Department, Faculty of Science, Ain Shams University, Cairo (Abbassieh), United Arab Republic. labranchiae atroparvus Van Thiel of temperate zones and the tropical An. gambiae Giles, species B, on the irritability of three other anopheline species to DDT water-dispersible powder, and on the relation be- tween aging and irritation by DDT in anopheline mosquitos. MATERIALS AND METHODS Apparatus The acoustic recording used in this work is a modification of the technique used by Jones (1964) for studying mosquito activity. The apparatus was set up in a double-walled room with very efficient sound-proofing; neither radio signals nor electrical circuits presented any serious interference problems. Two recording channels were operated simultane- ously. The apparatus contained two chambers, one of which was available for insecticide-treated papers and the other for the control or Risella-oil papers. Three series of tests were conducted; in the first two the insecticide was used as a water-dispersible powder 2533 - 917 - A. H. KASCHEF while in the last series it was dissolved in Risella oil. The water-dispersible powder DDT (w. d. p. DDT) or its non-insecticidal components (w. d. p. blank) were applied as 51 % emulsions to give 4 g/m2 deposition of theDDT powder (2 g/m2 of 70%DDT; 1.4 g/m2 active ingredient) and 2 g/m2 of the blank on control papers. The insecticide papers were treated with 3% DDT, 2% methoxychlor or 2% DDD (dichlorodiphenyl dichloroethane) dissolved in Shell Risella oil No. 17 at the rate of 5 mg/cm2 (DDT) or 7.5 mg/cm2 (methoxychlor or DDD). Chloroform was used as a spreading agent in the preparation of these papers. Both the temperature and the humidity of the experimental room were adjusted and kept constant for at least 2 days before any test. The temperature fluctuated by less than 1 deg C and the relative humidity by ±5%. Tests were carried out at 190C, 230C, 27°C and 32°C. The light regime (alternating 12 hours light and 12 hours dark) was controlled using a Venner time-switch. Methods The female mosquitos due to be tested were always transferred from the rearing insectary to the experi- mental room 12-24 hours before being tested. Except where otherwise stated, the females used were 1-2 days old. They were supplied with 15% sugar solution in a Petri dish with a cotton-wool wick from the first day of emergence. The sugar-fed females were placed singly in plastic tubes for half an hour to an hour before being tested. A precon- ditioning box, as described by Coluzzi (1963), was used for this purpose. The acoustic apparatus was switched on and left running for about half an hour before introducing one female into each of the two chambers. The number of take-offs and the duration of flight were recorded for 18 min, 20 min or 30 min as mentioned below. At the end of this period the insects were transferred to 3 in x 1 in (7.5 cm x 2.5 cm) glass tubes, loosely plugged with cotton- wool damped with a few drops of 15% sugar solution. After 24 hours, mortality was assessed. The test was then repeated, the control chamber with non- treated paper being substituted for either an insecti- cide chamber or the oil-paper-lined chamber. The record of each experiment was obtained as a strip of paper. A pulse-clock and event-marker provided a time trace of 1-minute intervals for the experiment. Another two event-markers recorded the flights in the insecticide and control (and Risella oil) chambers. This sort of record was easily analysed; the pen response showed a strict on/off response according to whether the tested mosquito had taken off, continued to fly or landed at the time. Mosquitos The following species of mosquito, including two members of the Anopheles gambiae species-complex, were tested in this work: Anopheles labranchiae atroparvus Van Thiel; An. gambiae Giles, species B (Bobo strain); An. gambiae Giles, species A (Ibadan strain); An. punctulatus farauti Laveran; An. pharoensis Theobald. The temperate An. 1. atroparvus has been reared since 1931 in the insectary of the Medical En- tomology Department, London School of Hygiene, at 25°C-27°C and 40%-80% relative humidity. The tropical An. gambiae, species B (Bobo strain), received in 1958, has been reared with the other three anopheline mosquitos in the Ross Institute at 26°C-27°C and 60Y%-80% relative humidity. All mosquitos were susceptible to DDT, with the excep- tion of An. pharoensis, which was resistant to both DDT and dieldrin. RESULTS Effect of age on flight activity Hecht et al. (1960) in their study on the behaviour of anopheline mosquitos in the presence of DDT used 2-day-old females without giving any reason for their choice. It was thought worth while to try to find the optimum age for ffight activity. The mean number of flights and mean flight duration over 20 min were the parameters used. An. 1. atroparvus was investigated at 27°C and 55% relative humidity. The response of females 1-2, 3-4, 5-6 and 7-8 days old to aqueous emulsions of w.d.p. DDT and to w.d.p. blank, as well as the activity of the control females were recorded. The results are given in Table 1 and the means of both number of take-offs and duration of flight are plotted against age in Fig. 1. Analysis of the results showed that, in general, both the number of take-offs and the duration of flight gave similar indications of mosquito activity. In the control insects there was a high flight activity in the young mosquitos aged 1-2 days, followed by a gradual but pronounced decrease as the females advanced in age. In the presence of w.d.p. DDT the flight activity in all age-groups was greater than for the controls but the intensity of extra activity declined with age. The highest flight activity was 918 EFFECT OF TEMPERATURE ON DDT IRRITABILITY IN ANOPHELINES TABLE 1 RELATION BETWEEN AGE AND FLIGHT ACTIVITY OF AN. L. ATROPARVUS IN THE PRESENCE OF DDT WATER-DISPERSIBLE POWDER DURING AN INVESTIGATION PERIOD OF 18 MINUTES AT 27°C AND 55% RELATIVE HUMIDITY Age Formulation Mortality a Number Duration (seconds) Mean duration(days) Formulation of take-offs of flight (seconds)(days)()(Mean±SE) (Mean±SE) of single flight w.d.p. DDT 91.7 (36) 81.89±6.89 b 127.00±10.49 b 1.59 1-2 w.d.p. blank 23.8 (21) 33.41+8.13 61.36± 9.88 1.84 control 11.1 (18) 30.38±5.23 70.81+ 9.77 2.36 w.d.p. DDT 96.67 (30) 57.00±5.20 b 112.86+14.55 b 1.98 3-4 w.d.p. blank 9.1 (11) 26.45±6.73 47.97± 9.92 1.81 control 5 (20) 26.63±6.09 46.17± 7.31 1.73 w.d.p. DDT 84 (25) 36.92±4.08 b 58.97±12.17 1.59 5-6 w.d.p. blank 15 (20) 21.83±6.88 32.74± 7.57 1.50 control 12 (25) 23.47±5.25 38.82± 8.55 1.65 w.d.p. DDT 91.3 (23) 18.91±4.22 31.10± 8.76 1.65 7-8 w.d.p. blank 28.1 (21) 15.38+4.44 26.21± 5.57 1.70 control 25 (20) 16.09±5.61 29.81± 7.09 1.85 a Values in parentheses Indicate numbers of mosquitos tested. b P<0.01 (differences between w.d.p. DDT and both w.d.p. blank and control highly signifi- cant). FIG. 1 RELATION BETWEEN AGE AND FLIGHT ACTIVITY OF AN. L. ATROPARVUS IN THE PRESENCE OF DDT WATER-DISPERSIBLE POWDER AT 27°C AND 55% RELATIVE HUMIDITY W.D.P. DDT - W.D.P. DDT W.D.P. BLANK CONTR L W.D.P. 200 100 90 80 70 60 50 40 30 20 I .1 5-6 7-8days 1-2 3-4 5-6 Age --I ~10 7-8days WH3 90626 919 200 , c .E 0 20 \ ,o E Da) 100 90 80 70 60 50 40 30 20 00c Q_ C 0 0 0 1-c_. 1-2 3-4 Age A. H. KASCHEF demonstrated by the 1-2-day-old females, which were used in all further tests. The activity of the mosquitos in the presence of the w.d.p. blank was not significantly different from that of the control mosquitos. Activity pattern in presence ofDDT water-dispersible powder Flight activity of three mosquito species, An. p. farauti, An. gambiae A (Ibadan strain) and An. pharoensis, was also investigated in the acoustic recording apparatus, using the normal procedure. Activity was recorded over 20 min for the first two DDT-susceptible mosquitos, and over 30 min for the DDT-resistant An. pharoensis. This series of experi- ments was carried out at 27°C and 55% relative humidity, using 1-2-two-day-old sugar-fed females. The results given in Table 2 showed that there was a noticeable increase in flight activity due to the w.d.p. DDT. The differences between the means of both number of take-offs and flight duration of w.d.p. DDT females and those of the control females were highly significant. A specific difference in flight activity (based on both parameters) was obvious, in the following order: slightly active, An. p. farauti; moderately active, An. pharoensis; highly active, An. gambiae A. In the normal course of a 20-min investigation, which is the usual investigation period of flight activity, An. pharoensis would be the least active of the three species and this is indicated by both the number of take-offs and duration of flight. It would be expected that the resistant mosquito would be the least irritable, as found by Busvine (1964). However, it was noticed in this investigation that the period of activity was starting about the end of the 20-min period; therefore it seemed worth while to extend the investigation period to 30 min, and this showed intermediate activity of An. pharoensis between that of An. jarauti and that of A. gambiae A. An. gambiae A was more active and showed a higher response to DDT than An. gambiae B (Bobo strain) although both strains were susceptible to DDT. The difference in flight activity, however, was not significant. The first flight activity of An. gambiae A occurred in the first minute and the peak of activity between the fifth and seventh minutes. The flight activity of An. p. farauti started in the seventh minute and the activity peak took place about 5 minutes later. With An. pharoensis, the first flight was not recorded until the fourteenth minute and the activity peak was delayed to the twentieth minute. The mortality after 24 hours of both An. gambiae A and An. p. farauti was high; that of the resistant An. pharoensis was comparatively low. Effect of temperature on the response to DDT and DD T-analogues The results obtained with w.d.p. DDT were satis- factory but it appeared somewhat difficult to make consistent deposits; and also the water-dispersible formulations of both methoxychlor and DDD may be different from that of DDT. Therefore, for consistency, all insecticides were dissolved in Risella oil. TABLE 2 ACTIVITY PATTERN OF AN. P. FARAUTI, AN. GAMBIAE A (IBADAN STRAIN) AND AN. PHAROENSIS IN THE PRESENCE OF DDT WATER-DISPERSIBLE POWDER AT 27°C AND 55% RELATIVE HUMIDITY Mosquito Inverigd Mortalitya Numberoftake-offs Duration (seconds) dMetin species Formutation pnertioa- Mo y N Mberotan±E-o) of flight (seconds)of(min) (Mean±SE) (sigensoflih An. p. farauti w.d.p. DDT 20 72 (25) 16.32±2.74 b 16.81±3.11 b 1.03 control 24 (25) 0.12±0.06 0.07±0.04 0.63 An. gambiae A w.d.p. DDT 20 80 (25) 74.36±6.63 b 69.57±8.03 b 0.94 control 7.7 (13) 13.77±4.22 16.22±8.26 1.18 An. pharoensis w.d.p. DDT 20 24 (25) 13.84±3.76 b 13.04±2.79 b 0.94 control 12 (25) 0.28±0.15 0.17±0.08 0.61 An. pharoensis w.d.p. DDT 30 24 (25) 40.12±6.37 a 36.48±5.67 b 0.91 control 12 (25) 0.92±0.55 0.63±0.36 0.68 a Values in parentheses indicate numbers of mosquitos tested. b P<0.01 (difference highly significant). 920 EFFECT OF TEMPERATURE ON DDT IRRITABILITY IN ANOPHELINES In this series of experiments the response of two anopheline mosquitos, the temperate-zone mosquito An. 1. atroparvus and the tropical An. gambiae B (Bobo strain), to DDT, methoxychlor and DDD was investigated over 20 min at four constant temper- atures- 9'C, 23°C, 27°C and 32°C, all at 55% rela- tive humidity. Investigation of one insecticide was completed before starting a test with a new one. This resulted in testing of female mosquitos from different generations with the three insecticides. But as will be seen from Table 3, results with the controls were reasonably consistent in the different sets of experiments. Furthermore, in one instance, the insecticide tests at 32°C were repeated for confirma- tion (as will be described shortly) and the second group of tests gave a very similar picture of relative activity. The means of both number of take-offs and duration of flight were calculated at each temper- ature. Once again, both parameters gave the same picture of activity. Aui. 1. atroparvus: reactions to DDT. The results given in Table 3 showed that the activity of control insects was increased by raising the temperature from 19 C to 23°C, then decreased gradually as the temperature was further raised to 32°C. Both the mean number of take-offs and mean duration of flight indicated a maximum activity for this temperate mosquito at 23°C (Fig. 2). In addition to the over-all reduction in activity, there was with DDT an additional effect at the highest temperature (32°C)-namely, a prolonged delay (8 min-10 min) before the first flight-whereas the control and the Risella-oil mosquiitos showed their highest activity in the first 5 min. The flight activity of the females on the Risella oil, compared with that of the control females, was reduced in intensity especially at 23°C and 27°C; Risella oil had a suppressing effect on flight activity. The female mosquitos in the presence of DDT in Risella oil presumably suffered the depressing effect of the oil; nevertheless, the irritant effect of DDT raised the activity significantly above the controls, except at the highest temperature of 320C. At 323C (see Table 3) the means of both the num- ber of take-offs and the ffight duration for the control insects were somewhat higher than those for the DDT-treated insects although the difference was not significant. At first it was thought that this might be due to the wide range of individual variation well known in mosquito behaviour even though the mosquitos tested were of the same age and feeding condition and the tests were carried out under similar environmental conditions. Therefore this experiment was repeated using 72 more females; the data are given in Table 4. The results confirmed the above observation. The means of both the number of take- offs and flight duration in the presence of DDT was about the same as those recorded by the control females. The highest flight activity in the presence of DDT appeared at the twentieth minute of the investigation period. The mean duration of a single flight ranged from about 1 second to 3 seconds. There did not seem to be a distinct difference between control insects or those exposed to either Risella oil or DDT. In all cases there was a tendency for the duration of flight to increase with temperature up to 32°C. A further experiment was carried out, also at 32°C, but the duration of activity recorded was extended to 30 min, using the same DDT chamber (3 % DDT in Risella oil). The results given in Table 4 showed that (a) although the flight activity was comparatively high, the difference between the means of both number of take-offs and ffight duration in the presence of DDT and of the control females was not significant; (b) DDT took effect after a latent period of 6-12 minutes whereas there was no latent period in the control; and (c) the flight activity peak appeared at the twentieth minute of the experi- ment while that of the control insects occurred at the third minute. An. 1. atroparvus: reactions to methoxychlor and DDD. This species showed a higher response both to methoxychlor and to DDD than to DDT at 23°C, 27°C and 32°C (see Table 3, Fig. 3 and Fig. 4). At 19°C methoxychlor was nearly as irritant as DDT, but DDD was more irritating than either DDT or methoxychlor. At 32°C the response to both methoxychlor and DDD was delayed until the fourth minute of the investigation period. At this tempera- ture the mosquito females showed an activity peak at the eighth to tenth minutes in the presence of methoxychlor and at the sixteenth to eighteenth minutes in the presence ofDDD. All other reactions to temperature, in the controls, with Risella oil, and in the presence ofDDT, were also observed with methoxychlor and DDD. These were: (a) the increase in flight activity as the temperature was raised to 23'C and its gradual decrease with fuirther raising of temperature to 32°C; (b) a tendency for the average duration of single flights to increase up to 32°C; and (c) the suppressing effect on flight activity by Risella oil alone 921 A. H. KASCHEF TABLE 3 EFFECT OF TEMPERATURE ON IRRITABILITY OF AN. L. ATROPARVUS EXPOSED TO DDT, METHOXYCHLOR OR DDD FOR 20 MINUTES Tern- Duain(eod) Mean pera- Formulation Mortality a Number of take-offs Duration (seconds) durationpteurrae- FormulatWon (%) (Mean±SE) ofefighE) (seconds) ofture (Mean±SE) single flight 3% DDT Insecticide 4.4 (23) 77.26+3.58 b 124.57± 7.62 b 1.62 190C Risella oil 0 (23) 11.26+2.59 22.28± 7.80 1.99 Control 0 (25) 32.43±3.96 31.35± 5.63 0.97 Insecticide 7.7 (26) 87.65±7.90 b 168.99±17.39 b 1.93 230C Risella oil 0 (25) 10.44±2.56 21.25+ 5.38 2.04 Control 0 (18) 53.99±7.48 88.79±13.01 1.65 Insecticide 4 (25) 73.44±4.49 b 122.88±11.27 b 1.67 27°C Risella oil 0 (25) 7.52±3.14 12.74± 4.87 1.7 Control 0 (25) 28.08±4.90 65.03±10.61 2.32 Insecticide 9.6 (45) 22.04±3.40 c 53.85± 7.88 c 2.44 32°C Risella oil 0 (25) 3.32±1.46 8.37± 3.43 2.52 Control 0 (20) 23.90±4.87 62.02±15.20 2.59 2 % Methoxychlor Insecticide 3.3 (30) 77.20±6.29 b 109.82±12.04 b 1.69 190C Risella oil 0 (20) 15.55±2.76 14.04±13.67 1.65 Control 0 (30) 31.53±2.97 33.37± 5.57 1.82 Insecticide 0 (35) 166.43±2.56 b 281.20±15.42 b 1.74 23°C Risella oil 0 (15) 16.40±3.93 28.71+ 6.77 1.75 Control 0 (16) 55.69±9.64 102.98±14.71 1.83 Insecticide 0 (35) 87.59±6.82 b 205.36±17.48 b 2.35 27°C Risella oil 0 (15) 8.73±3.06 16.41± 5.20 1.89 Control 0 (25) 32.48±5.96 62.70± 9.85 1.93 Insecticide 10 (30) 49.10±7.53 b 147.65±20.31 b 3.01 32°C Risella oil 0 (15) 5.07±2.77 12.35± 6.61 2.43 Control 5.6 (18) 24.79±6.89 34.99± 4.15 1.41 2% DDD Insecticide 12 (25) 88.16±4.05 b 131.70±11.35 b 1.49 190C Risella oi l 0 (15) 10.60±2.47 7.85± 2.26 0.74 Control 0 (20) 33.32±4.95 29.33± 5.69 0.88 Insecticide 0 (30) 127.17±8.19 b 232.92±12.69 b 1.84 23°C Risella oil 0 (15) 18.07±4.70 17.94± 4.55 0.94 Control 0 (20) 52.30±5.33 74.60±11.32 1.45 Insecticide 0 (25) 71.64±4.72 b 155.23±13.43 b 2.11 27°C Risella oil 0 (15) 15.00±4.03 15.15± 3.72 1.01 Control 0 (25) 23.68±3.24 67.39±11.38 2.84 Insecticide 3.3 (30) 50.87±4.04 130.89±13.90 b 2.87 32°C Risella oil 0 (17) 10.35±2.29 10.49± 2.14 1.01 Control 0 (25) 14.20±2.57 43.86±10.00 3.09 a Values in parentheses indicate numbers of mosquitos tested. b P<0.01 (differences between insecticide and both Risella oil and control highly significant). c P<0.01 (difference between insecticide and Risella oil highly significant). 922 EFFECT OF TEMPERATURE ON DDT IRRITABILITY IN ANOPHELINES FIG. 2 EFFECT OF TEMPERATURE ON FLIGHT ACTIVITY OF AN. L. ATROPARVUS IN THE PRESENCE OF DDT 200 100 - DDT CONTROL 40 ," " I, \\~~30 \ 0______ 20 10 R .OI L \ R.OIL 3 200 100 50 g a40 n Q- 30 a 020_. Co r:3 0 20 = 3 - U 3 19 23 27 I 19 23 27 320C 1t TABLE 4 CONFIRMATORY EXPERIMENT ON EFFECT OF TEMPERATURE ON FLIGHT ACTIVITY OF AN. L. ATROPARVUS IN THE PRESENCE OF DDT AT 32°C AND 55 % RELATIVE HUMIDITY Investigation Fm t Mortality a Number Duration (seconds)period Formulation o a y of take-offs of flight(min) (Mean±SE) (Mean±SE) DDT 4 (25) 28.16±3.51 b 61.39± 9.10 b 20 Risella oil 0 (25) 3.84±2.11 4.85+ 2.73 Control 0 (22) 26.75±3.44 59.12± 7.63 DDT 8 (25) 53.52±7.77 b 120.54±15.56 30 Risella oil 0 (13) 5.00±3.14 11.37± 6.73 Control 0 (12) 45.75±7.35 115.88±26.92 a Values in parentheses indicate numbers of mosquitos tested. b P<0.01 (difference between DDT and Risella oil highly significant). 923 In 0 0 a 0 04 -o E 0 a IV1 A. H. KASCHEF FIG. 3 EFFECT OF TEMPERATURE ON FLIGHT ACTIVITY OF AN. L. ATROPARVUS IN THE PRESENCE OF METHOXYCHLOR METHOXYCHLOR CONTROL 4 'I* 300 200 100 lo 50 0m 40 Q 0 0 30 = _. 20 '__ 20 10 I I 1 9 23 27 32C 1 9 23 27 32°C wmo 90628 An. 1. atroparvus: mortality. Mortality of An. 1. atroparv us after 24 hours did not exceed 12% after contact x ith DDT, methoxychlor or DDD dis- solved in Risella oil respectively for 20 min at the four temperatures tested (Table 3). This result was a further confirmation of what had been found by many authors (e.g., Brown, 1958; de Zulueta, 1959; Elliot, 1964) that there was no correlation between mosquito irritability due to insecticides and 24-hour mortality. Ani. gambiae, species B: reactions to DDT, mnethoxychlor and DDD. All three insecticides were tested at 23°C, 27°C and 32°C and DDT was investi- gated at one other temperature, 19°C. The three insecticides were tested over the same period of time, the DDT-chamber being substituted for either a methoxychlor- or a DDD-chamber. This allowed for recording the activity and response to insecticides of female mosquitos not only of the same age and feeding condition but also of the same generation. The results are given in Table 5 and the means of both number of take-offs and flight duration as well as the 24-hour mortality are represented in Fig. 5. As in the case of An. 1. atroparvus, there was a noticeable effect of temperature on the flight activity of An. gambiae B. Activity increased gradually as the temperature was raised from 19°C to 27°C, then decreased as it was raised further to 32°C. This was clearly demonstrated by both parameters. The activity of the An. gambiae females due to Risella oil alone was lower than that of the control females but the differences between the means of both number of take-offs and duration of flight were not significant. The response to each of the three insecticides was significantly higher than the baseline activity of both the control and Risella oil females at the four temperatures. The maximum flight activity, however, took place at 27°C, as in the controls. The flight activity peak of both controls and insecticide- 300 200 100 50 40 30 20 10 ,0% CONTROL .'I 924 O.. -la EFFECT OF TEMPERATURE ON DDT IRRITABILITY IN ANOPHELINES 925 FIG. 4 EFFECT OF TEMPERATURE ON FLIGHT ACTIVITY OF AN. L. ATROPARVUS IN THE PRESENCE OF DDD 300 - - 300 DDD 200 - 200 DDD 100 100 a CONTROLP--- - ' o. a ° 50 CONTROL ,s ,; 50 :5 40 40 R.01L R.01L 1 1 0 6~~~~~~~0 6 1 9 23 27 32C 1 9 23 27 32°C WHO 90629 exposed mosquitos was recorded between the thirteenth and seventeenth minutes of investigation, being progressively slightly delayed as the tempera- ture was raised from 19°C to 32°C. The intensity of activity in the case of An. gambiae B was lower than that of An. l. atroparvus. The mean number of take-offs at 27'C in the two species was 62 and 169 per 20 min respectively, and the mean duration of flight 51 and 281 seconds per 20 min, respectively. There is a very slight increase of dura- tion of a single flight up to 27°C or 32°C. In all cases, the duration of flight was considerably less than with An. 1. atroparvus. DISCUSSION Techniqiue In order to assess levels of mosquito irritability caused by DDT, many workers have used the techniques suggested by the WHO Expert Committee on Insecticides (1960) and the modifications sug- gested by Coluzzi.1 In either case, it was possible to rely on "Step A" (time to first take-off), "Step B" (number of flights by a single mosquito in 15 min) or "Alternative Step B" (number of flights by a batch of 5 mosquitos in 15 min). Brown (1964) examined these different criteria fairly exhaustively in experi- ments with a normal and DDT-resistant strain of Aedes aegypti. He concluded that "Alternative Step B" was the most satisfactory and that the best tech- nique combined elements of the WHO Expert Committee and Coluzzi methods. In the present study, the characteristics of the apparatus demand examination of single mosquitos. Most of the conclusions are based on mean number of flights in 20 min (and, occasionally, 30 min), thus approximating to Step B. In addition, the total period of flying (and hence the mean flight time) was 1 Coluzzi, M. (1962) An experimental method for deter- mining the irritability of adult mosquitos to insecticides(unpublished document WHO/Mal/329; WHO/Insecticides/ 130). 7 A. H. KASCHEF TABLE 5 EFFECT OF TEMPERATURE ON IRRITABILITY OF AN. GAMBIAE B (BOBO STRAIN) EXPOSED TO DDT, METHOXYCHLOR OR DDD FOR 20 MINUTES Tem- M rtalit a Number Duration (seconds) Mean durationpera- Formulation orality of take-offs of flight (seconds) ture (Mean±SE) (Mean±SE) of single flight DDT 8 (25) 21.92±3.08 b 7.96±3.08 b 0.37 19°C Risella oil 0 (15) 5.07±1.22 1.61±0.42 0.32 Control 0 (25) 6.16±0.60 1.80±0.17 0.29 DDT 3.3 (30) 34.83±3.97 b 14.58±2.29 b 0.42 Methoxychlor 28 (25) 41.04±2.21 b 32.13±4.71 b 0.79 230C DDD 24 (25) 19.88±2.05 b 14.09±1.95 b 0.71 Risella oil 0 (18) 10.17±1.20 3.41±0.56 0.33 Control 0 (30) 11.90±1.69 4.45±0.62 0.37 DDT 6.7 (30) 62.03±4.47 b 41.35±4.46 b 0.67 Methoxychlor 16.7 (30) 61.50±6.81 b 49.89±9.05 b 0.81 27°C DDD 20 (30) 49.40±4.68 b 50.97±6.33 b 1.03 Risella oil 0 (20) 16.25±1.50 6.85±0.65 0.42 Control 0 (25) 21.40±1.89 8.58±0.82 0.42 DDT 6.7 (30) 35.60±3.02 b 27.36±4.83 b 0.77 Methoxychlor 13.3 (30) 36.53±4.22 24.42±4.36 b 0.67 32°C DDD 13.3 (30) 29.00±3.47 b 27.11±4.39 b 0.94 Risella oil 0 (18) 12.78±1.10 5.00±0.50 0.32 Control 0 (30) 14.80±1.11 6.00±0.48 0.41 a Values in parentheses indicate numbers of mosquitos tested. b P<0.01 (differences between each of the three insecticides and both Risella oil and control highly significant). used for the first time. This last parameter gave results quite consistent with the number of take-offs; in fact, both seem to relate to the same mobility phenomenon. The tirne to first take-off (Step A) was not invariably employed, but it was noted in some instances. In the present study a delay in response to insecti- cides was first observed in An. 1. atroparvus at 320C. The delay lasted 8-10 min in the presence of DDT and 4 min in the presence of methoxychlor or DDD. Both An. gambiae B, tested at four different tempera- tures, and An. gambiae A, tested at 27°C, reacted in the first minute and did not show any delay in response to DDT. However, An. p. Jarauti and An. pharoensis reacted much later at 27°C-after the seventh and fourteenth minutes respectively. This more noticeable delay in response to DDT rather than to methoxychlor and DDD was interpreted as due to the fact that the latter two insecticides were stronger irritants than DDT. Resistance to DDT may also have accounted for the long-delayed re- sponse to DDT in An. pharoensis. Results Aging. Many authors have pointed out noticeable decreases in the flight activity of different insect species in the first or second week of adult life. Williams et al. (1943) and Wigglesworth (1948) reported similar age effects in Drosophila funebris and D. melanogaster. The flight performance increased rapidly during the first few days of adult life, reached a maximum level at the end of the second week, then 926 EFFECT OF TEMPERATURE ON DDT IRRITABILITY IN ANOPHELINES FIG. 5 EFFECT OF TEMPERATURE ON FLIGHT ACTIVITY OF AN. GAMBIAE B (BOBO STRAIN) IN THE PRESENCE OF DDT, METHOXYCHLOR AND DDD 100 50 40 R.OIL R.OIL 30 a) 20 D 0 10 (0 (a'_ I I I 19 23 27 32C dropped to a minimum by the third week. Leven- book & Williams (1956) found that the wing-beat frequency attained a maximum level in 7-day-old Phormia regina. Information on the effect of aging on the flight activity of mosquitos is slight. Hamon & Eyraud (1961) noted that adult An. gambiae and An. funestus over 10 days old were less irritable than 2-3-day-old mosquitos. Haufe (1962) found that the flight performance of Ae. aegypti stimulated by light, in the absence of insecticides, increased with age. Rowley & Graham (1968a) reported a sharp decrease at the end of the second week of adult life in the flight activity of Ae. aegypti females flown on a flight mill. In the present study an inverse correlation between mosquito age and irritability was recorded in An. l. atroparvus exposed to DDT water-dispersible pow- der. The mean number of take-offs decreased I I I I I I 19 23 27 32°C W9Q0 )L.3 gradually from 81.8 to 18.9 per 18 min in 1-2- and 7-8-day-old females respectively. The mean duration of flight also decreased from 127.1 seconds to 31.1 seconds in the young and comparatively old females respectively. Mosquito species. Hecht et al. (1960), investigating the irritability of three anophelines and Ae. aegypti to DDT, suspected that the more susceptible species were the most irritable. Mouchet & Cavalie (1961), however, found An. gambiae more irritable than An. funestus but less susceptible; also Bhatia & Deobhankar (1962) found An. culicifacies less irritable than An. stephensi but more susceptible to, DDT. Cullen & de Zulueta (1962) showed a decreasing irri- tability with increasing susceptibility in both An. gambiae (two strains) and An. funestus, but lowest irritability with the resistant Ae. aegypti. Busvine (1964), using the WHO irritability test kit, found that 100 50 40 30 , 20 E (N ? 10 01 a) 1-E C -0 E) 927 I A. H. KASCHEF the DDT-resistant An. albimanus, An. stephensi, Ae. aegypti and Culexfatigans were less irritated than the corresponding normal ones. Using the three alterna- tive steps in the WHO test method, Brown (1964) found that in all of them a DDT-resistant strain of Ae. aegypti demonstrated less irritability than a susceptible strain. The present study of irritability of An. p. farauti, An. gambiae A (Ibadan strain) and An. pharoensis to water-dispersible DDT powder over a 20-min investigation period showed that the DDT-resistant An. pharoensis was less irritable than either of the susceptible species An. gambiae A or An. p. farauti. When An. pharoensis was investigated over 30 min it proved to be more irritable than An. p. farauti. Type of insecticide. Hadaway & Barlow (1953), who exposed mosquitos to insecticide residues on mud blocks and who used the time to the first flight as a measure of irritability, found that mosquitos were irritated by residues ofDDT and a number of DDT- analogues and ofy-HCH and other HCH-analogues. It was interesting to note that irritability was not strictly related to toxicity in either case. They found that chlordane, dieldrin and toxaphene were non- irritant. Vigueras & Corzo (1960) exposed mosquitos to residues of insecticides from acetone solutions on paper and counted the number of flights over 20 min. They noted that diazinon as well as DDT were highly irritating, malathion was moderately irritating but chlordane and dieldrin were not. Elliott (1964), who designed a slightly more complex apparatus for studying the mosquito activity, pointed out that DDT had features in common with the repellents dimethyl phthalate and deet in stimulating mosquito flight, while fenthion and malathion did not stimu- late locomotor activity before a latent period had elapsed. The present study shows that in An. 1. atroparvus both methoxychlor and DDD were stronger irritants than DDT at 23°C, 27°C and 32°C. The three insecticides could be arranged in the following order: strongly irritant, methoxychlor; moderately irritant, DDD and comparatively less irritant, DDT (except at 19'C, at which DDD was slightly more irritant than either methoxychlor or DDT). In An. gambiae B (Bobo strain) methoxychlor also induced the strongest reaction although the differences be- tween the three insecticides were not significant (see Table 5). Temperature. The investigation closest to the present study is that of Hecht et al. (1960), who enclosed mosquitos in a plastic cylinder with the ends covered with paper. The experimental tube had papers with DDT deposits (apparently residues from acetone solutions) while a control tube had clean paper ends. The numbers of flights were observed for batches of 10 mosquitos at from 5°C to 38.5°C. Hecht et al. "corrected" the number of flights in the DDT tube by a modified Abbot's formula; but it is very doubtful whether there are sound statistical grounds for this. Abbott's formula is based on two competing probabilities and relates to proportions affected. In this case, actual numbers are recorded and it seems that the only way in which the "control flights" could interfere with "irritated flights" would be by the time actually spent in flight; but no information on ffight duration is given. Haufe (1964) measured the reactions of Ae. aegypti to combined changes in temperature and humidity using a small, copper, climate chamber. He applied probit transformations to estimate the times (log min) for 50% take-off and 50% immobilization at the end of the activity period. The period of activity was short at 16°C (14 min), very long over the range 20°C-28°C (over 1000 min) and then declined again at higher temperatures. Initial rates of take-off grew steadily to a maximum at 34°C, with a straight decline at 36°C. During the period of activity the rate fell off sharply at these high temperatures, but only slowly over the long activity periods in the lower temperature range. Rowley & Graham (1968b) found that in Ae. aegypti the maximum flight activity temperature was 21°C; the sustained tethered females flew further at 15°C-210C than at the commonly reported optimal temperature of 270C. Yurkiewicz & Smyth (1966) found that the wing-beat frequency of the blowfly Lucilia sericata increased as the temperature was raised from 150C to 30°C; but this is somewhat different from the activity measurements in this paper and the other literature cited. In the present study An. 1. atroparvus showed greater flight activity at 23°C, whereas the maximum flight activity of An. gambiae B occurred at 27°C. Both mosquitos showed increased flight activity and irritability to insecticides with an increase of tempera- ture from 190C to their respective maxima, then a gradual decrease as the temperature was further raised to 32°C. With DDT the fall-off in activity of An. 1. atropar- vus at high temperature was faster than with the control, suggesting that irritability declined more quickly than normal activity. With the other 928 EFFECT OF TEMPERATURE ON DDT IRRITABILITY IN ANOPHELINES mosquito-insecticide combinations the rates of fall- off in activity were about the same as in the controls. Risella oil and irritability. Cullen & de Zulueta (1962), using An. gambiae, found slightly less activity in controls on Risella-oil-impregnated paper than on clean filter-paper. On filter-paper blackened with India ink or on plain mud blocks the controls were very much less active. In contrast to these results, Gerold & Laarman (1964) found that Risella-oil- impregnated paper had an irritant effect on An. atroparvus, as judged by numbers of escapes from a cylinder of paper. The present results agree with those of Cullen & de Zulueta in showing a slight reduction in activity in An. gambiae B controls on Risella oil paper as compared with plain paper. With An. 1. atroparvus, however, the reduction is very clearly significant, especially at 23°C and 27°C. No explanation except the considerable differences in technique- can be offered for the results being diametrically opposed to those of Gerold & Laarman. Mortality. When An. l. atroparvus and An. gambiae B were exposed for 20 min to 3% DDT, 2% methoxychlor or 2% DDD dissolved in Risella oil the mortality after 24 hours was low (Tables 3 and 5) and strikingly similar to that noted by Brown (1958) with An. albimanus, by de Zulueta (1959) with An. atroparvus and An. labranchiae, by Mouchet & Cavali6 (1961) with An. funestus and Coluzzi & Coluzzi (1961) with An. stephensi, although the latter authors used 4% DDT paper. However, in the presence of an emulsion of water-dispersible DDT powder (2 g/m2 of 70% DDT; 1.4 g/m2 active ingredient) the 24-hour mortality at 27°C was very high in 1-2-day-old females of An. 1. atroparvus, An. p. farauti and An. gambiae A but comparatively low in An. pharoensis. The latter mosquito was resistant to DDT. ACKNOWLEDGEMENTS The author is very grateful to Professor D. S. Bertram for accommodating the work in his department, and to Professor J. R. Busvine for his interest, continuous encouragement and very valuable discussion and for reading the manuscript. RE-SUME INFLUENCE DE LA TEMPERATURE SUR L'IRRITABILITE D'ANOPHELINES EXPOSES AU DDT ET AUX ANALOGUES DU DDT On a pris comme criteres la duree du vol et le nombre d'envols pour evaluer l'action irritante sur des anopheles duDDT et de ses analogues en fonction de la temperature. On note une reaction retard&e aux insecticides chez Anopheles labranchiae atroparvus expose a 32'C et chez A. pharoensis resistant au DDT expose a 27°C. La durde de la periode de latence parait dependre ai la fois de facteurs intrinseques (espece de moustique, resistance aux insecticides, etc.) et de facteurs extrinseques comme la temperature. L'activite de vol et l'irritabilite sont inverse- ment proportionnelles 'a l'age du moustique. Mesuree sur une periode de 20 minutes, l'irritabilite d'A. pharoensis resistant au DDT est moins elev&e que celle des especes sensibles A. gambiae A et A. punctulatus farauti. Si l'observation est prolong6e pendant 30 minutes, A. pharoensis se revele plus irritable qu'A. punctulatus farauti. L'action irritante du methoxychlore et du DDD est plus marquee que celle du DDT. L'activite de vol d'A. labranchiae atroparvus et d'A. gambiae B augmente lorsque la temperature est portee de 19°C 'a 23°C-27'C, puis decroit lorsque la temperature atteint 32°C. La temperature optimale en ce qui conceme lPactivit6 de vol est de 23'C pour le premier de ces anopheles et de 270C pour le second. L'emploi au cours des epreuves de papier impregne d'huile minerale (Risella) a pour effet de diminuer dans une mesure sensible l'activite d'A. labranchiae atroparvus et d'A. gam- biae B. Les composants depourvus d'activit6 insecticide de la poudre de DDT dispersable dans l'eau n'ont aucune action sur l'activite de vol des moustiques. 929 930 A. H. KASCHEF REFERENCES Bhatia, S. C. & Deobhankar, R. B. (1962) Indian J. Ent., 24, 36 Brown, A. W. A. (1958) Bull. WldHlth Org., 19, 1053 Brown, A. W. A. (1964) Bull. Wid Hith Org., 30, 97 Busvine, J. R. (1964) Bull. Wld Hlth Org., 31, 645 Coluzzi, A. & Coluzzi, M. (1961) Riv. Malar., 40, 35 Coluzzi, M. (1963) Riv. Malar., 42, 189 Cullen, J. R. & Zulueta, J. de (1962) Bull. Wid Hlth Org., 27, 239 Elliot, R. (1964) Bull. Wld Hlth Org., 31, 657 Gerold, J. L. & Laarman, J. J. (1964) Nature (Lond.), 204, 500 Hadaway, A. B. & Barlow, F. (1953) Bull. ent. Res., 44, 255 Hamon, J. & Eyraud, M. (1961) Riv. Malar., 40, 219 Haufe, W. 0. (1962) Canad. J. Zool., 40, 53 Haufe, W. 0. (1964) Int. J. Biomet., 7, 245 Hecht, O., Mancera, 0. & Carvillo, G. R. (1960) Bol. C.N.E.P. (Mix.), 4, 222 Jones, M. D. R. (1964) J. 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