Resistance in insects: the role of metabolism and the possible use of synergists

SESSION V TOXIC EFFECTS PRODUCED IN INSECTS BY ANTICHOLINESTERASES Bull. Org. mond. Sant) 1971, 44, 195-202Bull. Wld Hlth Org. Resistance in Insects: the Role of Metabolism and the Possible Use of Synergists F. J. OPPENOORTH 1 The use of synergists to prevent detoxification raises two principal problems: (1) the importance of detoxification as a resistance mechanism and the extent to which synergists could contribute to a solution of the resistance problem, and (2) the role of detoxification as a cause of selectivity, and whether the loss in selectivity that might result from the use ofsynergists would be a disadvantage. Ifinsecticides could be combined with detoxification- blocking synergists, a much wider range of insecticides might become available for use. This paper also discusses the relative importance of different routes of detoxification, whether to use a constant synergist-insecticide ratio, and some recent work carried out in the author's laboratory on microsomal oxidation of paraoxon and on the synergistic action of P= S compounds. Several reviews have been published dealing with detoxification in insects and with the general aspects of synergistic action (Metcalf, 1967; Wilkinson, 1968; several chapters in Hodgson, 1968; Casida, 1969, 1970; and in particular many chapters in O'Brien & Yamamoto, 1970, amongst them being one by Plapp on the molecular biology of insecticide resistance and one by Hennessy on carbamate syn- ergists). The latter subject has also been dealt with by Wilkinson (1971). Consequently, this paper does not attempt to make a complete survey of the literature; instead, it presents some general considerations on detoxification as a factor determining the effective- ness of insecticides and on the importance of block- ing detoxification by synergists, and discusses some recent work carried out in the author's laboratory that seems relevant to this topic. PROBLEMS INVOLVED IN THE USE OF SYNERGISTS Three problems present themselves if we consider the use of synergists that block detoxification: (1) What is the importance of detoxification in resistance and to what extent could synergists con- tribute to a solution of resistance problems? 1 Laboratory for Research on Insecticides, Wagecningen, The Netherlands. (2) What is the importance of detoxification in causing selectivity, and would the use of synergists be disadvantageous since they might cause a loss of selectivity ? (3) Would large numbers of chemicals become available as insecticides if only they could be com- bined with synergists to prevent their degradation? Would such combinations be advantageous com- pared with insecticides that are comparatively resis- tant to detoxification themselves? Detoxification as a resistance mechanism Theoretically, resistance mechanisms could involve all of the many steps that are of importance in the intoxication process that finally leads to death. An example of the complexity of such factors is given by Winteringham (1969). There is evidence, how- ever, that some of these mechanisms are of far greater importance than others. This is especially true of an increased capacity for detoxification and an altered site of action; a reduced rate of penetra- tion of insecticide into the insect seems to be of less importance than these two mechanisms. Rather than enumerating the many more or less convincing examples of detoxification as a cause of resistance, this paper discusses some of the cases where it has become apparent that detoxification is not involved. If we hope to overcome resistance by blocking detoxification it seems more important to 2627 - 195 - F. J. OPPENOORTH know about definite cases where this attempt will fail than to have many examples of situations in .which it may be successful. To the author's knowledge there is only one well analysed example of resistance to modern insecticides resulting from alteration at the site of action. This is the resistance in some spider mites (Smissaert, 1964; Smissaert et al., 1970) and ticks (Wharton & Roul- ston, 1970) to inhibitors of acetylcholinesterase, resulting from a change in the enzyme that con- siderably decreases the rate of inhibition. It should be noted that this resistance mechanism has not been reported so far for insects, despite the fact that many species have developed resistance to cholinesterase inhibitors and that it is technically not difficult to study it. Smissaert and the author stud- ied the cholinesterase in ten strains of housefly resistant to organophosphorus compounds, many of which were obtained from Dr J. Keiding in Denmark, and which had different resistance charac- teristics. None of these strains showed a reduced rate of acetylcholinesterase inhibition when incu- bated with the PO-analogue of the insecticide to which it was resistant. The absence of an altered site of action of the cholinesterase inhibitors in insects is important and is promising in relation to the possibility of overcoming resistance by blocking detoxification. Detoxification is not involved in several impor- tant cases of resistance to organochlorine insecti- cides. This is so in houseflies carrying the gene kdr (knock-down-resistance), which is one of the main mechanisms for DDT-resistance. When such a strain was compared with two other DDT-resistant strains carrying genes for detoxification, it appeared that 18 hours after application 500% of a 1 -jzg dose of DDT persisted in the kdr flies and only 2% in the other strains (Oppenoorth, 1967). The failure of synergists that block the degradation of DDT to overcome resistance in the field is probably due to the selection of the kdr gene (see Metcalf, 1967). Resistance to cyclodiene compounds in houseflies and many other dipterous insects also does not depend on degradation or other factors that deter- mine the amount present in the insects (Wintering- ham & Harrison, 1959; Brooks, 1960). This type of resistance may be due to alterations at the site of action, but so far there are no clues as to their molecular nature and synergists are not effective. Plapp (in O'Brien & Yamamoto, 1970) reports an increased level ofglucose-6-phosphate dehydrogenase for housefly strains with the kdr gene and in those with the gene for dieldrin-resistance, but the relation- ship with resistance has not yet been proved. It must be borne in mind that the relative impor- tance of detoxification and changes of the site of action are determined by the resistance genes that are available in a population as well as by the kind of selection pressure. If detoxification provides for a high degree of resistance and genes for this mecha- nism are relatively frequent, other mechanisms will not become apparent. If detoxification were ruled out-e.g., by the use of synergists-selection would operate again and resistance mechanisms of a differ- ent nature could be sought. Unfortunately it is essentially unpredictable whether such a search would be successful. There are several examples of the effect of an increased capacity for detoxification in a resistant strain being augmented by secondary changes. Since it is important in the context of this paper to realize that blocking detoxification will also overcome the effect of other dynamic resistance factors, two exam- ples are discussed below. The best analysed example of the interaction of penetration factors with detoxification is that re- ported by Sawicki (1970). It appears that a factor for reduced penetration, which when present alone only slightly affects resistance to phosphorothioates, causes-when combined with a detoxification fac- tor-an increase in resistance of 5-10 times the increase caused by the detoxification factor alone. A second example is provided by the kdr gene for DDT resistance: when this gene is present in the heterozygous condition and when a factor for a low degree of DDT dehydrochlorination is also present, high resistance results, whereas neither the hetero- zygous kdr gene nor the gene for low detoxi- fication produces appreciable resistance alone (Gri- golo & Oppenoorth, 1966). In both examples the factors increase the effectiveness of the detoxification mechanism by prolonging the time during which insecticide can be degraded-in the first case since it takes longer for insecticide to accumulate in the insect and in the second since the insect can stand an otherwise toxic concentration for a longer period of time. Plapp (in O'Brien & Yamamoto, 1970) pointed out that the factor for decreased penetration of DDT increased resistance due to kdr. The author believes that this is also the result of detoxification, which is present at a low level even in susceptible strains and which, as mentioned above, is still able to metabolize 50% of a 1-,ug dose of DDT in 18 hours. This indicates that the blocking of detoxi- 196 RESISTANCE IN INSECTS: ROLE OF METABOLISM AND USE OF SYNERGISTS 197 fication, even if not itself a resistance mechanism, can be effective because of its interaction with other factors. Detoxification as a factor determining selectivity Differences in detoxification capacity certainly are important for selectivity (see O'Brien, 1967, for a review), and if synergists were to be applied, this selectivity could be lost. Selectivity between spe- cies of insect is probably less urgently required than that between arthropods and vertebrates. The author sees no reason to expect any problems greater-or smaller-than those that generally occur in this respect with insecticides alone. Selectivity of synergists is quite feasible, and it should be men- tioned here that mammals generally metabolize the 1,2-methylenedioxyphenyl compounds more rapidly than insects (Casida et al., 1966; Wilkinson & Hicks, 1969). Greater variety of insecticides when synergists are included Many more compounds would become available as insecticides if detoxification could be prevented by the use of synergists. A classic example is offered in the work of Brooks & Harrison (1964), showing that many cyclodiene analogues, which would not normally be considered to be insecticides, are quite toxic if their degradation is blocked by sesamex.t The same undoubtedly is true of other classes of insecti- cide. Carbamates vary considerably in their toxicity for different species, and the toxicity of many of them is low. However, Metcalf et al. (1960), Moorefield (1958), and Weiden & Moorefield (1965) have demonstrated that if detoxification is sup- pressed their toxicity is greatly increased and that a good correlation with their potency as inhibitors of cholinesterase is obtained for many, but not all, compounds. Summarizing, if efficient blocking of detoxifica- tion were possible we might hope to overcome some of the problems of resistance, since detoxification and related dynamic factors are responsible for quite a large number of these problems; further- more, a much wider choice of compounds that are relatively labile in insects might become available as insecticides. Of course, the latter would be effective only when combined with a synergist, and this might have an advantage in reducing residue prob- lems as well as obvious disadvantages. t Names against which this symbol appears are identified in the Glossary on pages 445-446. RELATIVE IMPORTANCE OF DIFFERENT ROUTES OF DETOXIFICATION Since organophosphates, carbamates, and pyre- thrins are all esters it might be expected that they would be readily degradable by esterases, but this appears not to be so. It should be pointed out that older reports of hydrolytic degradation based solely on the nature of the products found cannot be trusted, since it has been shown, mainly by the work of Nakatsugawa & Dahm (1968), that these products can also be derived by oxidation, in which case their formation is dependent on the presence of NADPH. Parathion thus is oxidized by micro- somes and the products found are diethyl phos- phorothioate and p-nitrophenol. Since these pro- ducts are not really of a higher oxidation level, the author believes that one should assume the formation of an intermediate that is oxidized, this intermediate then giving rise to the products mentioned. There is only one report (Matsumura & Hogendijk, 1964) of the hydrolytic detoxification of a P = S com- pound (parathion), but this finding could not be confirmed by Nakatsugawa et al. (1969) or by Welling (1971). P = S compounds seem not to be attacked hydrolytically, except where additional ester groups are present, as in malathion. Hydrolytic degradation of phosphates does occur and is a resistance mechanism, but its role seems to be smaller than was originally thought. Oppenoorth & van Asperen (1960) proposed the " mutant alies- terase" theory, which postulates the existence, in many organophosphate-resistant houseflies, of an organophosphate-hydrolysing enzyme replacing an esterase present in susceptible flies. This theory was severely criticized by O'Brien (1966) on the grounds that the rate of detoxification found would be insuf- ficient to explain the degree of resistance, and that enzymatic action had not been proved since pro- ducts of hydrolysis had not been studied. This has recently been done by Welling (1971) for paraoxon, and this study has proved beyond doubt that in parathion-resistant strains there is a phosphatase that slowly forms diethyl phosphate and p-nitro- phenol. The enzyme is not dependent on NADPH, as has been suggested by Casida (1970), but is a true hydrolytic enzyme, which can still be irre- versibly inhibited by some organophosphates (e.g., the n-propyl homologue of paraoxon). This enzyme may not be responsible for all of the resistance (a gene responsible for increased oxidation is present F. J. OPPENOORTH on the same chromosome as the gene a for phos- phatase degradation), but Table 1 clearly shows that it contributes significantly. Table 1 Influence of sesamex on susceptibility of houseflies to diazoxon and paraoxon a LDso (pg per fly) Degree Resistance factorStrain ~~~~~ofand sex without with without with sesamex sesamex synergism sesamex sesamex diazoxon acb 3 0.024 0.007 3 _ ; 0.060 0.012 5 - - Fc d 2.0 0.04 50 83 6 W 3.0 0.06 50 50 5 Ei d 0.7 0.14 5 29 20 ; 2.0 0.24 8 33 20 paraoxon acb d 0.06 0.006 10 - - $ 0.13 0.010 13 - - Fc S 0.20 0.010 20 3.3 1.7 + 0.27 0.012 23 2.1 1.2 El 0.5 0.10 5 8.3 17 1.0 0.15 7 7.7 15 a Key to strains: acb, a susceptible strain; Fc, a strain with oxidative degradation as a cause of resistance; El, a strain with hydrolytic degradation and possibly also some oxidative degrada- tion as a cause of resistance. The nature of this phosphatase seems to indicate that the formation of a hydrolytic enzyme degrad- ing organophosphates is " difficult ". An alteration from an esterase that is irreversibly inhibited by the organophosphate (and can be said therefore to detoxify one molecule per enzyme molecule) into one with a turnover of 0.5 per minute seems to be the maximum attainable. The alteration into the malathion- and malaoxon-attacking carboxylester- ase is more efficient: the turnover number is 50 for malathion, but still only 0.3 for malaoxon (Welling, personal communication). Carbamate-resistance has also been reported to depend on an altered esterase (Plapp et al., 1964), which would probably mean hydrolytic detoxifica- tion of the carbamate. Since the author found a hydrolytic enzyme in the same strain degrading diazoxon, it seems more likely that this is connected with the low esterase found, and that the carbamate- resistance is a completely separate matter. It seems likely that in this strain carbamate-resistance was due to increased oxidation, and that the strain has closely linked genes for organophosphate hydrolysis and carbamate oxidation, which would be in accor- dance with later observations that both genes are located on the second chromosome. Hydrolytic degradation has been reported for some more insects, but apart from the case of malathion-resistance, where the carboxylic ester group is attacked, such indications are scarce. Hydro- lysis therefore appears to play a smaller role then was originally thought. The better technical approach to the study of microsomal oxidation that has followed the work of Terriere (1968), Tsukamoto & Casida (1967), and others has demonstrated that oxidases are of fairly general importance in insects, which had been masked by the presence of inhibitors of this system in insect tissues. The same view appears from a relatively simple toxicological approach. The application of inhibitors of microsomal oxidation such as piperonyl butoxide and sesamex in combination with insecti- cides can provide information on the role of this route of detoxification. This approach involves some danger of misinterpretations, of course, since there is some risk in assuming that such substances never inhibit other detoxifications and are always effective against microsomal oxidations. In fact, Plapp (in O'Brien & Yamamoto, 1970) noticed the fact of " cross synergism ", the effect of synergists on differ- ent insecticides that are mainly degraded by different mechanisms. The easiest explanation of this cross synergism is to assume a lack of specificity in the effect on detoxification mechanisms. However, this method has contributed considerably to the under- standing of the importance of microsomal oxidation. In the author's laboratory resistance to parathion and diazinon in the housefly has been shown to be due to phosphatases as well as to microsomal oxida- tions (see below), and the role of each varies con- siderably in different strains. An example of the difference in the effect of sesamex on such strains is given in Table 1. The method has been used extensively by Moorefield and others to elucidate the role of microsomal oxidation of carbamates, and recently by Keiding (1969, 1970) to study the relative role of microsomal oxidation in houseflies resistant to many different organophosphorus com- pounds. 198 RESISTANCE IN INSECTS: ROLE OF METABOLISM AND USE OF SYNERGISTS A systematic and complete overall picture of the importance of different detoxification routes is cer- tainly not yet available. In addition to hydrolysis and microsomal oxidation there are such special features as a DDT-dehydrochlorination enzyme, which is glutathione-dependent, and dealkylation enzymes present in the soluble cell fraction, which transfer alkyl (mainly methyl) groups from organo- phosphates to glutathione (Lewis, 1969; Holling- worth, in O'Brien & Yamamoto, 1970). Whereas the former is the earliest and classic example of a detoxifying enzyme causing resistance, the role of the latter is only just becoming recognized and will require much further study. The production of synergism by the blocking of this degradation has not, to the author's knowledge, yet been described. Owing to the considerable improvement in tech- niques in recent years, much progress in elucidating the relative importance of the different detoxification routes is to be expected in the near future. At pres- ent, the author's view is that microsomal oxidation ranks first, with hydrolysis and other mechanisms next, but of course this depends on the insecticides and insects involved and is only an overall estimate. Although there are synergists that act against prac- tically all of the known detoxification mechanisms, those that act against microsomal oxidation are pat ticularly varied and have been widely studied. However, only a new type recently found in the author's laboratory will be discussed here. CONSTANT SYNERGIST-INSECTICIDE RATIO VERSUS FIXED AMOUNTS OF SYNERGIST It has become common practice to study the effect of synergists on the toxicity of different insecticides by applying them in a certain fixed ratio to the insecticides. This is done partly since, when syner- gists are used in practice, this is the only possible method of application. However, for the purpose of investigating the role of certain types of detoxi- fication, or for comparing the synergistic effect on insecticides of different toxicity, this method has disadvantages. The degree and duration of inhibi- tion depend on the amount and toxicity of the insecticide applied, which causes unneccessary com- plications. A better method is to use a nontoxic dose that completely blocks detoxification for the desired period (i.e., the period necessary to cause the death of the insect), or, if this cannot be attained-and it often cannot-to use the closest possible approximation to such a dose. Further- more, only the inhibition of detoxification is desired, and further interactions between synergist and insec- ticide should be minimized in this type of investiga- tion. Since the synergist sometimes has an effect on penetration, application on separate areas of the integument can be helpful. A comparison of dif- ferent potential synergists now becomes, in the first place, a comparison of the amounts needed for a sufficient and persistent block of detoxification. These amounts depend on the properties that deter- mine transport to the site of action, on affinity for the site, and on resistance to degradation. This procedure gives a better evaluation than a com- parison of the minimum LD50 that can be attained, since with any reasonably good synergist this should be a fixed level determined by the properties of the insecticide in the absence of detoxification. Fig. 1 Products of microsomal degradation of paraoxon * 10.000 7 5 0 0 5000 2500 C I :I0.000 7500 50 00 2500 I * Microsomes of 20 abdomens of Fc flies were incubated with 10 ug of paraoxon (specific activity 47 600 dpm/jg) in the presence of NADPH in a closed system consisting of 2 ml of the incubation mixture, 2 tubes containing a solution of 5,5-dimethyl-1,3-cyclo- hexanedione (" dimedon "), and air. After incubation the reaction was stopped by heating to 70°C for 5 minutes. Excess acetaldehyde (5 jAlitres) was added and the air was circulated through the incubation mixture and the " dimedon ". The incubation mixture was then extracted with chloroform, and the water phase was transferred to TLC silica gel plates and developed with 9: 1 aceto- nitrile-water acidified with a drop of acetic acid. The silica gel was scraped off in zones and counted in a liquid scintillation counter. In the upper graph the peaks correspond to acetic acid and deethyl paraoxon. The lower graph shows the suppression of acetic acid formation by the addition of 0.1 ,ilitre of acetaldehyde at the beginn 'ng of the incubation period. 199 F. J. OPPENOORTH MICROSOMAL OXIDATION OF PARAOXON AND SYNERGISTIC ACTION OF P = S COMPOUNDS In conclusion, the results of some recent work carried out in the author's laboratory will be dis- cussed. Some of these have been published previ- ously (Oppenoorth, 1971; Oppenoorth et al., 1971). It had been shown by ElBashir & Oppenoorth (1969) that some resistant strains of the housefly have factors for increased microsomal oxidation oforgano- phosphorus compounds. Microsomes of these strains degraded paraoxon and diazoxon in vitro at about 10 times the rate of degradation by microsomes from susceptible strains. Surprisingly, the in vitro capa- city for the activation of parathion and diazinon into the PO-analogues is also increased in the resistant strains. The fact that the overall result of the increased activation and detoxification is resistance is probably attributable to a limited supply of the P= S compounds to the site where they are acti- vated, so that the increased capacity does not show up. It was shown that if the supply was increased by injecting the insecticide, the more rapid activation into the inhibitor in the resistant strain became apparent, and the resistant flies were killed more rapidly. The degradation of paraoxon by these strains was further analysed. Anion-exchange and thin-layer chromatography of the in vitro incubates of micro- somes showed the presence of two 3H-labelled prod- ucts, which were identified in the two chromato- graphic systems by cochromatography with 14C-label- led acetic acid and with deethyl paraoxon. Since acetic acid is unlikely to be the primary product formed by microsomal oxidation, its possible forma- tion from acetaldehyde was investigated. It had been shown by Donninger (1967) to be a product of oxidation by rabbit liver microsomes. Fig. 1 shows Fig. 2 Inhibition of microsomal oxidation of paraoxon * without pre-incubation 0.5 0.4 - sesamex 3jg 0.3 0.2 -0.1 - , 0 CAL V a C: 0 x 0.5 ,0. S VI 0.05 tlg0 L0.4 S -.5ji 0.3 0.2 0.1 0 5 10 15 20 incubation time (min) with pre-incubation sesamex 3,jg S V, 0.03 pg _ - l i 0 5 10 15 pre-incubation time (min) *-* control activity *- inhibited activity * Microsomes of 10 abdomens were incubated at 27 c in 10 ml of phosphate buffer (pH 7.5) with 2 mg of NADPH and 20 mg of albumin fraction V. Paraoxon (10 jig) was added either (1) at the beginning of incubation, the incubation being ended after various periods of time (left), or (2) after varying periods of preincubation, after which the degradation during a 1 5-minute incubation period was measured(right). The left-hand and right-hand graphs do not correspond quantitatively, since they represent different experiments. 200 RESISTANCE IN INSECTS: ROLE OF METABOLISM AND USE OF SYNERGISTS 201 that acetaldehyde is indeed the precursor of acetic acid. Since measuring the rate of activation involves estimating the products, which might in turn be further degraded, paraoxon degradation was studied in the presence of parathion. Quite surprisingly a very strong inhibition was found, and it turned out that the '50 values of parathion and diazinon combined with 10 /tg or 60 ,ug of paraoxon were of the order of 0.1 jug. Since it seemed attractive to study a possible synergistic effect of P= S com- pounds on phosphates, without complications aris- ing from their own toxicity, a nontoxic analogue was synthesized, 0, 0-diethyl 0-phenyl phosphoro- thioate (SV1). It was found to inhibit paraoxon degradation by microsomes in vitro to an even greater extent than did parathion, the I) being 0.025 ,ug. Fig. 2 shows a comparison of inhibition by sesamex and by SV1 with and without preincuba- tion. From these and other experiments it appears that SV1 is by far the better inhibitor. The presence of albumin in the incubation mixture and preincu- bation with the inhibitor increase inhibition by sesamex to a greater extent than they increase inhibition by SV1. Table 2 Synergistic activity of sesamex and SVi LDso (ng per 3 fly)a Synergistic factor Insecticide Synergit.for the following strains for the following strainsInsectic de Synergist SRS Fc Nic SRS Fc Nic paraoxon sesamex 7 (2) 15 (10) 20 (10) 1.1 1 3 5 SVi 3 (2) 3 (10) 2 (10) 2.7 65 50 parathion sesamex 25 ( 1) 6 13 (110) 12 SVi 2 (5) 8 ( 1) 5 19 4 (10) 5 (10) 38 14 diazinon sesamex 50 (20) 30 SV, 6 (10) 250 Talcordt sesamex 40 (2) 150 (10) > 500 (10) 4 28 220 SVi 15 (2) 15 (10) 20 (10) 10 300 > 500 DDT sesamex 400 (10) > 25 SVi 200 (10) > 50 a The figures in parentheses indicate the amounts of synergist in pg. Table 2 shows a comparison of the synergistic action of sesamex and of SV1 on different insecti- cides in the susceptible strain SRS and in the two strains whose resistance results from microsomal oxidation, Fc and Nic. It was found that SV1 was superior in all cases tested. Talcord t was also found to be synergized in Blattella germanica. Phosphoro- thioates have been found to synergize carbamates in the housefly and the granary weevil; '- a promis- ing finding was that they did not synergize the carbamates in rats. The importance of the observation that phosphoro- thioates can be used as synergists that block micro- somal degradation is that they represent a large group of relatively cheap chemicals from which compounds with suitable physical and toxicological properties can be selected. Further tests with other members of the group and on more insects are, of course, required for a complete evaluation of their importance. There is evidence (Oppenoorth et al., 1971) that the site of the activation reaction is different from that of the detoxification reactions where the syner- gistic action is effected. 1 Schering (1962) Deutsches Patentamt Auslegeschrift 1128219 and 1137895 (Sch 28796 IV a/451 and 29236 IV a/45 1). 202 F. J. OPPENOORTH REFERENCES Brooks, G. T. (1960) Nature (Lond.), 186, 96-98 Brooks, G. T. & Harrison, A. (1964) Biochem. Pharmacol., 13, 827-840 Casida, J. E. (1969) In: Gillette, J. R. et al. Microsomes and drug oxidations, New York, Academic Press, pp. 517-531 Casida, J. E. (1970) J. agric. Food Chem., 18, 753-772 Casida, J. E., Engel, J. L., Essac, E. G., Kamienski, K. W. & Kuwatsuka, S. (1967) Science, 153, 1130-1133 Donninger, C. (1967) Biochem. J., 102, 26P-27P ElBashir, S. & Oppenoorth, F. J. (1969) Nature (Lond.), 223, 210-211 Grigolo, A. & Oppenoorth, F. J. (1966) Genetica, 37, 159-170 Hodgson, E., ed. (1968) Enzymatic oxidations oftoxicants, Raleigh, North Carolina State University Press Keiding, J. (1969) In: Annual report of the Danish Pest Infestation Laboratory, Lyngby Keiding, J. (1970) In: Annual report of the Danish Pest Infestation Laboratory, Lyngby Lewis, J. B. (1969) Nature (Lond.), 224, 917-918 Matsumura, F. & Hogendijk, C. J. (1964) J. agric. Food Chem., 12, 447-453 Metcalf, R. L. (1967) Ann. Rev. Ent., 12, 229-256 Metcalf, R. L., Fukuto, T. R. & Winton, M. Y. (1960) J. econ. Ent., 53, 828-832 Moorefield, H. H. (1958) Contrib. Boyce Thompson Inst., 19, 501-507 Nakatsugawa, T. & Dahm, P. A. (1968) Biochem. Pharmacol., 17, 1517-1528 Nakatsugawa, T., Tolman, N. M. & Dahm, P. A. (1969) J. econ. Ent., 62, 408-411 O'Brien, R. D. (1966) Ann. Rev. Ent., 11, 369-402 O'Brien, R. D. (1967) Insecticides, action and metabolism, New York, Academic Press O'Brien, R. D. & Yamamoto, I. (1970) Biochenmical toxicology of insecticides, New York, Academic Press Oppenoorth, F. J. (1967) Ent. exp. appl., 10, 75-86 Oppenoorth, F. J. (1971) In: NATO conference on toxicity of pesticides used on livestock, Lethbridge, Canada, 1970 (in press) Oppenoorth, F. J. & Asperen, K. van (1960) Science, 132, 298-299 Oppenoorth, F. J., Voerman, S., Welling, W., Houx, N. W. H. & Wouters van den Oudenweyer, J. (1971) Nature (Lond.) (in press) Plapp, F. W., Chapman, G. A. & Bigley, W. S. (1964) J. econ. Ent., 57, 692-695 Sawicki, R. M. (1970) Pestic. Sci., 1, 84-87 Smissaert, H. R. (1964) Science, 143, 129-131 Smissaert, H. R., Voerman, S., Oostenbrugge, L. & Renooy, N. (1970) J. agric. Food Chem., 18, 66-75 Terriere, L. C. (1968) Ann. Rev. Ent., 13, 75-98 Tsukamoto, M. & Casida, J. E. (1967) J. econ. Ent., 60, 617-619 Weiden, M. H. J. & Moorefield, H. H. (1965) J. agric. Food Chem., 13, 200-204 Welling, W. (1971) Pestic. Biochem. Physiol., 1 (in press) Wharton, R. H. & Roulston, W. J. (1970) Ann. Rev. Ent., 15, 381-404 Wilkinson, C. F. (1968) Wld Rev. Pest Control, 7, 155- 168 Wilkinson, C. F. (1971) Bull. Wld Hlth Org., 44, 171-190 Wilkinson, C. F. & Hicks, L. J. (1969) J. agric. Food Chem., 17, 829-836 Winteringham, F. P. W. (1969) Ann. Rev. Ent., 14, 409-442 Winteringham, F. P. W. & Harrison, A. (1959) Nature (Lond.), 184, 608-610 DISCUSSION DAUTERMAN: Is there any difference in the rates of degradation of SV1 in resistant and in susceptible house- flies ? OPPENOORTH: We do not know. SV1 is slightly toxic to susceptible flies and less so to resistant strains relying on microsomal oxidation, which indicates that there may be such a difference. HEILBRONN: Dr Dahm mentioned a cholinesterase- reactivating factor. Is there any evidence on the nature of this factor? Is it possible that it is simply spontaneous reactivation ? DAHM: This factor was suggested by Mengle & O'Brien (Biochem. J., 1960, 75, 201), who indicated that spontane- ous recovery of housefly-brain acetylcholinesterase did not occur after several organophosphorus compounds were applied to the tip of the housefly abdomen.