Insecticide-resistant strains of insects of public health importance

1t

ORDINARY MEETING of t h e ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE,

held at Manson House, 26, Portland Place, London, W.,

on Thursday, 13th December, 1956, at 7.30 p.m. The President, Professor R. M. GORDON, O.B.E., M.D., D.SC., F.R.C.P., in the Chair.

PAPER

INSECTICIDE-RESISTANT

STRAINS

OF

INSECTS

OF PUBLIC

HEALTH

IMPORTANCE BY

J. R. B U S V I N E , PH.D., D.se.

Reader in Entomology as Applied to Hygiene, London School of Hygiene and Tropical Medicine.

Insecticide resistance is becoming increasingly serious in all branches of pest control and there are several recent reviews of the subject from the entomological and toxicological standpoints : METCALF, 1955 ; HOSKINGSand GORDON, 1956 ; BUSVlNE,in press. This paper deals with the problem as it affects insects of public health importance. To appreciate the significance of resistance in this field, one must realize the extent to which public health relies on the newer insecticides, especially in the tropics, following the achievements of the past decade. D D T and other new chlorinated insecticides combine high activity and persistence with relatively low toxicity to man ; but above all, they are cheap and simple to apply, which has led to their use on a vast scale in poor, disease-infested and under-developed countries. An excellent illustration of this is provided by the report of the World Health Organization Regional Committee on Malaria held in November, 1954. In the countries of South-East Asia, antimalarial schemes based on residual insecticides are benefiting some 84 million people, at an annual cost of only about 1/6 per head. T h e excellent results of such campaigns, in reducing or eliminating malaria in many parts of the world, were summarized by PAMPANA(1951) and later by JASWANTSINGH (1954) for the Asian region. Only two years ago, the combination of past success and the fact that many nations were now conducting campaigns, led the Pan-American Sanitary Bureau to propose a vast programme to eradicate malaria from the entire western hemisphere. A similar attitude has produced an internationally co-ordinated campaign to eradicate Aedes aegypti from Central and South America (PINTO SEVERO, 1955). Striking results obtained with insecticides against other insects of public health importance, have been reviewed by SIMMONS and UPHOLT (1951) and by BUXTON (1952). T h e y showed that the experiences of early years after the war suggested that a complete answer had been found to louse-borne epidemics of typhus and relapsing fever, and to flea-borne typhus and plague. Further, there was great hope of eliminating onchocerciasis by anti-larval treatments against Sirnnlium, and leishmaniasis, by anti-adult treatments against Phlebotomus.

12

INSECTICIDE-RESISTANT STRAINS OF INSECTS

THE EMERGENCE OF RESISTANCE

Strictly speaking, resistance is not a new phenomenon. Racesof scale insects resistant to hydrogen cyanide were causing trouble in California over 40 years ago. Prior to the Second World War, however, it was a rarity, since only two or three species had developed resistance in the field. Resistance has become much more prevalent in the past decade and seems, therefore, to be connected with the wide use of new insecticides. This may be due to one or more of the following causes :--(i) there may be certain weak links in the mode of action of chlorinated synthetic insecticides, which permit the development of biological defence mechanisms ; (ii) the persistent residues of these insecticides may impose a type of selection on the insect population, which especially favours the elimination of the susceptible and survival of the resistant ; (iii) the use of insecticides has been on an unprecedented wide scale and therefore the populations of pests exposed to selection have been bigger than ever before. A circumstance that seems to support this last point is the high proportion of cases of resistance among insects of public health importance. Thus, WIESMANN (1954) lists 37 examples of possible resistance in pests of hygienic importance as compared with 25 examples from the far more numerous pests of agriculture, forestry and stored products. It is likely that insecticidal campaigns against disease-carrying insects have been more extensively conducted (often on a national basis) than measures against other pests. Incidentally, Wiesmann's lists call attention to the special prominence of certain orders of insects, in the development of resistance. Two-thirds of the hygienically important examples belong to the order Diptera ; and nearly half the agricultural cases belong to the Hemiptera. Since this paper concerns, specifically, pests of public health importance, the development of resistance in this field must be examined more closely. In 1946, soon after D D T became available for wide use, resistant strains of the common housefly emerged. Since then, signs of resistance have appeared in a stealthily growing number of species of public health importance. In the last year or so, some serious disease vectors have been added to the list and this has given rise to considerable anxiety. Early this year, Dr. Candau, Director-General of the World Health Organization, issued a warning that reports of resistance had arrived from no less than 32 nations. It is becoming evident that to proceed with our plans, and even to hold the ground we have won, we must re-think our strategy of attack against insect disease vectors. Unfortunately, it is not easy to give an exact picture of the growth in incidence of resistance in recent years. The difficulty can be illustrated by a comparison of various estimates of the numbers of resistant strains of public health importance :-No. species with res~tant strains in different years Author

_.~

. [94_7_4194881-~--1--~19491950195116 . . . .

31

Quarterman and Schoof (1957) Wiesmann (1955) Busvine

....

1952 1953 1954 1955

.. ..

1

1

2

5

8

10

11

32

34

37

3L:I::

It will be seen that my own estimates are considerably lower than the others for the following reasons :--(i) Other authors accept Musca domestica, M . vicina and M . nebulo

J. R. BUSVINE

13

and also Culex pi_piens, C. molestus and C. fatigans as separate species, whereas I have considered them as geographical races. (ii) Some authors include cases of " behaviouristic resistance," which I do not believe to be valid. The term behaviouristic resistance should be restricted to describing the " development of the ability to avoid a dose which would prove lethal." This would exclude the natural avoidence of D D T by Anopheles gambiae, A. melas, A. albitarsis, A. darlingi and A. pseudopunctipennis, each of which has appeared in lists of resistant strains. In fact, the only evidence for development of behaviouristie resistance in mosquitoes appears to be for the species Anopheles albimanus, in Panama (TRAelDO, 1952). (iii) The major source of discrepancy between my estimates and those of other experts, however, is in the nature of the evidence accepted. I have only included cases for which reports of resistance have been confirmed experimentally. Some of the reports included by other authors derive from field observations and so far as published information is concerned, it seems insufficient to establish the existence of resistant strains of the following: Anopheles rnaculipennis, A. superpictus arid Phlebotomus papatasii in Greece ; Triatoma infestans in Chile ; Anopheles quadrimaculatus in Tennessee ; Blatta orientalis in Italy; Anopheles philippinensis in India; Stomoxys calcitrans in Sweden; Fannia cannicularis in Spain ; and various species of flea. I wish to emphasize that I am excluding these reports, because they seem to be unproved, not because I believe them all incorrect. Therefore, it should be realized that my estimates err on the cautious side and that the true figure is somewhere intermediate. Whichever estimate is taken, it is clear that the number of species evading control because of resistance is growing from year to year.

Details of the present situation. Crude lists of resistant species cannot give much information about the relative seriousness of each case, which depends on the importance of the insect concerned and whether the resistance is localized or wide-spread. Some of the most important examples must be considered individually.

I.

THE HOUSEFLY

It is well known that the housefly (and its sub-tropical and tropical varieties) are, nearly everywhere, more or less resistant to all the newer synthetic chlorinated insecticides. It might be argued that this is more of a grievous disappointment than a disaster, for the housefly is not one of the most important disease vectors. Nevertheless, flies do carry disease ; and before the residual insecticides had become ineffective, WATTS and LINDSAY (1948) had shown how conveniently D D T could be used to reduce shigellosis in fly infested villages. The failure of chlorinated insecticides is felt keenly in hot dry climates. I know of at least one oil company, in the near East, which spends large sums on fogging machines, which belch forth expensive pyrethrin aerosol two or three times a week to reduce flies. Needless to say, this could not benefit the Egyptian peasant child suffering from fly-borne ophthalmic disease. A more practical measure against resistant flies is the use of organo-phosphorus insecticides. Several years ago, laboratory tests were begun to investigate the chances of resistance developing against these compounds. The results were moderately encouraging, for some 100 generations of selection only produced a mild degree of resistance (CHADWICK, 1954).

14

INSECTICIDE-RESISTANT

STRAINSOF

INSECTS

Similar levels of resistance have appeared recently in the field in two areas where organophosphorus compounds had been used for 3 to 4 years for practical control in dairy barns. One place is Florida, where these insecticides have been used as poison baits (LINDQmST, 1956) ; the other is Denmark, where they have been employedas residual treatments (KEIDING, 1956). Unfortunately, although the levels of resistance are low compared with those shown towards chlorinated insecticides, they are beginning to prevent effective control. Perhaps it is a little early to abandon hope of using organo-phorphorus insecticides for fly control ; but as regards the chlorinated compounds, they seem to have no future for fly control. This is all the more depressing in that we have known this type of resistance for nine years and a substantial amount of research has been done on the subject. The results, though of great theoretical interest, have born no practical fruit.

II.

ANOPHELINE MOSQUITOES

In the past year, three new cases of resistance among malaria vectors have been reported and two former claims confirmed. Despite these ominous warnings, the over-all situation is not entirely gloomy. Anopheline mosquitoes were some of the first insects to be systematically attacked by residual insecticides and many have been under continuous control with D D T for 8 to 10 years. Years of house spraying have evidently not changed the suscepfi= bility of Anopheles atroparvus in Spain, Sardinia and Italy, and of A. culicifacies in Ceylon and India, of A. pseudopunctipennis in Mexico or of A. darlingi in British Guiana. Nevertheless, where resistance has developed, it is sufficiently serious to prevent control by the insecticide concerned. (a) Anopheles sacharovi in Greece. D D T has been used in a national antimalarial campaign in Greece since 1946, both as a residual house spray and for aerial larviciding. Malaria was reduced from the pre-war incidence of one to two million cases a year to some 10 to 15 thousand per year. The first signs of resistance were reported to W.H.O. in December, 1951, and published later by LIVADAS and GEORGOPOULOS(1953). At present, resistance of A. sacharovi appears to be wide spread in the country, judging from tests in several localities. In recent years, chlordane and dieldrin have been used to some extent and A. sacharovi has become resistant to them also (GEORGOPOULOS, 1954 ; BUSVINE,in press). According to field observations, A. maculipennis and A. superplctus have also lost their susceptibility to D D T ; but this has not been confirmed. In spite of wide-spread prevalence of the important vector, A. sacharovi, owing to resistance, there has not been an appreciable rise in malaria. The disease has never been extinguished ; it continues to smoulder on in a number of villages, especially in the north western Peloponnese, in Epirus and in Macedonia and Thrace (BELIOS, 1955). Various reasons have been advanced for the " anophelism without malaria " which exists in many areas. Perhaps the partial immunity to malaria persists longer than was supposed. Perhaps the gametocyte carriers are too few and do not travel about. Alternatively, the selective effect of D D T has produced a more zoophilic race. I observed that A. sacharovitends to rest more outside houses than formerly ; but this may be due to the irritant effect of DDT, which may persist even if the mosquitoes are more resistant. The most sensible malaria policy for Greece now that insecticides have failed would

J. R. BUSVINE

15

appear to be the use of drugs in malarious villages. While antimalarial drugs may be impractical for the initial stages of a campaign, they may be valuable for what one may call " mopping up " operations. A. sacharovi in Lebanon. D D T has been used widely in the Lebanon since 1947. In 1954, Garrett-Jones and Gramiccia reported evidence of resistance in a village on the Syrian border. I myself confirmed this in 1955 ; but the resistance was not of a high order and does not yet seem to prevent adequate control. (b) Anopheles sundaicus in Java. In 1952, two residual spraying campaigns were initiated in Java. (Prior to that time there had been an indefinite amount of control by larvicides containing DDT.) Housespraying against the principal vector, A. sundaicus, began in a W.H.O. scheme in the south, along the coast near Tjilatjap ; and at the same time a programmedirected by I.C.A. (F.O.A.) began in the north, near Djakarta and Tjirebon. Results were not very satisfactory. In the Djakarta area there was no decline in malaria. In the south, there was a fall in malaria indices in 1953 in both treated and untreated areas ; but both rose substantially in 1954. It appears that one reason for these failures is that A. sundaicus is very readily irritated by D D T and tends to avoid contact with treated surfaces. Apart from this, however, resistant strains were discovered near Djakarta and Tjirebon (CRANDAL, 1954) and later high resistance was also proved at Semerang, also on the north coast (DAvlDSON, quoted by BUSVlSIE,in press). No evidence of D D T resistance has been reported for other local anophelines, such as A. subpictus or A. aconitus. The A. sundaicus were shown to be still susceptible to dieldrin and gamma BHC and the former has been employed since 1954, so far with success. (c) Anopheles gambiae in Nigeria In Western Sokoto (Northern Nigeria) a Malaria Control Pilot Project, depending on residual spraying, was initiated in June 1954. Some 300 square miles were allocated to treatment with dieldrin, mainly at 25 mg./sq, ft. After three sprayings at 6-monthly intervals had been completed, evidence of resistance was found and confirmed by measurements of susceptibility of mosquitoes from treated and untreated areas (ELLIOTT and RAMAKRISHNA, 1956). Subsequently, a colony of these resistant mosquitoes was established at the Ross Institute in London. This colony was found to be enormously resistant to dieldrin and similar insecticides, moderately resistant to gamma BHC, but not significantly resistant to D D T (DAVlDSON, 1956a). Further studies in London (DAVII)SON, 1956b) revealed a simple Mendelian basis of inheritance of resistance in this mosquito. It seems very likely that the comparatively low level of resistance, registered first in the field, was due to the heterogeneity of the population (mixed susceptible, resistant and heterozygotes) ; whereas the London colony seems to be a pure homozygous resistant strain. It is not clear yet whether A. gambiae will become resistant to dieldrin etc. in other parts of Africa. Many schemes relying on this insecticide are in progress, so far without signs of trouble. There may be two reasons for the emergence of resistance in Northern Nigeria : (i) a unique gene for resistance may be present only in the mosquito population in Western Sokoto ; (ii) conditions in the N. Nigerian scheme may favour early development

16

I N S E C T I C I D E - R E S I S T A N T STRAINS OF INSECTS

of resistance. For example, the very hot dry season forces mosquitoes into houses and it may be that a higher proportion of the population contacts the insecticide than in more humid parts of the mosquito's range. (d) Anopheles quadrimaculatus in U.S.A. D D T has been widely used in south-eastern U.S.A., for control of A. quadrimaculatus, since 1946. At one point it seemed that DDT-resistance had developed in the Tennessee Valley (KRuSE et al., 1952) ; but subsequent investigations showed that the failure of the D D T was due to causes other than resistance (HAWKINS and HALL, 1954). An indication of another type of resistance was obtained in 1953, when heavy applications of dieldrin failed to kill A. quadrimaculatus larvae in Mississippi. This year, definite proof has been obtained of high resistance to dieldrin, chlordane and gamma BHC, in this region ; however, the tests show normal susceptibility to D D T (MATHIS et al., 1956). It appears that this resistance has developed as a result of contamination of the breeding grounds during aircraft dusting with insecticides against cotton pests. The seriousness of this particular incidence of resistance is mitigated by the virtual eradication of malaria from the U.S.A. in recent times. (e) Anopheles stephensi in Saudi Arabia and India. D D T house spraying was first used against A. stephensi in Saudi Arabia in 1947, by the Government, in collaboration with the Arabian American Petroleum Company. The places treated were the two oases of Qatif, 10 miles north of Dhahran, and A1-Hasa, about 100 miles to the south, as well as some suburbs of Dhahran. About a quarter of a million people live in these localities, which were sprayed in alternate years, with the resulting improvement in malaria. In 1954 and 1955, however, Drs. Daggy and Peffly of " Aramco," reported an increase in malaria and the appearance of A. stephensi in DDT-treated houses. They suggested the possibility of resistance ; and this was confirmed by Mr. G. Davidson, who visited the country in November, 1955. He found, however, that the mosquito was normally susceptible to dieldrin and it is understood that this insecticide is now being used for house spraying in Saudi Arabia. DDT-resistant A. stephensi have also appeared locally in southern India, judging from tests on larvae (RAJAGOPALANet al., 1956). In the district concerned , D D T larvicide had been used regularly since 1947 : and in the year before signs of resistance were noted, two house-spraying campaigns had also been done. III.

CULICINE MOSQUITOES

The first mosquitoes to show signs of insecticide resistance were various culicine species in the U.S.A., which were being sprayed from the air with larvicides, because of the nuisance of their bites. Apart from these culicines, there have been scattered reports of resistance in members of the Culex pipiens complex, and, more recently, in Aedes aegypti. The last two cases will be considered first, because of their greater importance as disease vectors. (a) Aedes aegypti in Trinidad. Since this mosquito is so endophilic in habit, it has in many places come in contact with D D T residues which were primarily intended for killing malaria vectors. The result

J. R. BUSVINE

17

has usually been very satisfactory. Thus, the anti-anopheline campaign in Greece, Mauritius and British Guiana was effective in eradicating A. aegypti from these countries. PINTO SEVERO (1954) points out that anti-anopheline campaigns have simplified the task of eradicating A. aegypti from central and southern America. A very different story appears in the reports of the Medical Department of Trinidad. In this island, regular house spraying with D D T had been done as an antimalarial measure, with fairly satisfactory results. In the years prior to 1953, about 25,000 houses were sprayed annually, mainly it seems in rural areas. In 1953 to 1955, the amount of spraying was increased to include some suburbs of Port of Spain and altogether some 80,000 houses were treated. In spite of the residual house-spraying programme, A. aegypti was, throughout, plentiful in some areas. Observations of the malaria control officers in the course of their duties gave rise to concern and in 1950 an Aedes survey was made and an eradication plan proposed. At this time, however, there were no grounds for urgent action and the full programme was not put into effect. A limited campaign in the rather densely populated areas near Port of Spain was carried out in 195I and 1952. Four teams of men attempted to treat all breeding sites in the area with D D T wettable powder to give a dose of ½ p.p.m. DDT. The results, however, were not very satisfactory ; they gave control but no eradication. In 1954, a few cases of yellow fever were diagnosed, which gave riseto some anxiety and the authorities instituted an island-wide A. aegypti eradication scheme. With the obiect of perifocal treatment of all breeding sites, some 18 teams were at work in 1954 and about 30 in 1955. The results, again, were disappointing, for treatments of 1 p.p.m, gave no certain kill. However, numbers were kept low, and this, together with vaccination and residual house spraying, prevented a yellow fever epidemic. Following the failure of D D T anti-larval treatments in the field, laboratory colonies were started in Trinidad and experiments proving high resistance to D D T were later confirmed by tests on a sub-colony at Savannah, Ga., U.S.A. (BROWNand PERRY,1956). So far, there is no resistance to dieldrin or gamma BHC, which are still suitable for control measures. (b) Culex pipiens complex. Culex fatigans. Reports of the first tests of D D T as a residual house spray, in different parts of the world, all agreed that C. fatigans is a mosquito with high natural resistance to this insecticide. Treatments which killed high proportions of the local anophelines produced only low mortalities (10-30 per cent.)in C.fatigans. (cf. GIGLIOLI- British Guiana, 1946 ; WHARTON and REin--Malaya, 1950; DAVlDSON- - Kenya, 1952.) This initial low susceptibility seems to have been decreased even further by residual spray programmes. Thus, there is evidence of locally enhanced resistance to DDT, shown by tests with larvae, in R6union (HAMON and DUFOUR, 1954) and in tests with adults near Delhi (PAL et al., 1952). Since D D T gave poor results against C. fatigans, various field workers have used BHC or dieldrin, which initially gave good results. However, after about two years' use of BHC as a larvicide, strains resistant to BHC and dieldrin were developed in areas in South India (RNACOPALANet al., 1954) and Malaya (REID, 1955). The degree of resistance was moderate, being of the order of a sixfotd increase in India and a tenfold increase in Malaya ; however, it was enough to affect control measures. Both authors found that when they reared these strains in the laboratory they gradually lost their enhanced resistance.

18

INSECTICIDE-RESISTANT STRAINS OF INSECTS

Resistance to dieldrin in C. fatigans would seem to be particularly unfortunate, since CHARLES (1954) has shown that combined spraying and larviciding with dieldrin can virtually eradicate this mosquito, which, of course, is the classical vector of Wuchereria bancrofti.

Other members of the complex. MOSNA (1947) published an early claim that the D D T spraying in Italy had induced local resistance in C. molestus ; but his data are scanty. HADJINIKOLAOU (1954) reported that the resistance of this mosquito in Greece was found to be high in the earliest tests conducted. HESS (1953) published a personal communication from H. A. Crandell, to the effect that " Observations at Toledo, Ohio, with Culex pipiens have indicated that larvae collected from a heavily treated area showed significantly lower mortality when exposed to D D T than did larvae which were collected from untreated areas." AmUSTRONG(1955) describes some experiments in Cambridge, Mass., which gave the same results. (c) Culidne nuisance mosquitoes. Salt-marsh breeders in Florida. Salt marshes along the coasts of Florida produce hoards of mosquitoes which cause vexation from bites and are bad for the tourist business. From 1945, regular control measures have been employed, using D D T as a larvicide, sprayed from the air ; but after five seasons, these measures began to fail. Laboratory tests on larvae from sprayed and unsprayed areas, revealed resistance in two of the most important species, Aedes sollicitans and A. nigromaculis (DEONIERand GILBERT, 1950). According to BROWN (1954), " D D T resistance is now general throughout Florida in both larvae and adults of these species, which are now appreciably less susceptible to other hydrocarbon insecticides." Nevertheless, it is still possible to control them with appropriate doses of dieldrin, gamma BHC, chlordane, etc. as well as the organo-phosphorus insecticides EPN and malathion: In addition, some permanent improvement by drainage is being done. Flood-water breedersin California. In the agricultural districts of California, it is the practice to flood the fields periodically for irrigation purposes. This leads to the production of large numbers of culicine mosquitoes, especially A. nigromaculis, A. dorsalis and Culex tarsalis. All of them are annoying biters ; and, in addition, C. tarsalis is a vector of equine encephalitis. Control measures based on D D T larvicide were started in 1945 and used successfully for three years. In 1949, failures were reported and laboratory tests confirmed the existence of resistant strains (BoI-IARTand MURRAY,1950). Subsequently, other insecticides, such as toxaphene, BHC, chlordane, etc. were used on a large scale ; but by 1951 C. tarsalis had developed high resistance to these other compounds (GJULLIN and PETERS, 1952). Following the failure of chlorinated insecticides, mosquito abatement officers have turned to organo-phosphorus compounds, espeeially EPN, which GJULLIN et al. (1953) found highly effective against the resistant strains. Rice-field breeders in Mississippi. The introduction and rapid expansion of rice growing in Mississippi in 1950-51, created a serious mosquito pest problem. The principal nuisance to residents in the rice growing districts is Psorophora confinis, while smaller numbers of P. discolor are also troublesome, especially at night. Experiments in 1952-53 led to the adoption of pre-flood treatment of the rice fields with dieldrin, which gave good

j. R. B u s v i ~

19

control. However, in 1954, even a succession of dieldrin treatments failed to give adequate control. Comparative tests, with adults reared from larvae taken from treated and untreated districts, proved the existence of dieldrin resistance, but no difference in susceptibility to D D T (MATHIS et al., 1955). It seems rather unlikely that this intractable resistance could have resulted solely from the 1953 larvicide spraying. Probably, like the simultaneous development of resistance in A. quadrimaculatus in the same region, it is a consequence of wide-spread cotton dusting with dieldrin and similar insecticides. IV.

LICE OF MAN

Throughout this section, the lice referred to are the body-louse form, Pediculus human~ human~. There is no evidence of resistance in the head-louse form, P. h. capitis nor in the crab louse, Phthirus pubis. The part played by D D T dust in terminating the 1943 typhus epidemic in Naples is well known. Within a few years after the war, similar successes in checking typhus were obtained in Transvaal, Nigeria, Mexico, Chile and Spain ; while relapsing fever epidemics were quelled in Tunisia and Kenya (SIMMONSand UPHOLT,1951). The first failure of D D T to control lice occurred in P.O.W. camps in Korea ; and soon afterwards experiments proved the existence of a high level of resistance (HURBUTet al., 1952). Similar observations were made in Japan (KtTAOKA, 1952 ; BARNETT and KNOELOCK, 1952). A strain of lice collected in Cairo in 1950, and tested later in England, was found resistant (BUSVlNE, 1953) and further evidence of resistance was later obtained in other parts of Egypt (HuRBUT et al., 1954). All these cases of resistance occurred in areas where considerable amounts of D D T had been used in control measures against lice. Strictly speaking, however, there was no proof of actual loss of susceptibility. In 1953, W.H.O. inaugurated a survey of susceptibility levels of lice, throughout the world. Testing kits were supplied to public health authorities in countries which agreed to co-operate, with requests for information. By 1956, results of 137 complete tests from 35 countries had been received in Geneva ; these were compiled and collated by WRIGHT and BROWN(in press). These results are very variable, partly perhaps owing to small differences in technique (e.g. in the handling of lice and their age and state of nutrition). Furthermore, the test is not a sensitive one since a large increase in concentration frequently gives only a small rise in kill. Nevertheless, it is clear that there are wide differences in tolerance of lice. It is not clear whether this is entirely referable to locality, since a French study revealed variations in DDT-tolerance of lice from different individual hosts (NICOLI and SAUTET, 1955). However, that investigation was done on vagrants of mixed nationality in Marseilles. Wright and Brown found no clear correlation between resistance and a history of previous dusting campaigns. High levels of D D T resistance were found in many parts of the world (though only one in Europe) and a few cases of BHC resistance were also discovered. Indications of resistance to pyrethrins were very rare. The present position seems to be as follows. While D D T is still quite satisfactory for controlling lice in most places, it appears that failures might occur in regions outside Europe, so that it cannot be guaranteed as a universal anti-louse measure. So far, gamma BHC dust would be a reliable substitute nearly everywhere, though there are ominous suggestions of incipient resistance. Pyrethrum powders remain as the final line of insecticidal defence against louse-borne epidemics.

20

INSECTICIDE-RESISTANT STRAINS OF INSECTS

V.

BED BUtS

House spraying with D D T enormously simplified the task of destroying bed bugs in old houses. Campaigns to eradicate bugs from urban areas were undertaken in several countries, after the war. Most of this work was not published, but KEMPER (1952) describes the successful results obtained in Berlin. The first record of failure came from Hawaii in 1948 and subsequently reports of resistance have come from many countries, including (single localities in) Greece, Italy, Israel, U.S.A., the Belgian Congo and China. In addition, C. hemipterus appears to have become resistant in Hongkong, Formosa and Singapore. Most of these reports are merely bald statements based on field observations ; but some simple experiments seem to substantiate the observations in Hawaii (JOHNSONand HILL, 1948) and Israel (LEvINSON,1953). I myself have a colony of resistant C. hemipterus from Hongkong. Laboratory tests show that it is much more resistant to D D T than normal C. lectularius though slightly more susceptible to gamma BHC. No normal C. hemipterus are available for comparison. It is difficult to make general comments on the situation, as the data are so meagre. It may be noted that the trouble seems to have occurred in the warmer regions of the world. So far, the resistant bugs in Israel, Hongkong and Singapore appear to be normally susceptible to gamma BHC and dieldrin. VI.

FLEAS

In the period between 1944 and 1948, D D T dusts used against fleas, substantially reduced murine typhus in parts of U.S.A. and checked plague outbreaks in Dakar, Haifa, Ngamiland (South Africa), Peru and Ecuador (SIMMONS and UPImLT, 1951). Subsequently there has been a considerable number of reports of resistance, based on field observations. KILPATRICK and FAY (1952), referring to the summer of 1949, remark that " in several instances high populations of the cat flea Ctenocephalides felis persisted even after three applications of 5% D D T pyrophyllite dusts to infested premises. With the use of 5% chlordane dusts, the infestations dropped markedly or were completely eliminated." VERA (1953) states that D D T dust was of great value for anti-plague flea control in Ecuador for several years after 1945 ; but in 1950 failures were observed in two localities. The following species of fleas survived after thorough treatments, which should have been effective: Pulex irritans, Nosopsyllus londiniensis, N. fasciatus, Rhopalopsylla claviculus, Xenopsylla cheopis and Polygenis sp. HADJINIKOLAOU (1954) describes how he made collections of large numbers of P. irritans in DDT-sprayed houses and pigsties in Greece in 1951. It is not clear, however, whether D D T residual spraying of walls could be expected to eliminate fleas, though apparently it did so in the early years of the antimalaria campaign. It will be observed that the question of D D T resistance in fleas is nowhere backed by experimental proof nor even, in most cases, by documented field observations. The series of reports of failure of D D T in the field is, however, disquieting. VII.

COCKROACHES

It seems that cockroaches have a natural low susceptibility to D D T and for some years it has been the practice to attack them with other insecticides, such as gamma BHC or chlordane. Failure to control Blatella germanica with chlordane has been observed in

J. R. BUSVINE

21

parts of Texas since 1951. L a b o r a t o r y tests revealed a high level of resistance to chlordane and dieldrin and some increased tolerance of g a m m a B H C and D D T (HEAL et al., 1953 ; FISK and ISERT, 1953). So far, it is possible to obtain satisfactory control of these insects with a mixture of malathion and perthane (LAAKE, 1955).

ASSESSMENT AND INVESTIGATION OF RESISTANCE

While it is difficult to assess some of the reports of insecticide resistance, it is clear that this trouble is impeding the control of more and more insects of public health importance. So far, no effective way has been found of regaining the activity of an insecticide to which resistance has developed. Indeed, it m a y well be said that the problems of resistance are growing faster than our capacity to deal with them. T h i s was the opinion of the W . H . O . Expert C o m m i t t e e on Insecticides, expressed at a meeting in July 1956. I t was agreed that the matter is serious and the committee r e c o m m e n d e d that the W . H . O . should act as a co-ordinating body, to facilitate an international attack on the problem. T h e ways in which the Organization can help are as follows : (a) Delays in publication and other difficulties in rapid circulation of information about resistance prevent the most efficient organization of control campaigns and impede research at various levels. The W.H.O. will act as a repository of information and endeavour to circulate new data quickly. (b) Another way in which the Organization can help concerns the detection and measurement of resistance. Hitherto, a great variety of methods has been employed, some better than others ; but the main trouble is that the results are not comparable. Already some progress has been made in promoting standard methods to be used throughout the world, so that results of different workers can become comparable and more meaningful. As I have already mentioned, the W.H.O. test for susceptibility in body lice has been used in 35 countries. The Busvine-Nash test for adult mosquitoes has been employed in at least 12 countries and has recorded basic figures for seven culicine and a dozen anopheline species. An improved version of this test is being developed in conjunction with other panel members of the Committee. At the same time, a standard test for measuring resistanee in larval mosquitoes is reaching completion and suitable tests will be designed for other insect pests. These activities of W.H.O. should assist us to get a clearer and more reliable picture of the resistance situation at any moment and should facilitate the discovery of new cases in future. (c) The delineation of the resistance situation is, of course, only a first step. In order to understand what is happening and to predict future possibilities of resistance, more basic and applied research are required. The W.H.O. has already made a world survey of laboratories capable of contributing to this work and the results are not very reassuring. Only a proportion of the institutes which would be expected to contribute were, in fact, doing so ; and in most cases the research was on a part-time basis. The W.H.O. intends to draw attention to the need for this research and to foster it in various ways.

RESEARCH ON RESISTANCE A N D LACUNAE I N OUR K N O W L E D G E

I should like to try to show how our attempts to answer practical questions about resistance immediately expose our fundamental ignorance. W h a t we urgently need to know is whether resistance is liable to develop in every insect pest, towards any type of insecticide, if the latter is used extensively and for long enough. T h e happenings of recent years show that resistance has developed more rapidly and extensively in some situations than others ; but we do not know h o w m u c h this depends on the species concerned and how m u c h on the insecticide and the way it is used. Several years ago I suggested that the chances of developing resistance quickly depended on three things : - (a) the frequency and importance of genes conferring resistance in the original population of insects ;

22

INSECTICIDE-RESISTANT STRAINS OF INSECTS

(b) the intensity of selection (i.e. the size of the population exposed to insecticide and the proportion killed) ; (c) the number of generations per year. These three factors require further examination.

(a) Potentialities for developing resistance Extremely little is known about the occurrence of genes for resistance in natural wild populations of pest insects. There are some reasons for expecting them to be very rare before they are concentrated by selective mortality due to wide use of insecticides. Therefore we have to base our speculations largely on information about existing resistant strains. Some quite recent experiments with a dieldrin-resistant strain of A. gambiae seem to have given clear-cut results (DAVlDSON, 1956b) ; perhaps because this resistance is of a simple, but highly developed kind. On the other hand, results of a dozen different investigations on DDT-resistance in the housefly and about the same number concerning induced resistance in Drosophila melanogaster have produced a welter of confusion. Various authors have ascribed DDT-resistance in Musca to a single recessive gene, a single dominant gene, multiple genes, cytoplasmic inheritance and mixtures of several types (see BusvI~n~, in press). This confusion appears to be due to two causes : (i) there may be several factors concerned in resistance, which are inherited in different ways ; (ii) the subject is more difficult to investigate than it appears and the techniques both of genetics and of measuring resistance have not been adequately refined. It is to be hoped that future investigations of the subject will benefit from two reviews, shortly to be published by competent geneticists (CRow, in press ; MILANI, in press). In view of the fragmentary state of our knowledge at present some of the following remarks must be regarded as quite speculative. It seems quite feasible that there may be two kinds of resistant genes ; those giving generalized resistance and those conferring specific protection from a particular poison. (i) Generalized resistance has been called "vigour tolerance" by HOSKINS (1956). It may be due to various hereditable physiological characters (such as size, or resistance to starvation or desiccation) which would be expected to promote survival after treatment with any insecticide. Reduced permeability of the cuticle or increased lipoids in the body (WIESM~N, 1956) would raise resistance to a variety of hydrocarbon insecticides. On analogy with other variable biological characters, one might expect such attributes to be controlled by multiple genes and one would not expect the change to be very large. It would seem very likely that resistance depending on genes of this type may develop in any species, if it is subjected to selective mortality pressure by any toxicant, for long enough on a sufficient scale. From the practical point of view, the importance of such low-level generalized resistance, depends very much on the safety margin available with the particular control measure concerned. To give a concrete example: it unfortunately seems that the control of some mosquitoes by residual spray treatments has only a small margin of efficacy. It is true that some mosquitoes, like A. darlingi and A. maculatus, are very sensitive to D D T . But other forms, such as C. fatigans, have a naturally low susceptibility, so that a small additional increase will render them virtually immune. (ii) Genes conferring highly specific resistance to a particular type of poison are likely to promote a biochemical defenee mechanism. This could either mean a specific detoxifying enzyme or the introduction of a metabolic system unaffected by the poison.

j. R. BusvmE

23

Examples of both are known. The DDT-dehydrochlorinase enzyme in resistant houseflies is one mechanism which protects them from this poison. The hydrogen cyanide resistant scale insects are believed to have developed a cyanide-insensitive respiratory system. It seems that specific resistance mechanisms confer much higher resistance than vigour tolerance and, furthermore, they appear to be due, at least in some instances, to single genes. A characteristic of this type of resistant strain is that laboratory colonies of very high levels of immunity can be readily produced, presumably by selection of homozygous colonies. Whether these are liable to develop and remain stable in the field is another unanswered question. The nature of a biochemical defence mechanism is obviously specifically connected with a particular type of insecticide. It would be of great interest to know how widely distributed among different insects are the potentialities for developing those mechanisms we have already encountered. As a first step, it would be helpful to know if the strains of various insects which already show resistance, depend on similar defence mechanisms. Three general kinds of resistance have become prominent recently : (i) to D D T and analogous compounds ; (ii) to gamma BHC and compounds of the chlordane type ; (iii) to organophosphorus insecticides. (i) Types of DDT-resistance Since resistance in the housefly has been far more extensively investigated than that of any other species, it would seem reasonable to use this as a basis for comparison. So far as I can judge, DDT-resistanee in the housefly depends on more than one defence mechanism. Some excellent biochemical work has proved that a part of the resistance depends on an enzyme which converts D D T into the harmless compound DDE (2, 2-bis(p-chlorophenyl)-l, 1-dichlorethylene), (STERNBURG et al., 1950; see also review by METCALF, 1955). This enzyme seems to be present in all resistant strains and absent, or nearly so, in susceptible ones ; it is probably an important factor in fly resistance. Some of us, however, do not believe that this is the only cause of resistance. When investigating two resistant fly strains some years ago (BUSVINE, 1951), I noted that one colony was resistant to chemicals analogous to D D T in proportion to the ease of dehydrochlorination, in vitro (i.e. the degradation of D D T to DDE, by alkali). The other strain of flies, however, was more or less resistant to all DDT-like compounds. A critical test is the existence of resistance to compounds like " prolan " (1, 1-bis (p-chlorophenyl) -2-nitropropane) or " DANP " (dianisyl neopentane), which simulate the D D T molecule in shape but cannot be degraded by dehydrochlorination, because the core of their molecules contains no chlorine. Turning now to resistant strains of other insects, we find that a strain of body lice resistant to D D T was found to be also resistant to DANP (BuSVlNE, 1953). Biochemical tests show that both resistant and normal lice can degrade DDT, so that it is difficult to judge whether this is a cause of resistance or not. The D D T is changed to a water-soluble derivative, so that DDE is either not formed, or is changed to something else (PERRY, cited by BROWN, in press). Some biochemical experiments with DDT-resistant A. aegypti from Trinidad have shown that larvae of this mosquito can change D D T to DDE, in vivo (BRowN, 1956). However, it has not been possible to extract the enzyme and perform the conversion in vitro, as it has with houseflies (STERNBURGet al., 1954). Tests with analogues of D D T support the impression that the resistant Aedes depend on a dehydrochlorination mechanism.

24

INSECTICIDE-RESISTANT STRAINS OF INSECTS

Thus they are highly resistant to D D T and " B r - D D T " (1, 1-bis (p-bromophenyl) 2, 2, 2trichlorethane), both easily degraded; but n o t resistant to methoxychlor (difficult) or dianisyl-neopentane (impossible) to dehydroehlorinate. To summarize the information on dehydrochlorination of D D T ; this is an important factor in resistance of some houseflies, it seems to be essential to resistance of Aedes, but it is probably unimportant in some resistant lice. It may be remarked that this mechanism has been demonstrated in several normally susceptible species (see HOSKINS, 1956) ; SO that perhaps it is not rare or unique. It is obviously desirable to investigate how far this mechanism is concerned with DDT-resistance in other species, such as A. sundiacus and C. lectularius. (ii) Resistance to gamma BHC, chlordane, dieldrin etc. After the housefly had become resistant to DDT, other chlorinated insecticides were used ; but it was soon found that when a new resistance developed, either to chlordane or to gamma BHC, the flies had automatically acquired resistance to several other insecticides (BuSVINE, 1951). This group ~resistance was eventually found to include the following : gamma BHC, chlordane, heptachlor, dieldrin, aldrin, endrin, isodrin and toxaphene. It would seem that a single mechanism is responsible (at least, partly) for resistance to the whole series, though it may be more efficient against some than others. Some workers found that resistance developed highest to the compound used for selection (BRucE and DECKER, 1950 ; KEIDING, 1953) ; but my own research suggests that the order of resistance is basically independent of the chemical used for selection (BusvINE, 1953). Resistance in four colonies of resistant flies from different countries was, in all cases, developed as follows : chlordane = dieldrin = aldrin > gamma BHC > endrin = isodrin. Evidence of the same resistance to the same group of insecticides has been found in other insects as follows : Pest species

Exposed to

Resistance developed to

C. fatigans

gamma BHC

gamma BHC, dieldrin.

C. tarsalis

toxaphene, aldrin

toxaphene, aldrin, gamma BHC, heptachlor.

A. gambiae

dieldrin

dieldrin, aldrin, chlordane, gamma BHC, endrin, isodrin.

B. germanica

chlordane

chlordane, dieldrin (gamma BHC).

Boophilus decoloratus

gamma BHC

gamma BHC, chlordane, dieldrin, toxaphene.

In most of these cases, only approximate measurements of relative resistance were made ; however, the results with A. gambiae are most interesting (DAVIDSON, 1956a). The levels of resistance are in the following order ; chlordane > dieldrin ~ aldrin > endrin ~ isodrin > gamma BHC. The order is not greatly different from that shown by Musca, except that gamma BHC has fallen below isodrin and endrin. Another interesting fact is that a separate strain of A. gambiae from an adjacent area treated only with gamma BHC, has given the same " resistance spectrum" as the one from the dieldrin-treated zone. The conclusion seems to follow that resistance to the group of insecticides mentioned

j. R. BUSVlNE

25

above depends on a similar mechanism in several'different species. Verylittle can be said about the nature of this mechanism ; biochemical studies of it have been so far limited to gamma BHC, owing to technical difficulties in estimating minute quantities of the other insecticides. Evidence from two sources is accumulating to show that the isomers of BHC are absorbed more slowly and metabolized more quickly by resistant houseflies than susceptible ones. As a result, three or four times as much free gamma BHC may be present inside a normal fly, as compared to a resistant one exposed in the same way (BRADBURYand STANDEI'~, 1955, 1956a ; OPPENOORTH, 1956). These findings, however, do not seem to explain the very large change in susceptibility observed. Furthermore, recent experiments with resistant A. gambiae from N. Nigeria revealed no difference in the rate of degradation of BHC in comparison with normal mosquitoes (BRADBUR¥ and STANDEN, 1956b). The exact nature of the metabolic degradation of BHC is unknown, but it does not follow the dehydrochlorination to trichlorbenzene produced by alkali, in vitro. Pentachlor cyclopentane is, apparently, formed as an intermediate product (STERNBURGet al., I956), but the final metabolite is water soluble, both in M. domestica and in A. gambiae. It is, perhaps, difficult to imagine a biochemical process involving such different compounds as gamma BHC and dieldrin ; whereas, there is evidence of similar toxic action due to these and other members of the group, possibly due to a sterically similar toxophore (BusvlI,~E, 1953). This suggests that the common resistance factor may be the development of a metabolic path insensitive to this particular type of poisoning. (iii) Resistance to organo-phosphorus insecticides. Comparatively little is known about resistance to organo-phosphorus insecticides, perhaps because they have been less extensively used, especially for insects of public health importance. Measurements of relative resistance have been made on several fly strains taken from areas in Denmark where organo-phosphorus treatments were beginning to fail (KEIDIZ~G, 1956). The results showed that the use of parathion induced resistance to diazinon and to "Bayer 21/199" (3-chloro-4-methyl-umbelliferone-0, 0-diethyl thiophosphate) as well as to parathion. Whichever of these three compounds was used in the field, the flies seemed to have built up higher resistance to " Bayer 21/199 " than to the other pair, for which resistance increased only two or three times. A little more evidence comes from unpublished tests on a strain which I obtained from Professor L. E. Chadwick. Resistance had been developed by laboratory selection by DFP vapour over 100 generations. My results showed a low level of resistance to a wide range of organo-phosphorus compounds, of the order X3. Like CHADWICK(1954) I found that resistance was reduced if the insecticide was injected, which suggests that the cause is reduced penetration of the cuticle. Little helpful information derives from agricultural pest control. There are bare records of organo-phosphorus resistance among certain aphids in the U.S.A. and Switzerland (WIESMANN, 1954) ; and there is experimental evidence of resistance in mites of the genus Tetranychus. This evidence consists of the results of orchard and greenhouse sprayings which are not of sufficient precision to support many toxicological deductions. In general, they suggest that parathion induces resistance in mites to some organo-phosphorus compounds, but not all (e.g. resistant mites controlled by TEPP (SMITH and FVLTON, 1951) or by EPN and malathion (NEwcoMER and DEAN, 1953)).

26

INSECTICIDE-RESISTANT STRAINS OF INSECTS

(b) Intensity of selection and development of resistance. Generally speaking, a potential resistance will develop faster with more intense selection. The matter requires close examination; it involves both genetical and ecological considerations.

Genetical aspects. As CROW (1957) points out " t h e quantitative relation between selective intensity and rate of progress is complex and depends on the number of genes involved, dominance and epistasis, amount of environmental effect, counteracting effects of natural selection, etc. ; but qualitatively, the more intense the selection, the more rapid the progress." In other words, the greater the proportion killed in each generation, the more rapid the increase in resistance (excluding, of course, insecticidal effects giving 100 per cent. kill, which are exceptional, in practice). The results of KING (1954, 1955) concerning Drosophila are apparent exceptions, for he made greater progress by selecting at 50 per cent. rather than a 95 per cent. level. However, as Crow points out, the numbers of survivors from the 95 per cent. kills were small (sometimes on two flies) and this stringently curtailed genetic variability. Thus, the genetical potentialities of a small laboratory population may give results misleading in regard to happenings in the field. Another abnormal effect of selecting in small genetically homogeneous colonies is that the resistance genes selected may be specifically adapted to a unique gene assembly. In crossing with other resistant genes there may be interaction and loss of phenotypic resistance. This " c o m plementary epistasis " is only likely to occur in small colonies, whereas in large heterogeneous populations, resistant genes are all likely to be additive in effects, without interaction. Gene frequency will affect rate of emergence of resistance as well as the intensity of selection. When the genes are rare, which is probably the original condition in most cases, rate of selection will be slow ; but progress will accelerate later as they become more concentrated. When selection is relaxed, the return to susceptibility will follow the same course, in reverse. Unfortunately, the adverse effects associated with some resistance genes in the absence of insecticide are fairly mild ; so that the return to susceptibility will be slower than the rise of resistance. In particular, there should be a long period with resistant genes in a minority but not very rare ; and at this time they can rapidly be concentrated again if selection is reintroduced. This seems to be supported by evidence from laboratory colonies and also from the field. Where D D T has been used again, after being abandoned for three or four years, the latent resistance develops very rapidly indeed. In the course of laboratory investigations, certain checks to selection have been observed ; but comparable data from the field are lacking :--(i) a resistant gene may be linked to others causing deleterious biological effects (or the connection may be pleiotropie). The main deleterious effects observed with resistant genes have been slightly longer development cycles and, possibly, reduced fertility. (ii) When characters are induced by multiple genes, a progressive one may be linked with a retarding one, so that no further selection can be effective until crossing-over separates the two (e.g. bristle numbers in Drosophila : MATH•R and HARRISON, 1949). This phenomenon has not been actually demonstrated in experiments on resistance ; but it may be the explanation for the erratic progress sometimes observed. (iii) When resistance is due to a single dominator gene, it is difficult to produce a homozygous resistant strain by selection alone. Inbreeding is necessary (LIcHTW~T, 1956). Ecological aspects. There are only the most rudimentary quantitative data about the

J.

1t. B U S Y I N E

27

intensity of selection by insecticides in the field. Consider the effects of house spraying on mosquito populations. It is possible, by the use of huts with window traps, to estimate the probable mortality caused to mosquitoes which enter sprayed houses ; but this only has significance in relation to the total population of mosquitoes in the locality, the proportion which enter houses, other sources of mortality and the effects of insecticides on reproduction by the females. All these ecological questions require investigation to make even crude predictions of resistance potentials of mosquitoes. Several authorities have suggested that regular larviciding treatments against mosquitoes a r e prone to develop resistant strains more readily than measures against adults alone. This seems not unreasonable, since larvicides applied wholesale (e.g. by aircraft) would probably reach a greater proportion of a local population than imagicides. The adults contacting insecticide will be only that fraction (i) which have survived larval mortality from natural causes, which may have eliminated many individuals carrying resistance genes, (ii) which enter houses. Unless the proportion entering houses is high, the dilution effect of interbreeding with unselected mosquitoes will probably prevent the development of resistance. In passing, we may note that the selective effect of residual insecticides on mosquito adults may alter characters other than physiological resistance. Thus, where there is potentiality for developing a zoophilic or non-domestic race, the use of residual insecticides will tend to promote its selection. Perhaps this is what happened in Sardinia where, long after the end of the island-wide D D T spraying campaigns, the remaining Anopheles labranchiae seem to keep away from houses (AITKEN et al., 1954)' Alternatively, spray campaigns may produce a race with a changed reaction to insecticide, e.g. with abnormally developed avoidance of DDT. However, it should be emphasized that it is difficult to get evidence of such changes and very little exists at present. Insecticide persistence. So far, I have considered the intensity of selection solely from the biological standpoint. There is, however, the type of insecticide to be considered. Perhaps it is not mere chance that resistance is rare towards insecticides like pyrethrum which cannot easily give the persistent selective power of the chlorinated hydrocarbon type. Recent attempts to prolong the residual life of organo-phosphorus insecticides by adding chlorinated terphenyls (HoRNsTEI~ et al., 1955) may render them suitable for malaria control programmes. But, at the same time, this may increase their liability to develop resistance. (e) Number of generations per year.

When resistant genes exist in a population, it seems reasonable to suppose that the speed of selection of a resistant strain will depend on the numbers of generations per year. Many of the species which have developed resistance have short life cycles ; but some have not, as shown by the following rough estimates. (" Tropical " is taken as a mean temperature of 25 ° C. ; " mild temperate" as 6 months at 18 ° C. and 6 months at 8 ° C.). Approx. generations per year. Tropical Mild temperate A. aegypti, A. gambiae, A. M. domestica . . . . . P. humanus (on clothing) C. lectularius . . . . . B. germanica (indoors)

sundaicus, A. stephensi, etc. . . . . . . . . .

..

. . . . . . . . . .

.. .. ..

30 25 16

. . . . . . . . .

..

8

. . . . . . . . . .

..

2½

m

7 16 2 2

28

INSECTICIDE=RESISTANT STRAINS OF INSECTS

Since Blattella showed resistance no later than A. aegypti, it seems unlikely that reproduction rate is of overwhelming importance. However, where a species has a very wide range of distribution, one might expect it to show resistance first in tropical zones. This seems to be true in regard to C. lectularius. M. domestica, however, developed resistance mainly in sub-tropical areas. It may be recalled, also, that the first instance of D D T resistance was reported in Sweden.

COUNTER-MEASURES

AGAINST RESISTANCE

Three kinds of action to oppose resistance are possible :--(i) prevention ; (ii) early detection ; (iii) cure. (i) Prevention of resistance. The fragmentary nature of our basic knowledge of resistance makes it difficult to recommend sound practical ways of preventing the development of resistance. Some suggestions which have been put forward appear to have little or no evidence to support them. For example, it seems, sometimes, to be assumed that resistance originates in insufficient dosage or inadequate control measures. However, it seems quite probable that a potential resistance will develop more rapidly in response to intensified dosage. Indeed, I would like to put forward the opposite view and suggest that control measures should aim only at that level of mortality necessary to prevent transmission. Higher kills (short of complete extermination) seem not only wasteful, but potentially dangerous. It should be remembered that the only requirement of disease prevention is the removal (by death, or even merely by repelling) of insect vectors which have fed on infective human beings (see GABALDON, 1953). Possibly the only sure way of preventing development of resistance is by avoiding the use of a particular insecticide. Thus, it might be feasible to stop using D D T powder for sanitary control of lice and fleas and to save this valuable weapon for a typhus or plague epidemic. Alternatively, since D D T has already been widely employed, a ban might be placed on some good alternative insecticide, such as gamma BHC, in order to keep this in reserve for an epidemic. For some time, there have been suggestions that resistance could be prevented, or at least delayed, either by using mixtures of insecticides differing in mode of action or else by alternating from one to another. Unfortunately, there is little direct evidence on the matter, and theoretical speculations are based on untested assumptions. Nevertheless, since mixing or alternating insecticides are among the few readily practicable measures against resistance, I think we should consider the merits of each. Alternation. The value of alternation of insecticides depends largely on the rate of decline in resistance after selection pressure is removed. Unfortunately, the long life of residual insecticides like D D T makes it impossible to stop their selective action sharply. Furthermore, as we have already noted, the harmful effects of resistant genes in the absence of insecticide seem to be slight. Therefore, recession is likely to be very slow indeed after the initial fall. My impression is that a period of the order of 10 years is required before reversion to the use of D D T for controlling houseflies. At the end of this time, the best that could be hoped would be a 2-year effectiveness of DDT, comparable to its initial use in 1945.

J. R. BUSVlNE

29

Mixtures. The selective effect of a mixture of two insecticides depends very much on whether the susceptibility of the insects to the two compounds is positively correlated, negatively correlated or independent. If susceptibility to the second insecticide is positively correlated with that to the first, the mixture will merely act as a higher concentration of the original compound, so that selection for resistance will be intensified. In the somewhat unlikely event of the actions of the two poisons being inversely correlated, the effect will be highly synergistic. Since the survivors from compound " A " will be the individuals most sensitive to " B," it should be easy to obtain complete kills of all individuals contacting the mixture. Unfortunately, no such combination of poisons has been discovered. The most likely event is that resistance to B would be quite independent of A (since their modes of action are different). CROW(1952) believes that a mixture of poisons would be less satisfactory than alternation. His argument is as follows : If compounds A and B, at given rates, each cause 90 per cent. kill, they will together cause 99 per cent. mortality. Selection for resistance to either, will be at the 90 per cent. level. Alternatively, one could use a higher rate of A or B alone to give 99 per cent. kill. Crow calculates that 10 years' selection at the 90 per cent. level will produce one-third more increase in resistance than five years at 99 per cent., followed by a change to another compound. His calculations assume multiple factors for resistance, and also that the resistance levels of progeny of survivors will have a normal distribution. Both these points seem dubious to me and it seems that calculations of this kind will be more fruitful when we have more data on which to base them.

One interesting suggestion, which seems to fall between mixture and alternation, is to apply one type of insecticide as a larvicide and use another against the adults. (ii) Early detection of resistance Resistance is most likely to develop as a result of the widespread and systematic use of insecticide in organized control campaigns. In planning such schemes, therefore, adequate tests of susceptibility should be made before the start of operations and at intervals afterwards. Wherever possible, tests recommended by the World Health Organization should be used. In this way, reports of failure in the field can be speedily checked to determine whether they are due to a resistant strain or to some other cause. (iii) Overcoming existing resistance Where resistance is of a low-level " vigour tolerance " type, it is possible that increased dosage or a more efficient method of application will solve the problem. However, this is unlikely to be of help in combatting high-level specific resistance. If we had complete knowledge of the mode of action of insecticides and of the various defence mechanisms of resistant strains, we might be able to devise means of circumventing the protective system. Something like this was nearly achieved when so-called synergists were added to D D T to inhibit the detoxifying enzyme present in resistant houseflies. At first, this regained much of the original power of D D T ; but unfortunately, strains soon developed which were resistant to the DDT-synergist combination (probably because they depended on an alternative defence mechanism). In general, our ignorance of the toxicology of insecticides is such that anti-resistance measures cannot be based on fundamental knowledge. In fact, the only course open at the

30

INSECTICIDE-RESISTANT STRAINS OF INSECTS

moment is the obvious one of changing to a different type of insecticide. We cannot even forecast how long a new type of insecticide is likely to remain effective. The only evidence derives from the housefly, which developed a second type of resistance (to the chlordane, gamma BHC group) even more quickly than the original immunity to D D T . However, these data are too few to generalize. The possibilities of changing to alternative insecticides are more limited than is generally imagined. The three types of resistance described in the previous section embrace nearly all the effective new synthetic insecticides, developed by intensive testing in many countries in the past 25 years. Instead of changing to another insecticide, another possibility is to change to other measures. Prior to the introduction of cheap and highly effective residual insecticides, many pests were controlled by altering the breeding site to render it unfavourable, or by introducing parasites and predators. While these measures should be borne in mind, most of them are no adequate substitute for the insecticides, because, either on account of expense or for other reasons, they cannot be used over very large areas. However, certain measures may be feasible if resistance develops in the later stages of a campaign. Thus, where the use of insecticides for several years has drastically reduced a disease such as malaria, it may be possible to eliminate the residue by distribution of drugs, though that might not have been a feasible measure originally. Another method which might succeed after the insect population had been greatly reduced by insecticide consists of releasing males sterilized by radio activity, which in turn prevent the females with which they mate from breeding. This very interesting new method of pest control was successful in eradicating screw worms from the island of Curacao (BusFILAND et al., 1955). REFERENCES

AITKIN, T. H. G., MAIER,J. & TRAPIDO,H. (1954). Amer. J. Hyg., 60, 37.

ARMSTRONG, R. L. (1950). Proc. N.J. Mosq. Ext. Ass., 42, 137. BARNETT, H. C. & KNOBLOCK, E. C. (1952). U.S. Forces reed. J., 3, 297.

BELIOS, G. D. (1955). Riv. Malariol., 34, 1. BOHART,R. M. • MURRAY,W. D. (1950). Proc. 18 Ann. Conf. Californ. Mosq. Control Ass., p. 20. BRADBURY, F. R. & STANDEN,H. (1955). J. Sci. Fd. Agric., 6, 90. , (1956a). Ibid., 7, 389. , (1956b). Nature, Lond., 178, 1053. BROWN,A. W. A. (1954). Proc. N. J. Mosq. Ext. Ass., 41, 136. (1956). Bull. World Hlth Org., 14, 807. Ibid. (in press). & PERI~Y,A. S. (1956). Nature, Lond., 178, 368. BUSlJLAND,R. C., LINDQUIST,A. W. & KNIPLING, E. F. (1955). Science, 122, 287. BUSVlNE,J. R. (1951). Nature, Lond., 168, 193. (1953). Ibid., 171, 118. (1954). Ibid., 174, 783. Bull. World Hlth Org. (in press). BIrXTON, P. A. (1952). Trans. R. Soc. trop. Med. Hyg., 46, 213. CHADWICK,L. E, (1954). 1st Internat. Sympos. Control Insect Vectors of Disease, Rome. CHARLES,L. J. (1954). Proc. N. J. Mosq. Ext. Ass., 41, 166. CRANDEL, H. A. (1954). Mosquito News, 14, 194. CRow, J. (1952). National Academy of Science (National Research Council) Washington Publication, No. 219, p. 75. Ann, Rev. Ent. (in press). DAVlDSON,G. (1952). Nature, Lond., 170, 702. - (1956a). Ibid., 178, 705. (1956b). Ibid., 178, 861.

J. R. BUSVINE

31

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